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Master

Affiliation (Master)

  • Faculty of Medicine　Physiological Science　Anatomy

Affiliation (Master)

  • Faculty of Medicine　Physiological Science　Anatomy

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Profile and Settings

Profile and Settings

  • Name (Japanese)

    WATANABE

  • Name (Kana)

    Masahiko

  • Name

    200901014585072736

Alternate Names

Achievement

Research Interests

  • 包括脳ネットワーク・研究集会委員会   包括脳ネットワーク・リソース・技術開発委員会   包括脳ネットワーク・包括支援委員会・脳神経回路機能   包括脳ネットワーク   統合脳・神経回路機能   嗅神経被覆グリア   遺伝子欠損マウス   登上繊維   錐体細胞   ラット   GTP結合蛋白   GluRδ2   可塑性   シナプス可塑性   プルキン工細胞   バーグマングリア   グルタミン酸トランスポーター   3PGDH   星状膠細胞   海馬   NMDA受容体   NMDA受容体チャネル   グルタミン酸   脳   ノックアウトマウス   平行線維   シナプス   登上線維   免疫組織化学   マウス   シナプス形成   発達   プルキンエ細胞   小脳   グルタミン酸受容体   

Research Areas

  • Life sciences / Neuroscience - general
  • Life sciences / Neuroanatomy and physiology

Research Experience

  • 1998 - Today 北海道大学 医学(系)研究科(研究院) 教授
  • 1992 - 1998 Hokkaido University School of Medicine
  • 1990 - 1992 東北大学医学部 解剖学第２講座 助手
  • 1988 - 1989 Kanazawa University

Education

  • 1984/04 - 1988/03  University of Tsukuba  Graduate School of Medicine
  • 1978/04 - 1984/03  Tohoku University  School of Medicine

Awards

  • 2024/06 The Japanese Society of Microscopy Setou Award
     Molecular and cellular basis of activity-dependent synaptic circuit development
  • 2023/06 The Akiyama Life Science Foundation Akiyama Life Science Foundation Award
     Molecular-anatomical basis for synaptic circuit development by glutamatergic signaling system 
    受賞者: Masahiko Watanabe
  • 2019/10 伊藤医薬学術交流財 伊藤太郎学術賞
     グルタミン酸伝達系による神経回路発達の分子細胞基盤
  • 2015/07 日本神経科学学会 時実利彦記念賞
     「神経活動依存的な 神経回路発達と回路機能発現に関する分子解剖学的研究」 
    受賞者: 渡辺 雅彦
  • 2015/03 北海道大学 北海道大学総長研究賞（優秀賞）
     
    受賞者: 渡辺 雅彦
  • 2006/03 北海道大学医学研究科 北海道大学医学研究科優秀研究賞
     「小脳シナプス回路網の競合的発達を制御する分子機構の解明」 
    受賞者: 渡辺 雅彦
  • 1996/04 日本解剖学会 日本解剖学会奨励賞
     「中枢神経系の発達とNMDA型グルタミン酸受容体発現調節」 
    受賞者: 渡辺 雅彦
  • 1992/01 東北医学会 東北医学会奨励賞
     「14-3-3蛋白の遺伝子解析とその脳内発現」 
    受賞者: 渡辺 雅彦

Published Papers

  • Takafumi Kawai, Shin Morioka, Haruhiko Miyata, Rizki Tsari Andriani, Sharmin Akter, Gabriel Toma, Tatsuya Nakagawa, Yuki Oyama, Rie Iida-Norita, Junko Sasaki, Masahiko Watanabe, Kenji Sakimura, Masahito Ikawa, Takehiko Sasaki, Yasushi Okamura
    Nature Communications 15 (1) 2024/08/24
  • Hisako Nakayama, Taisuke Miyazaki, Manabu Abe, Maya Yamazaki, Yoshinobu Kawamura, Myeongjeong Choo, Kohtarou Konno, Shinya Kawata, Naofumi Uesaka, Kouichi Hashimoto, Mariko Miyata, Kenji Sakimura, Masahiko Watanabe, Masanobu Kano
    Communications biology 7 (1) 806 - 806 2024/07/03 
    Developmental synapse elimination is crucial for shaping mature neural circuits. In the neonatal mouse cerebellum, Purkinje cells (PCs) receive excitatory synaptic inputs from multiple climbing fibers (CFs) and synapses from all but one CF are eliminated by around postnatal day 20. Heterosynaptic interaction between CFs and parallel fibers (PFs), the axons of cerebellar granule cells (GCs) forming excitatory synapses onto PCs and molecular layer interneurons (MLIs), is crucial for CF synapse elimination. However, mechanisms for this heterosynaptic interaction are largely unknown. Here we show that deletion of AMPA-type glutamate receptor functions in GCs impairs CF synapse elimination mediated by metabotropic glutamate receptor 1 (mGlu1) signaling in PCs. Furthermore, CF synapse elimination is impaired by deleting NMDA-type glutamate receptors from MLIs. We propose that PF activity is crucial for CF synapse elimination by directly activating mGlu1 in PCs and indirectly enhancing the inhibition of PCs through activating NMDA receptors in MLIs.
  • Masami Arai, Etsuko Suzuki, Satoshi Kitamura, Momoyo Otaki, Kaori Kanai, Miwako Yamasaki, Masahiko Watanabe, Yuki Kambe, Koshi Murata, Yuuki Takada, Tetsu Arisawa, Kenta Kobayashi, Rei Tajika, Tomoyuki Miyazaki, Masahiro Yamaguchi, Michael Lazarus, Yu Hayashi, Shigeyoshi Itohara, Alban de Kerchove d'Exaerde, Hiroyuki Nawa, Ryang Kim, Haruhiko Bito, Toshihiko Momiyama, Daiki Masukawa, Yoshio Goshima
    The Journal of neuroscience : the official journal of the Society for Neuroscience 44 (11) 2024/03/13 
    Dopamine neurons play crucial roles in pleasure, reward, memory, learning, and fine motor skills and their dysfunction is associated with various neuropsychiatric diseases. Dopamine receptors are the main target of treatment for neurologic and psychiatric disorders. Antipsychotics that antagonize the dopamine D2 receptor (DRD2) are used to alleviate the symptoms of these disorders but may also sometimes cause disabling side effects such as parkinsonism (catalepsy in rodents). Here we show that GPR143, a G-protein-coupled receptor for L-3,4-dihydroxyphenylalanine (L-DOPA), expressed in striatal cholinergic interneurons enhances the DRD2-mediated side effects of haloperidol, an antipsychotic agent. Haloperidol-induced catalepsy was attenuated in male Gpr143 gene-deficient (Gpr143-/y ) mice compared with wild-type (Wt) mice. Reducing the endogenous release of L-DOPA and preventing interactions between GPR143 and DRD2 suppressed the haloperidol-induced catalepsy in Wt mice but not Gpr143-/y mice. The phenotypic defect in Gpr143-/y mice was mimicked in cholinergic interneuron-specific Gpr143-/y (Chat-cre;Gpr143flox/y ) mice. Administration of haloperidol increased the phosphorylation of ribosomal protein S6 at Ser240/244 in the dorsolateral striatum of Wt mice but not Chat-cre;Gpr143flox/y mice. In Chinese hamster ovary cells stably expressing DRD2, co-expression of GPR143 increased cell surface expression level of DRD2, and L-DOPA application further enhanced the DRD2 surface expression. Shorter pauses in cholinergic interneuron firing activity were observed after intrastriatal stimulation in striatal slice preparations from Chat-cre;Gpr143flox/y mice compared with those from Wt mice. Together, these findings provide evidence that GPR143 regulates DRD2 function in cholinergic interneurons and may be involved in parkinsonism induced by antipsychotic drugs.
  • Izumi Iida, Kohtarou Konno, Rie Natsume, Manabu Abe, Masahiko Watanabe, Kenji Sakimura, Miho Terunuma
    Scientific reports 14 (1) 4521 - 4521 2024/02/24 
    Kainate receptors (KARs) are one of the ionotropic glutamate receptors in the central nervous system (CNS) comprised of five subunits, GluK1-GluK5. There is a growing interest in the association between KARs and psychiatric disorders, and there have been several studies investigating the behavioral phenotypes of KAR deficient mice, however, the difference in the genetic background has been found to affect phenotype in multiple mouse models of human diseases. Here, we examined GluK1-5 single KO mice in a pure C57BL/6N background and identified that GluK3 KO mice specifically express anxiolytic-like behavior with an alteration in dopamine D2 receptor (D2R)-induced anxiety, and reduced D2R expression in the striatum. Biochemical studies in the mouse cortex confirmed that GluK3 subunits do not assemble with GluK4 and GluK5 subunits, that can be activated by lower concentration of agonists. Overall, we found that GluK3-containing KARs function to express anxiety, which may represent promising anti-anxiety medication targets.
  • Izumi Iida, Kohtarou Konno, Rie Natsume, Manabu Abe, Masahiko Watanabe, Kenji Sakimura, Miho Terunuma
    Scientific Reports 14 (1) 2024/02/24 
    Abstract Kainate receptors (KARs) are one of the ionotropic glutamate receptors in the central nervous system (CNS) comprised of five subunits, GluK1-GluK5. There is a growing interest in the association between KARs and psychiatric disorders, and there have been several studies investigating the behavioral phenotypes of KAR deficient mice, however, the difference in the genetic background has been found to affect phenotype in multiple mouse models of human diseases. Here, we examined GluK1-5 single KO mice in a pure C57BL/6N background and identified that GluK3 KO mice specifically express anxiolytic-like behavior with an alteration in dopamine D2 receptor (D2R)-induced anxiety, and reduced D2R expression in the striatum. Biochemical studies in the mouse cortex confirmed that GluK3 subunits do not assemble with GluK4 and GluK5 subunits, that can be activated by lower concentration of agonists. Overall, we found that GluK3-containing KARs function to express anxiety, which may represent promising anti-anxiety medication targets.
  • Tayo Katano, Kohtarou Konno, Keizo Takao, Manabu Abe, Akari Yoshikawa, Tsuyoshi Miyakawa, Kenji Sakimura, Masahiko Watanabe, Seiji Ito, Takuya Kobayashi
    Scientific reports 13 (1) 22027 - 22027 2023/12/12 
    Brain-enriched guanylate kinase-associated protein (BEGAIN) is highly enriched in the post-synaptic density (PSD) fraction and was identified in our previous study as a protein associated with neuropathic pain in the spinal dorsal horn. PSD protein complexes containing N-methyl-D-aspartate receptors are known to be involved in neuropathic pain. Since these PSD proteins also participate in learning and memory, BEGAIN is also expected to play a crucial role in this behavior. To verify this, we first examined the distribution of BEGAIN in the brain. We found that BEGAIN was widely distributed in the brain and highly expressed in the dendritic regions of the hippocampus. Moreover, we found that BEGAIN was concentrated in the PSD fraction of the hippocampus. Furthermore, immunoelectron microscopy confirmed that BEGAIN was localized at the asymmetric synapses. Behavioral tests were performed using BEGAIN-knockout (KO) mice to determine the contribution of BEGAIN toward learning and memory. Spatial reference memory and reversal learning in the Barns circular maze test along with contextual fear and cued fear memory in the contextual and cued fear conditioning test were significantly impaired in BEGAIN-KO mice compared to with those in wild-type mice. Thus, this study reveals that BEGAIN is a component of the post-synaptic compartment of excitatory synapses involved in learning and memory.
  • Yoshifumi Abe, Sho Yagishita, Hiromi Sano, Yuki Sugiura, Masanori Dantsuji, Toru Suzuki, Ayako Mochizuki, Daisuke Yoshimaru, Junichi Hata, Mami Matsumoto, Shu Taira, Hiroyoshi Takeuchi, Hideyuki Okano, Nobuhiko Ohno, Makoto Suematsu, Tomio Inoue, Atsushi Nambu, Masahiko Watanabe, Kenji F Tanaka
    Cell reports. Medicine 4 (10) 101208 - 101208 2023/10/17 
    Dyskinesia is involuntary movement caused by long-term medication with dopamine-related agents: the dopamine agonist 3,4-dihydroxy-L-phenylalanine (L-DOPA) to treat Parkinson's disease (L-DOPA-induced dyskinesia [LID]) or dopamine antagonists to treat schizophrenia (tardive dyskinesia [TD]). However, it remains unknown why distinct types of medications for distinct neuropsychiatric disorders induce similar involuntary movements. Here, we search for a shared structural footprint using magnetic resonance imaging-based macroscopic screening and super-resolution microscopy-based microscopic identification. We identify the enlarged axon terminals of striatal medium spiny neurons in LID and TD model mice. Striatal overexpression of the vesicular gamma-aminobutyric acid transporter (VGAT) is necessary and sufficient for modeling these structural changes; VGAT levels gate the functional and behavioral alterations in dyskinesia models. Our findings indicate that lowered type 2 dopamine receptor signaling with repetitive dopamine fluctuations is a common cause of VGAT overexpression and late-onset dyskinesia formation and that reducing dopamine fluctuation rescues dyskinesia pathology via VGAT downregulation.
  • Hirokazu Ishii, Kohei Otomo, Ching-Pu Chang, Miwako Yamasaki, Masahiko Watanabe, Hiroyuki Yokoyama, Tomomi Nemoto
    PLOS ONE 2023/08/24 [Refereed][Not invited]
  • Kohtarou Konno, Miwako Yamasaki, Taisuke Miyazaki, Masahiko Watanabe
    Science advances 9 (28) eadf7084  2023/07/14 
    The gold-standard fixative for immunohistochemistry is 4% formaldehyde; however, it limits antibody access to target molecules that are buried within specialized neuronal components, such as ionotropic receptors at the postsynapse and voltage-gated ion channels at the axon initial segment, often requiring additional antigen-exposing techniques to detect their authentic signals. To solve this problem, we used glyoxal, a two-carbon atom di-aldehyde. We found that glyoxal fixation greatly improved antibody penetration and immunoreactivity, uncovering signals for buried molecules by conventional immunohistochemical procedures at light and electron microscopic levels. It also enhanced immunosignals of most other molecules, which are known to be detectable in formaldehyde-fixed sections. Furthermore, we unearthed several specific primary antibodies that were once judged to be unusable in formaldehyde-fixed tissues, allowing us to successfully localize so far controversial synaptic adhesion molecule Neuroligin 1. Thus, glyoxal is a highly effective fixative for immunostaining, and a side-by-side comparison of glyoxal and formaldehyde fixation is recommended for routine immunostaining in neuroscience research.
  • Yuto Okuno, Kazuto Sakoori, Kyoko Matsuyama, Miwako Yamasaki, Masahiko Watanabe, Kouichi Hashimoto, Takaki Watanabe, Masanobu Kano
    Frontiers in Molecular Neuroscience 16 2023/06/22 
    Functionally mature neural circuits are shaped during postnatal development by eliminating redundant synapses formed during the perinatal period. In the cerebellum of neonatal rodents, each Purkinje cell (PC) receives synaptic inputs from multiple (more than 4) climbing fibers (CFs). During the first 3 postnatal weeks, synaptic inputs from a single CF become markedly larger and those from the other CFs are eliminated in each PC, leading to mono-innervation of each PC by a strong CF in adulthood. While molecules involved in the strengthening and elimination of CF synapses during postnatal development are being elucidated, much less is known about the molecular mechanisms underlying CF synapse formation during the early postnatal period. Here, we show experimental evidence that suggests that a synapse organizer, PTPδ, is required for early postnatal CF synapse formation and the subsequent establishment of CF to PC synaptic wiring. We showed that PTPδ was localized at CF-PC synapses from postnatal day 0 (P0) irrespective of the expression of Aldolase C (Aldoc), a major marker of PC that distinguishes the cerebellar compartments. We found that the extension of a single strong CF along PC dendrites (CF translocation) was impaired in global PTPδ knockout (KO) mice from P12 to P29-31 predominantly in PCs that did not express Aldoc [Aldoc (–) PCs]. We also demonstrated via morphological and electrophysiological analyses that the number of CFs innervating individual PCs in PTPδ KO mice were fewer than in wild-type (WT) mice from P3 to P13 with a significant decrease in the strength of CF synaptic inputs in cerebellar anterior lobules where most PCs are Aldoc (–). Furthermore, CF-specific PTPδ-knockdown (KD) caused a reduction in the number of CFs innervating PCs with decreased CF synaptic inputs at P10-13 in anterior lobules. We found a mild impairment of motor performance in adult PTPδ KO mice. These results indicate that PTPδ acts as a presynaptic organizer for CF-PC formation and is required for normal CF-PC synaptic transmission, CF translocation, and presumably CF synapse maintenance predominantly in Aldoc (–) PCs. Furthermore, this study suggests that the impaired CF-PC synapse formation and development by the lack of PTPδ causes mild impairment of motor performance.
  • Etsuko Tarusawa, Saki Hasegawa, Daisuke Noda, Nanami Kawamura, Yugo Fukazawa, Masahiko Watanabe, Takahiro Hirabayashi, Takeshi Yagi
    2023/04/15 
    SUMMARY Clustered protocadherins (cPcdhs) are candidates for the neural circuit formation; however, the localization of cPcdhs in pre- and post-synaptic compartments has not been well characterized. Here we examined the localization of cPcdhγ proteins in the mouse hippocampal CA1 region using light and electron microscopy. From postnatal day 7 to 21, cPcdhγ immunosignals were detected in approximately 40–60% of spines of pyramidal cells. SDS-digested freeze-fracture replica labelling revealed that cPcdhγ immunolabeling was found in 50% of PSD 95-positive postsynaptic profiles but only in less than 10% of vGluT1-positive pre-synaptic terminals. Interestingly, cPcdhγ-positive pre-synaptic terminal was exclusively accompanied by cPcdhγ-positive postsynaptic counterpart. In addition, electrophysiological investigations revealed that the miniature excitatory postsynaptic current frequency in cPcdhγ cKO mice was significantly higher than that in wild-type mice. These results suggest that cPcdhγ proteins are unequally distributed in the pre- and post-synaptic membrane during neural circuit development and regulate the number of excitatory synapses.
  • Raphaëlle Quillet, Allen C Dickie, Erika Polgár, Maria Gutierrez-Mecinas, Andrew M Bell, Luca Goffin, Masahiko Watanabe, Andrew J Todd
    Scientific reports 13 (1) 5891 - 5891 2023/04/11 
    Excitatory interneurons in the superficial dorsal horn (SDH) are heterogeneous, and include a class known as vertical cells, which convey information to lamina I projection neurons. We recently used pro-NPFF antibody to reveal a discrete population of excitatory interneurons that express neuropeptide FF (NPFF). Here, we generated a new mouse line (NPFFCre) in which Cre is knocked into the Npff locus, and used Cre-dependent viruses and reporter mice to characterise NPFF cell properties. Both viral and reporter strategies labelled many cells in the SDH, and captured most pro-NPFF-immunoreactive neurons (75-80%). However, the majority of labelled cells lacked pro-NPFF, and we found considerable overlap with a population of neurons that express the gastrin-releasing peptide receptor (GRPR). Morphological reconstruction revealed that most pro-NPFF-containing neurons were vertical cells, but these differed from GRPR neurons (which are also vertical cells) in having a far higher dendritic spine density. Electrophysiological recording showed that NPFF cells also differed from GRPR cells in having a higher frequency of miniature EPSCs, being more electrically excitable and responding to a NPY Y1 receptor agonist. Together, these findings indicate that there are at least two distinct classes of vertical cells, which may have differing roles in somatosensory processing.
  • Natsumi Hoshino*, Takashi Kanadome*(*co-first), Mizuho Itoh, Ryosuke Kaneko, Yukiko U. Inoue, Takayoshi Inoue, Takahiro Hirabayashi, Masahiko Watanabe, Tomoki Matsuda, Takeharu Nagai, Etsuko Tarusawa, Takeshi Yagi
    bioRxiv 2023/04 
    Abstract Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in CNS. However, its interactions have yet not been visualized in neurons. We previously reported PcdhγB2-FRET probes to be applicable only for cell lines. Herein, we newly designed PcdhγB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single PcdhγB2 molecule and succeeded in visualizing PcdhγB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals were observed at contact sites between neurites and eliminated by EGTA addition. Live imaging revealed that the FRET-negative γB2 signals were rapidly moving along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe would allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism. Significance Statement We visualize the Pcdh homophilic interaction using a novel FRET-based probe, and reveal that the homophilically interacting Pcdh proteins are found at contact sites between the neurites and roots of neurites from the soma, and are stable at a location. Additionally, in neurons, Pcdh proteins are located at synapses but rarely interact homophilically.
  • Yasuhiro Suzuki, Chihiro Nakamoto, Izumi Watanabe-Iida, Masahiko Watanabe, Tomonori Takeuchi, Toshikuni Sasaoka, Manabu Abe, Kenji Sakimura
    Neurochemistry international 165 105517 - 105517 2023/03/11 
    NMDA-type glutamate receptors (NMDARs) are tetrameric channel complex composed of two subunits of GluN1, which is encoded by a single gene and diversified by alternative splicing, and two subunits from four subtypes of GluN2, leading to various combinations of subunits and channel specificities. However, there is no comprehensive quantitative analysis of GluN subunit proteins for relative comparison, and their compositional ratios at various regions and developmental stages have not been clarified. Here we prepared six chimeric subunits, by fusing an N-terminal side of the GluA1 subunit with a C-terminal side of each of two splicing isoforms of GluN1 subunit and four GluN2 subunits, with which titers of respective NMDAR subunit antibodies could be standardized using common GluA1 antibody, thus enabling quantification of relative protein levels of each NMDAR subunit by western blotting. We determined relative protein amounts of NMDAR subunits in crude, membrane (P2) and microsomal fractions prepared from the cerebral cortex, hippocampus and cerebellum in adult mice. We also examined amount changes in the three brain regions during developmental stages. Their relative amounts in the cortical crude fraction were almost parallel to those of mRNA expression, except for some subunits. Interestingly, a considerable amount of GluN2D protein existed in adult brains, although its transcription level declines after early postnatal stages. GluN1 was larger in quantity than GluN2 in the crude fraction, whereas GluN2 increased in the membrane component-enriched P2 fraction, except in the cerebellum. These data will provide the basic spatio-temporal information on the amount and composition of NMDARs.
  • Premrudee Hemha, Surang Chomphoo, Yada Polsan, Kaoru Goto, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Histochemistry and cell biology 159 (3) 293 - 307 2023/03 
    Many signaling enzymes have multiple isozymes that are localized discretely at varying molecular levels in different compartments of cells where they play specific roles. In this study, among the various isozymes of phospholipase C (PLC) and diacylglycerol kinase (DGK), which work sequentially in the phosphoinositide cycle, both PLCβ3 and DGKι were found in renal brush-border microvilli, but found to replace each other along the proximal tubules: PLCβ3 in the proximal straight tubules (PST) of the outer stripe of the outer medulla (OSOM) and the medullary ray (MR), and DGKι in the proximal convoluted tubules (PCT) in the cortex and partially in the PST of the MR. Following daily injection of gentamicin for 1 week, the expression of PLCβ3 and DGKι was transiently enhanced, as demonstrated by western blot, and the increases were found to most likely occur in their original sites, that is, in the brush borders of the PST for PLCβ3 and in the PCT for DGKι. These findings showing differences in expression along the tubules suggest that the exertion of reabsorption and secretion through various ion channels and transporters in the microvillus membranes and the maintenance of microvillus turnover are regulated by a PLC-mediated signal with the balance shifted toward relative augmentation of the DAG function in the PST, and by a DGK-mediated signal with the balance shifted to relative augmentation of the phosphatidic acid function in the PCT. Our results also suggest the possibility that these isozymes are potential diagnostic signs for the early detection of acute kidney injury caused by gentamicin.
  • Sawetree Pakkarato, Hiroyuki Sakagami, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo, Surang Chomphoo
    Microscopy research and technique 86 (3) 351 - 358 2023/03 
    It is known that phosphatidylinositol phosphate 5 kinase (PIP5K) γ and phospholipase C (PLC) β3, working sequentially in the phosphoinositide cycle, are localized in dorsal root ganglion (DRG) somata and are involved in the regulation of pain and related sensations. However, the sites of their involvement have remained to be clarified. In the present study, immunoreactivity for PLCβ3 was distinct only in the central process of mouse DRG, but not in its peripheral process, in contrast to distinct PIP5Kγ-immunoreactivity in both peripheral and central DRG processes. No nerve terminals showing immunoreactivity for PLCβ3 were detected in any peripheral sensory fields, similar to PIP5Kγ-immunoreactivity. In DRG somata, PIP5Kγ-immunoreactivity was rather confined to the neurolemma in which dots and threads were discerned in 3D bright field light microscopy. This feature well corresponded to its discontinuous localization along the plasma membranes in immuno-electron microscopy. In contrast, PLCβ3-immunoreactivity occurred diffusely throughout the somata, but did not take distinct appearance of immunoreaction on neurolemma or plasma membranes, unlike PIP5Kγ-immunoreactivity. In addition, satellite glial cells were immunonegative for PLCβ3, but immunopositive for PIP5Kγ. The involvement of PLCβ3 in regulation of pain and related sensations is thus suggested to be mainly exerted at levels of the DRG soma and its upstream, but to be less significant in the peripheral sensory fields, similar to PIP5Kγ. The possibility is also suggested that PIP, PIP5Kγ-target, is localized heterogeneously, but PIP2, PLCβ3-target, is localized homogenously over the plane of the neuronal plasma membranes. RESEARCH HIGHLIGHTS: PIP5Kγ, different from PLCβ3, was localized heterogeneously on neuronal membranes, and this difference was demonstrated in 3D-bright field immuno-light and electron microscopy. Either PIP5Kγ or PLCβ3 was not detected in peripheral nerve terminals.
  • Maria Gutierrez-Mecinas, Éva Kókai, Erika Polgár, Raphaëlle Quillet, Heather F Titterton, Greg A Weir, Masahiko Watanabe, Andrew J Todd
    Neuroscience 510 60 - 71 2023/02/01 
    Gastrin-releasing peptide (GRP) in the spinal dorsal horn acts on the GRP receptor, and this signalling mechanism has been strongly implicated in itch. However, the source of GRP in the dorsal horn is not fully understood. For example, the BAC transgenic mouse line GRP::GFP only captures around 25% of GRP-expressing cells, and Grp mRNA is found in several types of excitatory interneuron. A major limitation in attempts to identify GRP-expressing neurons has been that antibodies against GRP cross-react with other neuropeptides, including some that are expressed by primary afferents. Here we have developed two antibodies raised against different parts of the precursor protein, pro-GRP. We show that labelling is specific, and that the antibodies do not cross-react with neuropeptides in primary afferents. Immunoreactivity was strongest in the superficial laminae, and the two antibodies labelled identical structures, including glutamatergic axons and cell bodies. The pattern of pro-GRP-immunoreactivity varied among different neurochemical classes of excitatory interneuron. Cell bodies and axons of all GRP-GFP cells were labelled, confirming reliability of the antibodies. Among the other populations, we found the highest degree of co-expression (>50%) in axons of NPFF-expressing cells, while this was somewhat lower (10-20%) in cells that expressed substance P and NKB, and much lower (<10%) in other classes. Our findings show that these antibodies reliably detect GRP-expressing neurons and axons, and that in addition to the GRP-GFP cells, excitatory interneurons expressing NPFF or substance P are likely to be the main source of GRP in the spinal dorsal horn.
  • Amy Cheung, Kotaro Konno, Yuka Imamura, Aya Matsui, Manabu Abe, Kenji Sakimura, Toshikuni Sasaoka, Takeshi Uemura, Masahiko Watanabe, Kensuke Futai
    eLife 12 2023/01/25 
    Extensive serotonin (5-hydroxytryptamine, 5-HT) innervation throughout the brain corroborates 5-HT's modulatory role in numerous cognitive activities. Volume transmission is the major mode for 5-HT transmission but mechanisms underlying 5-HT signaling are still largely unknown. Abnormal brain 5-HT levels and function have been implicated in autism spectrum disorder (ASD). Neurexin (Nrxn) genes encode presynaptic cell adhesion molecules important for the regulation of synaptic neurotransmitter release, notably glutamatergic and GABAergic transmission. Mutations in Nrxn genes are associated with neurodevelopmental disorders including ASD. However, the role of Nrxn genes in the 5-HT system is poorly understood. Here, we generated a mouse model with all three Nrxn genes disrupted specifically in 5-HT neurons to study how Nrxns affect 5-HT transmission. Loss of Nrxns in 5-HT neurons reduced the number of serotonin neurons in the early postnatal stage, impaired 5-HT release, and decreased 5-HT release sites and serotonin transporter expression. Furthermore, 5-HT neuron-specific Nrxn knockout reduced sociability and increased depressive-like behavior. Our results highlight functional roles for Nrxns in 5-HT neurotransmission, 5-HT neuron survival, and the execution of complex behaviors.
  • Takashi Kudo, Daigo Nakazawa, Kanako Watanabe-Kusunoki, Masatoshi Kanda, Satoka Shiratori-Aso, Nobuya Abe, Saori Nishio, Jun-Ichiro Koga, Sari Iwasaki, Takahiro Tsuji, Yuichiro Fukasawa, Miwako Yamasaki, Masahiko Watanabe, Sakiko Masuda, Utano Tomaru, Masaaki Murakami, Yasuaki Aratani, Akihiro Ishizu, Tatsuya Atsumi
    Arthritis & rheumatology (Hoboken, N.J.) 75 (1) 71 - 83 2023/01 
    OBJECTIVE: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis, in which ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial cell necrosis occur. Cyclophilin D (CypD) plays an important role in mediation of cell necrosis and inflammation via the opening of mitochondrial permeability transition pores. This study was undertaken to examine the role of CypD in AAV pathogenesis. METHODS: We assessed the role and mechanism of CypD in ANCA-stimulated neutrophils in vitro by immunostaining and electron microscopy observation. We performed a comprehensive RNA-sequencing analysis on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, we assessed disease features in CypD-knockout mice and wild-type mice using 2 different murine AAV models: anti-myeloperoxidase IgG transfer-induced AAV and spontaneous AAV. RESULTS: In vitro experiments showed that pharmacologic and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species and cytochrome c release from the mitochondria. RNA-sequencing analyses in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, with CypD deficiency reducing ANCA-induced alterations in gene expression. Furthermore, analyses of upstream regulators revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that the CypD-dependent opening of mitochondrial permeability transition pores is associated with ANCA-induced neutrophil activation and NETosis. In both AAV mouse models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis. CONCLUSION: CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.
  • Samu N Kurki, Pavel Uvarov, Alexey S Pospelov, Kalevi Trontti, Antje K Hübner, Rakenduvadhana Srinivasan, Masahiko Watanabe, Iiris Hovatta, Christian A Hübner, Kai Kaila, Mari A Virtanen
    Cerebral cortex (New York, N.Y. : 1991) 2022/12/27 
    The Na-K-2Cl cotransporter NKCC1 is widely expressed in cells within and outside the brain. However, our understanding of its roles in brain functions throughout development, as well as in neuropsychiatric and neurological disorders, has been severely hindered by the lack of reliable data on its developmental and (sub)cellular expression patterns. We provide here the first properly controlled analysis of NKCC1 protein expression in various cell types of the mouse brain using custom-made antibodies and an NKCC1 knock-out validated immunohistochemical procedure, with parallel data based on advanced mRNA approaches. NKCC1 protein and mRNA are expressed at remarkably high levels in oligodendrocytes. In immature neurons, NKCC1 protein was located in the somata, whereas in adult neurons, only NKCC1 mRNA could be clearly detected. NKCC1 immunoreactivity is also seen in microglia, astrocytes, developing pericytes, and in progenitor cells of the dentate gyrus. Finally, a differential expression of NKCC1 splice variants was observed, with NKCC1a predominating in non-neuronal cells and NKCC1b in neurons. Taken together, our data provide a cellular basis for understanding NKCC1 functions in the brain and enable the identification of major limitations and promises in the development of neuron-targeting NKCC1-blockers.
  • Ching-Pu Chang, Kohei Otomo, Yuichi Kozawa, Hirokazu Ishii, Miwako Yamasaki, Masahiko Watanabe, Shunichi Sato, Ryosuke Enoki, Tomomi Nemoto
    Scientific Reports 12 (1) 10468 - 10468 2022/12 [Refereed]
     
    Abstract Biological tissues and their networks frequently change dynamically across large volumes. Understanding network operations requires monitoring their activities in three dimensions (3D) with single-cell resolution. Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups, as well as deep expertise for microscopic technologies, resulting in a high threshold for biologists. In this study, we propose an easy-to-use light-needle creating device for conventional two-photon microscopy systems. By only installing the device in one position for a filter cube that conventional fluorescent microscopes have, single scanning of the excitation laser light beam excited fluorophores throughout over 200 μm thickness specimens simultaneously. Furthermore, the developed microscopy system successfully demonstrated single-scan visualization of the 3D structure of transparent YFP-expressing brain slices. Finally, in acute mouse cortical slices with a thickness of approximately 250 μm, we detected calcium activities with 7.5 Hz temporal resolution in the neuronal population.
  • Hitomi Sasamori, Toshiaki Asakura, Chiaki Sugiura, Youcef Bouchekioua, Naoya Nishitani, Masaaki Sato, Takayuki Yoshida, Miwako Yamasaki, Akira Terao, Masahiko Watanabe, Yu Ohmura, Mitsuhiro Yoshioka
    Scientific Reports 12 (1) 2022/12 
    Abstract Major psychiatric disorders such as attention-deficit/hyperactivity disorder and schizophrenia are often accompanied by elevated impulsivity. However, anti-impulsive drug treatments are still limited. To explore a novel molecular target, we examined the role of dopamine D5 receptors in impulse control using mice that completely lack D5 receptors (D5KO mice). We also measured spontaneous activity and learning/memory ability because these deficits could confound the assessment of impulsivity. We found small but significant effects of D5 receptor knockout on home cage activity only at specific times of the day. In addition, an analysis using the q-learning model revealed that D5KO mice displayed lower behavioral adjustment after impulsive actions. However, our results also showed that baseline impulsive actions and the effects of an anti-impulsive drug in D5KO mice were comparable to those in wild-type littermates. Moreover, unlike previous studies that used other D5 receptor-deficient mouse lines, we did not observe reductions in locomotor activity, working memory deficits, or severe learning deficits in our line of D5KO mice. These findings demonstrate that D5 receptors are dispensable for impulse control. Our results also indicate that time series analysis and detailed analysis of the learning process are necessary to clarify the behavioral functions of D5 receptors.
  • Megumu Takahashi, Tomoyo Kobayashi, Haruhi Mizuma, Kenta Yamauchi, Shinichiro Okamoto, Kazuki Okamoto, Yoko Ishida, Masato Koike, Masahiko Watanabe, Tadashi Isa, Hiroyuki Hioki
    Neuroscience research 190 92 - 106 2022/11/26 
    The claustrum coordinates the activities of individual cortical areas through abundant reciprocal connections with the cerebral cortex. Although these excitatory connections have been extensively investigated in three subregions of the claustrum-core region and dorsal and ventral shell regions-the contribution of GABAergic neurons to the circuitry in each subregion remains unclear. Here, we examined the distribution of GABAergic neurons and their dendritic and axonal arborizations in each subregion. Combining in situ hybridization with immunofluorescence histochemistry showed that approximately 10% of neuronal nuclei-positive cells expressed glutamic acid decarboxylase 67 mRNA across the claustral subregions. Approximately 20%, 30%, and 10% of GABAergic neurons were immunoreactive for parvalbumin (PV), somatostatin (SOM), and vasoactive intestinal polypeptide, respectively, in each subregion, and these neurochemical markers showed little overlap with each other. We then reconstructed PV and SOM neurons labeled with adeno-associated virus vectors. The dendrites and axons of PV and SOM neurons were preferentially localized to their respective subregions where their cell bodies were located. Furthermore, the axons were preferentially extended in a rostrocaudal direction, whereas the dendrites were relatively isotropic. The present findings suggest that claustral PV and SOM neurons might execute information processing separately within the core and shell regions.
  • Tadashi Tanioku, Masayuki Nishibata, Yasuyuki Tokinaga, Kohtaro Konno, Masahiko Watanabe, Hiroaki Hemmi, Yuri Fukuda-Ohta, Tsuneyasu Kaisho, Hidemasa Furue, Tomoyuki Kawamata
    Proceedings of the National Academy of Sciences of the United States of America 119 (45) e2121989119  2022/11/08 
    Persistent mechanical pain hypersensitivity associated with peripheral inflammation, surgery, trauma, and nerve injury impairs patients' quality of life and daily activity. However, the molecular mechanism and treatment are not yet fully understood. Herein, we show that chemical ablation of isolectin B4-binding (IB4+) afferents by IB4-saporin injection into sciatic nerves completely and selectively inhibited inflammation- and tissue injury-induced mechanical pain hypersensitivity while thermal and mechanical pain hypersensitivities were normal following nerve injury. To determine the molecular mechanism involving the specific types of mechanical pain hypersensitivity, we compared gene expression profiles between IB4+ neuron-ablated and control dorsal root ganglion (DRG) neurons. We identified Tmem45b as one of 12 candidate genes that were specific to somatosensory ganglia and down-regulated by IB4+ neuronal ablation. Indeed, Tmem45b was expressed predominantly in IB4+ DRG neurons, where it was selectively localized in the trans Golgi apparatus of DRG neurons but not detectable in the peripheral and central branches of DRG axons. Tmem45b expression was barely detected in the spinal cord and brain. Although Tmem45b-knockout mice showed normal responses to noxious heat and noxious mechanical stimuli under normal conditions, mechanical pain hypersensitivity was selectively impaired after inflammation and tissue incision, reproducing the pain phenotype of IB4+ sensory neuron-ablated mice. Furthermore, acute knockdown by intrathecal injection of Tmem45b small interfering RNA, either before or after inflammation induction, successfully reduced mechanical pain hypersensitivity. Thus, our study demonstrates that Tmem45b is essential for inflammation- and tissue injury-induced mechanical pain hypersensitivity and highlights Tmem45b as a therapeutic target for future treatment.
  • Li Zhou, Kohtarou Konno, Maya Yamazaki, Manabu Abe, Rie Natsume, Masahiko Watanabe, Hirohide Takebayashi, Kenji Sakimura
    International Journal of Molecular Sciences 23 (21) 12961 - 12961 2022/10/26 [Refereed]
     
    Nna1/CCP1 is generally known as a causative gene for a spontaneous autosomal recessive mouse mutation, Purkinje cell degeneration (pcd). There is enough evidence that the cytosolic function of the zinc carboxypeptidase (CP) domain at the C-terminus of the Nna1 protein is associated with cell death. On the other hand, this molecule’s two nuclear localization signals (NLSs) suggest some other functions exist. We generated exon 3-deficient mice (Nna1N KO), which encode a portion of the N-terminal NLS. Despite the frameshift occurring in these mice, there was an expression of the Nna1 protein lacking the N-terminal side. Surprisingly, the pcd phenotype did not occur in the Nna1N KO mouse. Behavioral analysis revealed that they were less anxious when assessed by the elevated plus maze and the light/dark box tests compared to the control. Furthermore, they showed impairments in context-dependent and sound stimulus-dependent learning. Biochemical analysis of Nna1N KO mice revealed a reduced level of the AMPA-type glutamine receptor GluA2 in the hippocampal synaptosomal fraction. In addition, the motor protein kinesin-1, which transports GluA2 to dendrites, was also decreased. These results indicate that Nna1 is also involved in emotion and memory learning, presumably through the trafficking and expression of synaptic signaling molecules, besides a known role in cell survival.
  • Yasuhiro Yamamoto, Ken Kadoya, Mohamad Alaa Terkawi, Takeshi Endo, Kohtarou Konno, Masahiko Watanabe, Satoshi Ichihara, Akira Hara, Kazuo Kaneko, Norimasa Iwasaki, Muneaki Ishijima
    Life science alliance 5 (10) 2022/10 
    Although inflammation is indispensable for the repair process in Wallerian degeneration (WD), the role of neutrophils in the WD repair process remains unclear. After peripheral nerve injury, neutrophils accumulate at the epineurium but not the parenchyma in the WD region because of the blood-nerve barrier. An increase or decrease in the number of neutrophils delayed or promoted macrophage infiltration from the epineurium into the parenchyma and the repair process in WD. Abundant neutrophil extracellular traps (NETs) were formed around neutrophils, and its inhibition dramatically increased macrophage infiltration into the parenchyma. Furthermore, inhibition of either MIF or its receptor, CXCR4, in neutrophils decreased NET formation, resulting in enhanced macrophage infiltration into the parenchyma. Moreover, inhibiting MIF for just 2 h after peripheral nerve injury promoted the repair process. These findings indicate that neutrophils delay the repair process in WD from outside the parenchyma by inhibiting macrophage infiltration via NET formation and that neutrophils, NETs, MIF, and CXCR4 are therapeutic targets for peripheral nerve regeneration.
  • Sawetree Pakkarato, Hiroyuki Sakagami, Kaoru Goto, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo, Surang Chomphoo
    Experimental eye research 223 109205 - 109205 2022/10 
    Based on the theory that the phosphoinositide (PI) signal is involved in the physiology of cornea and conjunctiva, we examined the localization in the mouse anterior ocular epithelia of immunoreactivities for phosphatidylinositol 4-phosphate 5-kinase (PIP5K), phospholipase C (PLC) and diacylglycerol kinase (DGK), enzymes that work sequentially in PI cycle. Immunoreactivity for PIP5Kγ in the corneal epithelium, including the limbus, was distinct in adults in contrast to faint or negligible immunoreactivity in the conjunctival epithelium in neonatal mice. This adult localization pattern was first recognized at the postnatal time of eyelid opening. Immunoreactivity for PLCβ3 was rather equally distinct throughout the entire corneal and conjunctival epithelia in adults. DGKζ-immunoreactive nuclei were mainly localized in the basal half domain of the corneal epithelium but in both basal and apical domains of the conjunctival epithelium in adults. This nuclear immunoreactivity was at weak or negligible levels in the peripheral and limbus cornea and in a considerable portion of the bulbar conjunctival epithelium continuous with the limbus. The adult patterns for PLCβ3 and DGKζ were already present at birth. The present findings suggest the following possibilities on the functional significance of the three enzyme molecules. PIP5Kγ is involved in cornea-specific functions such as bright-field vision, including corneal transparency, and in the stability of epithelial junctions, for which there seems to be a much higher requirement in the corneal epithelium than in the conjunctival epithelium. PLCβ3 is involved from birth in as-yet undefined functions exerted ubiquitously from birth in both corneal and conjunctival epithelia. DGKζ is involved in regulation from birth of the transcription in epithelial cells, including apoptosis as well as regulation of mitosis of epithelial cells in both cornea and conjunctiva, with the transcription involvement more apparent in the conjunctiva, although it does not work in stem cells of the corneal limbus.
  • Zhou L, Konno K, Yamazaki M, Abe M, Natsume R, Watanabe M, Takebayashi H, Sakimura K
    Int J Mol Sci. 23(21) (12961) 1 - 16 2022/10 [Refereed]
  • Kenta Yamauchi, Shinichiro Okamoto, Yoko Ishida, Kohtarou Konno, Kisara Hoshino, Takahiro Furuta, Megumu Takahashi, Masato Koike, Kaoru Isa, Masahiko Watanabe, Tadashi Isa, Hiroyuki Hioki
    Scientific reports 12 (1) 14807 - 14807 2022/09/12 
    Tyramide signal amplification (TSA) is a highly sensitive method for histochemical analysis. Previously, we reported a TSA system, biotinyl tyramine-glucose oxidase (BT-GO), for bright-filed imaging. Here, we develop fluorochromized tyramide-glucose oxidase (FT-GO) as a multiplex fluorescent TSA system. FT-GO involves peroxidase-catalyzed deposition of fluorochromized tyramide (FT) with hydrogen peroxide produced by enzymatic reaction between glucose and glucose oxidase. We showed that FT-GO enhanced immunofluorescence signals while maintaining low background signals. Compared with indirect immunofluorescence detections, FT-GO demonstrated a more widespread distribution of monoaminergic projection systems in mouse and marmoset brains. For multiplex labeling with FT-GO, we quenched antibody-conjugated peroxidase using sodium azide. We applied FT-GO to multiplex fluorescent in situ hybridization, and succeeded in labeling neocortical interneuron subtypes by coupling with immunofluorescence. FT-GO immunofluorescence further increased the detectability of an adeno-associated virus tracer. Given its simplicity and a staining with a high signal-to-noise ratio, FT-GO would provide a versatile platform for histochemical analysis.
  • Nobuya Abe, Masato Tarumi, Yuichiro Fujieda, Nobuhiko Takahashi, Kohei Karino, Mona Uchida, Michihito Kono, Yuki Tanaka, Rie Hasebe, Masaru Kato, Olga Amengual, Yoshiyuki Arinuma, Kenji Oku, Wakiro Sato, Khin Khin Tha, Miwako Yamasaki, Masahiko Watanabe, Tatsuya Atsumi, Masaaki Murakami
    Annals of the rheumatic diseases 81 (11) 1564 - 1575 2022/07/11 
    OBJECTIVES: The central nervous system disorder in systemic lupus erythematosus (SLE), called neuropsychiatric lupus (NPSLE), is one of the most severe phenotypes with various clinical symptoms, including mood disorder, psychosis and delirium as diffuse neuropsychological manifestations (dNPSLE). Although stress is one of the aggravating factors for neuropsychiatric symptoms, its role in the pathogenesis of dNPSLE remains to be elucidated. We aimed to investigate stress effects on the neuropsychiatric pathophysiology in SLE using lupus-prone mice and patients' data. METHODS: Sleep disturbance stress (SDS) for 2 weeks was placed on 6-8-week-old female MRL/lpr and control mice. Behavioural phenotyping, histopathological analyses and gene and protein expression analyses were performed to assess SDS-induced neuroimmunological alterations. We also evaluated cytokines of the cerebrospinal fluid and brain regional volumes in patients with dNPSLE and patients with non-dNPSLE. RESULTS: SDS-subjected MRL/lpr mice exhibited less anxiety-like behaviour, whereas stressed control mice showed increased anxiety. Furthermore, stress strongly activated the medial prefrontal cortex (mPFC) in SDS-subjected MRL/lpr. A transcriptome analysis of the PFC revealed the upregulation of microglial activation-related genes, including Il12b. We confirmed that stress-induced microglial activation and the upregulation of interleukin (IL) 12/23p40 proteins and increased dendritic spines in the mPFC of stressed MRL/lpr mice. IL-12/23p40 neutralisation and tyrosine kinase 2 inhibition mitigated the stress-induced neuropsychiatric phenotypes of MRL/lpr mice. We also found a higher level of cerebrospinal fluid IL-12/23p40 and more atrophy in the mPFC of patients with dNPSLE than those with non-dNPSLE. CONCLUSIONS: The microglial IL-12/23 axis in the mPFC might be associated with the pathogenesis and a promising therapeutic target for dNPSLE.
  • David Hauser, Katharina Behr, Kohtarou Konno, Dietmar Schreiner, Alexander Schmidt, Masahiko Watanabe, Josef Bischofberger, Peter Scheiffele
    Neuron 110 (13) 2094 - 2109 2022/07/06 
    The diversification of cell adhesion molecules by alternative splicing is proposed to underlie molecular codes for neuronal wiring. Transcriptomic approaches mapped detailed cell-type-specific mRNA splicing programs. However, it has been hard to probe the synapse-specific localization and function of the resulting protein splice isoforms, or "proteoforms," in vivo. We here apply a proteoform-centric workflow in mice to test the synapse-specific functions of the splice isoforms of the synaptic adhesion molecule Neurexin-3 (NRXN3). We uncover a major proteoform, NRXN3 AS5, that is highly expressed in GABAergic interneurons and at dendrite-targeting GABAergic terminals. NRXN3 AS5 abundance significantly diverges from Nrxn3 mRNA distribution and is gated by translation-repressive elements. Nrxn3 AS5 isoform deletion results in a selective impairment of dendrite-targeting interneuron synapses in the dentate gyrus without affecting somatic inhibition or glutamatergic perforant-path synapses. This work establishes cell- and synapse-specific functions of a specific neurexin proteoform and highlights the importance of alternative splicing regulation for synapse specification.
  • Mayuko Hashimoto, Masaki Fujimoto, Kohtarou Konno, Ming-Liang Lee, Yui Yamada, Koya Yamashita, Chitoku Toda, Michio Tomura, Masahiko Watanabe, Osamu Inanami, Hiroshi Kitamura
    The Journal of neuroscience : the official journal of the Society for Neuroscience 42 (23) 4607 - 4618 2022/06/08 
    Ubiquitin-specific protease 2 (USP2) participates in glucose metabolism in peripheral tissues such as the liver and skeletal muscles. However, the glucoregulatory role of USP2 in the CNS is not well known. In this study, we focus on USP2 in the ventromedial hypothalamus (VMH), which has dominant control over systemic glucose homeostasis. ISH, using a Usp2-specific probe, showed that Usp2 mRNA is present in VMH neurons, as well as other glucoregulatory nuclei, in the hypothalamus of male mice. Administration of a USP2-selective inhibitor ML364 (20 ng/head), into the VMH elicited a rapid increase in the circulating glucose level in male mice, suggesting USP2 has a suppressive role on glucose mobilization. ML364 treatment also increased serum norepinephrine concentration, whereas it negligibly affected serum levels of insulin and corticosterone. ML364 perturbated mitochondrial oxidative phosphorylation in neural SH-SY5Y cells and subsequently promoted the phosphorylation of AMP-activated protein kinase (AMPK). Consistent with these findings, hypothalamic ML364 treatment stimulated AMPKα phosphorylation in the VMH. Inhibition of hypothalamic AMPK prevented ML364 from increasing serum norepinephrine and blood glucose. Removal of ROS restored the ML364-evoked mitochondrial dysfunction in SH-SY5Y cells and impeded the ML364-induced hypothalamic AMPKα phosphorylation as well as prevented the elevation of serum norepinephrine and blood glucose levels in male mice. These results indicate hypothalamic USP2 attenuates perturbations in blood glucose levels by modifying the ROS-AMPK-sympathetic nerve axis.SIGNIFICANCE STATEMENT Under normal conditions (excluding hyperglycemia or hypoglycemia), blood glucose levels are maintained at a constant level. In this study, we used a mouse model to identify a hypothalamic protease controlling blood glucose levels. Pharmacological inhibition of USP2 in the VMH caused a deviation in blood glucose levels under a nonstressed condition, indicating that USP2 determines the set point of the blood glucose level. Modification of sympathetic nervous activity accounts for the USP2-mediated glucoregulation. Mechanistically, USP2 mitigates the accumulation of ROS in the VMH, resulting in attenuation of the phosphorylation of AMPK. Based on these findings, we uncovered a novel glucoregulatory axis consisting of hypothalamic USP2, ROS, AMPK, and the sympathetic nervous system.
  • Rie Hasebe, Kaoru Murakami, Masaya Harada, Nada Halaka, Hiroshi Nakagawa, Fuminori Kawano, Yoshinobu Ohira, Tadafumi Kawamoto, Fiona E Yull, Timothy S Blackwell, Junko Nio-Kobayashi, Toshihiko Iwanaga, Masahiko Watanabe, Nobuhiro Watanabe, Harumi Hotta, Toshihide Yamashita, Daisuke Kamimura, Yuki Tanaka, Masaaki Murakami
    The Journal of experimental medicine 219 (6) 2022/06/06 
    Neural circuits between lesions are one mechanism through which local inflammation spreads to remote positions. Here, we show the inflammatory signal on one side of the joint is spread to the other side via sensory neuron-interneuron crosstalk, with ATP at the core. Surgical ablation or pharmacological inhibition of this neural pathway prevented inflammation development on the other side. Mechanistic analysis showed that ATP serves as both a neurotransmitter and an inflammation enhancer, thus acting as an intermediary between the local inflammation and neural pathway that induces inflammation on the other side. These results suggest blockade of this neural pathway, which is named the remote inflammation gateway reflex, may have therapeutic value for inflammatory diseases, particularly those, such as rheumatoid arthritis, in which inflammation spreads to remote positions.
  • Takafumi Kawai, Hirotaka Narita, Kohtarou Konno, Sharmin Akter, Rizki Tsari Andriani, Hirohide Iwasaki, Shoji Nishikawa, Norihiko Yokoi, Yuko Fukata, Masaki Fukata, Pattama Wiriyasermkul, Pornparn Kongpracha, Shushi Nagamori, Keizo Takao, Tsuyoshi Miyakawa, Manabu Abe, Kenji Sakimura, Masahiko Watanabe, Atsushi Nakagawa, Yasushi Okamura
    The Biochemical journal 479 (11) 1127 - 1145 2022/05/16 
    Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.
  • Qin Zhao, Tetsufumi Ito, Chika Soko, Yoshie Hori, Takafumi Furuyama, Hiroyuki Hioki, Kohtarou Konno, Miwako Yamasaki, Masahiko Watanabe, Satoshi Ohtsuka, Munenori Ono, Nobuo Kato, Ryo Yamamoto
    eNeuro 9 (3) 2022/05/13 
    The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH) positive neurons which are regarded as dopaminergic (DA) neurons. These DA neurons in the DR and periaqueductal grey (PAG) region (DADR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DADR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with fluoro-gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DATDR-PAG neurons consisted of the following two subpopulations: TH+/VIP- and TH-/VIP+ neurons. The DAT+/TH-/VIP+ subpopulation would be non-DA non-canonical DAT neurons. Anterograde labeling of DATDR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these regions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH-/VIP+ subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DATDR-PAG neurons, TH+/VIP- and TH-/VIP+, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.Significance StatementDopaminergic (DA) neurons in the dorsal raphe (DR) and periaqueductal grey (PAG) regions have projections to the extended amygdala and have been reported to modulate various affective behaviors. These DA neurons are a subgroup of the A10 cluster, which is known to be heterogeneous. However, it remains unknown how heterogeneous subpopulations innervate the extended amygdala. We used the DA transporter as a DA neuron marker and found that the DAT DR-PAG neurons are composed of at least two subpopulations, DAT+/tyrosine hydroxylase (TH)+/vasoactive intestinal peptide (VIP)- putative DA neurons and DAT+/TH-/VIP+ putative non-DA glutamatergic neurons, innervating the extended amygdala similarly. These results indicate that the two subpopulations might differently modulate the affective behaviors controlled by the extended amygdala.
  • Éva Kókai, Wafa Aa Alsulaiman, Allen C Dickie, Andrew M Bell, Luca Goffin, Masahiko Watanabe, Maria Gutierrez-Mecinas, Andrew J Todd
    Molecular pain 18 17448069221119614 - 17448069221119614 2022/04 
    Projection neurons belonging to the anterolateral system (ALS) underlie the perception of pain, skin temperature and itch. Many ALS cells are located in laminae III-V of the dorsal horn and the adjacent lateral white matter. However, relatively little is known about the excitatory synaptic input to these deep ALS cells, and therefore about their engagement with the neuronal circuitry of the region. We have used a recently developed mouse line, Phox2a::Cre, to investigate a population of deep dorsal horn ALS neurons known as "antenna cells", which are characterised by dense innervation from peptidergic nociceptors, and to compare these with other ALS cells in the deep dorsal horn and lateral white matter. We show that these two classes differ, both in the density of excitatory synapses, and in the source of input at these synapses. Peptidergic nociceptors account for around two-thirds of the excitatory synapses on the antenna cells, but for only a small proportion of the input to the non-antenna cells. Conversely, boutons with high levels of VGLUT2, which are likely to originate mainly from glutamatergic spinal neurons, account for only ∼5% of the excitatory synapses on antenna cells, but for a much larger proportion of the input to the non-antenna cells. VGLUT1 is expressed by myelinated low-threshold mechanoreceptors and corticospinal axons, and these innervate both antenna and non-antenna cells. However, the density of VGLUT1 input to the non-antenna cells is highly variable, consistent with the view that these neurons are functionally heterogeneous.
  • Takeshi Uemura, Emi Suzuki-Kouyama, Shiori Kawase, Taiga Kurihara, Misato Yasumura, Tomoyuki Yoshida, Shuya Fukai, Maya Yamazaki, Peng Fei, Manabu Abe, Masahiko Watanabe, Kenji Sakimura, Masayoshi Mishina, Katsuhiko Tabuchi
    Cell Reports 39 (1) 110624 - 110624 2211-1247 2022/04
  • Yuka Kasahara, Daiki Masukawa, Kenta Kobayashi, Miwako Yamasaki, Masahiko Watanabe, Yoshio Goshima
    Stem cells (Dayton, Ohio) 40 (2) 215 - 226 2022/03/16 
    Neurogenesis occurs in the hippocampus throughout life and is implicated in various physiological brain functions such as memory encoding and mood regulation. L-3,4-dihydroxyphenylalanine (L-DOPA) has long been believed to be an inert precursor of dopamine. Here, we show that L-DOPA and its receptor, GPR143, the gene product of ocular albinism 1, regulate neurogenesis in the dentate gyrus (DG) in a dopamine-independent manner. L-DOPA at concentrations far lower than that of dopamine promoted proliferation of neural stem and progenitor cells in wild-type mice under the inhibition of its conversion to dopamine; this effect was abolished in GPR143 gene-deficient (Gpr143-/y) mice. Hippocampal neurogenesis decreased during development and adulthood, and exacerbated depression-like behavior was observed in adult Gpr143-/y mice. Replenishment of GPR143 in the DG attenuated the impaired neurogenesis and depression-like behavior. Our findings suggest that L-DOPA through GPR143 modulates hippocampal neurogenesis, thereby playing a role in mood regulation in the hippocampus.
  • Keita Mori, Michinori Koebis, Kazuki Nakao, Shizuka Kobayashi, Yuji Kiyama, Masahiko Watanabe, Toshiya Manabe, Yuichi Iino, Atsu Aiba
    Molecular brain 15 (1) 23 - 23 2022/03/12 
    Calsyntenins (CLSTNs) are important synaptic molecules whose molecular functions are not fully understood. Although mutations in calsyntenin (CLSTN) genes have been associated with psychiatric disorders in humans, their function is still unclear. One of the reasons why the function of CLSTNs in the nervous system has not been clarified is the functional redundancy among the three paralogs. Therefore, to investigate the functions of mammalian CLSTNs, we generated triple knockout (TKO) mice lacking all CLSTN paralogs and examined their behavior. The mutant mice tended to freeze in novel environments and exhibited hypersensitivity to stress. Consistent with this, glucose levels under stress were significantly higher in the mutant mice than in the wild-type controls. In particular, phenotypes such as decreased motivation, which had not been reported in single Clstn KO mice, were newly discovered. The TKO mice generated in this study represent an important mouse model for clarifying the function of CLSTN in the future.
  • Eriko Miura, Masahiko Watanabe
    Neuroscience research 2022/03/02 
    Calcineurin (CN) is a Ca2+/calmodulin-dependent serine/threonine protein phosphatase consisting of catalytic CNA and regulatory CNB subunits, and links activity-dependent Ca2+ signals to various neural functions. Here we studied CN expression in the mouse brain by producing subunit-specific probes and antibodies. Of five CN subunits. CNAα, CNAβ, and CNB1 mRNAs were predominantly expressed over the brain from early embryonic to adult stage, and all were high in the telencephalon and cerebellum. Protein localization was examined in the cerebellum by immunofluorescence with cellular and terminal markers and by preembedding silver-enhanced immunogold microscopy. CNB1 and CNAβ were co-distributed in subcellular and synaptic elements of various cerebellar neurons and glia, whereas CNAα was exclusive in granule cell elements, including parallel fiber terminals. The present study thus discloses that CNB1 subunit well coexists with one or two CNA subunits in various cerebellar compartments. Moreover, high CN contents are provided to parallel fiber-Purkinje cell synapses, i.e., CNAα, CNAβ, and CNB1 in their presynaptic side and CNAβ and CNB1 in their postsynaptic side. These findings will be the anatomical basis, at least partly, for the known regulatory roles of postsynaptic CNs in long-term depression and presynaptic CNs in transmitter release function.
  • Anussara Kamnate, Juthathip Sirisin, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo, Surang Chomphoo
    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 70 (3) 251 - 257 2022/03 
    Localization of cannabinoid receptor type 1 (CB1) immunoreactivity on mitochondrial membranes, at least their outer membranes distinctly, was detected in progesterone-producing cells characterized by mitochondria having tubular cristae and aggregations of lipid droplets in ovarian interstitial glands in situ of adult mice. Both immunoreactive and immunonegative mitochondria were contained in one and the same cell. Considering that the synthesis of progesterone is processed in mitochondria, the mitochondrial localization of CB1 in the interstitial gland cells suggests the possibility that endocannabinoids modulate the synthetic process of progesterone in the cells through CB1.
  • Czarina Ramos, Stefano Lutzu, Miwako Yamasaki, Yuchio Yanagawa, Kenji Sakimura, Susumu Tomita, Masahiko Watanabe, Pablo E Castillo
    The Journal of neuroscience : the official journal of the Society for Neuroscience 2022/02/22 
    Mossy cells (MCs) of the dentate gyrus (DG) are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place field encoding, pattern separation and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission, and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished in GluK2 KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus.SIGNIFICANCE STATEMENTHilar mossy cells (MCs) are an understudied population of hippocampal neurons that form an excitatory loop with dentate granule cells. MCs have been implicated in pattern separation, spatial navigation, and epilepsy. Despite their importance in hippocampal function and disease, little is known about how MC activity is recruited. Here, we show for the first time that MCs express extrasynaptic kainate receptors (KARs), a subtype of glutamate receptors critically involved in neuronal function and epilepsy. While we found no evidence for synaptic KARs in MCs, KAR activation induced strong action potential firing of MCs, raising the possibility that extracellular KARs regulate MC excitability in vivo and may also promote dentate gyrus hyperexcitability and epileptogenesis.
  • Ken Matsuura, Shizuka Kobayashi, Kohtarou Konno, Miwako Yamasaki, Takahiro Horiuchi, Takao Senda, Tomoatsu Hayashi, Kiyotoshi Satoh, Fumiko Arima-Yoshida, Kei Iwasaki, Lumi Negishi, Naomi Yasui-Shimizu, Kazuyoshi Kohu, Shigenori Kawahara, Yutaka Kirino, Tsutomu Nakamura, Masahiko Watanabe, Tadashi Yamamoto, Toshiya Manabe, Tetsu Akiyama
    The Journal of neuroscience : the official journal of the Society for Neuroscience 42 (12) 2448 - 2473 2022/02/04 
    SIPA1L1 (also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the PSD-95/NMDA-R-complex. However, its physiological role remains poorly understood. Here, we performed expression analyses using super-resolution microscopy in mouse brain and demonstrated that SIPA1L1 is mainly localized to general submembranous regions in neurons, but surprisingly, not to PSD. Our screening for physiological interactors of SIPA1L1 in mouse brain identified spinophilin and neurabin-1, regulators of GPCR signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1 -/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1 -/- mice showed aberrant responses to α2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation, and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Male mice were used for all experiments. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome.SIGNIFICANCE STATEMENTSIPA1L1 is thought to regulate essential synaptic functions as a component of the PSD-95/NMDA-R-complex. In our screening for physiological SIPA1L1-interactors, we identified GPCR-signaling regulators. Moreover, SIPA1L1 KO mice showed striking behavioral anomalies, which may be relevant to GPCR signaling. Our findings revealed an unexpected role of SIPA1L1, which may open new avenues for research on neuropsychiatric disorders that involve dysregulated GPCR signaling. Another important aspect of this paper is that we showed effective methods for checking PSD association and identifying native protein interactors that are difficult to solubilize. These results may serve as a caution for future claims about interacting proteins and PSD proteins, which could eventually save time and resources for researchers and avoid confusion in the field.
  • Soichiro Takamiya, Masahito Kawabori, Kazuyoshi Yamazaki, Sho Yamaguchi, Aki Tanimori, Koji Yamamoto, Shunsuke Ohnishi, Toshitaka Seki, Kotaro Konno, Khin Khin Tha, Daigo Hashimoto, Masahiko Watanabe, Kiyohiro Houkin, Miki Fujimura
    PloS one 17 (7) e0270606  2022 
    Spinal cord injury (SCI) is often accompanied by gastrointestinal dysfunction due to the disconnection of the spinal autonomic nervous system. Gastrointestinal dysfunction reportedly upregulates intestinal permeability, leading to bacterial translocation of the gut microbiome to the systemic circulation, which further activates systemic inflammation, exacerbating neuronal damage. Mesenchymal stem cells (MSC) reportedly ameliorate SCI. Here, we aimed to investigate their effect on the associated gastrointestinal dysfunction. Human amnion-derived MSC (AMSCs) were intravenously transplanted one day after a rat model of midthoracic SCI. Biodistribution of transplanted cells, behavioral assessment, and histological evaluations of the spinal cord and intestine were conducted to elucidate the therapeutic effect of AMSCs. Bacterial translocation of the gut microbiome was examined by in situ hybridization and bacterial culture of the liver. Systemic inflammations were examined by blood cytokines, infiltrating immune cells in the spinal cord, and the size of the peripheral immune tissue. AMSCs released various neurotrophic factors and were mainly distributed in the liver and lung after transplantation. AMSC-transplanted animals showed smaller spinal damage and better neurological recovery with preserved neuronal tract. AMSCs transplantation ameliorated intestinal dysfunction both morphologically and functionally, which prevented translocation of the gut microbiome to the systemic circulation. Systemic inflammations were decreased in animals receiving AMSCs in the chronic phase. Intravenous AMSC administration during the acute phase of SCI rescues both spinal damage and intestinal dysfunction. Reducing bacterial translocation may contribute to decreasing systemic inflammation.
  • Hideki Miwa, Ken Kobayashi, Shinobu Hirai, Mitsuhiko Yamada, Masahiko Watanabe, Haruo Okado, Yuchio Yanagawa
    Cerebellum (London, England) 2021/10/21 
    Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system, synthesized by two isoforms of glutamate decarboxylase (GAD): GAD65 and GAD67. GABA may act as a trophic factor during brain development, but its contribution to the development and maturation of cerebellar neural circuits is not known. To understand the roles of GABA in cerebellar organization and associated functions in motor coordination and balance, we examined GAD65 conventional knock out (KO) mice and mice in which GAD67 was eliminated in parvalbumin-expressing neurons (PV-Cre; GAD67flox/flox mice). We found aberrant subcellular localization of the Shaker-type K channel Kv1.1 in basket cell collaterals of PV-Cre; GAD67 flox/flox mice and abnormal projections from basket cells to Purkinje cells in both mouse strains. We also found that altered synaptic properties of basket cell terminals to Purkinje cells in PV-Cre; GAD67flox/flox mice. Furthermore, PV-Cre; GAD67 flox/flox mice exhibited abnormal motor coordination in the rotarod test. These results indicate that GABA signaling in the cerebellum is critical for establishing appropriate connections between basket cells and Purkinje cells and is associated with motor coordination in mice.
  • Taisuke Miyazaki, Megumi Morimoto-Tomita, Coralie Berthoux, Kotaro Konno, Yoav Noam, Tokiwa Yamasaki, Matthijs Verhage, Pablo E Castillo, Masahiko Watanabe, Susumu Tomita
    eLife 10 2021/10/18 
    Ionotropic neurotransmitter receptors at postsynapses mediate fast synaptic transmission upon binding of the neurotransmitter. Post- and trans-synaptic mechanisms through cytosolic, membrane, and secreted proteins have been proposed to localize neurotransmitter receptors at postsynapses. However, it remains unknown which mechanism is crucial to maintain neurotransmitter receptors at postsynapses. In this study, we ablated excitatory or inhibitory neurons in adult mouse brains in a cell-autonomous manner. Unexpectedly, we found that excitatory AMPA receptors remain at the postsynaptic density upon ablation of excitatory presynaptic terminals. In contrast, inhibitory GABAA receptors required inhibitory presynaptic terminals for their postsynaptic localization. Consistent with this finding, ectopic expression at excitatory presynapses of neurexin-3 alpha, a putative trans-synaptic interactor with the native GABAA receptor complex, could recruit GABAA receptors to contacted postsynaptic sites. These results establish distinct mechanisms for the maintenance of excitatory and inhibitory postsynaptic receptors in the mature mammalian brain.
  • Wafa A A Alsulaiman, Raphaelle Quillet, Andrew M Bell, Allen C Dickie, Erika Polgár, Kieran A Boyle, Masahiko Watanabe, R Brian Roome, Artur Kania, Andrew J Todd, Maria Gutierrez-Mecinas
    Scientific reports 11 (1) 17912 - 17912 2021/09/09 
    A recently developed Phox2a::Cre mouse line has been shown to capture anterolateral system (ALS) projection neurons. Here, we used this line to test whether Phox2a-positive cells represent a distinct subpopulation among lamina I ALS neurons. We show that virtually all lamina I Phox2a cells can be retrogradely labelled from injections targeted on the lateral parabrachial area (LPb), and that most of those in the cervical cord also belong to the spinothalamic tract. Phox2a cells accounted for ~ 50-60% of the lamina I cells retrogradely labelled from LPb or thalamus. Phox2a was preferentially associated with smaller ALS neurons, and with those showing relatively weak neurokinin 1 receptor expression. The Phox2a cells were also less likely to project to the ipsilateral LPb. Although most Phox2a cells phosphorylated extracellular signal-regulated kinases following noxious heat stimulation, ~ 20% did not, and these were significantly smaller than the activated cells. This suggests that those ALS neurons that respond selectively to skin cooling, which have small cell bodies, may be included among the Phox2a population. Previous studies have defined neurochemical populations among the ALS cells, based on expression of Tac1 or Gpr83. However, we found that the proportions of Phox2a cells that expressed these genes were similar to the proportions reported for all lamina I ALS neurons, suggesting that Phox2a is not differentially expressed among cells belonging to these populations. Finally, we used a mouse line that resulted in membrane labelling of the Phox2a cells and showed that they all possess dendritic spines, although at a relatively low density. However, the distribution of the postsynaptic protein Homer revealed that dendritic spines accounted for a minority of the excitatory synapses on these cells. Our results confirm that Phox2a-positive cells in lamina I are ALS neurons, but show that the Phox2a::Cre line preferentially captures specific types of ALS cells.
  • Tamás Wilheim, Krisztina Nagy, Mahendravarman Mohanraj, Kamil Ziarniak, Masahiko Watanabe, Joanna Sliwowska, Imre Kalló
    Brain structure & function 226 (7) 2387 - 2399 2021/09 
    The endocannabinoids have been shown to target the afferents of hypothalamic neurons via cannabinoid 1 receptor (CB1) and thereby to influence their excitability at various physiological and/or pathological processes. Kisspeptin (KP) neurons form afferents of multiple neuroendocrine cells and influence their activity via signaling through a variation of co-expressed classical neurotransmitters and neuropeptides. The differential potency of endocannabinoids to influence the release of classical transmitters or neuropeptides, and the ovarian cycle-dependent functioning of the endocannabinoid signaling in the gonadotropin-releasing hormone (GnRH) neurons initiated us to study whether (a) the different subpopulations of KP neurons express CB1 mRNAs, (b) the expression is influenced by estrogen, and (c) CB1-immunoreactivity is present in the KP afferents to GnRH neurons. The aim of the study was to investigate the site- and cell-specific expression of CB1 in female mice using multiple labeling in situ hybridization and immunofluorescent histochemical techniques. The results support that CB1 mRNAs are expressed by both the GABAergic and glutamatergic subpopulations of KP neurons, the receptor protein is detectable in two-thirds of the KP afferents to GnRH neurons, and the expression of CB1 mRNA shows an estrogen-dependency. The applied estrogen-treatment, known to induce proestrus, reduced the level of CB1 transcripts in the rostral periventricular area of the third ventricle and arcuate nucleus, and differently influenced its co-localization with vesicular GABA transporter or vesicular glutamate transporter-2 in KP neurons. This indicates a gonadal cycle-dependent role of endocannabinoid signaling in the neuronal circuits involving KP neurons.
  • Izumi Kaji, Joseph T Roland, Sudiksha Rathan-Kumar, Amy C Engevik, Andreanna Burman, Anna E Goldstein, Masahiko Watanabe, James R Goldenring
    JCI insight 6 (16) 2021/08/23 
    Functional loss of myosin Vb (MYO5B) induces a variety of deficits in intestinal epithelial cell function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). The impact of MYO5B loss on differentiated cell lineage choice has not been investigated. We quantified the populations of differentiated epithelial cells in tamoxifen-induced, epithelial cell-specific MYO5B-knockout (VilCreERT2 Myo5bfl/fl) mice utilizing digital image analysis. Consistent with our RNA-sequencing data, MYO5B loss induced a reduction in tuft cells in vivo and in organoid cultures. Paneth cells were significantly increased by MYO5B deficiency along with expansion of the progenitor cell zone. We further investigated the effect of lysophosphatidic acid (LPA) signaling on epithelial cell differentiation. Intraperitoneal LPA significantly increased tuft cell populations in both control and MYO5B-knockout mice. Transcripts for Wnt ligands were significantly downregulated by MYO5B loss in intestinal epithelial cells, whereas Notch signaling molecules were unchanged. Additionally, treatment with the Notch inhibitor dibenzazepine (DBZ) restored the populations of secretory cells, suggesting that the Notch pathway is maintained in MYO5B-deficient intestine. MYO5B loss likely impairs progenitor cell differentiation in the small intestine in vivo and in vitro, partially mediated by Wnt/Notch imbalance. Notch inhibition and/or LPA treatment may represent an effective therapeutic approach for treatment of MVID.
  • Miwako Yamasaki, Atsu Aiba, Masanobu Kano, Masahiko Watanabe
    Neuropharmacology 194 108629 - 108629 2021/08/15 
    The cerebellum is essential for the control, coordination, and learning of movements, and for certain aspects of cognitive function. Purkinje cells are the sole output neurons in the cerebellar cortex and therefore play crucial roles in the diverse functions of the cerebellum. The type 1 metabotropic glutamate receptor (mGluR1) is prominently enriched in Purkinje cells and triggers downstream signaling pathways that are required for functional and structural plasticity, and for synaptic responses. To understand how mGluR1 contributes to cerebellar functions, it is important to consider not only the operational properties of this receptor, but also its spatial organization and the molecular interactions that enable its proper functioning. In this review, we highlight how mGluR1 and its related signaling molecules are organized into tightly coupled microdomains to fulfill physiological functions. We also describe emerging evidence that altered mGluR1 signaling in Purkinje cells underlies cerebellar dysfunction in ataxias of human patients and mouse models.
  • Izumi Iida, Kohtarou Konno, Rie Natsume, Manabu Abe, Masahiko Watanabe, Kenji Sakimura, Miho Terunuma
    Behavioural brain research 405 113194 - 113194 2021/05/07 
    Kainate receptors (KARs) are members of the glutamate receptor family that regulate synaptic function in the brain. Although they are known to be associated with psychiatric disorders, how they are involved in these disorders remains unclear. KARs are tetrameric channels assembled from a combination of GluK1-5 subunits. Among these, GluK2 and GluK5 subunits are the major heteromeric subunits in the brain. To determine the functional similarities and differences between GluK2 and GluK5 subunits, we generated GluK2 KO and GluK5 KO mice on a C57BL/6N background, a well-characterized inbred strain, and compared their behavioral phenotypes. We found that GluK2 KO and GluK5 KO mice exhibited the same phenotypes in many tests, such as reduced locomotor activity, impaired motor function, and enhanced depressive-like behavior. No change was observed in motor learning, anxiety-like behavior, or sociability. Additionally, we identified subunit-specific phenotypes, such as reduced motivation toward their environment in GluK2 KO mice and an enhancement in the contextual memory in GluK5 KO mice. These results revealed that GluK2 and GluK5 subunits not only function in a coordinated manner but also have a subunit-specific role in regulating behavior. To summarize, we demonstrated subunit-specific and common behavioral effects of GluK2 and GluK5 subunits for the first time. Moreover, to the best of our knowledge, this is the first evidence of the involvement of the GluK5 subunit in the expression of depressive-like behavior and contextual memory, which strongly indicates its role in psychiatric disorders.
  • Shigeto Sato, Sachiko Noda, Satoru Torii, Taku Amo, Aya Ikeda, Manabu Funayama, Junji Yamaguchi, Takahiro Fukuda, Hiromi Kondo, Norihiro Tada, Satoko Arakawa, Masahiko Watanabe, Yasuo Uchiyama, Shigeomi Shimizu, Nobutaka Hattori
    Human molecular genetics 30 (6) 443 - 453 2021/04/30 
    Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurones, but the relationship between impaired autophagy and Lewy bodies (LBs) remains unknown. α-Synuclein and p62, components of LBs, are the defining characteristic of Parkinson's disease (PD). Until now, we have analyzed mice models and demonstrated p62 aggregates derived from an autophagic defect might serve as 'seeds' and can potentially be a cause of LB formation. P62 may be the key molecule for aggregate formation. To understand the mechanisms of LBs, we analyzed p62 homeostasis and inclusion formation using PD model mice. In PARK22-linked PD, intrinsically disordered mutant CHCHD2 initiates Lewy pathology. To determine the function of CHCHD2 for inclusions formation, we generated Chchd2-knockout (KO) mice and characterized the age-related pathological and motor phenotypes. Chchd2 KO mice exhibited p62 inclusion formation and dopaminergic neuronal loss in an age-dependent manner. These changes were associated with a reduction in mitochondria complex activity and abrogation of inner mitochondria structure. In particular, the OPA1 proteins, which regulate fusion of mitochondrial inner membranes, were immature in the mitochondria of CHCHD2-deficient mice. CHCHD2 regulates mitochondrial morphology and p62 homeostasis by controlling the level of OPA1. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system, in controlling intracellular inclusion body formation, and indicate that the pathologic processes associated with the mitochondrial proteolytic system are crucial for loss of DA neurones.
  • Toshikazu Kakizaki, Tomokazu Ohshiro, Makoto Itakura, Kohtarou Konno, Masahiko Watanabe, Hajime Mushiake, Yuchio Yanagawa
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 35 (2) e21224  2021/02 
    GABA is synthesized by glutamate decarboxylase (GAD), which has two isoforms, namely, GAD65 and GAD67, encoded by the Gad2 and Gad1 genes, respectively. GAD65-deficient (Gad2-/- ) mice exhibit a reduction in brain GABA content after 1 month of age and show spontaneous seizures in adulthood. Approximately 25% of Gad2-/- mice died by 6 months of age. Our Western blot analysis demonstrated that the protein expression ratio of GAD65 to GAD67 in the brain was greater in rats than in mice during postnatal development, suggesting that the contribution of each GAD isoform to GABA functions differs between these two species. To evaluate whether GAD65 deficiency causes different phenotypes between rats and mice, we generated Gad2-/- rats using TALEN genome editing technology. Western blot and immunohistochemical analyses with new antibodies demonstrated that the GAD65 protein was undetectable in the Gad2-/- rat brain. Gad2-/- pups exhibited spontaneous seizures and paroxysmal discharge in EEG at postnatal weeks 3-4. More than 80% of the Gad2-/- rats died at postnatal days (PNDs) 17-23. GABA content in Gad2-/- brains was significantly lower than those in Gad2+/- and Gad2+/+ brains at PND17-19. These results suggest that the low levels of brain GABA content in Gad2-/- rats may lead to epilepsy followed by premature death, and that Gad2-/- rats are more severely affected than Gad2-/- mice. Considering that the GAD65/GAD67 ratio in human brains is more similar to that in rat brains than in mouse brains, Gad2-/- rats would be useful for further investigating the roles of GAD65 in vivo.
  • Marta Valle-León, Luis F Callado, Ester Aso, María M Cajiao-Manrique, Kristoffer Sahlholm, Marc López-Cano, Concepció Soler, Xavier Altafaj, Masahiko Watanabe, Sergi Ferré, Víctor Fernández-Dueñas, José M Menchón, Francisco Ciruela
    Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 46 (3) 665 - 672 2021/02 
    According to the adenosine hypothesis of schizophrenia, the classically associated hyperdopaminergic state may be secondary to a loss of function of the adenosinergic system. Such a hypoadenosinergic state might either be due to a reduction of the extracellular levels of adenosine or alterations in the density of adenosine A2A receptors (A2ARs) or their degree of functional heteromerization with dopamine D2 receptors (D2R). In the present study, we provide preclinical and clinical evidences for this latter mechanism. Two animal models for the study of schizophrenia endophenotypes, namely the phencyclidine (PCP) mouse model and the A2AR knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native A2AR-D2R heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in A2AR knockout mice, similar to that observed in the PCP animal model of sensory gating impairment of schizophrenia, as well as a significant upregulation of striatal D2R without changes in A2AR expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal A2AR-D2R heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of A2AR-D2R heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both D2R and A2AR were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal A2AR-D2R heteromers. The degree of A2AR-D2R heteromer formation in schizophrenia might constitute a hallmark of the illness, which indeed should be further studied to establish possible correlations with chronic antipsychotic treatments.
  • Hajime Shiotani, Muneaki Miyata, Takeshi Kameyama, Kenji Mandai, Miwako Yamasaki, Masahiko Watanabe, Kiyohito Mizutani, Yoshimi Takai
    The Journal of comparative neurology 529 (2) 450 - 477 2021/02 [Refereed][Not invited]
     
    The medial habenula (MHb) receives afferents from the triangular septum and the medial septal complex, projects efferents to the interpeduncular nucleus (IPN) in the midbrain to regulate dopamine and serotonin levels, and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed that the cell adhesion molecule nectin-2α is localized at the boundary between adjacent somata of clustered cholinergic neurons and regulates the voltage-gated A-type K+ channel Kv4.2 localization at membrane specializations in the MHb. This adhesion apparatus, named nectin-2α spots, is not associated with the nectin-binding protein afadin or any classic cadherins and their binding proteins p120-catenin and β-catenin. We showed here that nectin-2α was additionally localized at cholinergic neuron dendrites in synaptic regions of the MHb. The genetic ablation of nectin-2 reduced the number of synapses in the MHb without affecting their morphology. Nectin-2α was associated with afadin, cadherin-8, p120-catenin, β-catenin, and αN-catenin, forming puncta adherentia junctions (PAJs). Nectin-2α was observed in the IPN, but not in the triangular septum or the medial septal complex. The genetic ablation of nectin-2 did not affect synapse formation in the IPN. These results indicate that nectin-2α forms two types of adhesion apparatus in the MHb, namely nectin-2α spots at neighboring somata and PAJs at neighboring dendrites, and that dendritic PAJs regulate synapse formation in the MHb.
  • Shigeo Miyata, Toshikazu Kakizaki, Kazuyuki Fujihara, Hideru Obinata, Touko Hirano, Junichi Nakai, Mika Tanaka, Shigeyoshi Itohara, Masahiko Watanabe, Kenji F Tanaka, Manabu Abe, Kenji Sakimura, Yuchio Yanagawa
    Molecular brain 14 (1) 5 - 5 2021/01/07 
    Reduced expression of glutamate decarboxylase 67 (GAD67), encoded by the Gad1 gene, is a consistent finding in postmortem brains of patients with several psychiatric disorders, including schizophrenia, bipolar disorder and major depressive disorder. The dysfunction of GAD67 in the brain is implicated in the pathophysiology of these psychiatric disorders; however, the neurobiological consequences of GAD67 dysfunction in mature brains are not fully understood because the homozygous Gad1 knockout is lethal in newborn mice. We hypothesized that the tetracycline-controlled gene expression/suppression system could be applied to develop global GAD67 knockdown mice that would survive into adulthood. In addition, GAD67 knockdown mice would provide new insights into the neurobiological impact of GAD67 dysfunction. Here, we developed Gad1tTA/STOP-tetO biallelic knock-in mice using Gad1STOP-tetO and Gad1tTA knock-in mice, and compared them with Gad1+/+ mice. The expression level of GAD67 protein in brains of Gad1tTA/STOP-tetO mice treated with doxycycline (Dox) was decreased by approximately 90%. The GABA content was also decreased in the brains of Dox-treated Gad1tTA/STOP-tetO mice. In the open-field test, Dox-treated Gad1tTA/STOP-tetO mice exhibited hyper-locomotor activity and decreased duration spent in the center region. In addition, acoustic startle responses were impaired in Dox-treated Gad1tTA/STOP-tetO mice. These results suggest that global reduction in GAD67 elicits emotional abnormalities in mice. These GAD67 knockdown mice will be useful for elucidating the neurobiological mechanisms of emotional abnormalities, such as anxiety symptoms associated with psychiatric disorders.
  • Anussara Kamnate, Juthathip Sirisin, Yada Polsan, Surang Chomphoo, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Journal of anatomy 238 (6) 1330 - 1340 2021/01/04 
    In order to understand the mechanism of the endocannabinoid (eCB) signal, which has so far been shown to work in oocyte genesis and maturation, it is critical to clarify detailed localization of the eCB synthesizing enzyme molecules as well as receptors for eCBs in oocytes in the ovary in situ. For this purpose, diacylglycerol lipase (DGL) α and β are involved in the synthesis of an eCB 2-arachidonoylglycerol (2-AG). DGLα/β and the cannabinoid receptor 1 (CB1) for 2-AG were shown to be localized to the primary oocytes of postnatal mice using immuno-light and electron microscopy. It was found that two types of localization existed: first, immunoreactivities for DGLα and β were weakly detected throughout the ooplasm in light microscopy for which the intracellular membranes of vesicles forming tiny scattered aggregates were responsible. Secondly, DGLβ-immunoreactivity was distinctly confined to the nuage of Balbiani bodies and small nuage-derivative structures; both amorphous materials and membranes of vesicles were responsible for their localization. On the other hand, the weak immunoreactivity for CB1 was localized in a pattern similar to the first one for DGLs, but not found in a pattern for the Balbiani nuage. Two routes of functional exertion of 2-AG synthesized by DGLs were suggested from the two types of localization: one was that the eCB synthesized at all the sites of DGLs is released from the oocytes and exerts paracrine or autocrine effects on adjacent intra-ovarian cells as well as the oocytes themselves. The other was that the eCB synthesized within the nuage was involved in the modulation of the posttranscriptional processing of oocytes. Owing to the failure in the detection of CB1 in the Balbiani nuage, however, the validity of the latter possibility remains to be elucidated.
  • Kohtarou Konno, Masahiko Watanabe
    Neuromethods 169 191 - 199 1940-6045 2021 
    Elucidating the molecular organization at synapses is essential for understanding brain function and plasticity. Immunohistochemistry is widely used as a sensitive and specific method in morphological studies. There are specific antibodies directed against receptors, ion channels, and their interacting molecules however, it is sometimes difficult and ineffective to visualize synaptic proteins by conventional immunocytochemistry. This is mainly owing to the fact that the cross-linking of proteins by chemical fixation hampers the accessibility of antibodies to antigen molecules. This is particularly true for receptors and ion channels condensed in the synaptic cleft, postsynaptic density, or trigger zone of action potentials. To overcome this problem, researchers have devised methods to improve immunohistochemical detection of proteins that are hidden or prone to be hidden in condensed molecular matrices. Of these methods, mild chemical fixation by low paraformaldehyde concentrations or fresh frozen sections is often effective in detecting such hidden proteins. Moreover, pretreatment of sections with proteases such as pepsin is a prerequisite to detect proteins embedded in the core of the postsynaptic density, for example, NMDA-type glutamate receptors and their interacting PSD-95 protein family. In this chapter, we introduce these improving techniques for light microscopic immunohistochemistry.
  • Miwako Yamasaki, Masahiko Watanabe
    Neuromethods 169 145 - 160 1940-6045 2021 
    We describe a simplified and efficient protocol for non-isotopic fluorescent in situ hybridization experiments, allowing the implementation of multiple probe analyses at a single-cell resolution. This protocol involves simultaneous and specific hybridization of hapten-labeled riboprobes for mRNAs, followed by probe visualization via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. This procedure can be used to detect virtually any combination of two or three mRNA populations and is therefore a powerful method to characterize neuronal populations expressing a target gene and to determine the expression of genes of interest in the same or distinct cells. A combination of fluorescent in situ hybridization with immunofluorescence and retrograde fluorescent tracer labeling further expands the benefit and utility of histology-based methods. We also provide representative results and troubleshooting for the important steps of this protocol. Once specific probes and tissue sections are obtained, the total length of the entire procedure is 2–3 days.
  • ラット脊髄損傷モデルにおけるFTY720の神経障害性疼痛に対する治療効果の検証
    山崎 和義, 川堀 真人, 関 俊隆, 高宮 宗一朗, 今野 幸太郎, 渡邉 雅彦
    日本脳神経外傷学会プログラム・抄録集 (一社)日本脳神経外傷学会 44回 117 - 117 2021/01
  • Kouichi Hashimoto, Yosuke Yamawaki, Kenji Yamaoka, Takayuki Yoshida, Kana Okada, Wanqin Tan, Miwako Yamasaki, Yoshiko Matsumoto-Makidono, Reika Kubo, Hisako Nakayama, Tsutomu Kataoka, Takashi Kanematsu, Masahiko Watanabe, Yasumasa Okamoto, Shigeru Morinobu, Hidenori Aizawa, Shigeto Yamawaki
    Brain communications 3 (4) fcab285  2021 
    Animals suffering from uncontrollable stress sometimes show low effort to escape stress (learned helplessness). Changes in serotonin (5-hydroxytryptamine) signalling are thought to underlie this behaviour. Although the release of 5-hydroxytryptamine is triggered by the action potential firing of dorsal raphe nuclei 5-hydroxytryptamine neurons, the electrophysiological changes induced by uncontrollable stress are largely unclear. Herein, we examined electrophysiological differences among 5-hydroxytryptamine neurons in naïve rats, learned helplessness rats and rats resistant to inescapable stress (non-learned helplessness). Five-week-old male Sprague Dawley rats were exposed to inescapable foot shocks. After an avoidance test session, rats were classified as learned helplessness or non-learned helplessness. Activity-dependent 5-hydroxytryptamine release induced by the administration of high-potassium solution was slower in free-moving learned helplessness rats. Subthreshold electrophysiological properties of 5-hydroxytryptamine neurons were identical among the three rat groups, but the depolarization-induced spike firing was significantly attenuated in learned helplessness rats. To clarify the underlying mechanisms, potassium (K+) channels regulating the spike firing were initially examined using naïve rats. K+ channels sensitive to 500 μM tetraethylammonium caused rapid repolarization of the action potential and the small conductance calcium-activated K+ channels produced afterhyperpolarization. Additionally, dendrotoxin-I, a blocker of Kv1.1 (encoded by Kcna1), Kv1.2 (encoded by Kcna2) and Kv1.6 (encoded by Kcna6) voltage-dependent K+ channels, weakly enhanced the spike firing frequency during depolarizing current injections without changes in individual spike waveforms in naïve rats. We found that dendrotoxin-I significantly enhanced the spike firing of 5-hydroxytryptamine neurons in learned helplessness rats. Consequently, the difference in spike firing among the three rat groups was abolished in the presence of dendrotoxin-I. These results suggest that the upregulation of dendrotoxin-I-sensitive Kv1 channels underlies the firing attenuation of 5-hydroxytryptamine neurons in learned helplessness rats. We also found that the antidepressant ketamine facilitated the spike firing of 5-hydroxytryptamine neurons and abolished the firing difference between learned helplessness and non-learned helplessness by suppressing dendrotoxin-I-sensitive Kv1 channels. The dendrotoxin-I-sensitive Kv1 channel may be a potential target for developing drugs to control activity of 5-hydroxytryptamine neurons.
  • Kazuyoshi Yamazaki, Masahito Kawabori, Toshitaka Seki, Soichiro Takamiya, Kotaro Konno, Masahiko Watanabe, Kiyohiro Houkin, Miki Fujimura
    Stem cells international 2021 9964877 - 9964877 2021 
    Stem cell therapy has been shown to reverse the sequelae of spinal cord injury (SCI). Although the ideal treatment route remains unknown, providing a large number of stem cells to the injured site using less invasive techniques is critical to achieving maximal recovery. This study was conducted to determine whether administration of bone marrow stem cell (BMSC) sheet made on its own without a scaffold is superior to intramedullary cell transplantation in a rat subacute SCI model. Adult female Sprague-Dawley rats were subjected to SCI by 30 g clip compression at the level of Th6 and Th7 and were administered BMSC cell sheet (7 × 104 cells, subdural), cell suspension (7 × 104 cells, intramedullary), or control seven days after the injury. Motor and sensory assessments, as well as histological evaluation, were performed to determine the efficacy of the different cell transplantation procedures. While both the cell sheet and cell intramedullary injection groups showed significant motor recovery compared to the control group, the cell sheet group showed better results. Furthermore, the cell sheet group displayed a significant sensory recovery compared to the other groups. A histological evaluation revealed that the cell sheet group showed smaller injury lesion volume, less inflammation, and gliosis compared to other groups. Sensory-related fibers of μ-opioid receptors (MOR, interneuron) and hydroxytryptamine transporters (HTT, descending pain inhibitory pathway), located around the dorsal horn of the spinal cord at the caudal side of the SCI, were preserved only in the cell sheet group. Stem cells could also be found inside the peri-injured spinal cord in the cell sheet group. BMSC cell sheets were able to promote functional recovery and palliate neuropathic pain more effectively than intramedullary injections, thus serving as a good treatment option for SCI.
  • Esther Suk King Lai, Hisako Nakayama, Taisuke Miyazaki, Takanobu Nakazawa, Katsuhiko Tabuchi, Kouichi Hashimoto, Masahiko Watanabe, Masanobu Kano
    Frontiers in neural circuits 15 676891 - 676891 2021 
    Neuroligin is a postsynaptic cell-adhesion molecule that is involved in synapse formation and maturation by interacting with presynaptic neurexin. Mutations in neuroligin genes, including the arginine to cystein substitution at the 451st amino acid residue (R451C) of neuroligin-3 (NLGN3), have been identified in patients with autism spectrum disorder (ASD). Functional magnetic resonance imaging and examination of post-mortem brain in ASD patients implicate alteration of cerebellar morphology and Purkinje cell (PC) loss. In the present study, we examined possible association between the R451C mutation in NLGN3 and synaptic development and function in the mouse cerebellum. In NLGN3-R451C mutant mice, the expression of NLGN3 protein in the cerebellum was reduced to about 10% of the level of wild-type mice. Elimination of redundant climbing fiber (CF) to PC synapses was impaired from postnatal day 10-15 (P10-15) in NLGN3-R451C mutant mice, but majority of PCs became mono-innervated as in wild-type mice after P16. In NLGN3-R451C mutant mice, selective strengthening of a single CF relative to the other CFs in each PC was impaired from P16, which persisted into juvenile stage. Furthermore, the inhibition to excitation (I/E) balance of synaptic inputs to PCs was elevated, and calcium transients in the soma induced by strong and weak CF inputs were reduced in NLGN3-R451C mutant mice. These results suggest that a single point mutation in NLGN3 significantly influences the synapse development and refinement in cerebellar circuitry, which might be related to the pathogenesis of ASD.
  • Toshinori Matsuoka, Miwako Yamasaki, Manabu Abe, Yukiko Matsuda, Hiroyuki Morino, Hideshi Kawakami, Kenji Sakimura, Masahiko Watanabe, Kouichi Hashimoto
    The Journal of physiology 599 (2) 547 - 569 2021/01 
    KEY POINTS: Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so-called 'resonant properties'). Here, we report that heterologous expression of mouse Kv11 voltage-dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties. Kv11.3-expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor. The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice. This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. ABSTRACT: The plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor-like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+ ) channels, encoded by ether-á-go-go-related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole-cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3-, Kv11.1(Merg1b)- and Kv11.1(Merg1a)-expressing cells were 2-6 Hz, 2-4 Hz, and 0.6-0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor-inductor-capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11-dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at -30 to -60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency-dependent responses in a subtype-dependent manner from resting to depolarized membrane potentials.
  • Péter Szocsics, Péter Papp, László Havas, Masahiko Watanabe, Zsófia Maglóczky
    Brain structure & function 226 (1) 281 - 296 2021/01 
    Betz cells-the gigantopyramidal neurons found in high amount in the primary motor cortex-are among of the most characteristic neuronal cells. A part of them contains the calcium-binding protein parvalbumin (PV) in primates. However, less is known about these cells in the human motor cortex despite their important role in different neurological disorders. Therefore, the aim of our study was to investigate the neurochemical features and perisomatic input properties of Betz cells in control human samples with short post-mortem interval. We used different microscopic techniques to investigate the primary motor cortex of both hemispheres. The soma size and density, and expression of PV of the Betz cells were investigated. Furthermore, we used confocal fluorescent and electron microscopy to examine their perisomatic input. The soma size and density showed moderate variability among samples and hemispheres. Post-mortem interval and hemispherical localization did not influence these features. Around 70% of Betz cells expressed PV, but in less intensity than the cortical interneurons. Betz neurons receive dense perisomatic input, which are mostly VIAAT- (vesicular inhibitory amino acid transporter) and PV immunopositive. In the electron microscope, we found PV-immunolabelled terminals with asymmetric-like synaptic structure, too. Terminals with morphologically similar synaptic specialisation were also found among vGluT2- (vesicular glutamate transporter type 2) immunostained terminals contacting Betz cells. Our data suggest that Betz cells' morphological properties showed less variability among subjects and hemispheres than the density of them. Their neurochemical and perisomatic input characteristics support their role in execution of fast and precise movements.
  • Motokazu Uchigashima, Kohtarou Konno, Emily Demchak, Amy Cheung, Takuya Watanabe, David G Keener, Manabu Abe, Timmy Le, Kenji Sakimura, Toshikuni Sasaoka, Takeshi Uemura, Yuka Imamura Kawasawa, Masahiko Watanabe, Kensuke Futai
    eLife 9 2020/12/23 
    Synapse formation and regulation require signaling interactions between pre- and postsynaptic proteins, notably cell adhesion molecules (CAMs). It has been proposed that the functions of neuroligins (Nlgns), postsynaptic CAMs, rely on the formation of trans-synaptic complexes with neurexins (Nrxns), presynaptic CAMs. Nlgn3 is a unique Nlgn isoform that localizes at both excitatory and inhibitory synapses. However, Nlgn3 function mediated via Nrxn interactions is unknown. Here we demonstrate that Nlgn3 localizes at postsynaptic sites apposing vesicular glutamate transporter 3-expressing (VGT3+) inhibitory terminals and regulates VGT3+ inhibitory interneuron-mediated synaptic transmission in mouse organotypic slice cultures. Gene expression analysis of interneurons revealed that the αNrxn1+AS4 splice isoform is highly expressed in VGT3+ interneurons as compared with other interneurons. Most importantly, postsynaptic Nlgn3 requires presynaptic αNrxn1+AS4 expressed in VGT3+ interneurons to regulate inhibitory synaptic transmission. Our results indicate that specific Nlgn-Nrxn signaling generates distinct functional properties at synapses.
  • Kazuyuki Fujihara, Kazuo Yamada, Yukio Ichitani, Toshikazu Kakizaki, Weiru Jiang, Shigeo Miyata, Takashi Suto, Daiki Kato, Shigeru Saito, Masahiko Watanabe, Yuki Kajita, Tomokazu Ohshiro, Hajime Mushiake, Yoshiki Miyasaka, Tomoji Mashimo, Hiroki Yasuda, Yuchio Yanagawa
    Translational psychiatry 10 (1) 426 - 426 2020/12/08 
    GABAergic dysfunctions have been implicated in the pathogenesis of schizophrenia, especially the associated cognitive impairments. The GABA synthetic enzyme glutamate decarboxylase 67-kDa isoform (GAD67) encoded by the GAD1 gene is downregulated in the brains of patients with schizophrenia. Furthermore, a patient with schizophrenia harboring a homozygous mutation of GAD1 has recently been discovered. However, it remains unclear whether loss of function of GAD1 leads to the symptoms observed in schizophrenia, including cognitive impairment. One of the obstacles faced in experimental studies to address this issue is the perinatal lethality of Gad1 knockout (KO) mice, which precluded characterization at the adult stage. In the present study, we successfully generated Gad1 KO rats using CRISPR/Cas9 genome editing technology. Surprisingly, 33% of Gad1 KO rats survived to adulthood and could be subjected to further characterization. The GABA concentration in the Gad1 KO cerebrum was reduced to ~52% of the level in wild-type rats. Gad1 KO rats exhibited impairments in both spatial reference and working memory without affecting adult neurogenesis in the hippocampus. In addition, Gad1 KO rats showed a wide range of behavioral alterations, such as enhanced sensitivity to an NMDA receptor antagonist, hypoactivity in a novel environment, and decreased preference for social novelty. Taken together, the results suggest that Gad1 KO rats could provide a novel model covering not only cognitive deficits but also other aspects of the disorder. Furthermore, the present study teaches an important lesson: differences between species should be considered when developing animal models of human diseases.
  • Erika Polgár, Andrew M Bell, Maria Gutierrez-Mecinas, Allen C Dickie, Oğuz Akar, Miruna Costreie, Masahiko Watanabe, Andrew J Todd
    Neuroscience 450 113 - 125 2020/12/01 
    The tachykinin peptide substance P (SP) is expressed by many interneurons and some projection neurons in the superficial dorsal horn of the spinal cord. We have recently shown that SP-expressing excitatory interneurons in lamina II correspond largely to a morphological class known as radial cells. However, little is known about their function, or their synaptic connectivity. Here we use a modification of the Brainbow technique to define the excitatory synaptic input to SP radial cells. We show that around half of their excitatory synapses (identified by expression of Homer) are from boutons with VGLUT2, which are likely to originate mainly from local interneurons. The remaining synapses presumably include primary afferents, which generally have very low levels of VGLUT2. Our results also suggest that the SP cells are preferentially innervated by a population of excitatory interneurons defined by expression of green fluorescent protein under control of the gene for gastrin-releasing peptide, and that they receive sparser input from other types of excitatory interneuron. We show that around 40% of lamina I projection neurons express Tac1, the gene encoding substance P. Finally, we show that silencing Tac1-expressing cells in the dorsal horn results in a significant reduction in reflex responses to cold and radiant heat, but does not affect withdrawal to von Frey hairs, or chloroquine-evoked itch.
  • ラット亜急性期脊髄損傷モデルに対する骨髄間葉系幹細胞シート移植の治療効果検証
    山崎 和義, 川堀 真人, 関 俊隆, 高宮 宗一朗, 今野 幸太郎, 渡辺 雅彦, 宝金 清博
    脳循環代謝 (一社)日本脳循環代謝学会 32 (1) 82 - 82 0915-9401 2020/11
  • Ryoji Fukabori, Yoshio Iguchi, Shigeki Kato, Kazumi Takahashi, Satoshi Eifuku, Shingo Tsuji, Akihiro Hazama, Motokazu Uchigashima, Masahiko Watanabe, Hiroshi Mizuma, Yilong Cui, Hirotaka Onoe, Keigo Hikishima, Yasunobu Yasoshima, Makoto Osanai, Ryo Inagaki, Kohji Fukunaga, Takuma Nishijo, Toshihiko Momiyama, Richard Benton, Kazuto Kobayashi
    The Journal of neuroscience : the official journal of the Society for Neuroscience 40 (43) 8367 - 8385 2020/10/21 [Not refereed][Not invited]
     
    The ability of animals to retrieve memories stored in response to the environment is essential for behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. Here, we developed a novel chemogenetic activation strategy exploiting insect olfactory ionotropic receptors (IRs), termed "IR-mediated neuronal activation," and used it for selective stimulation of NE neurons in the locus coeruleus (LC). Drosophila melanogaster IR84a and IR8a subunits were expressed in LC NE neurons in transgenic mice. Application of phenylacetic acid (a specific ligand for the IR84a/IR8a complex) at appropriate doses induced excitatory responses of NE neurons expressing the receptors in both slice preparations and in vivo electrophysiological conditions, resulting in a marked increase of NE release in the LC nerve terminal regions (male and female). Ligand-induced activation of LC NE neurons enhanced the retrieval process of conditioned taste aversion without affecting taste sensitivity, general arousal state, and locomotor activity. This enhancing effect on taste memory retrieval was mediated, in part, through α1- and β-adrenergic receptors in the basolateral nucleus of the amygdala (BLA; male). Pharmacological inhibition of LC NE neurons confirmed the facilitative role of these neurons in memory retrieval via adrenergic receptors in the BLA (male). Our findings indicate that the LC NE system, through projections to the BLA, controls the retrieval process of taste associative memory.SIGNIFICANCE STATEMENT Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation, but the role of the NE system in memory retrieval remains unclear. We developed a chemogenetic activation system based on insect olfactory ionotropic receptors and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons enhanced the retrieval of conditioned taste aversion, which was mediated, in part, through adrenoceptors in the basolateral amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC-amygdala pathway plays an important role in the recall of taste associative memory.
  • Hiroaki Sacai, Kazuto Sakoori, Kohtarou Konno, Kenichiro Nagahama, Honoka Suzuki, Takaki Watanabe, Masahiko Watanabe, Naofumi Uesaka, Masanobu Kano
    Nature communications 11 (1) 5140 - 5140 2020/10/12 
    Autism spectrum disorder (ASD) is thought to result from deviation from normal development of neural circuits and synaptic function. Many genes with mutation in ASD patients have been identified. Here we report that two molecules associated with ASD susceptibility, contactin associated protein-like 2 (CNTNAP2) and Abelson helper integration site-1 (AHI1), are required for synaptic function and ASD-related behavior in mice. Knockdown of CNTNAP2 or AHI1 in layer 2/3 pyramidal neurons of the developing mouse prefrontal cortex (PFC) reduced excitatory synaptic transmission, impaired social interaction and induced mild vocalization abnormality. Although the causes of reduced excitatory transmission were different, pharmacological enhancement of AMPA receptor function effectively restored impaired social behavior in both CNTNAP2- and AHI1-knockdown mice. We conclude that reduced excitatory synaptic transmission in layer 2/3 pyramidal neurons of the PFC leads to impaired social interaction and mild vocalization abnormality in mice.
  • Izumi Kaji, Joseph T Roland, Masahiko Watanabe, Amy C Engevik, Anna E Goldstein, Craig A Hodges, James R Goldenring
    Gastroenterology 159 (4) 1390 - 1405 2020/10 
    BACKGROUND & AIM: Myosin VB (MYO5B) is an essential trafficking protein for membrane recycling in gastrointestinal epithelial cells. The inactivating mutations of MYO5B cause the congenital diarrheal disease, microvillus inclusion disease (MVID). MYO5B deficiency in mice causes mislocalization of SGLT1 and NHE3, but retained apical function of CFTR, resulting in malabsorption and secretory diarrhea. Activation of lysophosphatidic acid (LPA) receptors can improve diarrhea, but the effect of LPA on MVID symptoms is unclear. We investigated whether LPA administration can reduce the epithelial deficits in MYO5B-knockout mice. METHODS: Studies were conducted with tamoxifen-induced, intestine-specific knockout of MYO5B (VilCreERT2;Myo5bflox/flox) and littermate controls. Mice were given LPA, an LPAR2 agonist (GRI977143), or vehicle for 4 days after a single injection of tamoxifen. Apical SGLT1 and CFTR activities were measured in Üssing chambers. Intestinal tissues were collected, and localization of membrane transporters was evaluated by immunofluorescence analysis in tissue sections and enteroids. RNA sequencing and enrichment analysis were performed with isolated jejunal epithelial cells. RESULTS: Daily administration of LPA reduced villus blunting, frequency of multivesicular bodies, and levels of cathepsins in intestinal tissues of MYO5B-knockout mice compared with vehicle administration. LPA partially restored the brush border height and the localization of SGLT1 and NHE3 in small intestine of MYO5B-knockout mice and enteroids. The SGLT1-dependent short-circuit current was increased and abnormal CFTR activities were decreased in jejunum from MYO5B-knockout mice given LPA compared with vehicle. CONCLUSIONS: LPA may regulate a MYO5B-independent trafficking mechanism and brush border maturation, and therefore be developed for treatment of MVID.
  • Sho Kakizawa, Yasushi Kishimoto, Shinichiro Yamamoto, Kazuko Onga, Kunihiko Yasuda, Yoshiaki Miyamoto, Masahiko Watanabe, Ryuichi Sakai, Nozomu Mori
    Scientific reports 10 (1) 14475 - 14475 2020/09/02 
    Intracellular Ca2+ levels are changed by influx from extracellular medium and release from intracellular stores. In the central nervous systems, Ca2+ release is involved in various physiological events, such as neuronal excitability and transmitter release. Although stable Ca2+ release in response to stimulus is critical for proper functions of the nervous systems, regulatory mechanisms relating to Ca2+ release are not fully understood in central neurons. Here, we demonstrate that ShcB, an adaptor protein expressed in central neurons, has an essential role in functional maintenance of Ca2+ store in cerebellar Purkinje cells (PCs). ShcB-knockout (KO) mice showed defects in cerebellar-dependent motor function and long-term depression (LTD) at cerebellar synapse. The reduced LTD was accompanied with an impairment of intracellular Ca2+ release. Although the expression of Ca2+ release channels and morphology of Ca2+ store looked intact, content of intracellular Ca2+ store and activity of sarco/endoplasmic reticular Ca2+-ATPase (SERCA) were largely decreased in the ShcB-deficient cerebellum. Furthermore, when ShcB was ectopically expressed in the ShcB-KO PCs, the Ca2+ release and its SERCA-dependent component were restored. These data indicate that ShcB plays a key role in the functional maintenance of ER Ca2+ store in central neurons through regulation of SERCA activity.
  • Kunimichi Suzuki, Jonathan Elegheert, Inseon Song, Hiroyuki Sasakura, Oleg Senkov, Keiko Matsuda, Wataru Kakegawa, Amber J Clayton, Veronica T Chang, Maura Ferrer-Ferrer, Eriko Miura, Rahul Kaushik, Masashi Ikeno, Yuki Morioka, Yuka Takeuchi, Tatsuya Shimada, Shintaro Otsuka, Stoyan Stoyanov, Masahiko Watanabe, Kosei Takeuchi, Alexander Dityatev, A Radu Aricescu, Michisuke Yuzaki
    Science (New York, N.Y.) 369 (6507) 2020/08/28 
    Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory-inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we designed and characterized CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1. CPTX can interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors and induced the formation of excitatory synapses both in vitro and in vivo. CPTX restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimer's disease, and spinal cord injury, respectively. Thus, CPTX represents a prototype for structure-guided biologics that can efficiently repair or remodel neuronal circuits.
  • Andrew M Bell, Maria Gutierrez-Mecinas, Anna Stevenson, Adrian Casas-Benito, Hendrik Wildner, Steven J West, Masahiko Watanabe, Andrew J Todd
    Scientific reports 10 (1) 13176 - 13176 2020/08/06 
    Dorsal horn excitatory interneurons that express gastrin-releasing peptide (GRP) are part of the circuit for pruritogen-evoked itch. They have been extensively studied in a transgenic line in which enhanced green fluorescent protein (eGFP) is expressed under control of the Grp gene. The GRP-eGFP cells are separate from several other neurochemically-defined excitatory interneuron populations, and correspond to a class previously defined as transient central cells. However, mRNA for GRP is widely distributed among excitatory interneurons in superficial dorsal horn. Here we show that although Grp mRNA is present in several transcriptomically-defined populations, eGFP is restricted to a discrete subset of cells in the GRP::eGFP mouse, some of which express the neuromedin receptor 2 and likely belong to a cluster defined as Glut8. We show that these cells receive much of their excitatory synaptic input from MrgA3/MrgD-expressing nociceptive/pruritoceptive afferents and C-low threshold mechanoreceptors. Although the cells were not innervated by pruritoceptors expressing brain natriuretic peptide (BNP) most of them contained mRNA for NPR1, the receptor for BNP. In contrast, these cells received only ~ 10% of their excitatory input from other interneurons. These findings demonstrate that the GRP-eGFP cells constitute a discrete population of excitatory interneurons with a characteristic pattern of synaptic input.
  • Kazuyoshi Yamazaki, Masahito Kawabori, Toshitaka Seki, Soichiro Takamiya, Takahiro Tateno, Kotaro Konno, Masahiko Watanabe, Kiyohiro Houkin
    Journal of neurotrauma 37 (15) 1720 - 1728 2020/08/01 [Refereed][Not invited]
     
    Neuropathic pain severely impairs rehabilitation and quality of life after spinal cord injury (SCI). The sphingosine-1-phosphate receptor agonist, FTY720, plays an important protective role in neuronal injury. This study aims to examine the effects of FTY720 in a rat acute SCI model, focusing on neuropathic pain. Female rats with SCI induced by 1-min clip compression were administered vehicle or 1.5 mg/kg of FTY720 24 h after the injury. Using the mechanical nociceptive threshold test, we monitored neuropathic pain and performed histological analysis of the pain pathway, including the μ opioid receptor (MOR), hydroxytryptamine transporter (HTT), and calcitonin gene-related peptide (CGRP). Motor score, SCI lesion volume, residual motor axons, inflammatory response, glial scar, and microvascular endothelial dysfunction were also compared between the two groups. FTY720 treatment resulted in significant attenuation of post-traumatic neuropathic pain. It also decreased systemic and local inflammation, thereby reducing the damaged areas and astrogliosis and resulting in motor functional recovery. Whereas there was no difference in the CGRP expression between the two groups, FTY720 significantly preserved the MOR in both the caudal and rostral areas of the spinal dorsal horn. Whereas HTT was preserved in the FTY720 group, it was significantly increased in the rostral side and decreased in the caudal side of the injury in the vehicle group. These results suggest that FTY720 ameliorates post-traumatic allodynia through regulation of neuroinflammation, maintenance of the blood-brain barrier, and inhibition of glial scar formation, thereby preserving the connectivity of the descending inhibitory pathway and reducing neuropathic pain.
  • Keitaro Yoshida, Iku Tsutsui-Kimura, Anna Kono, Akihiro Yamanaka, Kenta Kobayashi, Masahiko Watanabe, Masaru Mimura, Kenji F Tanaka
    Cell reports 31 (13) 107829 - 107829 2020/06/30 
    Medium spiny neurons (MSNs) of mice show opposing activities upon the initiation of a food-seeking lever press task. Ventromedial striatal (VMS)-MSNs are inhibited but ventrolateral striatal (VLS)-MSNs are activated; these activities mediate action selection and action initiation, respectively. To understand what input shapes the opposing MSN activities, here, we monitor cortical input activities at the cell population level and artificially reverse them. We demonstrate that the ventral hippocampus (vHP) and the insular cortex (IC) are major inputs to the VMS and VLS, both projections show silencing at the trial start time, and the vHP-VMS and IC-VLS pathways form functionally coupled input-output units during the task. Of note, the upstream IC silencing is converted to the downstream VLS-MSN activation. We find biased localization of striatal parvalbumin-expressing interneurons (PV INs) and verify PV IN-dependent feedforward architecture in the VLS. Our results reveal a distinct mode of cortico-striatal signal conveyance via feedforward disinhibition in behaving animals.
  • Shin-Ichiro Horigane, Shun Hamada, Satoshi Kamijo, Hirokazu Yamada, Miwako Yamasaki, Masahiko Watanabe, Haruhiko Bito, Toshihisa Ohtsuka, Sayaka Takemoto-Kimura
    Neuroscience research 169 17 - 26 2020/06/26 
    Increasing evidence has shown that voltage-gated L-type Ca2+ channels (LTCCs) are crucial for neurodevelopmental events, including neuronal differentiation/migration and neurite morphogenesis/extension. However, the time course of their functional maturation during the development of excitatory neurons remains unknown. Using a combination of fluorescence in situ hybridization and in utero electroporation-based labeling, we found that the transcripts of Cacna1c and Cacna1d, which encode the LTCC pore-forming subunits, were upregulated in the intermediate zone (IZ) during radial migration. Ca2+ imaging using GCaMP6s in acute brain slices showed spontaneous Ca2+ transients in migrating neurons throughout the IZ. Neurons in the IZ upper layer, especially in the multipolar-to-bipolar transition layer (TL), exhibited more frequent Ca2+ transients than adjacent layers and responded to FPL64176, a potent activator of LTCC. Consistently, nimodipine, an LTCC blocker, inhibited spontaneous Ca2+ transients in neurons in the TL. Collectively, we showed a hitherto unknown increased prevalence of LTCC-dependent Ca2+ transients in the TL of the IZ upper layer during the radial migration of excitatory neurons, which could be essential for the regulation of Ca2+-dependent neurodevelopmental processes.
  • Motokazu Uchigashima, Ming Leung, Takuya Watanabe, Amy Cheung, Timmy Le, Sabine Pallat, Alexandre Luis Marques Dinis, Masahiko Watanabe, Yuka Imamura Kawasawa, Kensuke Futai
    The Journal of biological chemistry 295 (25) 8589 - 8595 2020/06/19 
    Synapse formation is a dynamic process essential for the development and maturation of the neuronal circuitry in the brain. At the synaptic cleft, trans-synaptic protein-protein interactions are major biological determinants of proper synapse efficacy. The balance of excitatory and inhibitory synaptic transmission (E-I balance) stabilizes synaptic activity, and dysregulation of the E-I balance has been implicated in neurodevelopmental disorders, including autism spectrum disorders. However, the molecular mechanisms underlying the E-I balance remain to be elucidated. Here, using single-cell transcriptomics, immunohistochemistry, and electrophysiology approaches to murine CA1 pyramidal neurons obtained from organotypic hippocampal slice cultures, we investigate neuroligin (Nlgn) genes that encode a family of postsynaptic adhesion molecules known to shape excitatory and inhibitory synaptic function. We demonstrate that the NLGN3 protein differentially regulates inhibitory synaptic transmission in a splice isoform-dependent manner at hippocampal CA1 synapses. We also found that distinct subcellular localizations of the NLGN3 isoforms contribute to the functional differences observed among these isoforms. Finally, results from single-cell RNA-Seq analyses revealed that Nlgn1 and Nlgn3 are the major murine Nlgn genes and that the expression levels of the Nlgn splice isoforms are highly diverse in CA1 pyramidal neurons. Our results delineate isoform-specific effects of Nlgn genes on the E-I balance in the murine hippocampus.
  • Masahiro Fukaya, Takeyuki Sugawara, Yoshinobu Hara, Makoto Itakura, Masahiko Watanabe, Hiroyuki Sakagami
    The Journal of neuroscience : the official journal of the Society for Neuroscience 40 (22) 4277 - 4296 2020/05/27 
    Brefeldin A-resistant ArfGEF 2 (BRAG2) [or Iqsec1 (IQ motif and Sec7 domain-containing protein 1)] is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes. BRAG2 regulates Arf6-dependent endocytosis of AMPA receptors (AMPARs) through the direct interaction during the hippocampal long-term depression. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Herein, using mouse brains of both sexes, we demonstrated that BRAG2a, an alternative isoform with a long C-terminal insert containing a proline-rich domain and type I PDZ-binding motif, was selectively localized to the excitatory postsynaptic density (PSD). Using yeast two-hybrid screening, we identified PSD-95 and endophilin 1/3 as BRAG2a-binding partners in the brain. The interaction with PSD-95 was required for synaptic targeting of BRAG2a. In cultured hippocampal neurons, stimulation of group I metabotropic glutamate receptors (mGluRs) increased the interaction of BRAG2a with endophilin 3 and concomitant Arf6 activation in a time-dependent manner. Knockdown of BRAG2 in cultured hippocampal neurons blocked the mGluR-dependent decrease in surface AMPAR levels, which was rescued by introducing wild-type BRAG2a, but not wild-type BRAG2b or BRAG2a mutants lacking the ability to activate Arf6 or to interact with endophilin 3 or PSD-95. Further postembedding immunoelectron microscopic analysis revealed the preorganized lateral distribution of BRAG2a, Arf6, and endophilin 3 for efficient endocytosis at the postsynaptic membrane. Together, the present findings unveiled a novel molecular mechanism by which BRAG2a links AMPARs to the clathrin-dependent endocytic pathway through its interaction with PSD-95 and endophilin 3.SIGNIFICANCE STATEMENT BRAG2/Iqsec1 is a GDP/GTP exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes, and regulates Arf6-dependent endocytosis of AMPARs through direct interaction during hippocampal long-term depression, one of the mechanisms of synaptic plasticity related to learning and memory. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Here, we identified isoform-specific mechanisms of BRAG2-mediated AMPAR internalization. We demonstrated that the interaction of BRAG2a isoform with PSD-95 and endophilin 3 was required for the mGluR-dependent decrease in surface AMPARs in hippocampal neurons. These results unveiled a novel molecular mechanism by which BRAG2 links AMPARs to the clathrin-mediated endocytic machinery at postsynaptic sites.
  • Taisuke Miyazaki, Miwako Yamasaki, Kenji F Tanaka, Masahiko Watanabe
    Neuroscience 462 89 - 105 2020/05/26 
    Purkinje cells (PCs) are principal cerebellar neurons, and several classes of interneurons modulate their activity. Lugaro cells (LCs) are one such inhibitory interneuron with distinctive cytology and location, but still most enigmatic among cerebellar neurons. Here we serendipitously produced a novel transgenic mouse line, where a half of Yellow Cameleon (YC)(+) cells in the cerebellar cortex were judged to be LCs, and YC(+) LCs were estimated to constitute one-third of the total LC populations. Neurochemically, two-thirds of YC(+) LCs were dually GABAergic/glycinergic, with the rest being GABAergic. Beneath the PC layer, they extended a sheet of somatodendritic meshwork interconnected with neighboring LCs by adherens junctions, and received various inputs from climbing fibers, mossy fibers, granule cell axons, recurrent PC axons, Golgi cell axons, LC axons, and serotonergic fibers. Intriguingly, somatodendritic elements of individual LCs preferentially extended within a given cerebellar compartment defined by aldolase C expression. In turn, YC(+) LCs projected a dense lattice of ascending and transverse axons to the molecular layer, and innervated molecular layer interneurons (basket and stellate cells) and Golgi cells, but not PCs. Of note, ascending axons profusely innervated individual targets within a cerebellar compartment, while transverse axons ran across several compartments and innervated targets sparsely. This unique circuit configuration highlights that LCs integrate various excitatory, inhibitory, and modulatory inputs coming to the belonging cerebellar compartment and that, as an interneuron-selective interneuron, LCs can effectively disinhibit cerebellar cortical activities in a compartment-dependent manner through inhibition of inhibitory interneurons selectively targeting PCs and granule cells.
  • Atthapon Pidsaya, Anussara Kamnate, Juthathip Sirisin, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Archives of oral biology 113 104689 - 104689 2020/05 
    OBJECTIVE: The aim of this study was to clarify the mechanism of GABA (□-amino butyric acid)-signaling in the salivary glands by localization of vesicular inhibitory amino acid transporter, a key molecule in GABA-synthesis. DESIGN: Parotid, sublingual and submandibular glands of mice at various postnatal stages were examined in immuno-light and electron microscopy as well as immuno-blotting. RESULTS: Expression for vesicular inhibitory amino acid transporter was detected in parotid and sublingual glands of both sexes and female submandibular gland throughout postnatal development, while it was negligible in male submandibular glands at and after puberty. The expression in female submandibular glands attenuated after testosterone injection. The immunoreactivity was localized in striated ductal cells, but not acinar cells, in the salivary glands, and it occurred in association with intracellular and plasma membranes of the cells. It also occurred in myoepithelial and vascular smooth muscle cells. CONCLUSIONS: GABA-signaling was suggested to be a significant signaling pathway in salivary ductal cells, which was suppressed in male submandibular glands at and after puberty. The suppression in the submandibular duct was by testosterone. In addition, the participation of vesicular inhibitory amino acid transporter in GABA signaling through plasma membranes of the ductal cells was suggested. The significance of occurrence of the immunoreactivity in myoepithelial and smooth muscle cells remains to be further elucidated in terms of implication in GABA signaling.
  • Chihiro Nakamoto, Kohtarou Konno, Taisuke Miyazaki, Ena Nakatsukasa, Rie Natsume, Manabu Abe, Meiko Kawamura, Yugo Fukazawa, Ryuichi Shigemoto, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe
    The Journal of comparative neurology 528 (6) 1003 - 1027 2020/04 [Refereed][Not invited]
     
    In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD-95-expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS-digested freeze-fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular-anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions.
  • Erzsébet Farkas, Edina Varga, Balázs Kovács, Anett Szilvásy-Szabó, Antonieta Cote-Vélez, Zoltán Péterfi, Magdalini Matziari, Mónika Tóth, Dóra Zelena, Zsolt Mezriczky, Andrea Kádár, Dóra Kővári, Masahiko Watanabe, Masanobu Kano, Ken Mackie, Balázs Rózsa, Yvette Ruska, Blanka Tóth, Zoltán Máté, Ferenc Erdélyi, Gábor Szabó, Balázs Gereben, Ronald M Lechan, Jean-Louis Charli, Patricia Joseph-Bravo, Csaba Fekete
    iScience 23 (3) 100921 - 100921 2589-0042 2020/03/27 [Refereed][Not invited]
     
    Based on the type-I cannabinoid receptor (CB1) content of hypophysiotropic axons and the involvement of tanycytes in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis, we hypothesized that endocannabinoids are involved in the tanycyte-induced regulation of TRH release in the median eminence (ME). We demonstrated that CB1-immunoreactive TRH axons were associated to DAGLα-immunoreactive tanycyte processes in the external zone of ME and showed that endocannabinoids tonically inhibit the TRH release in this tissue. We showed that glutamate depolarizes the tanycytes, increases their intracellular Ca2+ level and the 2-AG level of the ME via AMPA and kainite receptors and glutamate transport. Using optogenetics, we demonstrated that glutamate released from TRH neurons influences the tanycytes in the ME. In summary, tanycytes regulate TRH secretion in the ME via endocannabinoid release, whereas TRH axons regulate tanycytes by glutamate, suggesting the existence of a reciprocal microcircuit between tanycytes and TRH terminals that controls TRH release.
  • B Anne Bannatyne, Zhao-Zhe Hao, Georgia M C Dyer, Masahiko Watanabe, David J Maxwell, Ari Berkowitz
    The Journal of neuroscience : the official journal of the Society for Neuroscience 40 (13) 2680 - 2694 2020/03/25 
    The spinal cord can appropriately generate diverse movements, even without brain input and movement-related sensory feedback, using a combination of multifunctional and behaviorally specialized interneurons. The adult turtle spinal cord can generate motor patterns underlying forward swimming, three forms of scratching, and limb withdrawal (flexion reflex). We previously described turtle spinal interneurons activated during both scratching and swimming (multifunctional interneurons), interneurons activated during scratching but not swimming (scratch-specialized interneurons), and interneurons activated during flexion reflex but not scratching or swimming (flexion reflex-selective interneurons). How multifunctional and behaviorally specialized turtle spinal interneurons affect downstream neurons was unknown. Here, we recorded intracellularly from spinal interneurons activated during these motor patterns in turtles of both sexes in vivo and filled each with dyes. We labeled motoneurons using choline acetyltransferase antibodies or earlier intraperitoneal FluoroGold injection and used immunocytochemistry of interneuron axon terminals to identify their neurotransmitter(s) and putative synaptic contacts with motoneurons. We found that multifunctional interneurons are heterogeneous with respect to neurotransmitter, with some glutamatergic and others GABAergic or glycinergic, and can directly contact motoneurons. Also, scratch-specialized interneurons are heterogeneous with respect to neurotransmitter and some directly contact motoneurons. Thus, scratch-specialized interneurons might directly excite motoneurons that are more strongly activated during scratching than forward swimming, such as hip-flexor motoneurons. Finally, and surprisingly, we found that some motoneurons are behaviorally specialized, for scratching or flexion reflex. Thus, either some limb muscles are only used for a subset of limb behaviors or some limb motoneurons are only recruited during certain limb behaviors.SIGNIFICANCE STATEMENT Both multifunctional and behaviorally specialized spinal cord interneurons have been described in turtles, but their outputs are unknown. We studied responses of multifunctional interneurons (activated during swimming and scratching) and scratch-specialized interneurons, filled each with dyes, and used immunocytochemistry to determine their neurotransmitters and contacts with motoneurons. We found that both multifunctional and scratch-specialized interneurons are heterogeneous with respect to neurotransmitter, with some excitatory and others inhibitory. We found that some multifunctional and some scratch-specialized interneurons directly contact motoneurons. Scratch-specialized interneurons may excite motoneurons that are more strongly activated during scratching than swimming, such as hip-flexor motoneurons, or inhibit their antagonists, hip-extensor motoneurons. Surprisingly, we also found that some motoneurons are behaviorally specialized, for scratching or for flexion reflex.
  • Atsara Rawangwong, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Acta histochemica 122 (2) 151497 - 151497 2020/02 [Refereed][Not invited]
     
    Our previous study demonstrated that, different from the parotid and sublingual glands, the submandibular glands of adult mice did not show an immunoblot band for PLCβ3 which is critical in the secretion mechanism by muscarinic cholinergic signaling. Therefore, the submandibular glands of mice at various stages of postnatal development were examined for this enzyme molecule in immunoblot and immunohistochemistry. In immunoblot, a weak band for PLCβ3-expression was detected only at early postnatal stages. In immunohistochemistry, PLCβ3-immunoreactivity was distinctly found in most basally located cells of immature ducts, while the immunoreactivity was weakly seen in terminal tubule cells without significant immunoreactivity in adjacent acinar cells. In contrast, the immunoreactivity was distinctly found in some basal cells of adult excretory ducts, and it was ultrastructurally localized densely in close association with bundles of tonofilaments in the cells. The present finding suggests the possibility that Ca2+ signaling governed by phospholipase Cβ3 is involved in the differentiation of ductal basal cells into apical cells through control of keratin molecule(s) in the cells.
  • Jun Yamada, Chihiro Sato, Kohtarou Konno, Masahiko Watanabe, Shozo Jinno
    The Journal of neuroscience : the official journal of the Society for Neuroscience 40 (4) 825 - 842 2020/01/22 [Refereed][Not invited]
     
    The extracellular glycan polysialic acid linked to neural cell adhesion molecule (PSA-NCAM) is principally expressed in the developing brain and the adult neurogenic regions. Although colocalization of PSA-NCAM with cholecystokinin (CCK) was found in the adult brain, the role of PSA-NCAM remains unclear. In this study, we aimed to elucidate the functional significance of PSA-NCAM in the CA1 region of the male mouse hippocampus. Combined fluorescence in situ hybridization and immunohistochemistry showed that few vesicular glutamate transporter 3-negative/CCK-positive (VGluT3-/CCK+) cells were colocalized with PSA-NCAM, but most of the VGluT3+/CCK+ cells were colocalized with PSA-NCAM. The somata of PSA-NCAM+/CCK+ cells were highly innervated by serotonergic boutons than those of PSA-NCAM-/CCK+ cells. The expression ratios of 5-HT3A receptors and p11, a serotonin receptor-interacting protein, were higher in PSA-NCAM+/CCK+ cells than in PSA-NCAM-/CCK+ cells. Pharmacological digestion of PSA-NCAM impaired the efficacy of antidepressant fluoxetine (FLX), a selective serotonin reuptake inhibitor, but not the efficacy of benzodiazepine anxiolytic diazepam. A Western blot showed that restraint stress decreased the expressions of p11 and mature brain-derived neurotrophic factor (BDNF), and FLX increased them. Interestingly, the FLX-induced elevation of expression of p11, but not mature BDNF, was impaired by the digestion of PSA-NCAM. Quantitative reverse transcription-polymerase chain reaction showed that restraint stress reduced the expression of polysialyltransferase ST8Sia IV and FLX elevated it. Collectively, PSA-NCAM colocalized with VGluT3+/CCK+ cells in the CA1 region of the hippocampus may play a unique role in the regulation of antidepressant efficacy via the serotonergic pathway.SIGNIFICANCE STATEMENT Polysialic acid (PSA) is composed of eight or more α2,8-linked sialic acids. Here, we examined the functional significance of polysialic acid linked to the neural cell adhesion molecule (PSA-NCAM) in the adult mouse hippocampus. Few vesicular glutamate transporter 3-negative/cholecystokinin-positive (VGluT3-/CCK+) cells were colocalized with PSA-NCAM, but most of the VGluT3+/CCK+ cells were colocalized with PSA-NCAM. The expression ratios of 5-HT3A receptors and p11, a serotonin receptor-interacting protein, were higher in PSA-NCAM+/CCK+ cells than in PSA-NCAM-/CCK+ cells. The efficacy of antidepressants, but not anxiolytics, was impaired by the digestion of PSA-NCAM. The antidepressant-induced increase in p11 expression was inhibited following PSA-NCAM digestion. We hence hypothesize that PSA-NCAM colocalized with VGluT3+/CCK+ cells may play a unique role in regulating antidepressant efficacy.
  • Chihiro Nakamoto, Meiko Kawamura, Ena Nakatsukasa, Rie Natsume, Keizo Takao, Masahiko Watanabe, Manabu Abe, Tomonori Takeuchi, Kenji Sakimura
    PloS one 15 (2) e0229288  2020 [Refereed][Not invited]
     
    The GluD1 gene is associated with susceptibility for schizophrenia, autism, depression, and bipolar disorder. However, the function of GluD1 and how it is involved in these conditions remain elusive. In this study, we generated a Grid1 gene-knockout (GluD1-KO) mouse line with a pure C57BL/6N genetic background and performed several behavioral analyses. Compared to a control group, GluD1-KO mice showed no significant anxiety-related behavioral differences, evaluated using behavior in an open field, elevated plus maze, a light-dark transition test, the resident-intruder test of aggression and sensorimotor gating evaluated by the prepulse inhibition test. However, GluD1-KO mice showed (1) higher locomotor activity in the open field, (2) decreased sociability and social novelty preference in the three-chambered social interaction test, (3) impaired memory in contextual, but not cued fear conditioning tests, and (4) enhanced depressive-like behavior in a forced swim test. Pharmacological studies revealed that enhanced depressive-like behavior in GluD1-KO mice was restored by the serotonin reuptake inhibitors imipramine and fluoxetine, but not the norepinephrine transporter inhibitor desipramine. In addition, biochemical analysis revealed no significant difference in protein expression levels, such as other glutamate receptors in the synaptosome and postsynaptic densities prepared from the frontal cortex and the hippocampus. These results suggest that GluD1 plays critical roles in fear memory, sociability, and depressive-like behavior.
  • Takafumi Kawai, Haruhiko Miyata, Hiroki Nakanishi, Souhei Sakata, Shin Morioka, Junko Sasaki, Masahiko Watanabe, Kenji Sakimura, Toyoshi Fujimoto, Takehiko Sasaki, Masahito Ikawa, Yasushi Okamura
    Proceedings of the National Academy of Sciences of the United States of America 116 (51) 26020 - 26028 2019/12/17 [Refereed][Not invited]
     
    The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P2 Sperm from VSP-/- mice show more Ca2+ influx upon capacitation than VSP+/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P2-sensitive K+ channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P2 distribution plays important roles in ion channel regulation as well as sperm motility.
  • Edina Varga, Erzsébet Farkas, Györgyi Zséli, Andrea Kádár, Alexandra Venczel, Dóra Kővári, Dorottya Németh, Zoltán Máté, Ferenc Erdélyi, András Horváth, Ottó Szenci, Masahiko Watanabe, Ronald M Lechan, Balázs Gereben, Csaba Fekete
    Thyroid : official journal of the American Thyroid Association 29 (12) 1858 - 1868 2019/12 [Refereed][Not invited]
     
    Background: Glycine is a classical neurotransmitter that has role in both inhibitory and excitatory synapses. To understand whether glycinergic inputs are involved in the regulation of the hypophysiotropic thyrotropin-releasing hormone (TRH) neurons, the central controllers of the hypothalamic-pituitary-thyroid axis, the glycinergic innervation of the TRH neurons was studied in the hypothalamic paraventricular nucleus (PVN). Methods: Double-labeling immunocytochemistry and patch-clamp electrophysiology were used to determine the role of glycinergic neurons in the regulation of TRH neurons in the PVN. Anterograde and retrograde tracing methods were used to determine the sources of the glycinergic input of TRH neurons. Results: Glycine transporter-2 (GLYT2), a marker of glycinergic neurons, containing axons were found to establish symmetric type of synapses on TRH neurons in the PVN. Furthermore, glycine receptor immunoreactivity was observed in these TRH neurons. The raphe magnus (RMg) and the ventrolateral periaqueductal gray (VLPAG) were found to be the exclusive sources of the glycinergic innervation of the TRH neurons within the PVN. Patch-clamp electrophysiology using sections of TRH-IRES-tdTomato mice showed that glycine hyperpolarized the TRH neurons and completely blocked the firing of these neurons. Glycine also markedly hyperpolarized the TRH neurons in the presence of tetrodotoxin demonstrating the direct effect of glycine. In more than 60% of the TRH neurons, spontaneous inhibitory postsynaptic currents (sIPSCs) were observed, even after the pharmacological inhibition of glutamatergic and GABAergic neuronal transmission. The glycine antagonist, strychnine, almost completely abolished these sIPSCs, demonstrating the inhibitory nature of the glycinergic input of TRH neurons. Conclusions: These data demonstrate that TRH neurons in the PVN receive glycinergic inputs from the RMg and the VLPAG. The symmetric type of synaptic connection and the results of the electrophysiological experiments demonstrate the inhibitory nature of these inputs.
  • Kelly M Smith, Tyler J Browne, Olivia C Davis, A Coyle, Kieran A Boyle, Masahiko Watanabe, Sally A Dickinson, Jacqueline A Iredale, Mark A Gradwell, Phillip Jobling, Robert J Callister, Christopher V Dayas, David I Hughes, Brett A Graham
    eLife 8 2019/11/12 [Refereed][Not invited]
     
    Nociceptive information is relayed through the spinal cord dorsal horn, a critical area in sensory processing. The neuronal circuits in this region that underpin sensory perception must be clarified to better understand how dysfunction can lead to pathological pain. This study used an optogenetic approach to selectively activate spinal interneurons that express the calcium-binding protein calretinin (CR). We show that these interneurons form an interconnected network that can initiate and sustain enhanced excitatory signaling, and directly relay signals to lamina I projection neurons. Photoactivation of CR interneurons in vivo resulted in a significant nocifensive behavior that was morphine sensitive, caused a conditioned place aversion, and was enhanced by spared nerve injury. Furthermore, halorhodopsin-mediated inhibition of these interneurons elevated sensory thresholds. Our results suggest that dorsal horn circuits that involve excitatory CR neurons are important for the generation and amplification of pain and identify these interneurons as a future analgesic target.
  • Maria Gutierrez-Mecinas, Andrew Bell, Erika Polgár, Masahiko Watanabe, Andrew J Todd
    Neuroscience 416 281 - 293 2019/09/15 
    The great majority of neurons in the superficial dorsal horn of the spinal cord are excitatory interneurons, and these are required for the normal perception of pain and itch. We have previously identified 5 largely non-overlapping populations among these cells, based on the expression of four different neuropeptides (cholecystokinin, neurotensin, neurokinin B and substance P) and of green fluorescent protein driven by the promoter for gastrin-releasing peptide (GRP) in a transgenic mouse line. Another peptide (neuropeptide FF, NPFF) has been identified among the excitatory neurons, and here we have used an antibody against the NPFF precursor (pro-NPFF) and a probe that recognises Npff mRNA to identify and characterise these cells. We show that they are all excitatory interneurons, and are separate from the five populations listed above, accounting for ~6% of the excitatory neurons in laminae I-II. By examining phosphorylation of extracellular signal-regulated kinases, we show that the NPFF cells can respond to different types of noxious and pruritic stimulus. Ablation of somatostatin-expressing dorsal horn neurons has been shown to result in a dramatic reduction in mechanical pain sensitivity, while somatostatin released from these neurons is thought to contribute to itch. Since the great majority of the NPFF cells co-expressed somatostatin, these cells may play a role in the perception of pain and itch.
  • Motokazu Uchigashima, Amy Cheung, Julie Suh, Masahiko Watanabe, Kensuke Futai
    The Journal of comparative neurology 527 (12) 1940 - 1965 2019/08/15 [Refereed][Not invited]
     
    Synapses, highly specialized membrane junctions between neurons, connect presynaptic neurotransmitter release sites and postsynaptic ligand-gated channels. Neurexins (Nrxns), a family of presynaptic adhesion molecules, have been characterized as major regulators of synapse development and function. Via their extracellular domains, Nrxns bind to different postsynaptic proteins, generating highly diverse functional readouts through their postsynaptic binding partners. Not surprisingly given these versatile protein interactions, mutations and deletions of Nrxn genes have been identified in patients with autism spectrum disorders, intellectual disabilities, and schizophrenia. Therefore, elucidating the expression profiles of Nrxns in the brain is of high significance. Here, using chromogenic and fluorescent in situ hybridization, we characterize the expression patterns of Nrxn isoforms throughout the brain. We found that each Nrxn isoform displays a unique expression profile in a region-, cell type-, and sensory system-specific manner. Interestingly, we also found that αNrxn1 and αNrxn2 mRNAs are expressed in non-neuronal cells, including astrocytes and oligodendrocytes. Lastly, we found diverse expression patterns of genes that encode Nrxn binding proteins, such as Neuroligins (Nlgns), Leucine-rich repeat transmembrane neuronal protein (Lrrtms) and Latrophilins (Adgrls), suggesting that Nrxn proteins can mediate numerous combinations of trans-synaptic interactions. Together, our anatomical profiling of Nrxn gene expression reflects the diverse roles of Nrxn molecules.
  • Ester Aso, Víctor Fernández-Dueñas, Marc López-Cano, Jaume Taura, Masahiko Watanabe, Isidre Ferrer, Rafael Luján, Francisco Ciruela
    Molecular neurobiology 56 (8) 5382 - 5391 2019/08 [Refereed][Not invited]
     
    At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ9-THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ9-THC-induced cognitive impairment in an adenosine A2A receptor (A2AR)-dependent manner. Furthermore, we reveal the existence of A2AR and cannabinoid CB1 receptor (CB1R) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent A2AR-CB1R functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal A2AR/CB1R complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of A2AR-CB1R interactions in the brain.
  • Maria Gutierrez-Mecinas, Andrew M Bell, Fraser Shepherd, Erika Polgár, Masahiko Watanabe, Takahiro Furuta, Andrew J Todd
    The Journal of comparative neurology 527 (11) 1857 - 1871 2019/08/01 [Refereed][Not invited]
     
    Excitatory interneurons account for the majority of dorsal horn neurons, and are required for perception of normal and pathological pain. We have identified largely non-overlapping populations in laminae I-III, based on expression of substance P, gastrin-releasing peptide, neurokinin B, and neurotensin. Cholecystokinin (CCK) is expressed by many dorsal horn neurons, particularly in the deeper laminae. Here, we have used immunocytochemistry and in situ hybridization to characterize the CCK cells. We show that they account for ~7% of excitatory neurons in laminae I-II, but between a third and a quarter of those in lamina III. They are largely separate from the neurokinin B, neurotensin, and gastrin-releasing peptide populations, but show limited overlap with the substance P cells. Laminae II-III neurons with protein kinase Cγ (PKCγ) have been implicated in mechanical allodynia following nerve injury, and we found that around 50% of CCK cells were PKCγ-immunoreactive. Neurotensin is also expressed by PKCγ cells, and among neurons with moderate to high levels of PKCγ, ~85% expressed CCK or neurotensin. A recent transcriptomic study identified mRNA for thyrotropin-releasing hormone in a specific subpopulation of CCK neurons, and we show that these account for half of the CCK/PKCγ cells. These findings indicate that the CCK cells are distinct from other excitatory interneuron populations that we have defined. They also show that PKCγ cells can be assigned to different classes based on neuropeptide expression, and it will be important to determine the differential contribution of these classes to neuropathic allodynia.
  • Víctor Fernández-Dueñas, Maricel Gómez-Soler, Marta Valle-León, Masahiko Watanabe, Isidre Ferrer, Francisco Ciruela
    International journal of molecular sciences 20 (14) 2019/07/23 [Refereed][Not invited]
     
    Background: Several biophysical techniques have been successfully implemented to detect G protein-coupled receptors (GPCRs) heteromerization. Although these approaches have made it possible to ascertain the presence of GPCR heteromers in animal models of disease, no success has been accomplished in pathological human post-mortem brains. The AlphaScreen technology has been consistently used to quantify small analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range and sensitivity exhibited by this technology support that it may be suitable to detect GPCR heteromers even under non-optimal conditions. Methods: Here, we describe the development of a new AlphaScreen assay to detect GPCR oligomers in human post-mortem brain. Results: Adenosine A2A-dopamine D2 receptor (A2AR/D2R) heteromer formation was monitored in caudate from healthy and Parkinson's disease (PD) subjects. The approach was first validated using striatal membranes from wild type and A2AR deficient mice. Secondly, we took advantage of the 6-hydroxydopamine hemiparkinsonian rat model to validate previous results. In addition, finally, A2AR/D2R heteromer formation was assessed in caudate membranes from human post-mortem brains. Importantly, our preliminary results revealed an increase in A2AR/D2R heteromer formation in PD brains. Conclusions: The new AlphaScreen assay allowed assessing GPCR heteromers in human post-mortem brains with high sensitivity.
  • Kieran A Boyle, Mark A Gradwell, Toshiharu Yasaka, Allen C Dickie, Erika Polgár, Robert P Ganley, Desmond P H Orr, Masahiko Watanabe, Victoria E Abraira, Emily D Kuehn, Amanda L Zimmerman, David D Ginty, Robert J Callister, Brett A Graham, David I Hughes
    Cell reports 28 (2) 526 - 540 2019/07/09 [Refereed][Not invited]
     
    Chronic pain presents a major unmet clinical problem. The development of more effective treatments is hindered by our limited understanding of the neuronal circuits underlying sensory perception. Here, we show that parvalbumin (PV)-expressing dorsal horn interneurons modulate the passage of sensory information conveyed by low-threshold mechanoreceptors (LTMRs) directly via presynaptic inhibition and also gate the polysynaptic relay of LTMR input to pain circuits by inhibiting lamina II excitatory interneurons whose axons project into lamina I. We show changes in the functional properties of these PV interneurons following peripheral nerve injury and that silencing these cells unmasks a circuit that allows innocuous touch inputs to activate pain circuits by increasing network activity in laminae I-IV. Such changes are likely to result in the development of tactile allodynia and could be targeted for more effective treatment of mechanical pain.
  • Harumi Nakao, Yasushi Kishimoto, Kouichi Hashimoto, Kazuo Kitamura, Miwako Yamasaki, Kazuki Nakao, Masahiko Watanabe, Masanobu Kano, Yutaka Kirino, Atsu Aiba
    Scientific reports 9 (1) 7353 - 7353 2019/05/14 [Refereed][Not invited]
     
    Classical eyeblink conditioning is a representative associative motor learning that requires both the cerebellar cortex and the deep cerebellar nucleus (DCN). Metabotropic glutamate receptor subtype 1 (mGluR1) is richly expressed in Purkinje cells (PCs) of the cerebellar cortex. Global mGluR1 knock-out (KO) mice show a significantly lower percentage of conditioned response (CR%) than wild-type mice in eyeblink conditioning, and the impaired CR% is restored by the introduction of mGluR1 in PCs. However, the specific roles of mGluR1 in major memory processes, including formation, storage and expression have not yet been defined. We thus examined the role of mGluR1 in these processes of eyeblink conditioning, using mGluR1 conditional KO (cKO) mice harboring a selective and reversible expression of mGluR1 in PCs. We have found that eyeblink memory is not latently formed in the absence of mGluR1 in adult mouse PCs. However, once acquired, eyeblink memory is expressed even after the depletion of mGluR1 in PCs. We thus conclude that mGluR1 in PCs is indispensable for the formation of eyeblink memory, while it is not required for the expression of CR.
  • Atsara Rawangwong, Atthapon Pidsaya, Wipawee Thoungseabyoun, Apussara Tachow, Tarinee Sawatpanich, Waraporn Sakaew, Miwako Yamasaki, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Acta histochemica 121 (4) 484 - 490 2019/05 [Refereed][Not invited]
     
    Phospholipase C (PLC)β has a role in saliva secretion by controlling intracellular Ca2+via its product, IP3. The present study was attempted to localize PLCβ isoforms in mouse salivary glands in situ. A single major band was detected for PLCβ3 in immunoblots of the parotid and sublingual glands (PG, SLG), while no such band was seen in the submandibular gland (SMG). No bands were detected for PLCβ1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCβ3 was seen in the cytoplasm including the plasmalemma of almost all ductal cells, while no distinct immunoreactivity was discerned in most acinar cells except for sublingual demilune cells. Numerous ductal cells exhibited higher immunoreactivity for PLCβ3 in their apical/supranuclear cell domain including the plasmalemma than in the basal/infranuclear domain, indicating an apico-basal polarity. In immuno-gold electron microscopy of PG ducts and SLG ducts and demilunes, most gold particles were found in association with plasma membranes as well as various intracellular membranes, most of which formed small oblong or flattened vesicles and vacuoles. A few particles were seen without association with any membranous structures. The present finding supports the previous physio-pharmacological result that Ca2+-signaling proteins as well as initial intracellular Ca2+ changes occur in the apical cell domain including the plasma membranes of the exocrine cells.
  • Atsara Rawangwong, Suthankamon Khrongyut, Surang Chomphoo, Kohtaro Konno, Miwako Yamasaki, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Archives of oral biology 100 14 - 22 2019/04 [Refereed][Not invited]
     
    We hypothesize variation in expression and localization, along the course of the glandular tubule, of muscarinic cholinergic receptor M1 which plays as a distinct contribution, though minor in comparison with M3 receptor, in saliva secretion. Localization of the M1 receptor was examined using immunohistochemistry in three major salivary glands. Although all glandular cells were more or less M1-immunoreactive, acinar cells were weakly immunoreactive, while ductal cells exhibited substantial M1-immunoreactivity. Many ductal cells exhibited clear polarity with higher immunoreactivity in their apical/supra-nuclear domain. However, some exhibited indistinct polarity because of additional higher immunoreactivity in their basal/infra-nuclear domain. A small group of cells with intense immunoreactivity was found, mostly located in the intercalated ducts or in portions of the striated ducts close to the intercalated ducts. In immuno-electron microscopy, the immunoreactive materials were mainly in the cytoplasm including various vesicles and vacuoles. Unexpectedly, distinct immunoreactivity on apical and basal plasma membranes was infrequent in most ductal cells. The heterogeneous localization of M1-immunoreactivity along the gland tubular system is discussed in view of possible modulatory roles of the M1 receptor in saliva secretion.
  • Waraporn Sakaew, Apussara Tachow, Wipawee Thoungseabyoun, Suthankamon Khrongyut, Atsara Rawangwong, Yada Polsan, Watanabe Masahiko, Hisatake Kondo, Wiphawi Hipkaeo
    Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft 222 21 - 27 2019/03 [Refereed][Not invited]
     
    Vesicular inhibitory amino acid transporter (VIAAT) is a transmembrane transporter which is responsible for the storage of gamma-aminobutyric acid (GABA) or glycine in synaptic vesicles. According to recent studies, GABA is known to be expressed in the kidney. For clear understanding of the intra-renal GABA signaling, the localization of VIAAT was examined in the present study. Intense immunoreactivity was found largely confined to the distal tubule epithelia, especially distinct in the inner medulla, although the immunoreactivity was discerned more or less in all tubules and glomeruli. No distinct immunoreactivity was seen in capillary endothelia or interstitial fibroblasts. In immuno-DAB and immuno-gold electron microscopy, the immunoreaction was found at the basal infoldings of plasma membranes and basal portions of the lateral plasma membranes, but not in any vesicles or vacuoles within the distal tubular cells. The significance of the enigmatic finding, localization of a vesicular molecule on selected portions of the plasma membrane of distal tubular cells, was discussed in view of the possibility of paracrine or autocrine effects of GABA on some other uriniferous tubular cells or interstitial cells.
  • Allen C Dickie, Andrew M Bell, Noboru Iwagaki, Erika Polgár, Maria Gutierrez-Mecinas, Rosalind Kelly, Heather Lyon, Kirsten Turnbull, Steven J West, Alexander Etlin, Joao Braz, Masahiko Watanabe, David L H Bennett, Allan I Basbaum, John S Riddell, Andrew J Todd
    Pain 160 (2) 442 - 462 0304-3959 2019/02 [Refereed][Not invited]
     
    Excitatory interneurons account for the majority of neurons in the superficial dorsal horn, but despite their presumed contribution to pain and itch, there is still limited information about their organisation and function. We recently identified 2 populations of excitatory interneuron defined by expression of gastrin-releasing peptide (GRP) or substance P (SP). Here, we demonstrate that these cells show major differences in their morphological, electrophysiological, and pharmacological properties. Based on their somatodendritic morphology and firing patterns, we propose that the SP cells correspond to radial cells, which generally show delayed firing. By contrast, most GRP cells show transient or single-spike firing, and many are likely to correspond to the so-called transient central cells. Unlike the SP cells, few of the GRP cells had long propriospinal projections, suggesting that they are involved primarily in local processing. The 2 populations also differed in responses to neuromodulators, with most SP cells, but few GRP cells, responding to noradrenaline and 5-HT; the converse was true for responses to the μ-opioid agonist DAMGO. Although a recent study suggested that GRP cells are innervated by nociceptors and are strongly activated by noxious stimuli, we found that very few GRP cells receive direct synaptic input from TRPV1-expressing afferents, and that they seldom phosphorylate extracellular signal-regulated kinases in response to noxious stimuli. These findings indicate that the SP and GRP cells differentially process somatosensory information.
  • Maria Gutierrez-Mecinas, Olivia Davis, Erika Polgár, Mahvish Shahzad, Keila Navarro-Batista, Takahiro Furuta, Masahiko Watanabe, David I Hughes, Andrew J Todd
    Neuroscience 398 171 - 181 2019/02/01 [Refereed][Not invited]
     
    Around 75% of neurons in laminae I-II of the mouse dorsal horn are excitatory interneurons, and these are required for normal pain perception. We have shown that four largely non-overlapping excitatory interneuron populations can be defined by expression of the neuropeptides neurotensin, neurokinin B (NKB), gastrin-releasing peptide (GRP) and substance P. In addition, we recently identified a population of excitatory interneurons in glabrous skin territory that express dynorphin. The calcium-binding protein calretinin is present in many excitatory neurons in this region, but we know little about its relation to these neuropeptide markers. Here we show that calretinin is differentially expressed, being present in the majority of substance P-, GRP- and NKB-expressing cells, but not in the neurotensin or dynorphin cells. Calretinin-positive cells have been implicated in detection of noxious mechanical stimuli, but are not required for tactile allodynia after neuropathic pain. Our findings are therefore consistent with the suggestion that neuropathic allodynia involves the neurotensin and/or dynorphin excitatory interneuron populations. Around a quarter of inhibitory interneurons in lamina I-II contain calretinin, and recent transcriptomic studies suggest that these co-express substance P. We confirm this, by showing that inhibitory Cre-expressing cells in a Tac1Cre knock-in mouse are calretinin-immunoreactive. Interestingly, there is evidence that these cells express low levels of peptidylglycine alpha-amidating monooxygenase, an enzyme required for maturation of neuropeptides. This may explain our previous finding that although the substance P precursor preprotachykinin A can be detected in some inhibitory interneurons, very few inhibitory axonal boutons are immunoreactive for substance P.
  • Toshihiko Iwanaga, Kohtarou Konno, Masahiko Watanabe
    Biomedical research (Tokyo, Japan) 40 (5) 207 - 214 2019 [Refereed][Not invited]
     
    Sensory circumventricular organs contain the subfornical organ, organum vasculosum of the lamina terminalis (OVLT), and area postrema. Here, immunostaining for GLUT3 in the murine brain selectively labeled the sobfornical organ and OVLT. The immunoreactive neural tract of the subfornical organ formed into thin bundles and extended ventro-rostrally over the anterior commissure. After turning over the commissure, the neural tract passed through the median preoptic nucleus (MnPO) and reached the OVLT; thus, a continuous neural tract expressing GLUT3 connected the subfornical organ, MnPO, and OVLT in the lamina terminalis. In the OVLT, GLUT3-immunoreactive fibers gathered in both the dorsal cap and lateral periventricular zone. Electron microscopically, the immunoreactive structures in the subfornical organ corresponded to nerve fibers or nerve terminals containing many small clear vesicles. The area postrema, another sensory organ, was immunonegative for GLUT3. This study not only presented a useful marker tracing the neural tract in the sensory sites of the lamina terminalis but also suggested a unique system for sensing and determining the metabolism of circulating glucose in the circumventricular organs.
  • Zoltán Péterfi, Imre Farkas, Raphael G P Denis, Erzsébet Farkas, Motokazu Uchigashima, Tamás Füzesi, Masahiko Watanabe, Ronald M Lechan, Zsolt Liposits, Serge Luquet, Csaba Fekete
    Molecular metabolism 18 120 - 133 2018/12 [Refereed][Not invited]
     
    OBJECTIVE: Neuropeptide Y (NPY) is one of the most potent orexigenic peptides. The hypothalamic paraventricular nucleus (PVN) is a major locus where NPY exerts its effects on energy homeostasis. We investigated how NPY exerts its effect within the PVN. METHODS: Patch clamp electrophysiology and Ca2+ imaging were used to understand the involvement of Ca2+ signaling and retrograde transmitter systems in the mediation of NPY induced effects in the PVN. Immuno-electron microscopy were performed to elucidate the subcellular localization of the elements of nitric oxide (NO) system in the parvocellular PVN. In vivo metabolic profiling was performed to understand the role of the endocannabinoid and NO systems of the PVN in the mediation of NPY induced changes of energy homeostasis. RESULTS: We demonstrated that NPY inhibits synaptic inputs of parvocellular neurons in the PVN by activating endocannabinoid and NO retrograde transmitter systems via mobilization of Ca2+ from the endoplasmic reticulum, suggesting that NPY gates the synaptic inputs of parvocellular neurons in the PVN to prevent the influence of non-feeding-related inputs. While intraPVN administered NPY regulates food intake and locomotor activity via NO signaling, the endocannabinoid system of the PVN selectively mediates NPY-induced decrease in energy expenditure. CONCLUSION: Thus, within the PVN, NPY stimulates the release of endocannabinoids and NO via Ca2+-influx from the endoplasmic reticulum. Both transmitter systems appear to have unique roles in the mediation of the NPY-induced regulation of energy homeostasis, suggesting that NPY regulates food intake, energy expenditure, and locomotor activity through different neuronal networks of this nucleus.
  • Li Zhou, M Ibrahim Hossain, Maya Yamazaki, Manabu Abe, Rie Natsume, Kohtaro Konno, Shun Kageyama, Masaaki Komatsu, Masahiko Watanabe, Kenji Sakimura, Hirohide Takebayashi
    Journal of neurochemistry 147 (4) 557 - 572 0022-3042 2018/11 [Refereed][Not invited]
     
    Purkinje cell degeneration (pcd) was first identified in a spontaneous mouse mutant showing cerebellar ataxia. In addition to cerebellar Purkinje cells (PCs), retinal photoreceptors, mitral cells in the olfactory bulb, and a discrete subpopulation of thalamic neurons also degenerate in the mutant brains. The gene responsible for the pcd mutant is Nna1, also known as ATP/GTP binding protein 1 or cytosolic carboxypeptidase-like 1, which encodes a zinc carboxypeptidase protein. To investigate pathogenesis of the pcd mutation in detail, we generated a conditional Nna1 allele targeting the carboxypeptidase domain at C-terminus. After Cre recombination and heterozygous crossing, we generated Nna1 knockout (KO) mice and found that the Nna1 KO mice began to show cerebellar ataxia at postnatal day 20 (P20). Most PCs degenerated until 4-week-old, except lobule X. Activated microglia and astrocytes were also observed in the Nna1 KO cerebellum. In the mutant brain, the Nna1 mRNA level was dramatically reduced, suggesting that nonsense-mediated mRNA decay occurs in it. Since the Nna1 protein acts as a de-glutamatase on the C-terminus of α-tubulin and β-tubulin, increased polyglutamylated tubulin was detected in the Nna1 KO cerebellum. In addition, the endoplasmic reticulum stress marker, C/EBP homologous protein (CHOP), was up-regulated in the mutant PCs. We report the generation of a functional Nna1 conditional allele and possible mechanisms of PC death in the Nna1 KO in the cerebellum. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
  • Tayo Katano, Keizo Takao, Manabu Abe, Maya Yamazaki, Masahiko Watanabe, Tsuyoshi Miyakawa, Kenji Sakimura, Seiji Ito
    Molecular brain 11 (1) 63 - 63 2018/10/25 [Refereed][Not invited]
     
    Calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1 (Caskin1) is a direct binding partner of the synaptic adaptor protein CASK. Because Caskin1 forms homo-multimers and binds not only CASK but also other neuronal proteins in vitro, it is anticipated to have neural functions; but its exact role in mammals remains unclear. Previously, we showed that the concentration of Caskin1 in the spinal dorsal horn increases under chronic pain. To characterize this protein, we generated Caskin1-knockout (Caskin1-KO) mice and specific anti-Caskin1 antibodies. Biochemical and immunohistochemical analyses demonstrated that Caskin1 was broadly distributed in the whole brain and spinal cord, and that it primarily localized at synapses. To elucidate the neural function of Caskin1 in vivo, we subjected Caskin1-KO mice to comprehensive behavioral analysis. The mutant mice exhibited differences in gait, enhanced nociception, and anxiety-like behavior relative to their wild-type littermates. In addition, the knockouts exhibited strong freezing responses, with or without a cue tone, in contextual and cued-fear conditioning tests as well as low memory retention in the Barnes Maze test. Taken together, these results suggest that Caskin1 contributes to a wide spectrum of behavioral phenotypes, including gait, nociception, memory, and stress response, in broad regions of the central nervous system.
  • Morató X, Luján R, Gonçalves N, Watanabe M, Altafaj X, Carvalho AL, Fernández-Dueñas V, Cunha RA, Ciruela F
    Human molecular genetics 27 (20) 3528 - 3541 0964-6906 2018/10 [Refereed][Not invited]
     
    The hippocampus is a key brain region for memory formation. Metabotropic glutamate type 5 receptors (mGlu5R) are strongly expressed in CA1 pyramidal neurons and fine-tune synaptic plasticity. Accordingly, mGlu5R pharmacological manipulation may represent an attractive therapeutic strategy to manage hippocampal-related neurological disorders. Here, by means of a membrane yeast two-hybrid screening, we identified contactin-associated protein 1 (Caspr1), a type I transmembrane protein member of the neurexin family, as a new mGlu5R partner. We report that mGlu5R and Caspr1 co-distribute and co-assemble both in heterologous expression systems and in rat brain. Furthermore, downregulation of Caspr1 in rat hippocampal primary cultures decreased mGlu5R-mediated signaling. Finally, silencing Caspr1 expression in the hippocampus impaired the impact of mGlu5R on spatial memory. Our results indicate that Caspr1 plays a pivotal role controlling mGlu5R function in hippocampus-dependent memory formation. Hence, this new protein-protein interaction may represent novel target for neurological disorders affecting hippocampal glutamatergic neurotransmission.
  • Natsumi Ageta-Ishihara, Kohtarou Konno, Maya Yamazaki, Manabu Abe, Kenji Sakimura, Masahiko Watanabe, Makoto Kinoshita
    Neurochemistry international 119 190 - 198 0197-0186 2018/10 [Refereed][Not invited]
     
    Configuration of tripartite synapses, comprising the pre-, post-, and peri-synaptic components (axon terminal or bouton, dendritic spine, and astroglial terminal process), is a critical determinant of neurotransmitter kinetics and hence synaptic transmission. However, little is known about molecular basis for the regulation of tripartite synapse morphology. Previous studies showed that CDC42EP4, an effector protein of a cell morphogenesis regulator CDC42, is expressed exclusively in Bergmann glia in the cerebellar cortex, that it forms tight complex with the septin heterooligomer, and that it interacts indirectly with the glutamate transporter GLAST and MYH10/nonmuscle myosin ΙΙB. Scrutiny of Cdc42ep4-/- mice had revealed that the CDC42EP4-septins-GLAST interaction facilitates glutamate clearance, while the role for CDC42EP4-septins-MYH10 interaction has remained unsolved. Here, we find anomalous configuration of the tripartite synapses comprising the parallel fiber boutons, dendritic spines of Purkinje cells, and Bergmann glial processes in Cdc42ep4-/- mice. The complex anomalies include 1) recession of Bergmann glial membranes from the nearest active zones, and 2) extension of nonactive synaptic contact around active zone. In line with the recession of Bergmann glial membranes by the loss of CDC42EP4, overexpression of CDC42EP4 in heterologous cells promotes cell spreading and partitioning of MYH10 to insoluble (i.e., active) fraction. Paradoxically, however, Cdc42ep4-/- cerebellum contained significantly more MYH10 and N-cadherin, which is attributed to secondary neuronal response mainly in Purkinje cells. Given cooperative actions of N-cadherin and MYH10 for adhesion between neurons, we speculate that their augmentation may reflect the extension of nonactive synaptic contacts in Cdc42ep4-/- cerebellum. Transcellular mechanism that links the absence of CDC42EP4 in Bergmann glia to the augmentation of N-cadherin and MYH10 in neurons is currently unknown, but the phenotypic similarity to GLAST-null mice indicates involvement of the glutamate intolerance. Together, the unique phenotype of Cdc42ep4-/- mice provides a clue to novel molecular network underlying tripartite synapse configuration.
  • Atsushi Yamagata, Sakurako Goto-Ito, Yusuke Sato, Tomoko Shiroshima, Asami Maeda, Masahiko Watanabe, Takashi Saitoh, Katsumi Maenaka, Tohru Terada, Tomoyuki Yoshida, Takeshi Uemura, Shuya Fukai
    Nature communications 9 (1) 3964 - 3964 2018/09/27 [Refereed][Not invited]
     
    Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) function as postsynaptic organizers that induce excitatory synapses. Neurexins (Nrxns) and heparan sulfate proteoglycans have been identified as presynaptic ligands for LRRTMs. Specifically, LRRTM1 and LRRTM2 bind to the Nrxn splice variant lacking an insert at the splice site 4 (S4). Here, we report the crystal structure of the Nrxn1β-LRRTM2 complex at 3.4 Å resolution. The Nrxn1β-LRRTM2 interface involves Ca2+-mediated interactions and overlaps with the Nrxn-neuroligin interface. Together with structure-based mutational analyses at the molecular and cellular levels, the present structural analysis unveils the mechanism of selective binding between Nrxn and LRRTM1/2 and its modulation by the S4 insertion of Nrxn.
  • Satoshi Nonomura, Kayo Nishizawa, Yutaka Sakai, Yasuo Kawaguchi, Shigeki Kato, Motokazu Uchigashima, Masahiko Watanabe, Ko Yamanaka, Kazuki Enomoto, Satomi Chiken, Hiromi Sano, Shogo Soma, Junichi Yoshida, Kazuyuki Samejima, Masaaki Ogawa, Kazuto Kobayashi, Atsushi Nambu, Yoshikazu Isomura, Minoru Kimura
    Neuron 99 (6) 1302 - 1314 0896-6273 2018/09/19 [Refereed][Not invited]
     
    The basal ganglia play key roles in adaptive behaviors guided by reward and punishment. However, despite accumulating knowledge, few studies have tested how heterogeneous signals in the basal ganglia are organized and coordinated for goal-directed behavior. In this study, we investigated neuronal signals of the direct and indirect pathways of the basal ganglia as rats performed a lever push/pull task for a probabilistic reward. In the dorsomedial striatum, we found that optogenetically and electrophysiologically identified direct pathway neurons encoded reward outcomes, whereas indirect pathway neurons encoded no-reward outcome and next-action selection. Outcome coding occurred in association with the chosen action. In support of pathway-specific neuronal coding, light activation induced a bias on repeat selection of the same action in the direct pathway, but on switch selection in the indirect pathway. Our data reveal the mechanisms underlying monitoring and updating of action selection for goal-directed behavior through basal ganglia circuits.
  • Notartomaso S, Nakao H, Mascio G, Scarselli P, Cannella M, Zappulla C, Madonna M, Motolese M, Gradini R, Liberatore F, Zonta M, Carmignoto G, Battaglia G, Bruno V, Watanabe M, Aiba A, Nicoletti F
    Scientific reports 8 (1) 13361 - 13361 2018/09 [Refereed][Not invited]
     
    In cerebellar Purkinje cells (PCs) type-1 metabotropic glutamate (mGlu1) receptors play a key role in motor learning and drive the refinement of synaptic innervation during postnatal development. The cognate mGlu5 receptor is absent in mature PCs and shows low expression levels in the adult cerebellar cortex. Here we found that mGlu5 receptors were heavily expressed by PCs in the early postnatal life, when mGlu1α receptors were barely detectable. The developmental decline of mGlu5 receptors coincided with the appearance of mGlu1α receptors in PCs, and both processes were associated with specular changes in CpG methylation in the corresponding gene promoters. It was the mGlu1 receptor that drove the elimination of mGlu5 receptors from PCs, as shown by data obtained with conditional mGlu1α receptor knockout mice and with targeted pharmacological treatments during critical developmental time windows. The suppressing activity of mGlu1 receptors on mGlu5 receptor was maintained in mature PCs, suggesting that expression of mGlu1α and mGlu5 receptors is mutually exclusive in PCs. These findings add complexity to the the finely tuned mechanisms that regulate PC biology during development and in the adult life and lay the groundwork for an in-depth analysis of the role played by mGlu5 receptors in PC maturation.
  • Kaplan E, Zubedat S, Radzishevsky I, Valenta AC, Rechnitz O, Sason H, Sajrawi C, Bodner O, Konno K, Esaki K, Derdikman D, Yoshikawa T, Watanabe M, Kennedy RT, Billard JM, Avital A, Wolosker H
    Proceedings of the National Academy of Sciences of the United States of America 115 (38) 9628 - 9633 0027-8424 2018/09 [Refereed][Not invited]
     
    d-serine is a physiologic coagonist of NMDA receptors, but little is known about the regulation of its synthesis and synaptic turnover. The amino acid exchangers ASCT1 (Slc1a4) and ASCT2 (Slc1a5) are candidates for regulating d-serine levels. Using ASCT1 and ASCT2 KO mice, we report that ASCT1, rather than ASCT2, is a physiologic regulator of d-serine metabolism. ASCT1 is a major d-serine uptake system in astrocytes and can also export l-serine via heteroexchange, supplying neurons with the substrate for d-serine synthesis. ASCT1-KO mice display lower levels of brain d-serine along with higher levels of l-alanine, l-threonine, and glycine. Deletion of ASCT1 was associated with neurodevelopmental alterations including lower hippocampal and striatal volumes and changes in the expression of neurodevelopmental-relevant genes. Furthermore, ASCT1-KO mice exhibited deficits in motor function, spatial learning, and affective behavior, along with changes in the relative contributions of d-serine vs. glycine in mediating NMDA receptor activity. In vivo microdialysis demonstrated lower levels of extracellular d-serine in ASCT1-KO mice, confirming altered d-serine metabolism. These alterations are reminiscent of some of the neurodevelopmental phenotypes exhibited by patients with ASCT1 mutations. ASCT1-KO mice provide a useful model for potential therapeutic interventions aimed at correcting the metabolic impairments in patients with ASCT1 mutations.
  • Tetsuya Itabashi, Yasunobu Arima, Daisuke Kamimura, Kotaro Higuchi, Yoshio Bando, Hiromi Takahashi-Iwanaga, Masaaki Murakami, Masahiko Watanabe, Toshihiko Iwanaga, Junko Nio-Kobayashi
    Neurochemistry International 118 176 - 184 1872-9754 2018/09/01 [Refereed][Not invited]
     
    Multiple sclerosis (MS) is an autoimmune disease in which pathogenic T cells play an important role, and an experimental autoimmune encephalomyelitis (EAE) is used as an animal model of MS. Galectins are β-galactoside-binding lectins and involved in various physiological and pathological events. Among fifteen members of galectins, galectin-1, -8, and -9 play immunosuppressive roles in MS and EAE however, the role of galectin-3 (gal-3) is complex and controversial. We examined expression of gal-3 in the spinal cord and nerve roots of EAE mice. No immunohistochemical signals were detected in naïve mice, whereas gal-3 appeared at lower lumbar levels of the spinal cord and nerve roots in EAE mice. In the spinal cord, gal-3-positive cells were activated microglia and/or infiltrating macrophages, which were round in shape and intensified for the lysosomal enzyme, cathepsin D, indicating elevated phagocytic activity. Gal-3-positive cells in the spinal cord were most abundant during the peak symptomatic period. In the recovery period, they disappeared from the spinal parenchyma but remained at moderate levels in the pia mater. Interestingly, gal-3-positive cells selectively appeared in ventral, but not dorsal, nerve roots running through the spinal canal, with expression peaking during the recovery period. In ventral nerve roots, the major cell type expressing gal-3 was a specific population of Schwann cells that surround unmyelinated axons and express the biosynthetic enzyme for L-serine, a potent neurotrophic amino acid. Gal-3 was also induced in Iba1/F4/80-positive macrophages, which engulf damaged myelin and axon debris. Thus, gal-3 is induced in distinct cell types that are engaged in removal of damaged axons and cell debris and axon regeneration and remyelination, suggesting a potential neuroprotective role of gal-3 in EAE mice.
  • Zhang MD, Su J, Adori C, Cinquina V, Malenczyk K, Girach F, Peng C, Ernfors P, Löw P, Borgius L, Kiehn O, Watanabe M, Uhlén M, Mitsios N, Mulder J, Harkany T, Hökfelt T
    The Journal of clinical investigation 128 (9) 3757 - 3768 0021-9738 2018/08 [Refereed][Not invited]
     
    Pain signals are transmitted by multisynaptic glutamatergic pathways. Their first synapse between primary nociceptors and excitatory spinal interneurons gates the sensory load. In this pathway, glutamate release is orchestrated by Ca2+-sensor proteins, with N-terminal EF-hand Ca2+-binding protein 2 (NECAB2) being particular abundant. However, neither the importance of NECAB2+ neuronal contingents in dorsal root ganglia (DRGs) and spinal cord nor the function determination by NECAB2 has been defined. A combination of histochemical analyses and single-cell RNA-sequencing showed NECAB2 in small- and medium-sized C- and Aδ D-hair low-threshold mechanoreceptors in DRGs, as well as in protein kinase C γ excitatory spinal interneurons. NECAB2 was downregulated by peripheral nerve injury, leading to the hypothesis that NECAB2 loss of function could limit pain sensation. Indeed, Necab2-/- mice reached a pain-free state significantly faster after peripheral inflammation than did WT littermates. Genetic access to transiently activated neurons revealed that a mediodorsal cohort of NECAB2+ neurons mediates inflammatory pain in the mouse spinal dorsal horn. Here, besides dampening excitatory transmission in spinal interneurons, NECAB2 limited pronociceptive brain-derived neurotrophic factor (BDNF) release from sensory afferents. Hoxb8-dependent reinstatement of NECAB2 expression in Necab2-/- mice then demonstrated that spinal and DRG NECAB2 alone could control inflammation-induced sensory hypersensitivity. Overall, we identify NECAB2 as a critical component of pronociceptive pain signaling, whose inactivation offers substantial pain relief.
  • Kano M, Watanabe T, Uesaka N, Watanabe M
    Cerebellum (London, England) 17 (6) 722 - 734 1473-4222 2018/07 [Refereed][Not invited]
     
    Functional neural circuits in the mature animals are shaped during postnatal development by elimination of unnecessary synapses and strengthening of necessary ones among redundant synaptic connections formed transiently around birth. In the cerebellum of neonatal rodents, excitatory synapses are formed on the somata of Purkinje cells (PCs) by climbing fibers (CFs) that originate from neurons in the contralateral inferior olive. Each PC receives inputs from multiple (~ five) CFs that have about equal synaptic strengths. Subsequently, a single CF selectively becomes stronger relative to the other CFs during the first postnatal week. Then, from around postnatal day 9 (P9), only the strongest CF ("winner" CF) extends its synaptic territory along PC dendrites. In contrast, synapses of the weaker CFs ("loser" CFs) remain on the soma and the most proximal portion of the dendrite together with somatic synapses of the "winner" CF. These perisomatic CF synapses are eliminated progressively during the second and the third postnatal weeks. From P6 to P11, the elimination proceeds independently of the formation of the synapses on PC dendrites by parallel fibers (PFs). From P12 and thereafter, the elimination requires normal PF-PC synapse formation and is presumably dependent on the PF synaptic inputs. Most PCs become mono-innervated by single strong CFs on their dendrites in the third postnatal week. In this review article, we will describe how adult-type CF mono-innervation of PC is established through these multiple phases of postnatal cerebellar development and make an overview of molecular/cellular mechanisms underlying them.
  • Hajime Shiotani, Muneaki Miyata, Yu Itoh, Shujie Wang, Aika Kaito, Akira Mizoguchi, Miwako Yamasaki, Masahiko Watanabe, Kenji Mandai, Hideki Mochizuki, Yoshimi Takai
    Journal of Comparative Neurology 526 (9) 1527 - 1549 1096-9861 2018/06/15 [Refereed][Not invited]
     
    The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin-like cell adhesion molecules (CAMs) nectin-2α and nectin-2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin-2α, but not nectin-2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin-2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin-2α at this boundary was not colocalized with the nectin-2α-binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin-2α forms a cell adhesion apparatus different from the Kv4.2-associated MSs. Genetic ablation of nectin-2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin-2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.
  • Wenjie Mao, Anna C. Salzberg, Motokazu Uchigashima, Yuto Hasegawa, Hanno Hock, Masahiko Watanabe, Schahram Akbarian, Yuka Imamura Kawasawa, Kensuke Futai
    Cell Reports 23 (11) 3209 - 3222 2211-1247 2018/06/12 [Refereed][Not invited]
     
    Homeostatic synaptic downscaling reduces neuronal excitability by modulating the number of postsynaptic receptors. Histone modifications and the subsequent chromatin remodeling play critical roles in activity-dependent gene expression. Histone modification codes are recognized by chromatin readers that affect gene expression by altering chromatin structure. We show that L3mbtl1 (lethal 3 malignant brain tumor-like 1), a polycomb chromatin reader, is downregulated by neuronal activity and is essential for synaptic response and downscaling. Genome-scale mapping of L3mbtl1 occupancies identified Ctnnb1 as a key gene downstream of L3mbtl1. Importantly, the occupancy of L3mbtl1 on the Ctnnb1 gene was regulated by neuronal activity. L3mbtl1 knockout neurons exhibited reduced Ctnnb1 expression. Partial knockdown of Ctnnb1 in wild-type neurons reduced excitatory synaptic transmission and abolished homeostatic downscaling, and transfecting Ctnnb1 in L3mbtl1 knockout neurons enhanced synaptic transmission and restored homeostatic downscaling. These results highlight a role for L3mbtl1 in regulating homeostasis of synaptic efficacy. Synaptic homeostasis is crucial for maintaining proper neuronal excitability and excitatory/inhibitory balance in the brain. Mao et al. report that an activity-dependent chromatin reader protein is required for homeostatic control of synaptic strength through the regulation of downstream target gene Ctnnb1.
  • Kikuchihara S, Sugio S, Tanaka KF, Watanabe T, Kano M, Yamazaki Y, Watanabe M, Ikenaka K
    Journal of neurochemistry 147 (3) 344 - 360 0022-3042 2018/06 [Refereed][Not invited]
     
    Mlc1 is a causative gene for megalencephalic leukoencephalopathy with subcortical cysts, and is expressed in astrocytes. Mlc1-over-expressing mice represent an animal model of early-onset leukoencephalopathy, which manifests as astrocytic swelling followed by myelin membrane splitting in the white matter. It has been previously reported that Mlc1 is highly expressed in Bergmann glia, while the cerebellar phenotypes of Mlc1-over-expressing mouse have not been characterized. Here, we examined the cerebellum of Mlc1-over-expressing mouse and found that the distribution of Bergmann glia (BG) was normally compacted along the Purkinje cell (PC) layer until postnatal day 10 (P10), while most BG were dispersed throughout the molecular layer by P28. Ectopic BG were poorly wrapped around somatodendritic elements of PCs and exhibited reduced expression of the glutamate transporter glutamate-aspartate transporter. Extraordinarily slow and small climbing fiber (CF)-mediated excitatory post-synaptic currents, which are known to be elicited under accelerated glutamate spillover, emerged at P20-P28 when BG ectopia was severe, but not at P9-P12 when ectopia was mild. Furthermore, maturation of CF wiring, which translocates the site of innervation from somata to proximal dendrites, was also impaired. Manipulations that restricted the Mlc1-over-expressing period successfully generated mice with and without BG ectopia, depending on the over-expressing period. Together, these findings suggest that there is a critical time window for mechanisms that promote the positioning of BG in the PC layer. Once normal positioning of BG is affected, the differentiation of BG is impaired, leading to insufficient glial wrapping, exacerbated glutamate spillover, and aberrant synaptic wiring in PCs. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14199.
  • Szabolcs Takács, Zsuzsanna Bardóczi, Katalin Skrapits, Balázs Göcz, Viktória Váczi, Zsófia Maglóczky, Iván Szűcs, Gergely Rácz, András Matolcsy, Waljit S. Dhillo, Masahiko Watanabe, Andrea Kádár, Csaba Fekete, Imre Kalló, Erik Hrabovszky
    Brain Structure and Function 223 (5) 2143 - 2156 1863-2661 2018/06/01 [Refereed][Not invited]
     
    Kisspeptin (KP) synthesizing neurons of the hypothalamic infundibular region are critically involved in the central regulation of fertility these cells regulate pulsatile gonadotropin-releasing hormone (GnRH) secretion and mediate sex steroid feedback signals to GnRH neurons. Fine structural analysis of the human KP system is complicated by the use of post mortem tissues. To gain better insight into the neuroanatomy of the somato-dendritic cellular compartment, we introduced the diolistic labeling of immunohistochemically identified KP neurons using a gene gun loaded with the lipophilic dye, DiI. Confocal microscopic studies of primary dendrites in 100-µm-thick tissue sections established that 79.3% of KP cells were bipolar, 14.1% were tripolar, and 6.6% were unipolar. Primary dendrites branched sparsely, contained numerous appendages (9.1 ± 1.1 spines/100 µm dendrite), and received rich innervation from GABAergic, glutamatergic, and KP-containing terminals. KP neuron synaptology was analyzed with immunoelectron microscopy on perfusion-fixed specimens. KP axons established frequent contacts and classical synapses on unlabeled, and on KP-immunoreactive somata, dendrites, and spines. Synapses were asymmetric and the presynaptic structures contained round and regular synaptic vesicles, in addition to dense-core granules. Although immunofluorescent studies failed to detect vesicular glutamate transporter isoforms in KP axons, ultrastructural characteristics of synaptic terminals suggested use of glutamatergic, in addition to peptidergic, neurotransmission. In summary, immunofluorescent and DiI labeling of KP neurons in thick hypothalamic sections and immunoelectron microscopic studies of KP-immunoreactive neurons in brains perfusion-fixed shortly post mortem allowed us to identify previously unexplored fine structural features of KP neurons in the mediobasal hypothalamus of humans.
  • Jing Huang, Erika Polgár, Hans Jürgen Solinski, Santosh K Mishra, Pang-Yen Tseng, Noboru Iwagaki, Kieran A Boyle, Allen C Dickie, Mette C Kriegbaum, Hendrik Wildner, Hanns Ulrich Zeilhofer, Masahiko Watanabe, John S Riddell, Andrew J Todd, Mark A Hoon
    Nature neuroscience 21 (6) 894 - 894 2018/06 
    In the version of this article initially published online, the labels were switched for the right-hand pair of bars in Fig. 4e. The left one of the two should be Chloroquine + veh, the right one Chloroquine + CNO. The error has been corrected in the print, HTML and PDF versions of the article.
  • Jing Huang, Erika Polgár, Hans Jürgen Solinski, Santosh K. Mishra, Pang-Yen Tseng, Noboru Iwagaki, Kieran A. Boyle, Allen C. Dickie, Mette C. Kriegbaum, Hendrik Wildner, Hanns Ulrich Zeilhofer, Masahiko Watanabe, John S. Riddell, Andrew J. Todd, Mark A. Hoon
    Nature Neuroscience 21 (5) 707 - 716 1546-1726 2018/05/01 [Refereed][Not invited]
     
    Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, particularly the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb+ neurons, and we demonstrate that Nppb+somatostatin+ cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR+ neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch and characterize a contrasting antinociceptive role for the peptide.
  • Hiroaki Yaguchi, Ichiro Yabe, Hidehisa Takahashi, Masashi Watanabe, Taichi Nomura, Takahiro Kano, Masahiko Watanabe, Shigetsugu Hatakeyama
    Journal of Neurology 265 (4) 962 - 965 1432-1459 2018/04/01 [Refereed][Not invited]
  • Rafael Luján, Carolina Aguado, Francisco Ciruela, Javier Cózar, David Kleindienst, Luis de la Ossa, Bernhard Bettler, Kevin Wickman, Masahiko Watanabe, Ryuichi Shigemoto, Yugo Fukazawa
    Brain Structure and Function 223 (3) 1565 - 1587 1863-2661 2018/04/01 [Refereed][Not invited]
     
    Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network.
  • Masanori Tachikawa, Hidetoh Toki, Masahiko Watanabe, Masatoshi Tomi, Ken-ichi Hosoya, Tetsuya Terasaki
    Anatomical Science International 93 (4) 1 - 8 1447-073X 2018/03/08 [Refereed][Not invited]
     
    The A-subclass of ATP-binding cassette (ABC) transporters is a highly conserved superfamily of potent lipid transporters. Although the ABCA1-like subgroup of ABCA1-4, and A7 have been shown to mediate the transport of endogenous lipids, the roles of the ABCA6-like subgroup transporters, which have been identified as a unique gene cluster on human chromosome 17q24 (ABCA5, A6, A8, A9, and A10) and mouse chromosome 11 (Abca5, a6, a8a, a8b, and a9), remains largely unknown. The purpose of the present study was to clarify the spatial and temporal expression profiles of Abca6-like subgroup transporters in embryonic and postnatal mouse brains by a combination of in situ hybridization and quantitative polymerase chain reaction (PCR) using magnetically isolated brain vascular endothelial cells. In embryonic brains, the transcripts of Abca5, a8a and a8b were detected predominantly in the mantle zone, where postmitotic neurons differentiate. At the postnatal stages, they were expressed in various nuclei and neuronal layers. Abca9 mRNA was detected diffusely in the embryonic and postnatal brains and sequential and/or strong spotted signals were detected in the leptomeninges on the brain surface. PCR detected expression of Abca8a and Abca9 mRNAs in isolated vascular endothelial cells. Expression signals for Abca6 mRNA were hardly observed at any stages examined. These distinct spatio-temporal expression patterns of Abca6-like subgroup transporters may reflect their functional significance and diversity to regulate lipid transport, particularly in neurons, leptomeningeal cells, and vascular endothelial cells.
  • Naofumi Uesaka, Manabu Abe, Kohtarou Konno, Maya Yamazaki, Kazuto Sakoori, Takaki Watanabe, Tzu-Huei Kao, Takayasu Mikuni, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    Neuron 97 (4) 796 - 805 0896-6273 2018/02/21 [Refereed][Not invited]
     
    Elimination of redundant synapses formed early in development and strengthening of necessary connections are crucial for shaping functional neural circuits. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs) with similar strengths. A single CF is strengthened whereas the other CFs are eliminated in each PC during postnatal development. The underlying mechanisms, particularly for the strengthening of single CFs, are poorly understood. Here we report that progranulin, a multi-functional growth factor implicated in the pathogenesis of frontotemporal dementia, strengthens developing CF synaptic inputs and counteracts their elimination from postnatal day 11 to 16. Progranulin derived from PCs acts retrogradely onto its putative receptor Sort1 on CFs. This effect is independent of semaphorin 3A, another retrograde signaling molecule that counteracts CF synapse elimination. We propose that progranulin-Sort1 signaling strengthens and maintains developing CF inputs, and may contribute to selection of single "winner" CFs that survive synapse elimination.
  • Valentin Kusch, Grit Bornschein, Desiree Loreth, Julia Bank, Johannes Jordan, David Baur, Masahiko Watanabe, Akos Kulik, Manfred Heckmann, Jens Eilers, Hartmut Schmidt
    Cell Reports 22 (8) 2094 - 2106 2211-1247 2018/02/20 [Refereed][Not invited]
     
    Spatial relationships between Cav channels and release sensors at active zones (AZs) are a major determinant of synaptic fidelity. They are regulated developmentally, but the underlying molecular mechanisms are largely unclear. Here, we show that Munc13-3 regulates the density of Cav2.1 and Cav2.2 channels, alters the localization of Cav2.1, and is required for the development of tight, nanodomain coupling at parallel-fiber AZs. We combined EGTA application and Ca2+-channel pharmacology in electrophysiological and two-photon Ca2+ imaging experiments with quantitative freeze-fracture immunoelectron microscopy and mathematical modeling. We found that a normally occurring developmental shift from release being dominated by Ca2+ influx through Cav2.1 and Cav2.2 channels with domain overlap and loose coupling (microdomains) to a nanodomain Cav2.1 to sensor coupling is impaired in Munc13-3-deficient synapses. Thus, at AZs lacking Munc13-3, release remained triggered by Cav2.1 and Cav2.2 microdomains, suggesting a critical role of Munc13-3 in the formation of release sites with calcium channel nanodomains. Central synapses shift from microdomain to nanodomain coupling between Ca2+ channels and release sensors during development. Kusch et al. show that Munc13-3 is a critical regulator of this process. Munc13-3 differentially regulates density and localization of Cav2.1 and Cav2.2 channels and establishes nanodomain coupling between Cav2.1 channels and release sensors.
  • Ryosuke Kaneko, Yusuke Takatsuru, Ayako Morita, Izuki Amano, Asahi Haijima, Itaru Imayoshi, Nobuaki Tamamaki, Noriyuki Koibuchi, Masahiko Watanabe, Yuchio Yanagawa
    Journal of Comparative Neurology 526 (3) 373 - 396 1096-9861 2018/02/15 [Refereed][Not invited]
     
    Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.
  • Ogawa Y, Kakumoto K, Yoshida T, Kuwako KI, Miyazaki T, Yamaguchi J, Konno A, Hata J, Uchiyama Y, Hirai H, Watanabe M, Darnell RB, Okano H, Okano HJ
    Scientific reports 8 (1) 2722 - 2722 2018/02 [Refereed][Not invited]
     
    Neuronal Elav-like (nElavl or neuronal Hu) proteins are RNA-binding proteins that regulate RNA stability and alternative splicing, which are associated with axonal and synaptic structures. nElavl proteins promote the differentiation and maturation of neurons via their regulation of RNA. The functions of nElavl in mature neurons are not fully understood, although Elavl3 is highly expressed in the adult brain. Furthermore, possible associations between nElavl genes and several neurodegenerative diseases have been reported. We investigated the relationship between nElavl functions and neuronal degeneration using Elavl3-/- mice. Elavl3-/- mice exhibited slowly progressive motor deficits leading to severe cerebellar ataxia, and axons of Elavl3-/- Purkinje cells were swollen (spheroid formation), followed by the disruption of synaptic formation of axonal terminals. Deficit in axonal transport and abnormalities in neuronal polarity was observed in Elavl3-/- Purkinje cells. These results suggest that nElavl proteins are crucial for the maintenance of axonal homeostasis in mature neurons. Moreover, Elavl3-/- mice are unique animal models that constantly develop slowly progressive axonal degeneration. Therefore, studies of Elavl3-/- mice will provide new insight regarding axonal degenerative processes.
  • Masanori Tachikawa, Masahiko Watanabe, Masahiro Fukaya, Kazuhisa Sakai, Tetsuya Terasaki, Ken-ichi Hosoya
    Neurochemical Research 43 (2) 500 - 510 1573-6903 2018/02/01 [Refereed][Not invited]
     
    Creatine is synthesized by S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT), and the creatine/phosphocreatine shuttle system mediated by creatine kinase (CK) is essential for storage and regeneration of high-energy phosphates in cells. Although the importance of this system in brain development is evidenced by the hereditary nature of creatine deficiency syndrome, the spatiotemporal cellular expression patterns of GAMT in developing brain remain unknown. Here we show that two waves of high GAMT expression occur in developing mouse brain. The first involves high expression in mitotic cells in the ventricular zone of the brain wall and the external granular layer of the cerebellum at the embryonic and neonatal stages. The second was initiated by striking up-regulation of GAMT in oligodendrocytes during the second and third postnatal weeks (i.e., the active myelination stage), which continued to adulthood. Distinct temporal patterns were also evident in other cell types. GAMT was highly expressed in perivascular pericytes and smooth muscle cells after birth, but not in adults. In neurons, GAMT levels were low to moderate in neuroblasts residing in the ventricular zone, increased during the second postnatal week when active dendritogenesis and synaptogenesis occur, and decreased to very low levels thereafter. Moderate levels were observed in astrocytes throughout development. The highly regulated, cell type-dependent expression of GAMT suggests that local creatine biosynthesis plays critical roles in certain phases of neural development. In accordance with this idea, we observed increased CK expression in differentiating neurons this would increase creatine/phosphocreatine shuttle system activity, which might reflect increased energy demand.
  • Sam A. Booker, Desiree Loreth, Annabelle L. Gee, Masahiko Watanabe, Peter C. Kind, David J.A. Wyllie, Ákos Kulik, Imre Vida
    Cell Reports 22 (1) 36 - 43 2211-1247 2018/01/02 [Refereed][Not invited]
     
    Inhibition provided by local GABAergic interneurons (INs) activates ionotropic GABAA and metabotropic GABAB receptors (GABABRs). Despite GABABRs representing a major source of inhibition, little is known of their function in distinct IN subtypes. Here, we show that, while the archetypal dendritic-inhibitory somatostatin-expressing INs (SOM-INs) possess high levels of GABABR on their somato-dendritic surface, they fail to produce significant postsynaptic inhibitory currents. Instead, GABABRs selectively inhibit dendritic CaV1.2 (L-type) Ca2+ channels on SOM-IN dendrites, leading to reduced calcium influx and loss of long-term potentiation at excitatory input synapses onto these INs. These data provide a mechanism by which GABABRs can contribute to disinhibition and control the efficacy of extrinsic inputs to hippocampal networks. Booker et al. show that GABAB receptors are highly expressed on somatostatin interneuron dendrites. Rather than activating Kir3 channels, they preferentially co-cluster with, and negatively couple to, L-type calcium channels inhibiting long-term potentiation at excitatory inputs.
  • Rafael Luján, Carolina Aguado, Francisco Ciruela, Xavier Morató Arus, Alejandro Martín-Belmonte, Rocío Alfaro-Ruiz, Jesús Martínez-Gómez, Luis de la Ossa, Masahiko Watanabe, John P Adelman, Ryuichi Shigemoto, Yugo Fukazawa
    Frontiers in cellular neuroscience 12 311 - 311 2018 [Refereed][Not invited]
     
    The small-conductance, Ca2+-activated K+ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca2+ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca2+: CaV2.1 channels and mGlu1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for CaV2.1 channels and mGlu1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to CaV2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca2+, and suggest that the ultrastructural association of SK2 with CaV2.1 and mGlu1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca2+ transients in PCs.
  • Uchida H, Matsumura S, Katano T, Watanabe M, Schlossmann J, Ito S
    Molecular pain 14 1744806918796409  2018/01 [Refereed][Not invited]
  • Haruko Kumamoto, Taku Yamaguchi, Kohtarou Konno, Takeshi Izumi, Takayuki Yoshida, Yu Ohmura, Masahiko Watanabe, Mitsuhiro Yoshioka
    Journal of Pharmacological Sciences 136 (1) 1 - 8 1347-8648 2018/01/01 [Refereed][Not invited]
     
    Childhood maltreatment is associated with impaired adult brain function, particularly in the hippocampus, and is not only a major risk factor for some psychiatric diseases but also affects early social development and social adaptation in later life. The aims of this study were to determine whether early postnatal stress affects social behavior and whether repeated fluvoxamine treatment reverses these changes. Rat pups were exposed to footshock stress during postnatal days 21–25 (at 3 weeks old: 3wFS). During the post-adolescent period (10–14 weeks postnatal), the social interaction test and Golgi-cox staining of dorsal hippocampal pyramidal neurons were performed. Following exposure to footshock stress, 3wFS rats showed an increase in social interaction time, which might be practically synonymous with hypersociability, and a decrease in spine density in the CA3 hippocampal subregion, but not in CA1. These behavioral and morphological changes were both recovered by repeated oral administration of fluvoxamine at a dose of 10 mg/kg/day for 14 days. These findings suggest that the vulnerability of the hippocampal CA3 region is closely related to social impairments induced by physical stress during the juvenile period and shed some light on therapeutic alternatives for early postnatal stress-induced emotional dysfunction.
  • Hiroaki Yaguchi, Ichiro Yabe, Hidehisa Takahashi, Masashi Watanabe, Taichi Nomura, Takahiro Kano, Masaki Matsumoto, Keiichi I Nakayama, Masahiko Watanabe, Shigetsugu Hatakeyama
    Biochemical and biophysical research communications 494 (1-2) 234 - 241 2017/12/09 
    Increasing evidence shows that immune-mediated mechanisms may contribute to the pathogenesis of central nervous system disorders including cerebellar ataxias, as indicated by the aberrant production of neuronal surface antibodies. We previously reported a patient with cerebellar ataxia associated with production of a new anti-neuronal antibody, anti-seizure-related 6 homolog like 2 (Sez6l2). Sez6l2 is a type 1 membrane protein that is highly expressed in the hippocampus and cerebellar cortex and mice lacking Sez6l2 protein family members develop ataxia. Here we used a proteomics-based approach to show that serum derived from this patient recognizes the extracellular domain of Sez6l2 and that Sez6l2 protein binds to both adducin (ADD) and glutamate receptor 1 (GluR1). Our results indicate that Sez6l2 is one of the auxiliary subunits of the AMPA receptor and acts as a scaffolding protein to link GluR1 to ADD. Furthermore, Sez6l2 overexpression upregulates ADD phosphorylation, whereas siRNA-mediated downregulation of Sez612 prevents ADD phosphorylation, suggesting that Sez6l2 modulates AMPA-ADD signal transduction.
  • Sam A. Booker, Daniel Althof, Claudius E. Degro, Masahiko Watanabe, Ákos Kulik, Imre Vida
    Brain Structure and Function 222 (8) 3677 - 3690 1863-2661 2017/11/01 [Refereed][Not invited]
     
    The perisomatic domain of cortical neurons is under the control of two major GABAergic inhibitory interneuron types: regular-spiking cholecystokinin (CCK) basket cells (BCs) and fast-spiking parvalbumin (PV) BCs. CCK and PV BCs are different not only in their intrinsic physiological, anatomical and molecular characteristics, but also in their presynaptic modulation of their synaptic output. Most GABAergic terminals are known to contain GABAB receptors (GABABR), but their role in presynaptic inhibition and surface expression have not been comparatively characterized in the two BC types. To address this, we performed whole-cell recordings from CCK and PV BCs and postsynaptic pyramidal cells (PCs), as well as freeze-fracture replica-based quantitative immunogold electron microscopy of their synapses in the rat hippocampal CA1 area. Our results demonstrate that while both CCK and PV BCs contain functional presynaptic GABABRs, their modulatory effects and relative abundance are markedly different at these two synapses: GABA release is dramatically inhibited by the agonist baclofen at CCK BC synapses, whereas a moderate reduction in inhibitory transmission is observed at PV BC synapses. Furthermore, GABABR activation has divergent effects on synaptic dynamics: paired-pulse depression (PPD) is enhanced at CCK BC synapses, but abolished at PV BC synapses. Consistent with the quantitative differences in presynaptic inhibition, virtually all CCK BC terminals were found to contain GABABRs at high densities, but only 40% of PV BC axon terminals contain GABABRs at detectable levels. These findings add to an increasing list of differences between these two interneuron types, with implications for their network functions.
  • Jean-Marc Good, Michael Mahoney, Taisuke Miyazaki, Kenji F. Tanaka, Kenji Sakimura, Masahiko Watanabe, Kazuo Kitamura, Masanobu Kano
    CELL REPORTS 21 (8) 2066 - 2073 2211-1247 2017/11 [Refereed][Not invited]
     
    Neural circuits undergo massive refinements during postnatal development. In the developing cerebellum, the climbing fiber (CF) to Purkinje cell (PC) network is drastically reshaped by eliminating early-formed redundant CF to PC synapses. To investigate the impact of CF network refinement on PC population activity during postnatal development, we monitored spontaneous CF responses in neighboring PCs and the activity of populations of nearby CF terminals using in vivo two-photon calcium imaging. Population activity is highly synchronized in newborn mice, and the degree of synchrony gradually declines during the first postnatal week in PCs and, to a lesser extent, in CF terminals. Knockout mice lacking P/Q-type voltage-gated calcium channel or glutamate receptor delta 2, in which CF network refinement is severely impaired, exhibit an abnormally high level of synchrony in PC population activity. These results suggest that CF network refinement is a structural basis for developmental desynchronization and maturation of PC population activity.
  • Kieran A. Boyle, Maria Gutierrez-Mecinas, Erika Polgar, Nicole Mooney, Emily O'Connor, Takahiro Furuta, Masahiko Watanabe, Andrew J. Todd
    NEUROSCIENCE 363 120 - 133 0306-4522 2017/11 [Refereed][Not invited]
     
    Around a quarter of neurons in laminae I-II of the dorsal horn are inhibitory interneurons. These play an important role in modulating somatosensory information, including that perceived as pain or itch. Previous studies in rat identified four largely non-overlapping neurochemical populations among these cells, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin. The galanin cells were subsequently shown to coexpress dynorphin. Several recent studies have used genetically modified mice to investigate the function of different interneuron populations, and it is therefore important to determine whether the same pattern applies in mouse, and to estimate the relative sizes of these populations. We show that the neurochemical organization of inhibitory interneurons in mouse superficial dorsal horn is similar to that in the rat, although a larger proportion of these neurons (33%) express NPY. Between them, these four populations account for similar to 75% of inhibitory cells in laminae I-II. Since similar to 25% of inhibitory interneurons in this region belong to a novel calretinin-expressing type, our results suggest that virtually all inhibitory interneurons in superficial dorsal horn can be assigned to one of these five neurochemical populations. Although our main focus was inhibitory neurons, we also identified a population of excitatory dynorphin-expressing cells in laminae I-II that are largely restricted to the medial part of the mid-lumbar dorsal horn, corresponding to glabrous skin territory. These findings are important for interpretation of studies using molecular-genetic techniques to manipulate the functions of interneuron populations to investigate their roles in somatosensory processing. (C) 2017 The Authors. Published by Elsevier Ltd on behalf of IBRO.
  • Zsuzsanna Bardoczi, Balazs Pal, Aron Koszeghy, Tamas Wilheim, Masahiko Watanabe, Laszlo Zaborszky, Zsolt Liposits, Imre Kallo
    JOURNAL OF NEUROSCIENCE 37 (39) 9534 - 9549 0270-6474 2017/09 [Refereed][Not invited]
     
    The basal forebrain (BF) receives afferents from brainstem ascending pathways, which has been implicated first by Moruzzi and Magoun (1949) to induce forebrain activation and cortical arousal/waking behavior; however, it is very little known about how brainstem inhibitory inputs affect cholinergic functions. In the current study, glycine, a major inhibitory neurotransmitter of brainstem neurons, and gliotransmitter of local glial cells, was tested for potential interaction with BF cholinergic (BFC) neurons in male mice. In the BF, glycine receptor alpha subunit-immunoreactive (IR) sites were localized in choline acetyltransferase (ChAT)-IR neurons. The effect of glycine on BFC neurons was demonstrated by bicuculline-resistant, strychnine-sensitive spontaneous IPSCs (sIPSCs; 0.81 +/- 0.25 +/- 10(-1) Hz) recorded in whole-cell conditions. Potential neuronal as well as glial sources of glycine were indicated in the extracellular space of cholinergic neurons by glycine transporter type 1 (GLYT1)- and GLYT2-IR processes found in apposition to ChAT-IR cells. Ultrastructural analyses identified synapses of GLYT2-positive axon terminals on ChAT-IR neurons, as well as GLYT1-positive astroglial processes, which were localized in the vicinity of synapses ofChAT-IRneurons. The brainstem raphe magnus was determined to be a major source of glycinergic axons traced retrogradely from the BF. Our results indicate a direct effect of glycine on BFC neurons. Furthermore, the presence of high levels of plasma membrane glycine transporters in the vicinity of cholinergic neurons suggests a tight control of extracellular glycine in the BF.
  • N. Shioda, Y. Yabuki, Y. Wang, M. Uchigashima, T. Hikida, T. Sasaoka, H. Mori, M. Watanabe, M. Sasahara, K. Fukunaga
    MOLECULAR PSYCHIATRY 22 (8) 1205 - 1222 1359-4184 2017/08 [Refereed][Not invited]
     
    Aberrant dopamine D-2 receptor (D2R) activity is associated with neuropsychiatric disorders, making those receptors targets for antipsychotic drugs. Here, we report that novel signaling through the intracellularly localized D2R long isoform (D2LR) elicits extracellular signal-regulated kinase (ERK) activation and dendritic spine formation through Rabex-5/ platelet-derived growth factor receptor-beta (PDGFR beta)-mediated endocytosis in mouse striatum. We found that D2LR directly binds to and activates Rabex-5, promoting early-endosome formation. Endosomes containing D2LR and PDGFR beta are then transported to the Golgi apparatus, where those complexes trigger Gai3-mediated ERK signaling. Loss of intracellular D2LR-mediated ERK activation decreased neuronal activity and dendritic spine density in striatopallidal medium spiny neurons (MSNs). In addition, dendritic spine density in striatopallidal MSNs significantly increased following treatment of striatal slices from wild-type mice with quinpirole, a D2R agonist, but those changes were lacking in D2LR knockout mice. Moreover, intracellular D2LR signaling mediated effects of a typical antipsychotic drug, haloperidol, in inducing catalepsy behavior. Taken together, intracellular D2LR signaling through Rabex-5/PDGFR beta is critical for ERK activation, dendritic spine formation and neuronal activity in striatopallidal MSNs of mice.
  • Tomonori Aikawa, Takaki Watanabe, Taisuke Miyazaki, Takayasu Mikuni, Minoru Wakamori, Miyano Sakurai, Hidenori Aizawa, Nobutaka Ishizu, Masahiko Watanabe, Masanobu Kano, Hidehiro Mizusawa, Kei Watase
    HUMAN MOLECULAR GENETICS 26 (16) 3094 - 3104 0964-6906 2017/08 [Refereed][Not invited]
     
    Alternative splicing (AS) that occurs at the final coding exon (exon 47) of the Ca(v)2.1 voltage-gated calcium channel (VGCC) gene produces two major isoforms in the brain, MPI and MPc. These isoforms differ in their splice acceptor sites; human MPI is translated into a polyglutamine tract associated with spinocerebellar ataxia type 6 (SCA6), whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail. To gain insight into the functional role of the AS in vivo and whether modulating the splice patterns at this locus can be a potential therapeutic strategy for SCA6, here we created knockin mice that exclusively express MPc by inserting the splice-site mutation. The resultant Cacna1a(CtmKO/CtmKO) mice developed non-progressive neurological phenotypes, featuring early-onset ataxia and absence seizure without significant alterations in the basic properties of the channel. Interactions of Ca(v)2.1 with Ca-v beta 4 and Rimbp2 were significantly reduced while those with GABA(B2) were enhanced in the cerebellum of Cacna1a(CtmKO/CtmKO) mice. Treatment with the GABA(B) antagonist CGP35348 partially rescued the motor impairments seen in Cacna1a(CtmKO/CtmKO) mice. These results suggest that the carboxyl-terminal domain of Ca(v)2.1 is not essential for maintaining the basic properties of the channel in the cerebellar Purkinje neurons but is involved in multiple interactions of Ca(v)2.1 with other proteins, and plays an essential role in preventing a complex neurological disease.
  • Myeongjeong Choo, Taisuke Miyazaki, Maya Yamazaki, Meiko Kawamura, Takanobu Nakazawa, Jianling Zhang, Asami Tanimura, Naofumi Uesaka, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    NATURE COMMUNICATIONS 8 (1) 195 - 195 2041-1723 2017/08 [Refereed][Not invited]
     
    Elimination of early-formed redundant synapses during postnatal development is essential for functional neural circuit formation. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs). A single CF is strengthened whereas the other CFs are eliminated in each PC dependent on postsynaptic activity in PC, but the underlying mechanisms are largely unknown. Here, we report that brain-derived neurotrophic factor (BDNF) from PC facilitates CF synapse elimination. By PC-specific deletion of BDNF combined with knockdown of BDNF receptors in CF, we show that BDNF acts retrogradely on TrkB in CFs, and facilitates elimination of CF synapses from PC somata during the third postnatal week. We also show that BDNF shares signaling pathway with metabotropic glutamate receptor 1, a key molecule that triggers a canonical pathway for CF synapse elimination. These results indicate that unlike other synapses, BDNF mediates punishment signal for synapse elimination in the developing cerebellum.
  • Norikazu Katayama, Saori Yamamori, Masahiro Fukaya, Shizuka Kobayashi, Masahiko Watanabe, Masami Takahashi, Toshiya Manabe
    SCIENTIFIC REPORTS 7 (1) 7996 - 7996 2045-2322 2017/08 [Refereed][Not invited]
     
    Neurotransmitter release is mediated by the SNARE complex, but the role of its phosphorylation has scarcely been elucidated. Although PKC activators are known to facilitate synaptic transmission, there has been a heated debate on whether PKC mediates facilitation of neurotransmitter release through phosphorylation. One of the SNARE proteins, SNAP-25, is phosphorylated at the residue serine-187 by PKC, but its physiological significance has been unclear. To examine these issues, we analyzed mutant mice lacking the phosphorylation of SNAP-25 serine-187 and found that they exhibited reduced release probability and enhanced presynaptic short-term plasticity, suggesting that not only the release process, but also the dynamics of synaptic vesicles was regulated by the phosphorylation. Furthermore, it has been known that the release probability changes with development, but the precise mechanism has been unclear, and we found that developmental changes in release probability of neurotransmitters were regulated by the phosphorylation. These results indicate that SNAP-25 phosphorylation developmentally facilitates neurotransmitter release but strongly inhibits presynaptic short-term plasticity via modification of the dynamics of synaptic vesicles in presynaptic terminals.
  • Tomoko Yoshikawa, Natsuko F. Inagaki, Seiji Takagi, Shigeru Kuroda, Miwako Yamasaki, Masahiko Watanabe, Sato Honma, Ken-ichi Honma
    SCIENTIFIC REPORTS 7 (1) 8210 - 8210 2045-2322 2017/08 [Refereed][Not invited]
     
    The circadian pacemaker in the suprachiasmatic nucleus (SCN) yields photoperiodic response to transfer seasonal information to physiology and behavior. To identify the precise location involved in photoperiodic response in the SCN, we analyzed circadian Period1 and PERIOD2 rhythms in horizontally sectioned SCN of mice exposed to a long or short day. Statistical analyses of bioluminescence images with respective luciferase reporters on pixel level enabled us to identify the distinct localization of three oscillating regions; a large open-ring-shape region, the region at the posterior end and a sharply demarcated oval region at the center of the SCN. The first two regions are the respective sites for the so-called evening and morning oscillators, and the third region is possibly a site for mediating photic signals to the former oscillators. In these regions, there are two classes of oscillating cells in which Per1 and Per2 could play differential roles in photoperiodic responses.
  • Xavier Morato, Rafael Lujan, Marc Lopez-Cano, Jorge Gandia, Igor Stagljar, Masahiko Watanabe, Rodrigo A. Cunha, Victor Fernandez-Duenas, Francisco Ciruela
    SCIENTIFIC REPORTS 7 (1) 9452 - 9452 2045-2322 2017/08 [Refereed][Not invited]
     
    G protein-coupled receptor 37 (GPR37) is an orphan receptor associated to Parkinson's disease (PD) neuropathology. Here, we identified GPR37 as an inhibitor of adenosine A(2A) receptor (A(2A)R) cell surface expression and function in vivo. In addition, we showed that GPR37 and A(2A)R do oligomerize in the striatum. Thus, a close proximity of GPR37 and A(2A)R at the postsynaptic level of striatal synapses was observed by double-labelling post-embedding immunogold detection. Indeed, the direct receptor-receptor interaction was further substantiated by proximity ligation in situ assay. Interestingly, GPR37 deletion promoted striatal A(2A)R cell surface expression that correlated well with an increased A(2A)R agonist-mediated cAMP accumulation, both in primary striatal neurons and nerve terminals. Furthermore, GPR37-/-mice showed enhanced A(2A)R agonist-induced catalepsy and an increased response to A(2A)R antagonist-mediated locomotor activity. Overall, these results revealed a key role for GPR37 controlling A(2A)R biology in the striatum, which may be relevant for PD management.
  • Yoon-Mi Oh, Fuyuki Karube, Susumu Takahashi, Kenta Kobayashi, Masahiko Takada, Motokazu Uchigashima, Masahiko Watanabe, Kayo Nishizawa, Kazuto Kobayashi, Fumino Fujiyama
    BRAIN STRUCTURE & FUNCTION 222 (5) 2359 - 2378 1863-2653 2017/07 [Refereed][Not invited]
     
    In the present study, we generated a novel parvalbumin (PV)-Cre rat model and conducted detailed morphological and electrophysiological investigations of axons from PV neurons in globus pallidus (GP). The GP is considered as a relay nucleus in the indirect pathway of the basal ganglia (BG). Previous studies have used molecular profiling and projection patterns to demonstrate cellular heterogeneity in the GP; for example, PV-expressing neurons are known to comprise approximately 50% of GP neurons and represent majority of prototypic neurons that project to the subthalamic nucleus and/or output nuclei of BG, entopeduncular nucleus and substantia nigra (SN). The present study aimed to identify the characteristic projection patterns of PV neurons in the GP (PV-GP neurons) and determine whether these neurons target dopaminergic or GABAergic neurons in SN pars compacta (SNc) or reticulata (SNr), respectively. We initially found that (1) 57% of PV neurons co-expressed Lim-homeobox 6, (2) the PVGP terminals were preferentially distributed in the ventral part of dorsal tier of SNc, (3) PV-GP neurons formed basket-like appositions with the somata of tyrosine hydroxylase, PV, calretinin and cholecystokinin immunoreactive neurons in the SN, and (4) in vitro wholecell recording during optogenetic photo-stimulation of PVGP terminals in SNc demonstrated that PV-GP neurons strongly inhibited dopamine neurons via GABA(A) receptors. These results suggest that dopamine neurons receive direct focal inputs from PV-GP prototypic neurons. The identification of high-contrast inhibitory systems on dopamine neurons might represent a key step toward understanding the BG function.
  • Hiromi Takahashi-Iwanaga, Shunsuke Kimura, Kohtarou Konno, Masahiko Watanabe, Toshihiko Iwanaga
    AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY 313 (1) F20 - F29 1931-857X 2017/07 [Refereed][Not invited]
     
    The natriuretic hormone CCK exhibits its gene transcripts in total kidney extracts. To test the possibility of CCK acting as an intrarenal mediator of sodium excretion, we examined mouse kidneys by 1) an in situ hybridization technique for CCK mRNA in animals fed a normal- or a high-sodium diet; 2) immuno-electron microscopy for the CCK peptide, 3) an in situ hybridization method and immunohistochemistry for the CCK-specific receptor CCKAR; 4) confocal image analysis of receptor-mediated Ca2+ responses in isolated renal tubules; and 5) metabolic cage experiments for the measurement of urinary sodium excretion in high-salt-fed mice either treated or untreated with the CCKAR antagonist lorglumide. Results showed the CCK gene to be expressed intensely in the inner medulla and moderately in the inner stripe of the outer medulla, with the expression in the latter being enhanced by high sodium intake. Immunoreactivity for the CCK peptide was localized to the rough endoplasmic reticulum of the medullary interstitial cells in corresponding renal regions, confirming it to be a secretory protein. Gene transcripts, protein products, and the functional activity for CCKAR were consistently localized to the late proximal tubule segments (S2 and S3) in the medullary rays, and the outer stripe of the outer medulla. Lorglumide significantly diminished natriuretic responses of mice to a dietary sodium load without altering the glomerular filtration rate. These findings suggest that the medullary interstitial cells respond to body fluid expansion by CCK release for feedback regulation of the late proximal tubular reabsorption.
  • Taisuke Miyazaki, Miwako Yamasaki, Kouichi Hashimoto, Kazuhisa Kohda, Michisuke Yuzaki, Keiko Shimamoto, Kohichi Tanaka, Masanobu Kano, Masahiko Watanabe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 (28) 7438 - 7443 0027-8424 2017/07 [Refereed][Not invited]
     
    Astrocytes regulate synaptic transmission through controlling neurotransmitter concentrations around synapses. Little is known, however, about their roles in neural circuit development. Here we report that Bergmann glia (BG), specialized cerebellar astrocytes that thoroughly enwrap Purkinje cells (PCs), are essential for synaptic organization in PCs through the action of the L-glutamate/L-aspartate transporter (GLAST). In GLAST-knockout mice, dendritic innervation by the main ascending climbing fiber (CF) branch was significantly weakened, whereas the transverse branch, which is thin and non-synaptogenic in control mice, was transformed into thick and synaptogenic branches. Both types of CF branches frequently produced aberrant wiring to proximal and distal dendrites, causing multiple CF-PC innervation. Our electrophysiological analysis revealed that slow and small CF-evoked excitatory postsynaptic currents (EPSCs) were recorded from almost all PCs in GLAST-knockout mice. These atypical CF-EPSCs were far more numerous and had significantly faster 10-90% rise time than those elicited by glutamate spillover under pharmacological blockade of glial glutamate transporters. Innervation by parallel fibers (PFs) was also affected. PF synapses were robustly increased in the entire dendritic trees, leading to impaired segregation of CF and PF territories. Furthermore, lamellate BG processes were retracted from PC dendrites and synapses, leading to the exposure of these neuronal elements to the extracellular milieus. These synaptic and glial phenotypes were reproduced in wild-type mice after functional blockade of glial glutamate transporters. These findings highlight that glutamate transporter function by GLAST on BG plays important roles in development and maintenance of proper synaptic wiring and wrapping in PCs.
  • Yasukazu Hozumi, Hiroki Fujiwara, Kenya Kaneko, Satoshi Fujii, Matthew K. Topham, Masahiko Watanabe, Kaoru Goto
    CELL AND TISSUE RESEARCH 368 (3) 441 - 458 0302-766X 2017/06 [Refereed][Not invited]
     
    Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate. Diacylglycerol kinase (DGK) phosphorylates DG to produce phosphatidic acid, another second messenger. Of the DGK family, DGK epsilon is the only DGK isoform that exhibits substrate specificity for DG with an arachidonoyl acyl chain at the sn-2 position. Recently, we demonstrated that hydrophobic residues in the N-terminus of DGK epsilon play an important role in targeting the endoplasmic reticulum in transfected cells. However, its cellular expression and subcellular localization in the brain remain elusive. In the present study, we investigate this issue using specific DGK epsilon antibody. DGK epsilon was richly expressed in principal neurons of higher brain regions, including pyramidal cells in the hippocampus and neocortex, medium spiny neurons in the striatum and Purkinje cells in the cerebellum. In Purkinje cells, DGK epsilon was localized to the subsurface cisterns and colocalized with inositol 1,4,5-trisphosphate receptor-1 in dendrites and axons. In dendrites of Purkinje cells, DGK epsilon was also distributed in close apposition to DG lipase-alpha, which catalyzes arachidonoyl-DG to produce 2-arachidonoyl glycerol, a major endocannabinoid in the brain. Behaviorally, DGK epsilon-knockout mice exhibited hyper-locomotive activities and impaired motor coordination and learning. These findings suggest that DGK epsilon plays an important role in neuronal and brain functions through its distinct neuronal expression and subcellular localization and also through coordinated arrangement with other molecules involving the phosphoinositide signaling pathway.
  • Takafumi Miki, Walter A. Kaufmann, Gerardo Malagon, Laura Gomez, Katsuhiko Tabuchi, Masahiko Watanabe, Ryuichi Shigemoto, Alain Marty
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 (26) E5246 - E5255 0027-8424 2017/06 [Refereed][Not invited]
     
    Many central synapses contain a single presynaptic active zone and a single postsynaptic density. Vesicular release statistics at such "simple synapses" indicate that they contain a small complement of docking sites where vesicles repetitively dock and fuse. In this work, we investigate functional and morphological aspects of docking sites at simple synapses made between cerebellar parallel fibers and molecular layer interneurons. Using immunogold labeling of SDS-treated freeze-fracture replicas, we find that Ca(v)2.1 channels form several clusters per active zone with about nine channels per cluster. The mean value and range of intersynaptic variation are similar for Ca(v)2.1 cluster numbers and for functional estimates of docking-site numbers obtained from the maximum numbers of released vesicles per action potential. Both numbers grow in relation with synaptic size and decrease by a similar extent with age between 2 wk and 4 wk postnatal. Thus, the mean docking-site numbers were 3.15 at 2 wk (range: 1-10) and 2.03 at 4 wk (range: 1-4), whereas the mean numbers of Ca(v)2.1 clusters were 2.84 at 2 wk (range: 1-8) and 2.37 at 4 wk (range: 1-5). These changes were accompanied by decreases of miniature current amplitude (from 93 pA to 56 pA), active-zone surface area (from 0.0427 mu m(2) to 0.0234 mu m(2)), and initial success rate (from 0.609 to 0.353), indicating a tightening of synaptic transmission with development. Altogether, these results suggest a close correspondence between the number of functionally defined vesicular docking sites and that of clusters of voltage-gated calcium channels.
  • Nobuhiro Nakai, Masatoshi Nagano, Fumihito Saitow, Yasuhito Watanabe, Yoshinobu Kawamura, Akiko Kawamoto, Kota Tamada, Hiroshi Mizuma, Hirotaka Onoe, Yasuyoshi Watanabe, Hiromu Monai, Hajime Hirase, Jin Nakatani, Hirofumi Inagaki, Tomoyuki Kawada, Taisuke Miyazaki, Masahiko Watanabe, Yuka Sato, Shigeo Okabe, Kazuo Kitamura, Masanobu Kano, Kouichi Hashimoto, Hidenori Suzuki, Toru Takumi
    SCIENCE ADVANCES 3 (6) e1603001  2375-2548 2017/06 [Refereed][Not invited]
     
    Serotonin is a critical modulator of cortical function, and its metabolism is defective in autism spectrum disorder (ASD) brain. How serotonin metabolism regulates cortical physiology and contributes to the pathological and behavioral symptoms of ASD remains unknown. We show that normal serotonin levels are essential for the maintenance of neocortical excitation/inhibition balance, correct sensory stimulus tuning, and social behavior. Conversely, low serotonin levels in 15q dup mice (a model for ASD with the human 15q11-13 duplication) result in impairment of the same phenotypes. Restoration of normal serotonin levels in 15q dup mice revealed the reversibility of a subset of ASD-related symptoms in the adult. These findings suggest that serotonin may have therapeutic potential for discrete ASD symptoms.
  • Junko Kono, Kohtarou Konno, Ashraf Hossain Talukder, Toshimitsu Fuse, Manabu Abe, Katsuya Uchida, Shuhei Horio, Kenji Sakimura, Masahiko Watanabe, Keiichi Itoi
    BRAIN STRUCTURE & FUNCTION 222 (4) 1705 - 1732 1863-2653 2017/05 [Refereed][Not invited]
     
    We examined the morphological features of corticotropin-releasing factor (CRF) neurons in a mouse line in which modified yellow fluorescent protein (Venus) was expressed under the CRF promoter. We previously generated the CRF-Venus knock-in mouse, in which Venus is inserted into the CRF gene locus by homologous recombination. In the present study, the neomycin phosphotransferase gene (Neo), driven by the pgk-1 promoter, was deleted from the CRF-Venus mouse genome, and a CRF-Venusa dagger Neo mouse was generated. Venus expression is much more prominent in the CRF-Venusa dagger Neo mouse when compared to the CRF-Venus mouse. In addition, most Venus-expressing neurons co-express CRF mRNA. Venus-expressing neurons constitute a discrete population of neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) that project to the median eminence. Venus-expressing neurons were also found in brain regions outside the neuroendocrine PVH, including the olfactory bulb, the piriform cortex (Pir), the extended amygdala, the hippocampus, the neocortices, Barrington's nucleus, the midbrain/pontine dorsal tegmentum, the periaqueductal gray, and the inferior olivary nucleus (IO). Venus-expressing perikarya co-expressing CRF mRNA could be observed clearly even in regions where CRF-immunoreactive perikarya could hardly be identified. We demonstrated that the CRF neurons contain glutamate in the Pir and IO, while they contain gamma-aminobutyric acid in the neocortex, the bed nucleus of the stria terminalis, the hippocampus, and the amygdala. A population of CRF neurons was demonstrated to be cholinergic in the midbrain tegmentum. The CRF-Venusa dagger Neo mouse may be useful for studying the structural and functional properties of CRF neurons in the mouse brain.
  • Sam A. Booker, Daniel Althof, Anna Gross, Desiree Loreth, Johanna Mueller, Andreas Unger, Bernd Fakler, Andrea Varro, Masahiko Watanabe, Martin Gassmann, Bernhard Bettler, Ryuichi Shigemoto, Imre Vida, Akos Kulik
    CEREBRAL CORTEX 27 (3) 2318 - 2334 1047-3211 2017/03 [Refereed][Not invited]
     
    Cholecystokinin-expressing interneurons (CCK-INs) mediate behavior state-dependent inhibition in cortical circuits and themselves receive strong GABAergic input. However, it remains unclear to what extent GABA(B) receptors (GABA(B)Rs) contribute to their inhibitory control. Using immunoelectron microscopy, we found that CCK-INs in the rat hippocampus possessed high levels of dendritic GABABRs and KCTD12 auxiliary proteins, whereas postsynaptic effector Kir3 channels were present at lower levels. Consistently, whole-cell recordings revealed slow GABA(B)R-mediated inhibitory postsynaptic currents (IPSCs) in most CCK-INs. In spite of the higher surface density of GABABRs in CCK-INs than in CA1 principal cells, the amplitudes of IPSCs were comparable, suggesting that the expression of Kir3 channels is the limiting factor for the GABA(B)R currents in these INs. Morphological analysis showed that CCK-INs were diverse, comprising perisomatic-targeting basket cells (BCs), as well as dendrite-targeting (DT) interneurons, including a previously undescribed DT type. GABA(B)R-mediated IPSCs in CCK-INs were large in BCs, but small in DT subtypes. In response to prolonged activation, GABA(B)R-mediated currents displayed strong desensitization, which was absent in KCTD12-deficient mice. This study highlights that GABA(B)Rs differentially control CCK-IN subtypes, and the kinetics and desensitization of GABABR-mediated currents are modulated by KCTD12 proteins.
  • Maria Gutierrez-Mecinas, Andrew M. Bell, Alina Marin, Rebecca Taylor, Kieran A. Boyle, Takahiro Furuta, Masahiko Watanabe, Erika Polgar, Andrew J. Todd
    PAIN 158 (3) 440 - 456 0304-3959 2017/03 [Refereed][Not invited]
     
    The superficial dorsal horn, which is the main target for nociceptive and pruritoceptive primary afferents, contains a high density of excitatory interneurons. Our understanding of their roles in somatosensory processing has been restricted by the difficulty of distinguishing functional populations among these cells. We recently defined 3 nonoverlapping populations among the excitatory neurons, based on the expression of neurotensin, neurokinin B, and gastrin-releasing peptide. Here we identify and characterise another population: neurons that express the tachykinin peptide substance P. We show with immunocytochemistry that its precursor protein (preprotachykinin A, PPTA) can be detected in; similar to 14% of lamina I-II neurons, and these are concentrated in the outer part of lamina II. Over 80% of the PPTA-positive cells lack the transcription factor Pax2 (which determines an inhibitory phenotype), and these account for similar to 15% of the excitatory neurons in this region. They are different from the neurotensin, neurokinin B, or gastrin-releasing peptide neurons, although many of them contain somatostatin, which is widely expressed among superficial dorsal horn excitatory interneurons. We show that many of these cells respond to noxious thermal and mechanical stimuli and to intradermal injection of pruritogens. Finally, we demonstrate that these cells can also be identified in a knock-in Cre mouse line (Tac1(Cre)), although our findings suggest that there is an additional population of neurons that transiently express PPTA. This population of substance P-expressing excitatory neurons is likely to play an important role in the transmission of signals that are perceived as pain and itch.
  • Akiyo Natsubori, Iku Tsutsui-Kimura, Hiroshi Nishida, Youcef Bouchekioua, Hiroshi Sekiya, Motokazu Uchigashima, Masahiko Watanabe, Alban de Kerchove d'Exaerde, Masaru Mimura, Norio Takata, Kenji F. Tanaka
    JOURNAL OF NEUROSCIENCE 37 (10) 2723 - 2733 0270-6474 2017/03 [Refereed][Not invited]
     
    The ventral striatum is involved in motivated behavior. Akin to the dorsal striatum, the ventral striatum contains two parallel pathways: the striatomesencephalic pathway consisting of dopamine receptor Type 1-expressing medium spiny neurons (D1-MSNs) and the striatopallidal pathway consisting of D2-MSNs. These two genetically identified pathways are thought to encode opposing functions in motivated behavior. It has also been reported that D1/D2 genetic selectivity is not attributed to the anatomical discrimination of two pathways. We wanted to determine whether D1-and D2-MSNs in the ventral striatum functioned in an opposing manner as previous observations claimed, and whether D1/D2 selectivity corresponded to a functional segregation in motivated behavior of mice. To address this question, we focused on the lateral portion of ventral striatum as a region implicated in food-incentive, goal-directed behavior, and recorded D1 or D2-MSN activity by using a gene-encoded ratiometric Ca2+ indicator and by constructing a fiberphotometry system, and manipulated their activities via optogenetic inhibition during ongoing behaviors. We observed concurrent event-related compoundCa(2+) elevations in ventrolateral D1-and D2-MSNs, especially at trial start cue-related and first lever press-related times. D1 or D2 selective optogenetic inhibition just after the trial start cue resulted in a reduction of goal-directed behavior, indicating a shared coding of motivated behavior by both populations at this time. Only D1-selective inhibition just after the first lever press resulted in the reduction of behavior, indicating D1-MSN-specific coding at that specific time. Our data did not support opposing encoding by both populations in food-incentive, goal-directed behavior.
  • Hisataka Fujimoto, Kotaro Konno, Masahiko Watanabe, Shozo Jinno
    JOURNAL OF COMPARATIVE NEUROLOGY 525 (4) 868 - 884 0021-9967 2017/03 [Refereed][Not invited]
     
    The inferior colliculus (IC) is partitioned into three subdivisions: the dorsal and lateral cortices (DC and LC) and the central nucleus (ICC), and serves as an integration center of auditory information. Recent studies indicate that a certain population of IC neurons may represent the non-GABAergic phenotype, while they express well-established cortical/hippocampal GABAergic neuron markers. In this study we used the optical disector to investigate the phenotype of IC neurons expressing parvalbumin (PV) and/or nitric oxide synthase (NOS) in C57BL/6J mice during the late postnatal period. Four major types of IC neurons were defined by the presence (+) or absence (-) of PV, NOS, and glutamic acid decarboxylase 67 (GAD67): PV+/NOS-/GAD67(+), PV+/NOS+/GAD67(+), PV+/NOS-/GAD67(-), and PV-/NOS+/GAD67(-). Fluorescent in situ hybridization for vesicular glutamate transporter 2 mRNA indicated that almost all GAD67(-) IC neurons represented the glutamatergic phenotype. The numerical densities (NDs) of total GAD67(+) IC neurons remained unchanged in all subdivisions. The NDs of PV+/NOS-/GAD67(+) neurons and PV-/NOS+/GAD67(-) neurons were reduced with age in the ICC, while they remained unchanged in the DC and LC. By contrast, the NDs of PV+/NOS+/GAD67(+) neurons and PV+/NOS-/GAD67(-) neurons were increased with age in the ICC, although there were no changes in the DC and LC. The cell body size of GAD67(+) IC neurons did not vary according to the expression of PV with or without NOS. The present findings indicate that the expression of PV and NOS may shift with age within the GABAergic and glutamatergic phenotypes of IC neurons during the late postnatal period. J. Comp. Neurol. 525:868-884, 2017. (c) 2016 Wiley Periodicals, Inc.
  • Iku Tsutsui-Kimura, Hiroyuki Takiue, Keitaro Yoshida, Ming Xu, Ryutaro Yano, Hiroyuki Ohta, Hiroshi Nishida, Youcef Bouchekioua, Hideyuki Okano, Motokazu Uchigashima, Masahiko Watanabe, Norio Takata, Michael R. Drew, Hiromi Sano, Masaru Mimura, Kenji F. Tanaka
    Nature Communications 8 14304  2041-1723 2017/02 [Refereed][Not invited]
     
    Impaired motivation is present in a variety of neurological disorders, suggesting that decreased motivation is caused by broad dysfunction of the nervous system across a variety of circuits. Based on evidence that impaired motivation is a major symptom in the early stages of Huntington's disease, when dopamine receptor type 2-expressing striatal medium spiny neurons (D2-MSNs) are particularly affected, we hypothesize that degeneration of these neurons would be a key node regulating motivational status. Using a progressive, time-controllable, diphtheria toxin-mediated cell ablation/dysfunction technique, we find that loss-of-function of D2-MSNs within ventrolateral striatum (VLS) is sufficient to reduce goal-directed behaviours without impairing reward preference or spontaneous behaviour. Moreover, optogenetic inhibition and ablation of VLS D2-MSNs causes, respectively, transient and chronic reductions of goal-directed behaviours. Our data demonstrate that the circuitry containing VLS D2-MSNs control motivated behaviours and that VLS D2-MSN loss-of-function is a possible cause of motivation deficits in neurodegenerative diseases.
  • Jamie R. Flynn, Victoria L. Conn, Kieran A. Boyle, David I. Hughes, Masahiko Watanabe, Tomoko Velasquez, Martyn D. Goulding, Robert J. Callister, Brett A. Graham
    FRONTIERS IN NEUROANATOMY 11 5 - 5 1662-5129 2017/02 [Refereed][Not invited]
     
    Long descending propriospinal neurons (LDPNs) are interneurons that form direct connections between cervical and lumbar spinal circuits. LDPNs are involved in interlimb coordination and are important mediators of functional recovery after spinal cord injury (SCI). Much of what we know about LDPNs comes from a range of species, however, the increased use of transgenic mouse lines to better define neuronal populations calls for a more complete characterisation of LDPNs in mice. In this study, we examined the cell body location, inhibitory neurotransmitter phenotype, developmental provenance, morphology and synaptic inputs of mouse LDPNs throughout the cervical and upper thoracic spinal cord. LDPNs were retrogradely labelled from the lumbar spinal cord to map cell body locations throughout the cervical and upper thoracic segments. Ipsilateral LDPNs were distributed throughout the dorsal, intermediate and ventral grey matter as well as the lateral spinal nucleus and lateral cervical nucleus. In contrast, contralateral LDPNs were more densely concentrated in the ventromedial grey matter. Retrograde labelling in GlyT2(GFP) and GAD67(GFP) mice showed the majority of inhibitory LDPNs project either ipsilaterally or adjacent to the midline. Additionally, we used several transgenic mouse lines to define the developmental provenance of LDPNs and found that V2b positive neurons form a subset of ipsilaterally projecting LDPNs. Finally, a population of Neurobiotin (NB) labelled LDPNs were assessed in detail to examine morphology and plot the spatial distribution of contacts from a variety of neurochemically distinct axon terminals. These results provide important baseline data in mice for future work on their role in locomotion and recovery from SCI.
  • Hiromi Takahashi-Iwanaga, Shunsuke Kimura, Kohtarou Konno, Masahiko Watanabe, Toshihiko Iwanaga
    American Journal of Physiology - Renal Physiology 313 (1) F20 - F29 1522-1466 2017 [Refereed][Not invited]
     
    The natriuretic hormone CCK exhibits its gene transcripts in total kidney extracts. To test the possibility of CCK acting as an intrarenal mediator of sodium excretion, we examined mouse kidneys by 1) an in situ hybridization technique for CCK mRNA in animals fed a normal- or a high-sodium diet 2) immuno-electron microscopy for the CCK peptide, 3) an in situ hybridization method and immunohistochemistry for the CCK-specific receptor CCKAR 4) confocal image analysis of receptor-mediated Ca2+ responses in isolated renal tubules and 5) metabolic cage experiments for the measurement of urinary sodium excretion in high-salt-fed mice either treated or untreated with the CCKAR antagonist lorglumide. Results showed the CCK gene to be expressed intensely in the inner medulla and moderately in the inner stripe of the outer medulla, with the expression in the latter being enhanced by high sodium intake. Immunoreactivity for the CCK peptide was localized to the rough endoplasmic reticulum of the medullary interstitial cells in corresponding renal regions, confirming it to be a secretory protein. Gene transcripts, protein products, and the functional activity for CCKAR were consistently localized to the late proximal tubule segments (S2 and S3) in the medullary rays, and the outer stripe of the outer medulla. Lorglumide significantly diminished natriuretic responses of mice to a dietary sodium load without altering the glomerular filtration rate. These findings suggest that the medullary interstitial cells respond to body fluid expansion by CCK release for feedback regulation of the late proximal tubular reabsorption.
  • Fukudome Y, Ohno-Shosaku T, Matsui M, Omori Y, Watanabe M, Kano M
    Eur J Nuerosci. 19 (10) 2682 - 2692 2017 [Refereed][Not invited]
  • Shouta Sugio, Koujiro Tohyama, Shinichiro Oku, Kanehiro Fujiyoshi, Takeshi Yoshimura, Keigo Hikishima, Ryutaro Yano, Takahiro Fukuda, Masaya Nakamura, Hideyuki Okano, Masahiko Watanabe, Masaki Fukata, Kazuhiro Ikenaka, Kenji F Tanaka
    Glia 65 (1) 150 - 168 2017/01 [Refereed][Not invited]
     
    Astrocytes have recently been shown to provide physiological support for various brain functions, although little is known about their involvement in white matter integrity. Several inherited infantile-onset leukoencephalopathies, such as Alexander disease and megalencephalic leukoencephalopathy with subcortical cysts (MLC), implicate astrocytic involvement in the formation of white matter. Several mouse models of MLC had been generated by knocking out the Mlc1 gene; however, none of those models was reported to show myelin abnormalities prior to formation of the myelin sheath. Here we generated a new Mlc1 knockout mouse and a Mlc1 overexpressing mouse, and demonstrate that astrocyte-specific Mlc1 overexpression causes infantile-onset abnormalities of the white matter in which astrocytic swelling followed by myelin membrane splitting are present, whereas knocking out Mlc1 does not, and only shows myelin abnormalities after 12 months of age. Biochemical analyses demonstrated that MLC1 interacts with the Na+ /K+ ATPase and that overexpression of Mlc1 results in decreased activity of the astrocytic Na+ /K+ pump. In contrast, no changes in Na+ /K+ pump activity were observed in Mlc1 KO mice, suggesting that the reduction in Na+ /K+ pump activity resulting from Mlc1 overexpression causes astrocytic swelling. Our infantile-onset leukoencephalopathy model based on Mlc1 overexpression may provide an opportunity to further explore the roles of astrocytes in white matter development and structural integrity. We established a novel mouse model for infantile-onset leukoencephalopathy by the overexpression of Mlc1. Mlc1 overexpression reduced activity of the astrocytic sodium pump, which may underlie white matter edema followed by myelin membrane splitting. GLIA 2016 GLIA 2017;65:150-168.
  • Victoria E. Abraira, Emily D. Kuehn, Anda M. Chirila, Mark W. Springel, Alexis A. Toliver, Amanda L. Zimmerman, Lauren L. Orefice, Kieran A. Boyle, Ling Bai, Bryan J. Song, Karleena A. Bashista, Thomas G. O'Neill, Justin Zhuo, Connie Tsan, Jessica Hoynoski, Michael Rutlin, Laura Kus, Vera Niederkofler, Masahiko Watanabe, Susan M. Dymecki, Sacha B. Nelson, Nathaniel Heintz, David I. Hughes, David D. Ginty
    CELL 168 (1-2) 295 - + 0092-8674 2017/01 [Refereed][Not invited]
     
    The deep dorsal horn is a poorly characterized spinal cord region implicated in processing low-threshold mechanoreceptor (LTMR) information. We report an array of mouse genetic tools for defining neuronal components and functions of the dorsal horn LTMR-recipient zone (LTMR-RZ), a role for LTMR-RZ processing in tactile perception, and the basic logic of LTMR-RZ organization. We found an unexpectedly high degree of neuronal diversity in the LTMR-RZ: seven excitatory and four inhibitory subtypes of interneurons exhibiting unique morphological, physiological, and synaptic properties. Remarkably, LTMRs form synapses on between four and 11 LTMR-RZ interneuron subtypes, while each LTMR-RZ interneuron subtype samples inputs from at least one to three LTMR classes, as well as spinal cord interneurons and corticospinal neurons. Thus, the LTMR-RZ is a somatosensory processing region endowed with a neuronal complexity that rivals the retina and functions to pattern the activity of ascending touch pathways that underlie tactile perception.
  • Eiichi Tokuda, Itsuki Anzai, Takao Nomura, Keisuke Toichi, Masahiko Watanabe, Shinji Ohara, Seiji Watanabe, Koji Yamanaka, Yuta Morisaki, Hidemi Misawa, Yoshiaki Furukawa
    MOLECULAR NEURODEGENERATION 12 (1) 2 - 2 1750-1326 2017/01 [Refereed][Not invited]
     
    Background: Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS) with accumulation of misfolded SOD1 proteins as intracellular inclusions in spinal motor neurons. Oligomerization of SOD1 via abnormal disulfide crosslinks has been proposed as one of the misfolding pathways occurring in mutant SOD1; however, the pathological relevance of such oligomerization in the SOD1-ALS cases still remains obscure. Methods: We prepared antibodies exclusively recognizing the SOD1 oligomers cross-linked via disulfide bonds in vitro. By using those antibodies, immunohistochemical examination and ELISA were mainly performed on the tissue samples of transgenic mice expressing mutant SOD1 proteins and also of human SOD1-ALS cases. Results: We showed the recognition specificity of our antibodies exclusively toward the disulfide-crosslinked SOD1 oligomers by ELISA using various forms of purified SOD1 proteins in conformationally distinct states in vitro. Furthermore, the epitope of those antibodies was buried and inaccessible in the natively folded structure of SOD1. The antibodies were then found to specifically detect the pathological SOD1 species in the spinal motor neurons of the SOD1-ALS patients as well as the transgenic model mice. Conclusions: Our findings here suggest that the SOD1 oligomerization through the disulfide-crosslinking associates with exposure of the SOD1 structural interior and is a pathological process occurring in the SOD1-ALS cases.
  • Sonoko Hasegawa, Makiko Kumagai, Mitsue Hagihara, Hiroshi Nishimaru, Keizo Hirano, Ryosuke Kaneko, Atsushi Okayama, Teruyoshi Hirayama, Makoto Sanbo, Masumi Hirabayashi, Masahiko Watanabe, Takahiro Hirabayashi, Takeshi Yagi
    FRONTIERS IN MOLECULAR NEUROSCIENCE 9 155 - 155 1662-5099 2016/12 [Refereed][Not invited]
     
    The clustered protocadherin (Pcdh) genes are divided into the Pcdhα, Pcdhβ, and Pcdhγ clusters. Gene-disruption analyses in mice have revealed the in vivo functions of the Pcdhα and Pcdhγ clusters. However, all Pcdh protein isoforms form combinatorial cis-hetero dimers and enter trans-homophilic interactions. Here we addressed distinct and cooperative functions in the Pcdh clusters by generating six cluster-deletion mutants (Δα, Δβ, Δγ, Δαβ, Δβγ, and Δαβγ) and comparing their phenotypes: Δα, Δβ, and Δαβ mutants were viable and fertile; Δγ mutants lived less than 12 h; and Δβγ and Δαβγ mutants died shortly after birth. The Pcdhα, Pcdhβ, and Pcdhγ clusters were individually and cooperatively important in olfactory-axon targeting and spinal-cord neuron survival. Neurodegeneration was most severe in Δαβγ mutants, indicating that Pcdhα and Pcdhβ function cooperatively for neuronal survival. The Pcdhα, Pcdhβ, and Pcdhγ clusters share roles in olfactory-axon targeting and neuronal survival, although to different degrees.
  • Naoyuki Kitajima, Takuro Numaga-Tomita, Masahiko Watanabe, Takuya Kuroda, Akiyuki Nishimura, Kei Miyano, Satoshi Yasuda, Koichiro Kuwahara, Yoji Sato, Tomomi Ide, Lutz Birnbaumer, Hideki Sumimoto, Yasuo Mori, Motohiro Nishida
    Scientific Reports 6 37001 - 37001 2045-2322 2016/11/11 [Refereed][Not invited]
     
    Reactive oxygen species (ROS) produced by NADPH oxidase 2 (Nox2) function as key mediators of mechanotransduction during both physiological adaptation to mechanical load and maladaptive remodeling of the heart. This is despite low levels of cardiac Nox2 expression. The mechanism underlying the transition from adaptation to maladaptation remains obscure, however. We demonstrate that transient receptor potential canonical 3 (TRPC3), a Ca 2+-permeable channel, acts as a positive regulator of ROS (PRROS) in cardiomyocytes, and specifically regulates pressure overload-induced maladaptive cardiac remodeling in mice. TRPC3 physically interacts with Nox2 at specific C-terminal sites, thereby protecting Nox2 from proteasome-dependent degradation and amplifying Ca 2+-dependent Nox2 activation through TRPC3-mediated background Ca 2+ entry. Nox2 also stabilizes TRPC3 proteins to enhance TRPC3 channel activity. Expression of TRPC3 C-terminal polypeptide abolished TRPC3-regulated ROS production by disrupting TRPC3-Nox2 interaction, without affecting TRPC3-mediated Ca 2+ influx. The novel TRPC3 function as a PRROS provides a mechanistic explanation for how diastolic Ca 2+ influx specifically encodes signals to induce ROS-mediated maladaptive remodeling and offers new therapeutic possibilities.
  • Shintaro Otsuka, Kohtarou Konno, Manabu Abe, Junko Motohashi, Kazuhisa Kohda, Kenji Sakimura, Masahiko Watanabe, Michisuke Yuzaki
    JOURNAL OF NEUROSCIENCE 36 (46) 11801 - 11816 0270-6474 2016/11 [Refereed][Not invited]
     
    The cerebellum is thought to be involved in cognitive functions in addition to its well established role in motor coordination and motor learning in humans. Cerebellin 1 (Cbln1) is predominantly expressed in cerebellar granule cells and plays a crucial role in the formation and function of parallel fiber-Purkinje cell synapses. Although genes encoding Cbln1 and its postsynaptic receptor, the delta2 glutamate receptor (GluD2), are suggested to be associated with autistic-like traits and many psychiatric disorders, whether such cognitive impairments are caused by cerebellar dysfunction remains unclear. In the present study, we investigated whether and how Cbln1 signaling is involved in non-motor functions in adult mice. We show that acquisition and retention/retrieval of cued and contextual fear memory were impaired in Cbln1-null mice. In situ hybridization and immunohistochemical analyses revealed that Cbln1 is expressed in various extracerebellar regions, including the retrosplenial granular cortex and the hippocampus. In the hippocampus, Cbln1 immunoreactivity was present at the molecular layer of the dentate gyrus and the stratum lacunosum-moleculare without overt mRNA expression, suggesting that Cbln1 is provided by perforant path fibers. Retention/retrieval, but not acquisition, of cued and contextual fear memory was impaired in forebrain-predominant Cbln1-null mice. Spatial learning in the radial arm water maze was also abrogated. In contrast, acquisition of fear memory was affected in cerebellum-predominant Cbln1-null mice. These results indicate that Cbln1 in the forebrain and cerebellum mediates specific aspects of fear conditioning and spatial memory differentially and that Cbln1 signaling likely regulates motor and non-motor functions in multiple brain regions.
  • Kitaro Okamoto, Miwako Yamasaki, Keizo Takao, Shingo Soya, Monica Iwasaki, Koh Sasaki, Kenta Magoori, Iori Sakakibara, Tsuyoshi Miyakawa, Michihiro Mieda, Masahiko Watanabe, Juro Sakai, Masashi Yanagisawa, Takeshi Sakurai
    PLOS ONE 11 (11) e0164716  1932-6203 2016/11 [Refereed][Not invited]
     
    How the hypothalamus transmits hunger information to other brain regions to govern whole brain function to orchestrate feeding behavior has remained largely unknown. Our present study suggests the importance of a recently found lateral hypothalamic neuropeptide, QRFP, in this signaling. Qrfp(-/-) mice were hypophagic and lean, and exhibited increased anxiety-like behavior, and were hypoactive in novel circumstances as compared with wild type littermates. They also showed decreased wakefulness time in the early hours of the dark period. Histological studies suggested that QRFP neurons receive rich innervations from neurons in the arcuate nucleus which is a primary region for sensing the body's metabolic state by detecting levels of leptin, ghrelin and glucose. These observations suggest that QRFP is an important mediator that acts as a downstream mediator of the arcuate nucleus and regulates feeding behavior, mood, wakefulness and activity.
  • Ming-Dong Zhang, Swapnali Barde, Edit Szodorai, Anna Josephson, Nicholas Mitsios, Masahiko Watanabe, Johannes Attems, Gert Lubec, Gábor G. Kovács, Mathias Uhlén, Jan Mulder, Tibor Harkany, Tomas Hökfelt
    Brain Structure and Function 221 (7) 3843  1863-2661 2016/09/01 [Refereed][Not invited]
     
    The original article was published with an error in the author name. The author name Gábor M. Kovács should be changed to Gábor G. Kovács. The original article has been updated accordingly.
  • Ming-Dong Zhang, Swapnali Barde, Edit Szodorai, Anna Josephson, Nicholas Mitsios, Masahiko Watanabe, Johannes Attems, Gert Lubec, Gabor G. Kovacs, Mathias Uhlen, Jan Mulder, Tibor Harkany, Tomas Hokfelt
    BRAIN STRUCTURE & FUNCTION 221 (7) 3803 - 3823 1863-2653 2016/09 [Refereed][Not invited]
     
    Neuronal calcium-binding protein 1 and -2 (NECAB1/2) localize to multiple excitatory neuron populations in the mouse spinal cord. Here, we analyzed rat and human spinal cord, combining in situ hybridization and immunohistochemistry, complementing newly collated data on mouse spinal cord for direct comparisons. Necab1/2 mRNA transcripts showed complementary distribution in rodent's spinal cord. Multiple-labeling fluorescence histochemistry with neuronal phenotypic markers localized NECAB1 to a dense fiber plexus in the dorsal horn, to neurons mainly in superficial layers and to commissural interneurons in both rodent species. NECAB1-positive (+) motor neurons were only found in mice. NECAB1 distribution in the human spinal cord was similar with the addition of NECAB1-like immunoreactivity surrounding myelinated axons. NECAB2 was mainly present in excitatory synaptic boutons in the dorsal horn of all three species, and often in calbindin-D28k(+) neuronal somata. Rodent ependymal cells expressed calbindin-D28k. In humans, they instead were NECAB2(+) and/or calretinin(+). Our results reveal that the association of NECAB2 to excitatory neuronal circuits in the spinal cord is evolutionarily conserved across the mammalian species investigated so far. In contrast, NECAB1 expression is more heterogeneous. Thus, our study suggests that the phenotypic segregation of NECAB1 and -2 to respective excitatory and inhibitory spinal systems can underpin functional modalities in determining the fidelity of synaptic neurotransmission and neuronal responsiveness, and might bear translational relevance to humans.
  • Madoka Narushima, Motokazu Uchigashima, Yuki Yagasaki, Takeshi Harada, Yasuyuki Nagumo, Naofumi Uesaka, Kouichi Hashimoto, Atsu Aiba, Masahiko Watanabe, Mariko Miyata, Masanobu Kano
    NEURON 91 (5) 1097 - 1109 0896-6273 2016/09 [Refereed][Not invited]
     
    Neural circuits formed during postnatal development have to be maintained stably thereafter, but their mechanisms remain largely unknown. Here we report that the metabotropic glutamate receptor subtype 1 (mGluR1) is essential for the maintenance of mature synaptic connectivity in the dorsal lateral geniculate nucleus (dLGN). In mGluR1 knockout (mGluR1-KO) mice, strengthening and elimination at retinogeniculate synapses occurred normally until around postnatal day 20 (P20). However, during the subsequent visual-experience-dependent maintenance phase, weak retinogeniculate synapses were newly recruited. These changes were similar to those of wild-type (WT) mice that underwent visual deprivation or inactivation of mGluR1 in the dLGN from P21. Importantly, visual deprivation was ineffective in mGluR1-KO mice, and the changes induced by visual deprivation in WT mice were rescued by pharmacological activation of mGluR1 in the dLGN. These results demonstrate that mGluR1 is crucial for the visual-experience-dependent maintenance of mature synaptic connectivity in the dLGN.
  • Tomonori Takeuchi, Adrian J. Duszkiewicz, Alex Sonneborn, Patrick A. Spooner, Miwako Yamasaki, Masahiko Watanabe, Caroline C. Smith, Guillen Fernandez, Karl Deisseroth, Robert W. Greene, Richard G. M. Morris
    NATURE 537 (7620) 357 - + 0028-0836 2016/09 [Refereed][Not invited]
     
    The retention of episodic-like memory is enhanced, in humans and animals, when something novel happens shortly before or after encoding. Using an everyday memory task in mice, we sought the neurons mediating this dopamine-dependent novelty effect, previously thought to originate exclusively from the tyrosine-hydroxylase-expressing (TH+) neurons in the ventral tegmental area. Here we report that neuronal firing in the locus coeruleus is especially sensitive to environmental novelty, locus coeruleus TH+ neurons project more profusely than ventral tegmental area TH+ neurons to the hippocampus, optogenetic activation of locus coeruleus TH+ neurons mimics the novelty effect, and this novelty-associated memory enhancement is unaffected by ventral tegmental area inactivation. Surprisingly, two effects of locus coeruleus TH+ photoactivation are sensitive to hippocampal D-1/D-5 receptor blockade and resistant to adrenoceptor blockade: memory enhancement and long-lasting potentiation of synaptic transmission in CA1 ex vivo. Thus, locus coeruleus TH+ neurons can mediate post-encoding memory enhancement in a manner consistent with possible co-release of dopamine in the hippocampus.
  • Tayo Katano, Masafumi Fukuda, Hidemasa Furue, Maya Yamazaki, Manabu Abe, Masahiko Watanabe, Kazuhiko Nishida, Ikuko Yao, Akihiro Yamada, Yutaka Hata, Nobuaki Okumura, Takanobu Nakazawa, Tadashi Yamamoto, Kenji Sakimura, Toshifumi Takao, Seiji Ito
    eNeuro 3 (5) 2373-2822 2016/09/01 [Refereed][Not invited]
     
    Maintenance of neuropathic pain caused by peripheral nerve injury crucially depends on the phosphorylation of GluN2B, a subunit of the N-methyl-d-aspartate (NMDA) receptor, at Tyr1472 (Y1472) and subsequent formation of a postsynaptic density (PSD) complex of superficial spinal dorsal horn neurons. Here we took advantage of comparative proteomic analysis based on isobaric stable isotope tags (iTRAQ) between wild-type and knock-in mice with a mutation of Y1472 to Phe of GluN2B (Y1472F-KI) to search for PSD proteins in the spinal dorsal horn that mediate the signaling downstream of phosphorylated Y1472 GluN2B. Among several candidate proteins, we focused on brain-enriched guanylate kinase-associated protein (BEGAIN), which was specifically up-regulated in wild-type mice after spared nerve injury (SNI). Immunohistochemical analysis using the generated antibody demonstrated that BEGAIN was highly localized at the synapse of inner lamina II in the spinal dorsal horn and that its expression was up-regulated after SNI in wild-type, but not in Y1472F-KI, mice. In addition, alteration of the kinetics of evoked excitatory postsynaptic currents for NMDA but not those for _-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in spinal lamina II was demonstrated by BEGAIN deletion. We demonstrated that mechanical allodynia, a condition of abnormal pain induced by innocuous stimuli, in the SNI model was significantly attenuated in BEGAIN-deficient mice. However, there was no significant difference between naive wild-type and BEGAIN-knockout mice in terms of physiological threshold for mechanical stimuli. These results suggest that BEGAIN was involved in pathological pain transmission through NMDA receptor activation by the phosphorylation of GluN2B at Y1472 in spinal inner lamina II.
  • Shizuka Kobayashi, Yamato Hida, Hiroyoshi Ishizaki, Eiji Inoue, Miki Tanaka-Okamoto, Miwako Yamasaki, Taisuke Miyazaki, Masahiro Fukaya, Isao Kitajima, Yoshimi Takai, Masahiko Watanabe, Toshihisa Ohtsuka, Toshiya Manabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 44 (5) 2272 - 2284 0953-816X 2016/09 [Refereed][Not invited]
     
    Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size, and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system.
  • Maria Gutierrez-Mecinas, Emily D. Kuehn, Victoria E. Abraira, Erika Polgar, Masahiko Watanabe, Andrew J. Todd
    NEUROSCIENCE 329 171 - 181 0306-4522 2016/08 [Refereed][Not invited]
     
    The spinal dorsal horn processes somatosensory information before conveying it to the brain. The neuronal organization of the dorsal horn is still poorly understood, although recent studies have defined several distinct populations among the interneurons, which account for most of its constituent neurons. All primary afferents, and the great majority of neurons in laminae I-III are glutamatergic, and a major factor limiting our understanding of the synaptic circuitry has been the difficulty in identifying glutamatergic synapses with light microscopy. Although there are numerous potential targets for antibodies, these are difficult to visualize with immunocytochemistry, because of protein cross-linking following tissue fixation. Although this can be overcome by antigen retrieval methods, these lead to difficulty in detecting other antigens. The aim of this study was to test whether the postsynaptic protein Homer can be used to reveal glutamatergic synapses in the dorsal horn. Immunostaining for Homer gave punctate labeling when viewed by confocal microscopy, and this was restricted to synapses at the ultrastructural level. We found that Homer puncta were colocalized with the AMPA receptor GluR2 subunit, but not with the inhibitory synapse-associated protein gephyrin. We also examined several populations of glutamatergic axons and found that most boutons were in contact with at least one Homer punctum. These results suggest that Homer antibodies can be used to reveal the great majority of glutamatergic synapses without antigen retrieval. This will be of considerable value in tracing synaptic circuits, and also in investigating plasticity of glutamatergic synapses in pain states. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of IBRO.
  • Yuki Sugaya, Maya Yamazaki, Motokazu Uchigashima, Kenta Kobayashi, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    CELL REPORTS 16 (5) 1405 - 1415 2211-1247 2016/08 [Refereed][Not invited]
     
    Endocannabinoid signaling is considered to suppress excessive excitability of neural circuits and to protect the brain from seizures. However, the precise mechanisms of this effect are poorly understood. Here, we report that 2-arachidonoylglycerol (2-AG), one of the two major endocannabinoids, is crucial for suppressing seizures. We found that kainate-induced seizures in mice lacking the 2-AG synthesizing enzyme, diacylglycerol lipase alpha, were much more severe compared with those in cannabinoid CB1 receptor knockout mice and were comparable to those in mice lacking both CB1- and CB2-receptor-mediated signaling. In the dentate gyrus, 2-AG suppressed excitatory input around the inner and middle molecular layers through CB1 and presumably CB2 receptors, respectively. This 2-AG-mediated suppression contributed to decreased granule cell excitability and the dampening of seizures. Furthermore, lack of 2-AG signaling enhanced kindling epilepto-genesis and spontaneous seizures after kainate-induced status epilepticus. These results highlight critical roles of 2-AG signaling in the suppression of epileptic seizures.
  • Christoph Straub, Yoav Noam, Toshihiro Nomura, Miwako Yamasaki, Dan Yan, Herman B. Fernandes, Ping Zhang, James R. Howe, Masahiko Watanabe, Anis Contractor, Susumu Tomita
    CELL REPORTS 16 (2) 531 - 544 2211-1247 2016/07 [Refereed][Not invited]
     
    Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.
  • Yoshiko Matsumoto-Makidono, Hisako Nakayama, Miwako Yamasaki, Taisuke Miyazaki, Kazuto Kobayashi, Masahiko Watanabe, Masanobu Kano, Kenji Sakimura, Kouichi Hashimoto
    CELL REPORTS 16 (4) 994 - 1004 2211-1247 2016/07 [Refereed][Not invited]
     
    Some neurons have the ability to enhance output voltage to input current with a preferred frequency, which is called resonance. Resonance is thought to be a basis for membrane potential oscillation. Although ion channels responsible for resonance have been reported, the precise mechanisms by which these channels work remain poorly understood. We have found that resonance is reduced but clearly present in the inferior olivary neurons of Cav3.1 T-type voltage-dependent Ca2+ channel knockout (KO) mice. The activation of Cav3.1 channels is strongly membrane potential dependent, but less frequency dependent. Residual resonance in Cav3.1 KO mice is abolished by a hyper-polarization-activated cyclic nucleotide-gated (HCN) channel blocker, ZD7288, and is partially suppressed by voltage-dependent K+ channel blockers. Resonance is inhibited by ZD7288 in wild-type mice and impaired in HCN1 KO mice, suggesting that the HCN1 channel is essential for resonance. The ZD7288-sensitive current is nearly sinusoidal and strongly frequency dependent. These results suggest that Cav3.1 and HCN1 channels act as amplifying and resonating conductances, respectively.
  • Chiaki Itami, Jui-Yen Huang, Miwako Yamasaki, Masahiko Watanabe, Hui-Chen Lu, Fumitaka Kimura
    JOURNAL OF NEUROSCIENCE 36 (26) 7039 - 7054 0270-6474 2016/06 [Refereed][Not invited]
     
    The formation and refinement of thalamocortical axons (TCAs) is an activity-dependent process (Katz and Shatz, 1996), but its mechanism and nature of activity are elusive. We studied the role of spike timing-dependent plasticity (STDP) in TCA formation and refinement in mice. At birth (postnatal day 0, P0), TCAs invade the cortical plate, from which layers 4 (L4) and L2/3 differentiate at P3-P4. A portion of TCAs transiently reach toward the pia surface around P2-P4 (Senft and Woolsey, 1991; Rebsam et al., 2002) but are eventually confined below the border between L2/3 and L4. We previously showed that L4-L2/3 synapses exhibit STDP with only potentiation (timing-dependent long-term potentiation [t-LTP]) during synapse formation, then switch to a Hebbian form of STDP. Here we show that TCA-cortical plate synapses exhibit robust t-LTP in neonates, whose magnitude decreased gradually after P4-P5. After L2/3 is differentiated, TCA-L2/3 gradually switched to STDP with only depression (t-LTD) after P7-P8, whereas TCA-L4 lost STDP. t-LTP was dependent on NMDA receptor and PKA, whereas t-LTD was mediated by Type 1 cannabinoid receptors (CB1Rs) probably located at TCA terminals, revealed by global and cortical excitatory cell-specific knock-out of CB1R. Moreover, we found that administration of CB1R agonists, including Delta(9)-tetrahydrocannabinol, caused substantial retraction of TCAs. Consistent with this, individual thalamocortical axons exuberantly innervated L2/3 at P12 in CB1R knock-outs, indicating that endogenous cannabinoid signaling shapes TCA projection. These results suggest that the developmental switch in STDP and associated appearance of CB1R play important roles in the formation and refinement of TCAs.
  • Izumi Kaji, Yasutada Akiba, Kohtarou Konno, Masahiko Watanabe, Shunsuke Kimura, Toshihiko Iwanaga, Ayaka Kuri, Ken-ichi Iwamoto, Atsukazu Kuwahara, Jonathan D. Kaunitz
    JOURNAL OF PHYSIOLOGY-LONDON 594 (12) 3339 - 3352 0022-3751 2016/06 [Refereed][Not invited]
     
    The proximal colonic mucosa is constantly exposed to high concentrations of microbially-produced short-chain fatty acids (SCFAs). Although luminal SCFAs evoke electrogenic anion secretion and smooth muscle contractility via neural and non-neural cholinergic pathways in the colon, the involvement of the SCFA receptor free fatty acid receptor (FFA)3, one of the free fatty acid receptor family members, has not been clarified. We investigated the contribution of FFA3 to cholinergic-mediated secretory responses in rat proximal colon. FFA3 was immunolocalized to enteroendocrine cells and to the enteric neural plexuses. Most FFA3-immunoreactive nerve fibres and nerve endings were cholinergic, colocalized with protein gene product (PGP)9.5, the vesicular ACh transporter, and the high-affinity choline transporter CHT1. In Ussing chambered mucosa-submucosa preparations (including the submucosal plexus) of rat proximal colon, carbachol (CCh)-induced Cl- secretion was decreased by TTX, hexamethonium, and the serosal FFA3 agonists acetate or propionate, although not by an inactive analogue 3-chloropropionate. Serosal application of a selective FFA3 agonist (N-[2-methylphenyl]-[4-furan-3-yl]-2-methyl-5-oxo-1,4,5,6,7,8-hexahydro-quinoline-3-carboxamide; MQC) dose-dependently suppressed the response to CCh but not to forskolin, with an IC50 of 13m. Pretreatment with MQC inhibited nicotine-evoked but not bethanechol-evoked secretion. The inhibitory effect of MQC was reversed by pretreatment with pertussis toxin, indicating that FFA3 acts via the G(i/o) pathway. Luminal propionate induced Cl- secretion via the cholinergic pathway, which was reduced by MQC, as well as by TTX, hexamethonium or removal of the submucosal plexus. These results suggest that the SCFA-FFA3 pathway has a novel anti-secretory function in that it inhibits cholinergic neural reflexes in the enteric nervous system.
  • Yu Ohmura, Takayuki Yoshida, Kohtarou Konno, Masabumi Minami, Masahiko Watanabe, Mitsuhiro Yoshioka
    INTERNATIONAL JOURNAL OF NEUROPSYCHOPHARMACOLOGY 19 (6) 1461-1457 2016/06 [Refereed][Not invited]
     
    Background: Patients with posttraumatic stress disorder or panic disorder are often troubled by inappropriate retrieval of fear memory. Moreover, these disorders are often comorbid with irritable bowel syndrome. The main aim of the present study is to elucidate the involvement of hippocampal serotonergic systems in fear memory retrieval and stress-induced defecation. Methods and Results: Microinjection of serotonin 7 receptor antagonist, but not other serotonin receptor antagonists (serotonin (1A), (2A), (2C), (3), (4), and (6)), into the rat ventral hippocampus significantly suppressed the expression of freezing behavior, an index of fear memory retrieval, and decreased the amount of feces, an index of stress-induced defecation, in the contextual fear conditioning test. Electrophysiological data indicated that the serotonin 7 receptor agonist increased the frequency of action potentials in the ventral hippocampal CA3 pyramidal neuron via the activation of the hyperpolarization-activated nonselective cation current I-h. Moreover, in situ hybridization demonstrated that Htr7 mRNA was abundantly expressed in the CA3 compared with other subregions of the hippocampus and that these Htr7 mRNA-positive cells coexpressed hyperpolarization-activated cyclic nucleotide-gated channel 2 and 4 mRNAs, which are components of the I-h channel. Conclusions: These results indicated that the released serotonin activates the serotonin 7 receptor in the CA3 ventral hippocampus subregion, enhances the sensitivity to inputs via hyperpolarization-activated cyclic nucleotide 2 and 4 channels, and thereby facilitates fear memory retrieval. The serotonin 7 receptor might be a target of drug development for the treatment of mental disorders involving fear memory and gastrointestinal problems.
  • Gireesh Gangadharan, Jonghan Shin, Seong-Wook Kim, Angela Kim, Afshin Paydar, Duk-Soo Kim, Taisuke Miyazaki, Masahiko Watanabe, Yuchio Yanagawa, Jinhyun Kim, Yeon-Soo Kim, Daesoo Kim, Hee-Sup Shin
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 (23) 6550 - 6555 0027-8424 2016/06 [Refereed][Not invited]
     
    Exploratory drive is one of the most fundamental emotions, of all organisms, that are evoked by novelty stimulation. Exploratory behavior plays a fundamental role in motivation, learning, and well-being of organisms. Diverse exploratory behaviors have been described, although their heterogeneity is not certain because of the lack of solid experimental evidence for their distinction. Here we present results demonstrating that different neural mechanisms underlie different exploratory behaviors. Localized Ca(v)3.1 knockdown in the medial septum (MS) selectively enhanced object exploration, whereas the null mutant (KO) mice showed enhanced-object exploration as well as open-field exploration. In MS knockdown mice, only type 2 hippocampal theta rhythm was enhanced, whereas both type 1 and type 2 theta rhythm were enhanced in KO mice. This selective effect was accompanied by markedly increased excitability of septo-hippocampal GABAergic projection neurons in the MS lacking T-type Ca2+ channels. Furthermore, optogenetic activation of the septo-hippocampal GABAergic pathway in WT mice also selectively enhanced object exploration behavior and type 2 theta rhythm, whereas inhibition of the same pathway decreased the behavior and the rhythm. These findings define object exploration distinguished from open-field exploration and reveal a critical role of T-type Ca2+ channels in the medial septal GABAergic projection neurons in this behavior.
  • Keiko Matsuda, Timotheus Budisantoso, Nikolaos Mitakidis, Yuki Sugaya, Eriko Miura, Wataru Kakegawa, Miwako Yamasaki, Kohtarou Konno, Motokazu Uchigashima, Manabu Abe, Izumi Watanabe, Masanobu Kano, Masahiko Watanabe, Kenji Sakimura, A. Radu Aricescu, Michisuke Yuzaki
    NEURON 90 (4) 752 - 767 0896-6273 2016/05 [Refereed][Not invited]
     
    Postsynaptic kainate-type glutamate receptors (KARs) regulate synaptic network activity through their slow channel kinetics, most prominently at mossy fiber (MF)-CA3 synapses in the hippocampus. Nevertheless, how KARs cluster and function at these synapses has been unclear. Here, we show that C1q-like proteins C1ql2 and C1ql3, produced by MFs, serve as extracellular organizers to recruit functional postsynaptic KAR complexes to the CA3 pyramidal neurons. C1ql2 and C1ql3 specifically bound the amino-terminal domains of postsynaptic GluK2 and GluK4 KAR subunits and the presynaptic neurexin 3 containing a specific sequence in vitro. In C1ql2/3 double-null mice, CA3 synaptic responses lost the slow, KAR-mediated components. Furthermore, despite induction of MF sprouting in a temporal lobe epilepsy model, KARs were not recruited to postsynaptic sites in C1ql2/3 double-null mice, leading to reduced recurrent circuit activities. C1q family proteins, broadly expressed, are likely to modulate KAR function throughout the brain and represent promising antiepileptic targets.
  • Takanobu Nakazawa, Ryota Hashimoto, Kazuto Sakoori, Yuki Sugaya, Asami Tanimura, Yuki Hashimotodani, Kazutaka Ohi, Hidenaga Yamamori, Yuka Yasuda, Satomi Umeda-Yano, Yuji Kiyama, Kohtarou Konno, Takeshi Inoue, Kazumasa Yokoyama, Takafumi Inoue, Shusuke Numata, Tohru Ohnuma, Nakao Iwata, Norio Ozaki, Hitoshi Hashimoto, Masahiko Watanabe, Toshiya Manabe, Tadashi Yamamoto, Masatoshi Takeda, Masanobu Kano
    NATURE COMMUNICATIONS 7 11466  2041-1723 2016/04 [Refereed][Not invited]
  • Miwako Yamasaki, Masahiro Fukaya, Maya Yamazaki, Hirotsugu Azechi, Rie Natsume, Manabu Abe, Kenji Sakimura, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 36 (15) 4296 - 4312 0270-6474 2016/04 [Refereed][Not invited]
     
    The number of AMPA-type glutamate receptors (AMPARs) at synapses is the major determinant of synaptic strength and varies from synapse to synapse. To clarify the underlying molecular mechanisms, the density of AMPARs, PSD-95, and transmembrane AMPAR regulatory proteins (TARPs) were compared at Schaffer collateral/commissural (SCC) synapses in the adult mouse hippocampal CA1 by quantitative immunogold electron microscopy using serial sections. We examined four types of SCC synapses: perforated and nonperforated synapses on pyramidal cells and axodendritic synapses on parvalbumin-positive (PV synapse) and pravalbumin-negative interneurons (non-PV synapse). SCC synapses were categorized into those expressing high-density (perforated and PV synapses) or low-density (nonperforated and non-PV synapses) AMPARs. Although the density of PSD-95 labeling was fairly constant, the density and composition of TARP isoforms was highly variable depending on the synapse type. Of the three TARPs expressed in hippocampal neurons, the disparity inTARP gamma-2 labeling was closely related to that of AMPAR labeling. Importantly, AMPAR density was significantly reduced at perforated and PV synapses in TARP gamma-2-knock-out (KO) mice, resulting in a virtual loss of AMPAR disparity among SCC synapses. In comparison, TARP gamma-8 was the only TARP expressed at nonperforated synapses, where AMPAR labeling further decreased to a background level in TARP gamma-8-KO mice. These results show that synaptic inclusion of TARP gamma-2 potently increases AMPAR expression and transforms low-density synapses into high-density ones, whereas TARP gamma-8 is essential for low-density or basal expression of AMPARs at nonperforated synapses. Therefore, these TARPs are critically involved in AMPAR density control at SCC synapses.
  • Ryoichi Ichikawa, Kenji Sakimura, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 36 (17) 4846 - 4858 0270-6474 2016/04 [Refereed][Not invited]
     
    Although injured axons usually do not regenerate in the adult CNS, parallel fibers (PFs) regenerate synaptic connections onto cerebellar Purkinje cells (PCs). In this study, we investigated the role of GluD2 in this regenerative process after PF transection using GluD2-knock-out (KO) mice. All dendritic spines on distal dendrites were innervated by PFs in sham-operated wild-type controls, whereas one-third were devoid of innervation in GluD2-KO mice. In both genotypes, a steep drop in the number of PF synapses occurred with a reciprocal surge in the number of free spines on postlesion day 1, when the PF territory aberrantly expanded toward the proximal dendrites. In wild-type mice, the territory and number of PF synapses were nearly fully restored to normal on postlesion day 7, although PF density remained low. Moreover, presynaptic and postsynaptic elements were markedly enlarged, and the PF terminal-to-PC spine contact ratio increased from 1:1 to 1:2 at most synapses. On postlesion day 30, the size and contact ratio of PF synapses returned to sham-operated control values and PF density recovered through the sprouting and elongation of PF collaterals. However, GluD2-KO mice showed neither a hypertrophic response nor territorial restoration 7 d postlesion, nor the recovery of PF axons or synapses on postlesion day 30. This suggests that PF wiring regenerates initially by inducing hypertrophic responses in surviving synaptic elements (hypertrophic phase), followed by collateral formation by PF axons and retraction of PF synapses (remodeling phase). Without GluD2, no transition to these regenerative phases occurs.
  • Motokazu Uchigashima, Toshihisa Ohtsuka, Kazuto Kobayashi, Masahiko Watanabe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 (15) 4206 - 4211 0027-8424 2016/04 [Refereed][Not invited]
     
    Midbrain dopamine neurons project densely to the striatum and form so-called dopamine synapses on medium spiny neurons (MSNs), principal neurons in the striatum. Because dopamine receptors are widely expressed away from dopamine synapses, it remains unclear how dopamine synapses are involved in dopaminergic transmission. Here we demonstrate that dopamine synapses are contacts formed between dopaminergic presynaptic and GABAergic postsynaptic structures. The presynaptic structure expressed tyrosine hydroxylase, vesicular monoamine transporter-2, and plasmalemmal dopamine transporter, which are essential for dopamine synthesis, vesicular filling, and recycling, but was below the detection threshold for molecules involving GABA synthesis and vesicular filling or for GABA itself. In contrast, the postsynaptic structure of dopamine synapses expressed GABAergic molecules, including postsynaptic adhesion molecule neuroligin-2, postsynaptic scaffolding molecule gephyrin, and GABA(A) receptor alpha 1, without any specific clustering of dopamine receptors. Of these, neuroligin-2 promoted presynaptic differentiation in axons of midbrain dopamine neurons and striatal GABAergic neurons in culture. After neuroligin-2 knockdown in the striatum, a significant decrease of dopamine synapses coupled with a reciprocal increase of GABAergic synapses was observed on MSN dendrites. This finding suggests that neuroligin-2 controls striatal synapse formation by giving competitive advantage to heterologous dopamine synapses over conventional GABAergic synapses. Considering that MSN dendrites are preferential targets of dopamine synapses and express high levels of dopamine receptors, dopamine synapse formation may serve to increase the specificity and potency of dopaminergic modulation of striatal outputs by anchoring dopamine release sites to dopamine-sensing targets.
  • 一酸化窒素誘導Ca2+放出 1型リアノジン受容体のS-ニトロシル化による神経細胞死(Nitric oxide-induced calcium release: S -nitrosylation of the type 1 ryanodine receptor and neuronal cell death)
    Mikami Yoshinori, Kanemaru Kazunori, Oda Yasuhiro, Kakizawa Sho, Shibata Kazuki, Sugiyama Hiroki, Koyama Ryuta, Ito Akihiro, Murayama Takashi, Yamazawa Toshiko, Watanabe Masahiko, Ikageya Yuji, Saito Nobuhito, Sakurai Takashi, Iino Masamitsu
    The Journal of Physiological Sciences 66 (Suppl.1) S182 - S182 1880-6546 2016/03
  • Noboru Iwagaki, Robert P. Ganley, Allen C. Dickie, Erika Polgar, David I. Hughes, Patricia Del Rio, Yulia Revina, Masahiko Watanabe, Andrew J. Todd, John S. Riddell
    PAIN 157 (3) 598 - 612 0304-3959 2016/03 [Refereed][Not invited]
     
    The spinal dorsal horn contains numerous inhibitory interneurons that control transmission of somatosensory information. Although these cells have important roles in modulating pain, we still have limited information about how they are incorporated into neuronal circuits, and this is partly due to difficulty in assigning them to functional populations. Around 15% of inhibitory interneurons in laminae I-III express neuropeptide Y (NPY), but little is known about this population. We therefore used a combined electrophysiological/morphological approach to investigate these cells in mice that express green fluorescent protein (GFP) under control of the NPY promoter. We show that GFP is largely restricted to NPY-immunoreactive cells, although it is only expressed by a third of those in lamina I-II. Reconstructions of recorded neurons revealed that they were morphologically heterogeneous, but never islet cells. Many NPY-GFP cells (including cells in lamina III) appeared to be innervated by C fibres that lack transient receptor potential vanilloid-1, and consistent with this, we found that some lamina III NPY-immunoreactive cells were activated by mechanical noxious stimuli. Projection neurons in lamina III are densely innervated by NPY-containing axons. Our results suggest that this input originates from a small subset of NPY-expressing interneurons, with the projection cells representing only a minority of their output. Taken together with results of previous studies, our findings indicate that somatodendritic morphology is of limited value in classifying functional populations among inhibitory interneurons in the dorsal horn. Because many NPY-expressing cells respond to noxious stimuli, these are likely to have a role in attenuating pain and limiting its spread.
  • Andras Szonyi, Marton I. Mayer, Csaba Cserep, Virag T. Takacs, Masahiko Watanabe, Tamas F. Freund, Gabor Nyiri
    BRAIN STRUCTURE & FUNCTION 221 (2) 735 - 751 1863-2653 2016/03 [Refereed][Not invited]
     
    The median raphe region (MRR) is thought to be serotonergic and plays an important role in the regulation of many cognitive functions. In the hippocampus (HIPP), the MRR exerts a fast excitatory control, partially through glutamatergic transmission, on a subpopulation of GABAergic interneurons that are key regulators of local network activity. However, not all receptors of this connection in the HIPP and in synapses established by MRR in other brain areas are known. Using combined anterograde tracing and immunogold methods, we show that the GluN2A subunit of the NMDA receptor is present in the synapses established by MRR not only in the HIPP, but also in the medial septum (MS) and in the medial prefrontal cortex (mPFC) of the mouse. We estimated similar amounts of NMDA receptors in these synapses established by the MRR and in local adjacent excitatory synapses. Using retrograde tracing and confocal laser scanning microscopy, we found that the majority of the projecting cells of the mouse MRR contain the vesicular glutamate transporter type 3 (vGluT3). Furthermore, using double retrograde tracing, we found that single cells of the MRR can innervate the HIPP and mPFC or the MS and mPFC simultaneously, and these double-projecting cells are also predominantly vGluT3-positive. Our results indicate that the majority of the output of the MRR is glutamatergic and acts through NMDA receptor-containing synapses. This suggests that key forebrain areas receive precisely targeted excitatory input from the MRR, which is able to synchronously modify activity in those regions via individual MRR cells with dual projections.
  • Maria Gutierrez-Mecinas, Takahiro Furuta, Masahiko Watanabe, Andrew J. Todd
    MOLECULAR PAIN 12 1744-8069 2016/03 [Refereed][Not invited]
     
    Background: Excitatory interneurons account for the majority of neurons in laminae I-III, but their functions are poorly understood. Several neurochemical markers are largely restricted to excitatory interneuron populations, but we have limited knowledge about the size of these populations or their overlap. The present study was designed to investigate this issue by quantifying the neuronal populations that express somatostatin (SST), neurokinin B (NKB), neurotensin, gastrin-releasing peptide (GRP) and the gamma isoform of protein kinase C (PKC gamma), and assessing the extent to which they overlapped. Since it has been reported that calretinin- and SST-expressing cells have different functions, we also looked for co-localisation of calretinin and SST. Results: SST, preprotachykinin B (PPTB, the precursor of NKB), neurotensin, PKC gamma or calretinin were detected with antibodies, while cells expressing GRP were identified in a mouse line (GRP-EGFP) in which enhanced green fluorescent protein (EGFP) was expressed under control of the GRP promoter. We found that SST-, neurotensin-, PPTB-and PKC gamma-expressing cells accounted for 44%, 7%, 12% and 21% of the neurons in laminae I-II, and 16%, 8%, 4% and 14% of those in lamina III, respectively. GRP-EGFP cells made up 11% of the neuronal population in laminae I-II. The neurotensin, PPTB and GRP-EGFP populations showed very limited overlap, and we estimate that between them they account for similar to 40% of the excitatory interneurons in laminae I-II. SST which is expressed by similar to 60% of excitatory interneurons in this region, was found in each of these populations, as well as in cells that did not express any of the other peptides. Neurotensin and PPTB were often found in cells with PKC gamma, and between them, constituted around 60% of the PKC gamma cells. Surprisingly, we found extensive co-localisation of SST and calretinin. Conclusions: These results suggest that cells expressing neurotensin, NKB or GRP form largely non-overlapping sets that are likely to correspond to functional populations. In contrast, SST is widely expressed by excitatory interneurons that are likely to be functionally heterogeneous.
  • Gukhan Kim, Rafael Lujan, Jochen Schwenk, Melissa H. Kelley, Carolina Aguado, Masahiko Watanabe, Bernd Fakler, James Maylie, John P. Adelman
    ELIFE 5 2050-084X 2016/02 [Refereed][Not invited]
     
    Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca2+ influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.
  • Hiroshi Ueda, Shingo Nakamura, Taro Nakamura, Kaoru Inada, Takashi Okubo, Naohiro Furukawa, Reiichi Murakami, Shigeo Tsuchida, Yonathan Zohar, Kotaro Konno, Masahiko Watanabe
    SCIENTIFIC REPORTS 6 2045-2322 2016/02 [Refereed][Not invited]
     
    The olfactory hypothesis for salmon imprinting and homing to their natal stream is well known, but the endocrine hormonal control mechanisms of olfactory memory formation in juveniles and retrieval in adults remain unclear. In brains of hatchery-reared underyearling juvenile chum salmon (Oncorhynchus keta), thyrotropin-releasing hormone gene expression increased immediately after release from a hatchery into the natal stream, and the expression of the essential NR1 subunit of the N-methyl-D-aspartate receptor increased during downstream migration. Gene expression of salmon gonadotropin-releasing hormone (sGnRH) and NR1 increased in the adult chum salmon brain during homing from the Bering Sea to the natal hatchery. Thyroid hormone treatment in juveniles enhanced NR1 gene activation, and GnRHa treatment in adults improved stream odour discrimination. Olfactory memory formation during juvenile downstream migration and retrieval during adult homing migration of chum salmon might be controlled by endocrine hormones and could be clarified using NR1 as a molecular marker.
  • Ryoichi Ichikawa, Kouichi Hashimoto, Taisuke Miyazaki, Motokazu Uchigashima, Miwako Yamasaki, Atsu Aiba, Masanobu Kano, Masahiko Watanabe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 (8) 2282 - 2287 0027-8424 2016/02 [Refereed][Not invited]
     
    In Purkinje cells (PCs) of the cerebellum, a single "winner" climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9-15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase C gamma (PKC gamma), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1 alpha into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKC gamma signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites.
  • Takanobu Nakazawa, Ryota Hashimoto, Kazuto Sakoori, Yuki Sugaya, Asami Tanimura, Yuki Hashimotodani, Kazutaka Ohi, Hidenaga Yamamori, Yuka Yasuda, Satomi Umeda-Yano, Yuji Kiyama, Kohtarou Konno, Takeshi Inoue, Kazumasa Yokoyama, Takafumi Inoue, Shusuke Numata, Tohru Ohnuma, Nakao Iwata, Norio Ozaki, Hitoshi Hashimoto, Masahiko Watanabe, Toshiya Manabe, Tadashi Yamamoto, Masatoshi Takeda, Masanobu Kano
    NATURE COMMUNICATIONS 7 2041-1723 2016/02 [Refereed][Not invited]
     
    Intracellular trafficking of receptor proteins is essential for neurons to detect various extracellular factors during the formation and refinement of neural circuits. However, the precise mechanisms underlying the trafficking of neurotrophin receptors to synapses remain elusive. Here, we demonstrate that a brain-enriched sorting nexin, ARHGAP33, is a new type of regulator for the intracellular trafficking of TrkB, a high-affinity receptor for brain-derived neurotrophic factor. ARHGAP33 knockout (KO) mice exhibit reduced expression of synaptic TrkB, impaired spine development and neuropsychiatric disorder-related behavioural abnormalities. These deficits are rescued by specific pharmacological enhancement of TrkB signalling in ARHGAP33 KO mice. Mechanistically, ARHGAP33 interacts with SORT1 to cooperatively regulate TrkB trafficking. Human ARHGAP33 is associated with brain phenotypes and reduced SORT1 expression is found in patients with schizophrenia. We propose that ARHGAP33/SORT1-mediated TrkB trafficking is essential for synapse development and that the dysfunction of this mechanism may be a new molecular pathology of neuropsychiatric disorders.
  • Taku Nagai, Shinichi Nakamuta, Keisuke Kuroda, Sakura Nakauchi, Tomoki Nishioka, Tetsuya Takano, Xinjian Zhang, Daisuke Tsuboi, Yasuhiro Funahashi, Takashi Nakano, Junichiro Yoshimoto, Kenta Kobayashi, Motokazu Uchigashima, Masahiko Watanabe, Masami Miura, Akinori Nishi, Kazuto Kobayashi, Kiyofumi Yamada, Mutsuki Amano, Kozo Kaibuchi
    NEURON 89 (3) 550 - 565 0896-6273 2016/02 [Refereed][Not invited]
     
    Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors.
  • Masahiko Watanabe
    Essentials of Cerebellum and Cerebellar Disorders: A Primer for Graduate Students 195 - 199 2016/01/01 [Refereed][Not invited]
     
    Santiago Ramón y Cajal provided a defi nitive description of the basket cells of the cerebellum. Ramón y Cajal discovered a characteristic terminal plexus of basket cells around Purkinje cell somata, naming this the pericellular nest or nid. This was the fi rst clear observation of an axon terminal in the central nervous system the discovery cultivated his ideas that nerve cells need only be in contact, not in continuity, with one another to transmit nerve impulse, and that the fl ow of the impulse is directed from the axon of one cell to the cell body of another. These ideas later came to fruition as his Neuron Doctrine (Palay S, Chan-Palay V Cerebellar cortex cytology and organization. Springer, Berlin, 1974).
  • Izumi Watanabe-Iida, Kohtarou Konno, Kaori Akashi, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura
    JOURNAL OF NEUROCHEMISTRY 136 (2) 295 - 305 0022-3042 2016/01 [Refereed][Not invited]
     
    Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KARsubunits are predominant in the brain, with distinct subunitcombinations between the hippocampus and cerebellum.
  • Noemi Holderith, Andrea Lorincz, Gergely Katona, Balazs Rozsa, Akos Kulik, Masahiko Watanabe, Zoltan Nusser
    NATURE NEUROSCIENCE 19 (1) 172 - 172 1097-6256 2016/01 [Refereed][Not invited]
  • Morino H, Matsuda Y, Muguruma K, Miyamoto R, Ohsawa R, Ohtake T, Otobe R, Watanabe M, Maruyama H, Hashimoto K, Kawakami H
    Molecular brain 8 89 - 89 1756-6606 2015/12 [Refereed][Not invited]
     
    Background: Spinocerebellar ataxia (SCA) is a genetically heterogeneous disease. To date, 36 dominantly inherited loci have been reported, and 31 causative genes have been identified.Results: In this study, we analyzed a Japanese family with autosomal dominant SCA using linkage analysis and exome sequencing, and identified CACNA1G, which encodes the calcium channel CaV3.1, as a new causative gene. The same mutation was also found in another family with SCA. Although most patients exhibited the pure form of cerebellar ataxia, two patients showed prominent resting tremor in addition to ataxia. CaV3.1 is classified as a low-threshold voltage-dependent calcium channel (T-type) and is expressed abundantly in the central nervous system, including the cerebellum. The mutation p.Arg1715His, identified in this study, was found to be located at S4 of repeat IV, the voltage sensor of the CaV3.1. Electrophysiological analyses revealed that the membrane potential dependency of the mutant CaV3.1 transfected into HEK293T cells shifted toward a positive potential. We established induced pluripotent stem cells (iPSCs) from fibroblasts of the patient, and to our knowledge, this is the first report of successful differentiation from the patient-derived iPSCs into Purkinje cells. There was no significant difference in the differentiation status between control-and patient-derived iPSCs.Conclusions: To date, several channel genes have been reported as causative genes for SCA. Our findings provide important insights into the pathogenesis of SCA as a channelopathy.
  • Natsumi Ageta-Ishihara, Maya Yamazaki, Kohtarou Konno, Hisako Nakayama, Manabu Abe, Kenji Hashimoto, Tomoki Nishioka, Kozo Kaibuchi, Satoko Hattori, Tsuyoshi Miyakawa, Kohichi Tanaka, Fathul Huda, Hirokazu Hirai, Kouichi Hashimoto, Masahiko Watanabe, Kenji Sakimura, Makoto Kinoshita
    NATURE COMMUNICATIONS 6 10090  2041-1723 2015/12 [Refereed][Not invited]
     
    The small GTPase-effector proteins CDC42EP1-5/BORG1-5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4(-/-) mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (gamma DGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1-5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance.
  • Yasukazu Hozumi, Ryo Akimoto, Akihito Suzuki, Koichi Otani, Masahiko Watanabe, Kaoru Goto
    CELL AND TISSUE RESEARCH 362 (2) 295 - 305 0302-766X 2015/11 [Refereed][Not invited]
     
    Adrenal glands play a central role in the secretion of steroid hormones and catecholamines. Previous studies have revealed that molecules engaged in phosphoinositide (PI) turnover are expressed in the adrenal gland, suggesting the importance of PI signaling in adrenal signal transduction. Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DG), a major second messenger in the PI signaling cascade. The DGK family is expressed in distinct patterns in endocrine organs at the mRNA and protein levels. Nevertheless, little is known about the characteristics and morphological aspects of DGKs in the adrenal gland. We have performed immunohistochemical analyses to investigate the expression and localization of DGK isozymes, together with PI signaling molecules, in the adrenal gland at the protein level. Our results show that the DGK family and a set of PI signaling molecules are expressed intensely in zona glomerulosa cells and medullary chromaffin cells in the adrenal gland. In adrenal cells, DGK gamma localizes to the Golgi complex, DGK epsilon to the plasma membrane, and DGK zeta to the nucleus. These findings show the distinct expression and subcellular localization of DGK isozymes and PI signaling molecules in the adrenal gland, suggesting that each DGK isozyme has a role in signal transduction in adrenal cells, especially in the zona glomerulosa and medulla.
  • Darren Cameron, Erika Polgar, Maria Gutierrez-Mecinas, Maria Gomez-Lima, Masahiko Watanabe, Andrew J. Todd
    PAIN 156 (10) 2061 - 2071 0304-3959 2015/10 [Refereed][Not invited]
     
    The anterolateral tract (ALT), which originates from neurons in lamina I and the deep dorsal horn, represents a major ascending output through which nociceptive information is transmitted to brain areas involved in pain perception. Although there is detailed quantitative information concerning the ALT in the rat, much less is known about this system in the mouse, which is increasingly being used for studies of spinal pain mechanisms because of the availability of genetically modified lines. The aim of this study was therefore to determine the extent to which information about the ALT in the rat can be extrapolated to the mouse. Our results suggest that as in the rat, most lamina I ALT projection neurons in the lumbar enlargement can be retrogradely labelled from the lateral parabrachial area, that the majority of these cells (similar to 90%) express the neurokinin 1 receptor (NK1r), and that these are larger than other NK1r-expressing neurons in this lamina. This means that many lamina I spinoparabrachial cells can be identified in NK1r-immunostained sections from animals that have not received retrograde tracer injections. However, we also observed certain species differences, in particular we found that many spinoparabrachial cells in laminae III and IV lack the NK1r, meaning that they cannot be identified based solely on the expression of this receptor. We also provide evidence that the majority of spinoparabrachial cells are glutamatergic and that some express substance P. These findings will be important for studies designed to unravel the complex neuronal circuitry that underlies spinal pain processing.
  • Daniel Althof, David Baehrens, Masahiko Watanabe, Noboru Suzuki, Bernd Fakler, Ákos Kulik
    Frontiers in Cellular Neuroscience 9 (AUGUST) 1662-5102 2015/08/11 [Refereed][Not invited]
     
    Tuning of the time course and strength of inhibitory and excitatory neurotransmitter release is fundamental for the precise operation of cortical network activity and is controlled by Ca2+ influx into presynaptic terminals through the high voltage-activated P/Q-type Ca2+ (Cav2.1) channels. Proper channel-mediated Ca2+-signaling critically depends on the topographical arrangement of the channels in the presynaptic membrane. Here, we used high-resolution SDS-digested freeze-fracture replica immunoelectron microscopy together with automatized computational analysis of Cav2.1 immunogold labeling to determine the precise subcellular organization of Cav2.1 channels in both inhibitory and excitatory terminals. Immunoparticles labeling the pore-forming α1 subunit of Cav2.1 channels were enriched over the active zone of the boutons with the number of channels (3–62) correlated with the area of the synaptic membrane. Detailed analysis showed that Cav2.1 channels are non-uniformly distributed over the presynaptic membrane specialization where they are arranged in clusters of an average five channels per cluster covering a mean area with a diameter of about 70 nm. Importantly, clustered arrangement and cluster properties did not show any significant difference between GABAergic and glutamatergic terminals. Our data demonstrate a common nano-architecture of Cav2.1 channels in inhibitory and excitatory boutons in stratum radiatum of the hippocampal CA1 area suggesting that the cluster arrangement is crucial for the precise release of transmitters from the axonal boutons.
  • Yoshinori Shirai, Masahiko Watanabe, Hiroyuki Sakagami, Tatsuo Suzuki
    JOURNAL OF NEUROCHEMISTRY 134 (3) 578 - 589 0022-3042 2015/08 [Refereed][Not invited]
     
    General transcription factor II-I (Gtf2i) is a transcription factor and one of the genes implicated in Willams-Beuren syndrome, an autism spectrum disorder. In this study, we investigated splice variants of the Gtf2i gene in both the 5'untranslated region (5'UTR) and the coding region. To search for novel 5'UTRs of Gtf2i, we utilized the cap analysis gene expression database of the mouse. We identified seven novel Gtf2i transcripts with alternatively spliced 5'UTRs in the rat brain. We also identified four novel splice variants in the coding sequence of Gtf2i. Furthermore, we identified a selective usage of certain types of 5'UTR by coding variants. In situ hybridization demonstrated a differential pattern of expression of Gtf2i mRNAs with alternatively spliced 5'UTRs among neuronal cells, and the localization of one of the variants in neuronal dendrites in the rat brain. Immunohistochemistry also demonstrated a distribution of Gtf2i-immunoreactivity in the dendrites. These results suggest multiple pathways of expression of Gtf2i gene in the brain. The expression patterns may be under the control of alternative promoters coupled to the alternative splicing in the coding region. Differential localization of mRNA to neuronal dendrites suggests spatiotemporal-specific translation at the post-synaptic sites that is involved in transfer of synaptic activity to expression of specific sets of genes in the nucleus.
  • Kerise Lyttle, Yu Ohmura, Kohtarou Konno, Takayuki Yoshida, Takeshi Izumi, Masahiko Watanabe, Mitsuhiro Yoshioka
    BRAIN RESEARCH 1616 88 - 100 0006-8993 2015/08 [Refereed][Not invited]
     
    Human studies have suggested that early life stress such as child abuse could enhance susceptibility to depressive disorders. Moreover, the abnormalities of the prefrontal cortex have been associated with depression. Although clinical studies have implied the negative effects of early life stress on brain development, the causality and the detailed morphogenetic changes has not been clearly elucidated. In the present study, we determined the effect of juvenile stress exposure on the presentation of depressive-like behavior and the neural mechanisms involved using a rodent model. Rat pups were exposed to footshock stress during postnatal days 21-25 followed by repeated oral administration of fluvoxamine (0 or 10 mg/kg/d x 14 days), which is a selective serotonin reuptake inhibitor. At the postadolescent stage forced swim test assessment of depressive-like behavior and Golgi Cox staining of medial prefrontal cortex pyramidal neurons followed by morphological analyses were carried out. Post-adolescent behavioral and morphological studies identified the presentation of increased depressive-like behaviors and reduced spine densities and dendritic lengths of layer II/III pyramidal neuron in the infralimbic cortex, but not in the prelimbic cortex of rats exposed to juvenile stress. Repeated fluvoxamine treatment recovered the increased depressive-like behavior and reduced spine densities/dendritic lengths observed in rats exposed to footshock stress. Cortical thicknesses in the infralimbic cortex and prelimbic cortex were also reduced by juvenile stress, but these reductions were not recovered by fluvoxamine treatment. The results demonstrate cortical sensitivities to stress exposures during the juvenile stage which mediate behavioral impairments, and provide a clue to find therapeutics for early life stress-induced emotional dysfunctions. (C) 2015 Elsevier B.V. All rights reserved.
  • Sang-Hun Lee, Marco Ledri, Blanka Toth, Ivan Marchionni, Christopher M. Henstridge, Barna Dudok, Kata Kenesei, Laszlo Barna, Szilard I. Szabo, Tibor Renkecz, Michelle Oberoi, Masahiko Watanabe, Charles L. Limoli, George Horvai, Ivan Soltesz, Istvan Katona
    JOURNAL OF NEUROSCIENCE 35 (27) 10039 - 10057 0270-6474 2015/07 [Refereed][Not invited]
     
    Persistent CB1 cannabinoid receptoractivity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapsespecific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cellpatch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverseagonist AM251[N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS0327 [8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[2,3]cyclohepta[2,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184(4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy) methyl] piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), elicited a robust increase in 2-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic 2-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic intracellular membrane cisternae at perisomatic GABAergic symmetrical synapses. Interestingly, neither AM251, JZL184, nor PF3845 affected CB1-positive dendritic interneuron synapses. Together, these findings are consistent with the possibility that constitutively active CB1 receptors substantially influence perisomatic GABA release probability and indicate that the synaptic effects of tonic 2-AG release are tightly controlled by presynaptic MGL activity and also by postsynaptic endovanilloid signaling and FAAH activity.
  • Wiphawi Hipkaeo, Masahiko Watanabe, Hisatake Kondo
    INTERNATIONAL JOURNAL OF MORPHOLOGY 33 (2) 695 - 700 0717-9502 2015/06 [Refereed][Not invited]
     
    In view of the fact that human marijuana users often show dry mouth symptom, the present study was attempted to examine the localization of CB1, which was originally identified in brain, in the submandibular and sublingual salivary glands of postnatal developing male mice by immunohistochemistry. In submandibular gland, CB1-immunoreactivity was positive in a majority of acinar cells in forms of granular appearance in their apical cytoplasm, while it was negative in the ducts at newborn stage. The immunoreactivity decreased in the acinar cells at P1W and no immunoreactivity was detected in the acinar cells at P3W and thereafter. The immunoreactivity was positive in ductal cells at P3W and it remained positive thereafter until P8W stage. The immunoreaction was distinct on the apical plasmalemma of the intercalated ductal cells, while it was distinct on the basal plasmalemma of the granular convoluted ductal cells. The enhanced immunostaining on the lateral plasmalemma of the granular ductal cells was discerned only on P6W. In sublingual gland, CB1-immunoreactivity was detected in the demilune acinar cells and ductal cells only on P4W. Furthermore, CB1-immunoreactivity was shown to occur in the salivary ganglionic neurons, suggesting the CB1-inhibitory action in the saliva secretion through the parasympathetic nervous transmission.
  • Hiroyuki Takeda, Tomio Ogasawara, Tatsuhiko Ozawa, Atsushi Muraguchi, Pei-Ju Jih, Ryo Morishita, Motokazu Uchigashima, Masahiko Watanabe, Toyoshi Fujimoto, Takahiro Iwasaki, Yaeta Endo, Tatsuya Sawasaki
    SCIENTIFIC REPORTS 5 11333  2045-2322 2015/06 [Refereed][Not invited]
     
    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.
  • Ivan Soltesz, Bradley E. Alger, Masanobu Kano, Sang-Hun Lee, David M. Lovinger, Takako Ohno-Shosaku, Masahiko Watanabe
    NATURE REVIEWS NEUROSCIENCE 16 (6) 264 - 277 1471-003X 2015/06 [Not refereed][Not invited]
  • Robert P. Ganley, Noboru Iwagaki, Patricia del Rio, Najma Baseer, Allen C. Dickie, Kieran A. Boyle, Erika Polgar, Masahiko Watanabe, Victoria E. Abraira, Amanda Zimmerman, John S. Riddell, Andrew J. Todd
    JOURNAL OF NEUROSCIENCE 35 (19) 7626 - 7642 0270-6474 2015/05 [Refereed][Not invited]
     
    The superficial dorsal horn of the spinal cord contains numerous inhibitory interneurons, which regulate the transmission of information perceived as touch, pain, or itch. Despite the importance of these cells, our understanding of their roles in the neuronal circuitry is limited by the difficulty in identifying functional populations. One group that has been identified and characterized consists of cells in the mouse that express green fluorescent protein (GFP) under control of the prion protein (PrP) promoter. Previous reports suggested that PrP-GFP cells belonged to a single morphological class (central cells), received inputs exclusively from unmyelinated primary afferents, and had axons that remained in lamina II. However, we recently reported that the PrP-GFP cells expressed neuronal nitric oxide synthase (nNOS) and/or galanin, and it has been shown that nNOS-expressing cells are more diverse in their morphology and synaptic connections. We therefore used a combined electrophysiological, pharmacological, and anatomical approach to reexamine the PrP-GFP cells. We provide evidence that they are morphologically diverse (corresponding to "unclassified" cells) and receive synaptic input from a variety of primary afferents, with convergence onto individual cells. We also show that their axons project into adjacent laminae and that they target putative projection neurons in lamina I. This indicates that the neuronal circuitry involving PrP-GFP cells is more complex than previously recognized, and suggests that they are likely to have several distinct roles in regulating the flow of somatosensory information through the dorsal horn.
  • Yanyong Toomsan, Surang Chomphoo, Sawetree Pakkarato, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    INTERNATIONAL JOURNAL OF MORPHOLOGY 33 (1) 113 - 118 0717-9502 2015/03 [Refereed][Not invited]
     
    According to recent studies, it is highly possible that the occurrence of vesicular inhibitory amino acid transporter (VIAAT) is a good marker of GABA-signaling not only in the brain, but also in extra-brain tissue cells containing GABA and GAD. In view of this, the present study was attempted to localize VIAAT-immunoreactivity in the submandibular gland of mice. In the present study, the submandibular glands of male mice at various postnatal developmental stages were examined for detailed localization of VIAAT-immunoreactivity in immunohistochemistry at light microscopic level. The immunoreactivity for VIAAT was localized in epithelial cells of proximal and distal excretory ducts with the striated portion more intensely immunopositive at young postnatal stages. No significant immunoreactivity was seen in the acinar cells throughout the postnatal development. In addition, the immunoreactivity for VIAAT was detected in the salivary parasympathetic ganglionic neurons, but not in any nerve fibers surrounding the glandular cells. Furthermore, VIAAT-immunoreactivity was found in smooth muscle cells forming the outermost layer of intralobular arterioles. From the present findings, it is possible that GABA plays roles as paracrine and autocrine regulators in the saliva secretion as well as the gland development.
  • Kohtarou Konno, Hiromi Takahashi-Iwanaga, Motokazu Uchigashima, Kyoko Miyasaka, Akihiro Funakoshi, Masahiko Watanabe, Toshihiko Iwanaga
    HISTOCHEMISTRY AND CELL BIOLOGY 143 (3) 301 - 312 0948-6143 2015/03 [Refereed][Not invited]
     
    Information concerning the cellular localization of cholecystokinin (CCK)-1 receptors has been discrepant and remained scanty at ultrastructural levels. The present immunohistochemical study at light and electron microscopic levels revealed the distinct localization of CCK1 receptors in visceral organs. Immunohistochemistry by use of a purified antibody against mouse CCK1 receptor was applied to fixed tissue sections of the pancreas, gallbladder, stomach, and intestine of mice. A silver-intensified immunogold method revealed the subcellular localization under electron microscope. The immunoreactivity for CCK1 receptors was selectively found in the basolateral membrane of pancreatic acinar cells and gastric chief cells but was absent in pancreatic islets and gastric D cells. Another intense expression in the gut was seen in the myenteric nerve plexus of the antro-duodenal region and some populations of c-Kit-expressing pacemaker cells in the duodenal musculature. The gallbladder contained smooth muscle fibers with an intense immunoreactivity of CCK1 receptors on cell surfaces. The restricted localization of CCK1 receptors on the basolateral membrane of pancreatic acinar cells and gastric chief cells, along with their absence in the islets of Langerhans and gastric D cells, provides definitive information concerning the regulatory mechanism by circulating CCK. Especially, the subcellular localization in the acinar cells completes the investigation for the detection of circulating CCK by the basolateral membrane.
  • Yuki Omiya, Motokazu Uchigashima, Kohtarou Konno, Miwako Yamasaki, Taisuke Miyazaki, Takayuki Yoshida, Ichiro Kusumi, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 35 (10) 4215 - 4228 0270-6474 2015/03 [Refereed][Not invited]
     
    Invaginating synapses in the basal amygdala are a unique type of GABAergic synapses equipped with molecular-anatomical organization specialized for 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid signaling. Cholecystokinin (CCK)-positive basket cell terminals protrude into pyramidal cell somata and form invaginating synapses, where apposing presynaptic and postsynaptic elements are highly loaded with cannabinoid receptor CB1 or 2-AG synthetic enzyme diacylglycerol lipase-alpha (DGL alpha), respectively. The present study scrutinized their neurochemical and neuroanatomical phenotypes in adult mouse telencephalon. In the basal amygdala, vesicular glutamate transporter-3 (VGluT3) was transcribed in one-fourth of CB1-expressing GABAergic interneurons. The majority of VGluT3-positive CB1-expressing basket cell terminals apposed DGL alpha clusters, whereas the majority of VGluT3-negative ones did not. Importantly, VGluT3-positive basket cell terminals selectively constructed invaginating synapses. GABA(A) receptors accumulated on the postsynaptic membrane of invaginating synapses, whereas metabotropic glutamate receptor-5 (mGluR5) was widely distributed on the somatodendritic surface of pyramidal cells. Moreover, CCK2 receptor (CCK2R) was highly transcribed in pyramidal cells. In cortical regions, pyramidal cells equipped with such VGluT3/CB1/DGL alpha-accumulated invaginating synapses were found at variable frequencies depending on the subregions. Therefore, in addition to extreme proximity of CB1- and DGL alpha-loaded presynaptic and postsynaptic elements, tripartite transmitter phenotype of GABA/glutamate/CCK is the common neurochemical feature of invaginating synapses, suggesting that glutamate, CCK, or both can promote 2-AG synthesis through activating G alpha(q/11) protein-coupled mGluR5 and CCK2R. These molecular configurations led us to hypothesize that invaginating synapses might be evolved to provide some specific mechanisms of induction, regulation, and cooperativity for 2-AG-mediated retrograde signaling in particular cortical and cortex-like amygdaloid regions.
  • Taisuke Miyazaki, Masahiko Watanabe
    Immunocytochemistry and Related Techniques 299 - 311 2015/02/24 [Refereed][Not invited]
     
    Neuronal tracing ( neurotracing ) using anterograde and retrograde tracers is widely used to study the projections between different brain regions and the wiring between individual neurons . Neurotracing is a technique essential not only for examining the connectivity of complex neuronal networks but also for providing the neuroanatomical basis for electrophysiological, pharmacological and behavioral experiments. If neurotracing is combined with immunocytochemical labeling, the combined technique can characterize the neurochemical properties, postsynaptic targets and innervation modes of neurons. The utility and versatility of this approach can be further extended by adopting appropriate cellular and subcellular markers for immunocytochemistry, by applying the approach to animal models generated by advanced genemanipulation technology, and by using single-cell labeling techniques, e.g., after viral transfection of fluorescent proteins or in utero / in vivo electroporation . In this chapter, we introduce the methods for combined immunocytochemistry and neurotracing at both light and electron microscopic levels. We have developed and employed these combined approaches to study the mechanisms underlying the development and refinement of climbing fiber mono-innervation in cerebellar Purkinje cells . Therefore, we present some examples of the images obtained in this experimental context.
  • David Baur, Grit Bornschein, Daniel Althof, Masahiko Watanabe, Akos Kulik, Jens Eilers, Hartmut Schmidt
    JOURNAL OF NEUROSCIENCE 35 (5) 1858 - 1871 0270-6474 2015/02 [Refereed][Not invited]
     
    Tight coupling between Ca2+ channels and the sensor for vesicular transmitter release at the presynaptic active zone (AZ) is crucial for high-fidelity synaptic transmission. It has been hypothesized that a switch from a loosely coupled to a tightly coupled transmission mode is a common step in the maturation of CNS synapses. However, this hypothesis has never been tested at cortical synapses. We addressed this hypothesis at a representative small cortical synapse: the synapse connecting mouse cerebellar cortical parallel fibers to Purkinje neurons. We found that the slow Ca2+ chelator EGTA affected release significantly stronger at immature than at mature synapses, while the fast chelator BAPTA was similarly effective in both groups. Analysis of paired-pulse ratios and quantification of release probability (p(r)) with multiple-probability fluctuation analysis revealed increased facilitation at immature synapses accompanied by reduced pr. Cav2.1 Ca2+ channel immunoreactivity, assessed by quantitative high-resolution immuno-electron microscopy, was scattered over immature boutons but confined to putative AZs at mature boutons. Presynaptic Ca2+ signals were quantified with two-photon microscopy and found to be similar between maturation stages. Models adjusted to fit EGTA dose-response curves as well as differential effects of the Ca2+ channel blocker Cd2+ indicate looser and less homogenous coupling at immature terminals compared with mature ones. These results demonstrate functionally relevant developmental tightening of influx-release coupling at a single AZ cortical synapse and corroborate developmental tightening of coupling as a prevalent phenomenon in the mammalian brain.
  • Naofumi Uesaka, Motokazu Uchigashima, Takayasu Mikuni, Hirokazu Hirai, Masahiko Watanabe, Masanobu Kano
    CEREBELLUM 14 (1) 4 - 7 1473-4222 2015/02 [Refereed][Not invited]
     
    Neurons form exuberant synapses with target cells early in development. Then, necessary synapses are selectively strengthened whereas unnecessary connections are weakened and eventually eliminated during postnatal development. This process is known as synapse elimination and is a crucial step for shaping immature neural circuits into functionally mature versions. Accumulating evidence suggests that retrograde signaling from postsynaptic cells regulates synapse elimination, but the underlying mechanisms remain unknown. Here, we show that semaphorin3A (Sema3A) and semaphorin7A (Sema7A) mediate retrograde signals for elimination of redundant climbing fiber (CF) to Purkinje cell (PC) synapses in the developing cerebellum, a representative model of synapse elimination in the central nervous system. We picked up candidate retrograde signaling molecules that are expressed in PCs during the period of CF synapse elimination and the receptors of these candidate molecules that are present in CFs. We then assessed the effects of lentivirus-mediated RNAi-knockdown of these molecules on CF synapse elimination. By this systematic screening, we found that knockdown of Sema3A in PCs or its co-receptor, plexinA4 (PlxnA4), in CFs accelerated CF synapse elimination and decreased CF-mediated synaptic inputs. Conversely, knockdown of Sema7A in PCs or either of the two receptors for Sema7A, plexinC1 (PlxnC1) and integrinB1 (ItgB1), in CFs impaired CF synapse elimination. Importantly, the effect of Sema7A involves signaling by type 1 metabotropic glutamate receptor (mGluR1), a canonical pathway in PCs for the final stage of CF synapse elimination. These results demonstrate that specific semaphorins act as retrograde signaling molecules and regulate distinct processes of CF synapse elimination during postnatal cerebellar development.
  • Izumi Kaji, Toshihiko Iwanaga, Masahiko Watanabe, Paul H. Guth, Eli Engel, Jonathan D. Kaunitz, Yasutada Akiba
    AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY 308 (3) G188 - G197 0193-1857 2015/02 [Refereed][Not invited]
     
    Bacterial or ingested food-derived short-chain fatty acids (SCFAs) are present in the duodenal lumen. Acetate, the most abundant SCFA in the foregut lumen, is absorbed immediately after ingestion, although the mechanism by which this absorption occurs is not fully understood. We investigated the distribution and function of candidate SCFA transporters in rat duodenum. The Na+ -coupled monocarboxylate transporter-1 (SMCT1) was localized to the brush border, whereas the pH-dependent monocarboxylate transporter (MCT) 1 and MCT4 were localized to the duodenocyte basolateral membrane. In Ussing chambered duodenal mucosa, luminal acetate dose-dependently increased short-circuit current (I-sc) in the presence of serosal bumetanide and indomethacin by a luminal Na+ -dependent, ouabain-sensitive mechanism. The Isc response was inhibited dose-dependently by the SMCT1 nonsubstrate inhibitor ibuprofen, consistent with net electrogenic absorption of acetate via SMCT1. Other SCFAs and lactate also increased Isc. Furthermore, duodenal loop perfusion of acetate increased portal venous acetate concentration, inhibited by coperfusion of ibuprofen or a MCT inhibitor. Luminal acetate perfusion increased duodenal HCO3 secretion via capsaicin-sensitive afferent nerve activation and cyclooxygenase activity, consistent with absorption-mediated HCO3- secretion. These results suggest that absorption of luminal SCFA via SMCT1 and MCTs increases duodenal HCO3- secretion. In addition to SCFA sensing via free fatty acid receptors, the presence of rapid duodenal SCFA absorption may be important for the suppression of luminal bacterial colonization and implicated in the generation of functional dyspepsia due to bacterial overgrowth.
  • Barna Dudok, László Barna, Marco Ledri, Szilárd I. Szabó, Eszter Szabadits, Balázs Pintér, Stephen G. Woodhams, Christopher M. Henstridge, Gyula Y. Balla, Rita Nyilas, Csaba Varga, Sang-Hun Lee, Máté Matolcsi, Judit Cervenak, Imre Kacskovics, Masahiko Watanabe, Claudia Sagheddu, Miriam Melis, Marco Pistis, Ivan Soltesz, István Katona
    Nature Neuroscience 18 (1) 75 - 86 1546-1726 2015/01/01 [Refereed][Not invited]
     
    A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell type-and subcellular compartment-specific manner. We developed a new approach to this problem by combining cell-specific physiological and anatomical characterization with super-resolution imaging and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically projecting GABAergic interneurons possessed increased CB 1 receptor number, active-zone complexity and receptor/effector ratio compared with dendritically projecting interneurons, consistent with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ9-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked marked CB 1 downregulation in a dose-dependent manner. Full receptor recovery required several weeks after the cessation of Δ9-tetrahydrocannabinol treatment. These findings indicate that cell type-specific nanoscale analysis of endogenous protein distribution is possible in brain circuits and identify previously unknown molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction.
  • Katiuscia Martinello, Zhuo Huang, Rafael Lujan, Baouyen Tran, Masahiko Watanabe, Edward C. Cooper, David A. Brown, Mala M. Shah
    NEURON 85 (2) 346 - 363 0896-6273 2015/01 [Refereed][Not invited]
     
    Acetylcholine critically influences hippocampal-dependent learning. Cholinergic fibers innervate hippocampal neuron axons, dendrites, and somata. The effects of acetylcholine on axonal information processing, though, remain unknown. By stimulating cholinergic fibers and making electrophysiological recordings from hippocampal dentate gyrus granule cells, we show that synaptically released acetylcholine preferentially lowered the action potential threshold, enhancing intrinsic excitability and synaptic potential-spike coupling. These effects persisted for at least 30 min after the stimulation paradigm and were due to muscarinic receptor activation. This caused sustained elevation of axonal intracellular Ca2+ via T-type Ca2+ channels, as indicated by two-photon imaging. The enhanced Ca2+ levels inhibited an axonal K(V)7/M current, decreasing the spike threshold. In support, immunohistochemistry revealed muscarinic M1 receptor, Ca(V)3.2, and K(V)7.2/7.3 subunit localization in granule cell axons. Since alterations in axonal signaling affect neuronal firing patterns and neurotransmitter release, this is an unreported cellular mechanism by which acetylcholine might, at least partly, enhance cognitive processing.
  • Victor Fernandez-Duenas, Jaume J. Taura, Martin Cottet, Maricel Gomez-Soler, Marc Lopez-Cano, Catherine Ledent, Masahiko Watanabe, Eric Trinquet, Jean-Philippe Pin, Rafael Lujan, Thierry Durroux, Francisco Ciruela
    DISEASE MODELS & MECHANISMS 8 (1) 57 - 63 1754-8403 2015/01 [Refereed][Not invited]
     
    Parkinson's disease (PD) is a dopaminergic-related pathology in which functioning of the basal ganglia is altered. It has been postulated that a direct receptor-receptor interaction - i.e. of dopamine D-2 receptor (D2R) with adenosine A(2A) receptor (A(2A)R) (forming D2R-A(2A)R oligomers) - finely regulates this brain area. Accordingly, elucidating whether the pathology prompts changes to these complexes could provide valuable information for the design of new PD therapies. Here, we first resolved a long-standing question concerning whether D2R-A(2A)R assembly occurs in native tissue: by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D2R-A(2A)R oligomers in rat striatum. Subsequently, we determined that, under pathological conditions (i.e. in a rat PD model), D2R-A(2A)R interaction was impaired. Collectively, these results provide definitive evidence for alteration of native D2R-A(2A)R oligomers in experimental parkinsonism, thus conferring the rationale for appropriate oligomer-based PD treatments.
  • Claire Piochon, Alexander D. Kloth, Giorgio Grasselli, Heather K. Titley, Hisako Nakayama, Kouichi Hashimoto, Vivian Wan, Dana H. Simmons, Tahra Eissa, Jin Nakatani, Adriana Cherskov, Taisuke Miyazaki, Masahiko Watanabe, Toru Takumi, Masanobu Kano, Samuel S. -H. Wang, Christian Hansel
    NATURE COMMUNICATIONS 6 6014  2041-1723 2015/01 [Refereed][Not invited]
  • Mahnaz Mansouri, Yu Kasugai, Yugo Fukazawa, Federica Bertaso, Fabrice Raynaud, Julie Perroy, Laurent Fagni, Walter A. Kaufmann, Masahiko Watanabe, Ryuichi Shigemoto, Francesco Ferraguti
    EUROPEAN JOURNAL OF NEUROSCIENCE 41 (2) 157 - 167 0953-816X 2015/01 [Refereed][Not invited]
     
    Type 1 metabotropic glutamate (mGlu1) receptors play a pivotal role in different forms of synaptic plasticity in the cerebellar cortex, e.g. long-term depression at glutamatergic synapses and rebound potentiation at GABAergic synapses. These various forms of plasticity might depend on the subsynaptic arrangement of the receptor in Purkinje cells that can be regulated by protein-protein interactions. This study investigated, by means of the freeze-fracture replica immunogold labelling method, the subcellular localization of mGlu1 receptors in the rodent cerebellum and whether Homer proteins regulate their subsynaptic distribution. We observed a widespread extrasynaptic localization of mGlu1 receptors and confirmed their peri-synaptic enrichment at glutamatergic synapses. Conversely, we detected mGlu1 receptors within the main body of GABAergic synapses onto Purkinje cell dendrites. Although Homer proteins are known to interact with the mGlu1 receptor C-terminus, we could not detect Homer3, the most abundant Homer protein in the cerebellar cortex, at GABAergic synapses by pre-embedding and post-embedding immunoelectron microscopy. We then hypothesized a critical role for Homer proteins in the peri-junctional localization of mGlu1 receptors at glutamatergic synapses. To disrupt Homer-associated protein complexes, mice were tail-vein injected with the membrane-permeable dominant-negative TAT-Homer1a. Freeze-fracture replica immunogold labelling analysis showed no significant alteration in the mGlu1 receptor distribution pattern at parallel fibre-Purkinje cell synapses, suggesting that other scaffolding proteins are involved in the peri-synaptic confinement. The identification of interactors that regulate the subsynaptic localization of the mGlu1 receptor at neurochemically distinct synapses may offer new insight into its trafficking and intracellular signalling.
  • Norihiko Yokoi, Yuko Fukata, Daisuke Kase, Taisuke Miyazaki, Martine Jaegle, Toshika Ohkawa, Naoki Takahashi, Hiroko Iwanari, Yasuhiro Mochizuki, Takao Hamakubo, Keiji Imoto, Dies Meijer, Masahiko Watanabe, Masaki Fukata
    NATURE MEDICINE 21 (1) 19 - 26 1078-8956 2015/01 [Refereed][Not invited]
     
    Epilepsy is one of the most common and intractable brain disorders. Mutations in the human gene LGI1, encoding a neuronal secreted protein, cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). However, the pathogenic mechanisms of LGI1 mutations remain unclear. We classified 22 reported LGI1 missense mutations as either secretion defective or secretion competent, and we generated and analyzed two mouse models of ADLTE encoding mutant proteins representative of the two groups. The secretion-defective LGI1(E383A) protein was recognized by the ER quality-control machinery and prematurely degraded, whereas the secretable LGI1(S473L) protein abnormally dimerized and was selectively defective in binding to one of its receptors, ADAM22. Both mutations caused a loss of function, compromising intracellular trafficking or ligand activity of LGI1 and converging on reduced synaptic LGI1-ADAM22 interaction. A chemical corrector, 4-phenylbutyrate (4PBA), restored LGI1(E383A) folding and binding to ADAM22 and ameliorated the increased seizure susceptibility of the LGI 1(E383A) model mice. This study establishes LGI1-related epilepsy as a conformational disease and suggests new therapeutic options for human epilepsy.
  • Kazumasu Sasaki, Akira Sumiyoshi, Hiroi Nonaka, Yoshiyuki Kasahara, Kazutaka Ikeda, F. Scott Hall, George R. Uhl, Masahiko Watanabe, Ryuta Kawashima, Ichiro Sora
    BRITISH JOURNAL OF PHARMACOLOGY 172 (2) 654 - 667 0007-1188 2015/01 [Refereed][Not invited]
     
    Background and Purpose Opioid receptor knockout (MOP-KO) mice display several behavioural differences from wild-type (WT) littermates including differential responses to nociceptive stimuli. Brain structural changes have been tied to behavioural alterations noted in transgenic mice with targeting of different genes. Hence, we assess the brain structure of MOP-KO mice. Experimental ApproachMagnetic resonance imaging (MRI) voxel-based morphometry (VBM) and histological methods were used to identify structural differences between extensively backcrossed MOP-KO mice and WT mice. Key ResultsMOP-KO mice displayed robust increases in regional grey matter volume in olfactory bulb, several hypothalamic nuclei, periaqueductal grey (PAG) and several cerebellar areas, most confirmed by VBM analysis. The largest increases in grey matter volume were detected in the glomerular layer of the olfactory bulb, arcuate nucleus of hypothalamus, ventrolateral PAG (VLPAG) and cerebellar regions including paramedian and cerebellar lobules. Histological analyses confirm several of these results, with increased VLPAG cell numbers and increased thickness of the olfactory bulb granule cell layer and cerebellar molecular and granular cell layers. Conclusions and ImplicationsMOP deletion causes previously undescribed structural changes in specific brain regions, but not in all regions with high MOP receptor densities (e.g. thalamus, nucleus accumbens) or that exhibit adult neurogenesis (e.g. hippocampus). Volume differences in hypothalamus and PAG may reflect behavioural changes including hyperalgesia. Although the precise relationship between volume change and MOP receptor deletion was not determined from this study alone, these findings suggest that levels of MOP receptor expression may influence a broader range of neural structure and function in humans than previously supposed. Linked ArticlesThis article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit
  • Ayumi Tanaka-Hayashi, Shuuhei Hayashi, Ran Inoue, Tomokazu Ito, Kohtarou Konno, Tomoyuki Yoshida, Masahiko Watanabe, Tohru Yoshimura, Hisashi Mori
    AMINO ACIDS 47 (1) 79 - 86 0939-4451 2015/01 [Refereed][Not invited]
     
    d-Aspartate is an endogenous free amino acid in the brain, endocrine tissues, and exocrine tissues in mammals, and it plays several physiological roles. In the testis, d-aspartate is detected in elongate spermatids, Leydig cells, and Sertoli cells, and implicated in the synthesis and release of testosterone. In the hippocampus, d-aspartate strongly enhances N-methyl-d-aspartate receptor-dependent long-term potentiation and is involved in learning and memory. The existence of aspartate racemase, a candidate enzyme for d-aspartate production, has been suggested. Recently, mouse glutamic-oxaloacetic transaminase 1-like 1 (Got1l1) has been reported to synthesize substantially d-aspartate from l-aspartate and to be involved in adult neurogenesis. In this study, we investigated the function of Got1l1 in vivo by generating and analyzing Got1l1 knockout (KO) mice. We also examined the enzymatic activity of recombinant Got1l1 in vitro. We found that Got1l1 mRNA is highly expressed in the testis, but it is not detected in the brain and submandibular gland, where d-aspartate is abundant. The d-aspartate contents of wild-type and Got1l1 KO mice were not significantly different in the testis and hippocampus. The recombinant Got1l1 expressed in mammalian cells showed l-aspartate aminotransferase activity, but lacked aspartate racemase activity. These findings suggest that Got1l1 is not the major aspartate racemase and there might be an as yet unknown d-aspartate-synthesizing enzyme.
  • Wataru Kakegawa, Nikolaos Mitakidis, Eriko Miura, Manabu Abe, Keiko Matsuda, Yukari H. Takeo, Kazuhisa Kohda, Junko Motohashi, Akiyo Takahashi, Soichi Nagao, Shin-ichi Muramatsu, Masahiko Watanabe, Kenji Sakimura, A. Radu Aricescu, Michisuke Yuzaki
    NEURON 85 (2) 316 - 329 0896-6273 2015/01 [Refereed][Not invited]
     
    Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.
  • Ayuko Kishimoto, Toshina Ishiguro-Oonuma, Ritei Takahashi, Mamiko Maekawa, Kiyotaka Toshimori, Masahiko Watanabe, Toshihiko Iwanaga
    BIOMEDICAL RESEARCH-TOKYO 36 (4) 225 - U59 0388-6107 2015 [Refereed][Not invited]
     
    Lactate represents a preferential energy substrate of germ cells rather than glucose. Testicular Sertoli cells are believed to produce lactate and pyruvate and to supply these to germ cells, particularly spermatocytes and spermatids. Monocarboxylate transporter (MCT), responsible for the transport of lactate and other monocarboxylates via the cell membrane, is abundant in the testes and sperm (MCT1, MCT2, and MCT4). For the uptake of glucose, germ cells within the seminiferous tubules and sperm have been known to intensely express GLUT3. The present study investigated expression profiles of MCTs and GLUTs and revealed their cellular and subcellular localization in the mouse and rat testis. An in situ hybridization analysis showed significant expressions of MCT1, MCT2, and GLUT3 mRNA in the testis. Immunohistochemically, spermatogonia, spermatocytes, and spermatids expressed MCT1 on their cell surfaces in a stage-dependent manner: in some seminiferous tubules, an intense expression of MCT1 was unique to the spermatogonia. MCT2 was restricted to the tails of elongated spermatids and sperm. An intense immunoreactivity for GLUT3 was shared by spermatocytes, spermatids, and sperm. Sertoli cells were devoid of any immunoreactivities for MCT1, MCT2, and GLUT3. The predominant energy source of germ cells may be lactate and other monocarboxylates-especially for spermatogonia, but glucose and other hexoses may be responsible for an energy supply to spermatocytes and spermatids.
  • Yasukazu Hozumi, Kenichi Kakefuda, Miwako Yamasaki, Masahiko Watanabe, Hideaki Hara, Kaoru Goto
    BRAIN RESEARCH 1594 36 - 45 0006-8993 2015/01 [Refereed][Not invited]
     
    Spine formation, a salient feature underlying neuronal plasticity to adapt to a changing environment, is regulated by complex machinery involving membrane signal transduction. The diacylglycerol kinase (DGK) family, which is involved in membrane lipid metabolism, catalyzes the phosphorylation of a lipid second messenger, diacylglycerol (DG). Of the DGKs, DGK beta is characterized by predominant expression in a specific brain region: the striatum. We previously demonstrated that DGK beta is expressed selectively in medium spiny neurons (MSNs) and that it is highly enriched in the perisynaptic membrane on dendritic spines contacted with excitatory terminals. Moreover, DGK beta regulates spinogenesis through actin-based remodeling in an activity-dependent manner. However, the detailed mechanisms of spinogenesis regulation and its functional significance remain unclear. To address these issues, we performed Golgi-Cox staining to examine morphological aspects of MSNs in the striatum of DGK beta-knockout (KO) mice. Results show that striatal MSNs of DGK beta-KO mice exhibited lower dendritic spine density at distal dendrites than wild-type mice did. We also sought protein targets that interact with DGK beta and identified the GluA2 AMPA receptor subunit as a novel DGK beta binding partner. In addition, DGK beta-deficient brain exhibits significant reduction of TARP gamma-8, which represents a transmembrane AMPA receptor regulatory protein. These findings suggest that DGK beta regulates the spine formation at distal dendrites in MSNs. (C) 2014 Elsevier B.V. All rights reserved.
  • Natsuya Katada, Jiro Shibuya, Takafumi Ichikawa, Kei Hosoda, Keishi Yamashita, Masahiko Watanabe
    ESOPHAGUS 12 (1) 65 - 72 1612-9059 2015/01 [Refereed][Not invited]
     
    The objectives of this study were to delineate differences in the morphologic characteristics of reflux esophagitis between a rat gastroesophageal reflux (GER) model and a duodenoesophageal reflux (DER) model and to evaluate the effects of H2-receptor antagonists on morphologic characteristics of reflux esophagitis in DER model. Wistar rats were divided into 3 groups: GER model group (Group G), DER model group (Group D), and control group (Group C). Rats in each group were sacrificed 1 or 12 weeks after surgery. Intraesophageal pH was measured, and the excised esophagus was examined macroscopically and histologically. Subgroups of rats in Group D were given famotidine (10 mg/kg) or lafutidine (30 mg/kg) orally once daily for 1 week after surgery. The rats were then sacrificed, and histological findings were compared. Intraesophageal pH was significantly lower in Group G than in Group C. At 12 weeks, the epithelium of the lower esophagus in Groups G and D was significantly thicker than that in Group C and showed remarkable hyperplastic changes in Group D. The thickness of the epithelium in Group D + famotidine did not differ significantly from that in Group D. In contrast, the epithelium was significantly thinner in Group D + lafutidine than in Group D. As a rat model of reflux esophagitis, DER causes severer damage to the esophageal epithelium, including hyperplastic changes, than does GER. Famotidine had no apparent effect on esophageal epithelial damage caused by DER, whereas lafutidine was suggested to attenuate such damage.
  • Shinichiro Tsutsumi, Maya Yamazaki, Taisuke Miyazaki, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano, Kazuo Kitamura
    JOURNAL OF NEUROSCIENCE 35 (2) 843 - 852 0270-6474 2015/01 [Refereed][Not invited]
     
    Simple and regular anatomical structure is a hallmark of the cerebellar cortex. Parasagittally arrayed alternate expression of aldolase C/zebrin II in Purkinje cells (PCs) has been extensively studied, but surprisingly little is known about its functional significance. Here we found a precise structure-function relationship between aldolase C expression and synchrony of PC complex spike activities that reflect climbing fiber inputs to PCs. We performed two-photon calcium imaging in transgenic mice in which aldolase C compartments can be visualized in vivo, and identified highly synchronous complex spike activities among aldolase C-positive or aldolase C-negative PCs, but not across these populations. The boundary of aldolase C compartments corresponded to that of complex spike synchrony at single-cell resolution. Sensory stimulation evoked aldolase C compartment-specific complex spike responses and synchrony. This result further revealed the structure-function segregation. In awake animals, complex spike synchrony both within and between PC populations across the aldolase C boundary were enhanced in response to sensory stimuli, in a way that two functionally distinct PC ensembles are coactivated. These results suggest that PC populations characterized by aldolase C expression precisely represent distinct functional units of the cerebellar cortex, and these functional units can cooperate to process sensory information in awake animals.
  • Tomoaki Sobajima, Shin-ichiro Yoshimura, Tomohiko Iwano, Masataka Kunii, Masahiko Watanabe, Nur Atik, Sotaro Mushiake, Eiichi Morii, Yoshihisa Koyama, Eiji Miyoshi, Akihiro Harada
    BIOLOGY OPEN 4 (1) 86 - 94 2046-6390 2015/01 [Refereed][Not invited]
     
    The small GTPase Rab11 plays an important role in the recycling of proteins to the plasma membrane as well as in polarised transport in epithelial cells and neurons. We generated conditional knockout mice deficient in Rab11a. Rab11a-deficient mice are embryonic lethal, and brain-specific Rab11a knockout mice show no overt abnormalities in brain architecture. In contrast, intestine-specific Rab11a knockout mice begin dying approximately 1 week after birth. Apical proteins in the intestines of knockout mice accumulate in the cytoplasm and mislocalise to the basolateral plasma membrane, whereas the localisation of basolateral proteins is unaffected. Shorter microvilli and microvillus inclusion bodies are also observed in the knockout mice. Elevation of a serum starvation marker was also observed, likely caused by the mislocalisation of apical proteins and reduced nutrient uptake. In addition, Rab8a is mislocalised in Rab11a knockout mice. Conversely, Rab11a is mislocalised in Rab8a knockout mice and in a microvillus atrophy patient, which has a mutation in the myosin Vb gene. Our data show an essential role for Rab11a in the localisation of apical proteins in the intestine and demonstrate functional relationships between Rab11a, Rab8a and myosin Vb in vivo.
  • Maria Gutierrez-Mecinas, Masahiko Watanabe, Andrew J. Todd
    MOLECULAR PAIN 10 1744-8069 2014/12 [Refereed][Not invited]
     
    Background: Gastrin-releasing peptide (GRP) and its receptor have been shown to play an important role in the sensation of itch. However, although GRP immunoreactivity has been detected in the spinal dorsal horn, there is debate about whether this originates from primary afferents or local excitatory interneurons. We therefore examined the relation of GRP immunoreactivity to that seen with antibodies that label primary afferent or excitatory interneuron terminals. We tested the specificity of the GRP antibody by preincubating with peptides with which it could potentially cross-react. We also examined tissue from a mouse line in which enhanced green fluorescent protein (EGFP) is expressed under control of the GRP promoter. Results: GRP immunoreactivity was seen in both primary afferent and non-primary glutamatergic axon terminals in the superficial dorsal horn. However, immunostaining was blocked by pre-incubation of the antibody with substance P, which is present at high levels in many nociceptive primary afferents. EGFP(+) cells in the GRP-EGFP mouse did not express Pax2, and their axons contained the vesicular glutamate transporter 2 (VGLUT2), indicating that they are excitatory interneurons. In most cases, their axons were also GRP-immunoreactive. Multiple-labelling immunocytochemical studies indicated that these cells did not express either of the preprotachykinin peptides, and that they generally lacked protein kinase C gamma, which is expressed by a subset of the excitatory interneurons in this region. Conclusions: These results show that GRP is expressed by a distinct population of excitatory interneurons in laminae I-II that are likely to be involved in the itch pathway. They also suggest that the GRP immunoreactivity seen in primary afferents in previous studies may have resulted from cross-reaction of the GRP antibody with substance P or the closely related peptide neurokinin A.
  • Carmen Ballesteros-Merino, Jose Martinez-Hernandez, Carolina Aguado, Masahiko Watanabe, John P. Adelman, Rafael Lujan
    FRONTIERS IN NEUROANATOMY 8 1662-5129 2014/12 [Refereed][Not invited]
     
    Small-conductance, Ca2+-activated K+ (SK) channels regulate neuronal excitability in a variety of ways. To understand their roles in different neuronal subtypes it is important to determine their precise subcellular distribution. Here, we used biochemical, light microscopy immunohistochemical and immunoelectron microscopy techniques, combined with quantitative approaches, to reveal the expression and subcellular localization patterns of SK2 in the developing cerebellum. Using western blots, the SK2 protein showed a progressive increase during postnatal development. At the light microscopic level, SK2 immunoreactivity was very prominent in the developing Purkinje cells (PC), particularly in the molecular layer (ML). Electron microscopy revealed that throughout development SK2 was mostly detected at the extrasynaptic and perisynaptic plasma membrane of dendritic shafts and dendritic spines of PCs. However, there was some localization at axon terminals as well. Quantitative analyses and 3D reconstructions further revealed a progressive developmental change of SK2 on the surface of PCs from dendritic shafts to dendritic spines. Together, these results indicate that SK2 channels undergo dynamic spatial regulation during cerebellar development, and this process is associated with the formation and maturation of excitatory synaptic contacts to PCs.
  • Víctor Fernández-Dueñas, Maricel Gómez-Soler, Marc López-Cano, Jaume J. Taura, Catherine Ledent, Masahiko Watanabe, Kenneth A. Jacobson, Jean-Pierre Vilardaga, Francisco Ciruela
    ACS Chemical Biology 9 (11) 2496 - 2501 1554-8937 2014/11/21 [Refereed][Not invited]
     
    Caffeine, the most consumed psychoactive substance worldwide, may have bene fi cial effects on Parkinson's disease (PD) therapy. The mechanism by which caffeine contributes to its antiparkinsonian effects by acting as either an adenosine A< inf> 2A< /inf> receptor (A< inf> 2A< /inf> R) neutral antagonist or an inverse agonist is unresolved. Here we show that caffeine is an A< inf> 2A< /inf> R inverse agonist in cell-based functional studies and in experimental parkinsonism. Thus, we observed that caffeine triggers a distinct mode, opposite to A< inf> 2A< /inf> R agonist, of the receptor's activation switch leading to suppression of its spontaneous activity. These inverse agonist-related effects were also determined in the striatum of a mouse model of PD, correlating well with increased caffeine-mediated motor effects. Overall, caffeine A< inf> 2A< /inf> R inverse agonism may be behind some of the well-known physiological effects of this substance both in health and disease. This information might have a critical mechanistic impact for PD pharmacotherapeutic design. (Chemical Equation Presented).
  • Najma Baseer, Abdullah S. Al-Baloushi, Masahiko Watanabe, Safa A. S. Shehab, Andrew J. Todd
    PAIN 155 (11) 2291 - 2300 0304-3959 2014/11 [Refereed][Not invited]
     
    Fine myelinated (A delta) nociceptors are responsible for fast, well-localised pain, but relatively little is known about their postsynaptic targets in the spinal cord, and therefore about their roles in the neuronal circuits that process nociceptive information. Here we show that transganglionically transported cholera toxin B subunit (CTb) labels a distinct set of afferents in lamina I that are likely to correspond to A delta nociceptors, and that most of these lack neuropeptides. The vast majority of lamina I projection neurons can be retrogradely labelled from the lateral parabrachial area, and these can be divided into 2 major groups based on expression of the neurokinin 1 receptor (NK1r). We show that CTb-labelled afferents form contacts on 43% of the spinoparabrachial lamina I neurons that lack the NK1r, but on a significantly smaller proportion (26%) of those that express the receptor. We also confirm with electron microscopy that these contacts are associated with synapses. Among the spinoparabrachial neurons that received contacts from CTb-labelled axons, contact density was considerably higher on NK1r-lacking cells than on those with the NK1r. By comparing the density of CTb contacts with those from other types of glutamatergic bouton, we estimate that nonpeptidergic A delta nociceptors may provide over half of the excitatory synapses on some NK1r-lacking spinoparabrachial cells. These results provide further evidence that synaptic inputs to dorsal horn projection neurons are organised in a specific way. Taken together with previous studies, they suggest that both NK1r(+) and NK1r-lacking lamina I projection neurons are directly innervated by A delta nociceptive afferents. (C) 2014 The Authors. Published by Elsevier B.V. on behalf of International Association for the Study of Pain. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
  • Claire Piochon, Alexander D. Kloth, Giorgio Grasselli, Heather K. Titley, Hisako Nakayama, Kouichi Hashimoto, Vivian Wan, Dana H. Simmons, Tahra Eissa, Jin Nakatani, Adriana Cherskov, Taisuke Miyazaki, Masahiko Watanabe, Toru Takumi, Masanobu Kano, Samuel S. -H. Wang, Christian Hansel
    NATURE COMMUNICATIONS 5 2041-1723 2014/11 [Refereed][Not invited]
     
    A common feature of autism spectrum disorder (ASD) is the impairment of motor control and learning, occurring in a majority of children with autism, consistent with perturbation in cerebellar function. Here we report alterations in motor behaviour and cerebellar synaptic plasticity in a mouse model (patDp/+) for the human 15q11-13 duplication, one of the most frequently observed genetic aberrations in autism. These mice show ASD-resembling social behaviour deficits. We find that in patDp/+ mice delay eyeblink conditioning-a form of cerebellum-dependent motor learning-is impaired, and observe deregulation of a putative cellular mechanism for motor learning, long-term depression (LTD) at parallel fibre-Purkinje cell synapses. Moreover, developmental elimination of surplus climbing fibres-a model for activity-dependent synaptic pruning-is impaired. These findings point to deficits in synaptic plasticity and pruning as potential causes for motor problems and abnormal circuit development in autism.
  • Shun Hamada, Itone Ogawa, Miwako Yamasaki, Yuji Kiyama, Hidetoshi Kassai, Ayako M. Watabe, Kazuki Nakao, Atsu Aiba, Masahiko Watanabe, Toshiya Manabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 40 (8) 3136 - 3146 0953-816X 2014/10 [Refereed][Not invited]
     
    The N-methyl-d-aspartate receptor (NMDAR) plays various physiological and pathological roles in neural development, synaptic plasticity and neuronal cell death. It is composed of two GluN1 and two GluN2 subunits and, in the neonatal hippocampus, most synaptic NMDARs are GluN2B-containing receptors, which are gradually replaced with GluN2A-containing receptors during development. Here, we examined whether GluN2A could be substituted for GluN2B in neural development and functions by analysing knock-in (KI) mice in which GluN2B is replaced with GluN2A. The KI mutation was neonatally lethal, although GluN2A-containing receptors were transported to the postsynaptic membrane even without GluN2B and functional at synapses of acute hippocampal slices of postnatal day 0, indicating that GluN2A-containing NMDARs could not be substituted for GluN2B-containing NMDARs. Importantly, the synaptic -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluA1 was increased, and the transmembrane AMPAR regulatory protein, which is involved in AMPAR synaptic trafficking, was increased in KI mice. Although the regulation of AMPARs by GluN2B has been reported in cultured neurons, we showed here that AMPAR-mediated synaptic responses were increased in acute KI slices, suggesting differential roles of GluN2A and GluN2B in AMPAR expression and trafficking in vivo. Taken together, our results suggest that GluN2B is essential for the survival of animals, and that the GluN2B-GluN2A switching plays a critical role in synaptic integration of AMPARs through regulation of GluA1 in the whole animal.
  • Xiaohong Song, Miwako Yamasaki, Taisuke Miyazaki, Kohtarou Konno, Motokazu Uchigashima, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 40 (5) 2797 - 2810 0953-816X 2014/09 [Refereed][Not invited]
     
    Slc4a10 was originally identified as a Na+-driven Cl-/HCO3- exchanger NCBE that transports extracellular Na+ and HCO3- in exchange for intracellular Cl-, whereas other studies argue against a Cl--dependence for Na+-HCO3- transport, and thus named it the electroneutral Na+/HCO3- cotransporter NBCn2. Here we investigated Slc4a10 expression in adult mouse brains by in situ hybridization and immunohistochemistry. Slc4a10 mRNA was widely expressed, with higher levels in pyramidal cells in the hippocampus and cerebral cortex, parvalbumin-positive interneurons in the hippocampus, and Purkinje cells (PCs) in the cerebellum. Immunohistochemistry revealed an uneven distribution of Slc4a10 within the somatodendritic compartment of cerebellar neurons. In the cerebellar molecular layer, stellate cells and their innervation targets (i.e. PC dendrites in the superficial molecular layer) showed significantly higher labeling than basket cells and their targets (PC dendrites in the basal molecular layer and PC somata). Moreover, the distal dendritic trees of PCs (i.e. parallel fiber-targeted dendrites) had significantly greater labeling than the proximal dendrites (climbing fiber-targeted dendrites). These observations suggest that Slc4a10 expression is regulated in neuron type- and input pathway-dependent manners. Because such an elaborate regulation is also found for K+-Cl- cotransporter KCC2, a major neuronal Cl- extruder, we compared their expression. Slc4a10 and KCC2 overlapped in most somatodendritic elements. However, relative abundance was largely complementary in the cerebellar cortex, with particular enrichments of Slc4a10 in PC dendrites and KCC2 in molecular layer interneurons, granule cells and PC somata. These properties might reflect functional redundancy and distinction of these transporters, and their differential requirements by individual neurons and respective input domains.
  • Yuka Miyoshi, Yoshichika Yoshioka, Kinuko Suzuki, Taisuke Miyazaki, Minako Koura, Kazumasa Saigoh, Naoko Kajimura, Yoko Monobe, Susumu Kusunoki, Junichiro Matsuda, Masahiko Watanabe, Naoto Hayasaka
    PLOS ONE 9 (9) e107867 - e107867 1932-6203 2014/09 [Refereed][Not invited]
     
    Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)-PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants.
  • Elena Dragicevic, Christina Poetschke, Johanna Duda, Falk Schlaudraff, Stephan Lammel, Julia Schiemann, Michael Fauler, Andrea Hetzel, Masahiko Watanabe, Rafael Lujan, Robert C. Malenka, Joerg Striessnig, Birgit Liss
    BRAIN 137 2287 - 2302 0006-8950 2014/08 [Refereed][Not invited]
     
    D2 autoreceptors and L-type calcium channels are both implicated in Parkinson's disease, but how they interact is unclear. Dragicevic et al. reveal that L-type calcium channels can modulate D2-autoreceptor responses via the neuronal calcium sensor NCS-1. This dopamine-dependent signalling network is altered in Parkinson's disease and could represent a therapeutic target.Dopamine midbrain neurons within the substantia nigra are particularly prone to degeneration in Parkinson's disease. Their selective loss causes the major motor symptoms of Parkinson's disease, but the causes for the high vulnerability of SN DA neurons, compared to neighbouring, more resistant ventral tegmental area dopamine neurons, are still unclear. Consequently, there is still no cure available for Parkinson's disease. Current therapies compensate the progressive loss of dopamine by administering its precursor l-DOPA and/or dopamine D2-receptor agonists. D2-autoreceptors and Ca(v)1.3-containing L-type Ca2+ channels both contribute to Parkinson's disease pathology. L-type Ca2+ channel blockers protect SN DA neurons from degeneration in Parkinson's disease and its mouse models, and they are in clinical trials for neuroprotective Parkinson's disease therapy. However, their physiological functions in SN DA neurons remain unclear. D2-autoreceptors tune firing rates and dopamine release of SN DA neurons in a negative feedback loop through activation of G-protein coupled potassium channels (GIRK2, or KCNJ6). Mature SN DA neurons display prominent, non-desensitizing somatodendritic D2-autoreceptor responses that show pronounced desensitization in PARK-gene Parkinson's disease mouse models. We analysed surviving human SN DA neurons from patients with Parkinson's disease and from controls, and detected elevated messenger RNA levels of D2-autoreceptors and GIRK2 in Parkinson's disease. By electrophysiological analysis of postnatal juvenile and adult mouse SN DA neurons in in vitro brain-slices, we observed that D2-autoreceptor desensitization is reduced with postnatal maturation. Furthermore, a transient high-dopamine state in vivo, caused by one injection of either l-DOPA or cocaine, induced adult-like, non-desensitizing D2-autoreceptor responses, selectively in juvenile SN DA neurons, but not ventral tegmental area dopamine neurons. With pharmacological and genetic tools, we identified that the expression of this sensitized D2-autoreceptor phenotype required Ca(v)1.3 L-type Ca2+ channel activity, internal Ca2+, and the interaction of the neuronal calcium sensor NCS-1 with D2-autoreceptors. Thus, we identified a first physiological function of Ca(v)1.3 L-type Ca2+ channels in SN DA neurons for homeostatic modulation of their D2-autoreceptor responses. L-type Ca2+ channel activity however, was not important for pacemaker activity of mouse SN DA neurons. Furthermore, we detected elevated substantia nigra dopamine messenger RNA levels of NCS-1 (but not Ca(v)1.2 or Ca(v)1.3) after cocaine in mice, as well as in remaining human SN DA neurons in Parkinson's disease. Thus, our findings provide a novel homeostatic functional link in SN DA neurons between Ca(v)1.3- L-type-Ca2+ channels and D2-autoreceptor activity, controlled by NCS-1, and indicate that this adaptive signalling network (Ca(v)1.3/NCS-1/D2/GIRK2) is also active in human SN DA neurons, and contributes to Parkinson's disease pathology. As it is accessible to pharmacological modulation, it provides a novel promising target for tuning substantia nigra dopamine neuron activity, and their vulnerability to degeneration.
  • Miwako Yamasaki, Rieko Okada, Chihiro Takasaki, Shima Toki, Masahiro Fukaya, Rie Natsume, Kenji Sakimura, Masayoshi Mishina, Tetsuo Shirakawa, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 34 (35) 11534 - 11548 0270-6474 2014/08 [Refereed][Not invited]
     
    Development of correct topographical connections between peripheral receptors and central somatosensory stations requires activity-dependent synapse refinement, in which the NMDA type of glutamate receptors plays a key role. Here we compared functional roles of GluN2B (GluR epsilon 2 or NR2B) and GluN2D (GluR epsilon 4 or NR2D), two major regulatory subunits of neonatal NMDA receptors, in development of whisker-related patterning at trigeminal relay stations. Compared with control littermates, both the appearance of whisker-related patterning and the termination of the critical period, as assessed by unilateral infraorbital nerve transection, were delayed by nearly a day in the somatosensory cortex of GluN2B(+/-) mice but advanced by nearly a day in GluN2D(-/-) mice. Similar temporal shifts were found at subcortical relay stations in the thalamus and brainstem of GluN2B(+/-) and GluN2D(-/-) mice. In comparison, the magnitude of lesion-induced critical period plasticity in the somatosensory cortex, as assessed following row-C whisker removal, was normal in both mutants. Thus, GluN2B and GluN2D play counteractive roles in temporal development and maturation of somatosensory maps without affecting the magnitude of critical period plasticity. To understand the opposing action, we then examined neuronal and synaptic expressions of the two subunits along the trigeminal pathway. At each trigeminal station, GluN2B was predominant at asymmetrical synapses of non-GABAergic neurons, whereas GluN2D was selective to asymmetrical synapses of GABAergic neurons. Together, our findings suggest that GluN2B expressed at glutamatergic synapses on glutamatergic projection neurons facilitates refinement of ascending pathway synapses directly, whereas GluN2D expressed at glutamatergic synapses on GABAergic interneurons delays it indirectly.
  • Shinya Kawata, Taisuke Miyazaki, Maya Yamazaki, Takayasu Mikuni, Miwako Yamasaki, Kouichi Hashimoto, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    CELL REPORTS 8 (4) 1119 - 1129 2211-1247 2014/08 [Refereed][Not invited]
     
    Synapse elimination is crucial for precise neural circuit formation during postnatal development. We examined how relative differences in synaptic strengths among competing inputs and/or absolute synaptic strengths contribute to climbing fiber (CF) to Purkinje cell (PC) synapse elimination in the cerebellum. We generated mice with PC-selective deletion of stargazin (TARP gamma-2), the major AMPA receptor auxiliary subunit in PCs (gamma-2 PC-KO mice). Whereas relative differences between "strong'' and "weak'' CF-mediated postsynaptic response are preserved, absolute strengths of CF inputs are scaled down globally in PCs of gamma-2 PC-KO mice. Although the early phase of CF elimination is normal, dendritic translocation of the strongest CF and the late phase of CF elimination that requires Ca2+-dependent activation of Arc/Arg3.1 in PCs are both impaired in gamma-2 PC-KO mice. We conclude that, although relative differences in CF synaptic inputs are initially essential, proper synaptic scaling is crucial for accomplishing CF synapse elimination.
  • Adam P. Kardon, Erika Polgar, Junichi Hachisuka, Lindsey M. Snyder, Darren Cameron, Sinead Savage, Xiaoyun Cai, Sergei Karnup, Christopher R. Fan, Gregory M. Hemenway, Carcha S. Bernard, Erica S. Schwartz, Hiroshi Nagase, Christoph Schwarzer, Masahiko Watanabe, Takahiro Furuta, Takeshi Kaneko, H. Richard Koerber, Andrew J. Todd, Sarah E. Ross
    NEURON 82 (3) 573 - 586 0896-6273 2014/05 [Refereed][Not invited]
     
    Menthol and other counterstimuli relieve itch, resulting in an antipruritic state that persists for minutes to hours. However, the neural basis for this effect is unclear, and the underlying neuromodulatory mechanisms are unknown. Previous studies revealed that Bhlhb5(-/-) mice, which lack a specific population of spinal inhibitory interneurons (B5-I neurons), develop pathological itch. Here we characterize B5-I neurons and show that they belong to a neurochemically distinct subset. We provide cause-and-effect evidence that B5-I neurons inhibit itch and show that dynorphin, which is released from B5-I neurons, is a key neuromodulator of pruritus. Finally, we show that B5-I neurons are innervated by menthol-, capsaicin-, and mustard oil-responsive sensory neurons and are required for the inhibition of itch by menthol. These findings provide a cellular basis for the inhibition of itch by chemical counterstimuli and suggest that kappa opioids may be a broadly effective therapy for pathological itch.
  • Kohtarou Konno, Keiko Matsuda, Chihiro Nakamoto, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Kenji Sakimura, Michisuke Yuzaki, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 34 (22) 7412 - 7424 0270-6474 2014/05 [Refereed][Not invited]
     
    Of the two members of the delta subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum and regulates their structural and functional connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. In the present study, we investigated this issue by producing specific and sensitive histochemical probes for GluD1 and analyzing cerebellar synaptic circuits in GluD1-knock-out mice. GluD1 was widely expressed in the adult mouse brain, with high levels in higher brain regions, including the cerebral cortex, striatum, limbic regions (hippocampus, nucleus accumbens, lateral septum, bed nucleus stria terminalis, lateral habenula, and central nucleus of the amygdala), and cerebellar cortex. In the cerebellar cortex, GluD1 mRNA was expressed at the highest level in molecular layer interneurons and its immunoreactivity was concentrated at PF synapses on interneuron somata. In GluD1-knock-out mice, the density of PF synapses on interneuron somata was significantly reduced and the size and number of interneurons were significantly diminished. Therefore, GluD1 is common to GluD2 in expression at PF synapses, but distinct from GluD2 in neuronal expression in the cerebellar cortex; that is, GluD1 in interneurons and GluD2 in PCs. Furthermore, GluD1 regulates the connectivity of PF-interneuron synapses and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function.
  • Naofumi Uesaka, Motokazu Uchigashima, Takayasu Mikuni, Takanobu Nakazawa, Harumi Nakao, Hirokazu Hirai, Atsu Aiba, Masahiko Watanabe, Masanobu Kano
    SCIENCE 344 (6187) 1020 - 1023 0036-8075 2014/05 [Refereed][Not invited]
     
    Neural circuits are shaped by elimination of early-formed redundant synapses during postnatal development. Retrograde signaling from postsynaptic cells regulates synapse elimination. In this work, we identified semaphorins, a family of versatile cell recognition molecules, as retrograde signals for elimination of redundant climbing fiber to Purkinje cell synapses in developing mouse cerebellum. Knockdown of Sema3A, a secreted semaphorin, in Purkinje cells or its receptor in climbing fibers accelerated synapse elimination during postnatal day 8 (P8) to P18. Conversely, knockdown of Sema7A, a membrane-anchored semaphorin, in Purkinje cells or either of its two receptors in climbing fibers impaired synapse elimination after P15. The effect of Sema7A involves signaling by metabotropic glutamate receptor 1, a canonical pathway for climbing fiber synapse elimination. These findings define how semaphorins retrogradely regulate multiple processes of synapse elimination.
  • Linde Kegel, Martine Jaegle, Siska Driegen, Eerik Aunin, Kris Leslie, Yuko Fukata, Masahiko Watanabe, Masaki Fukata, Dies Meijer
    DEVELOPMENT 141 (8) 1749 - 1756 0950-1991 2014/04 [Refereed][Not invited]
     
    The cellular interactions that drive the formation and maintenance of the insulating myelin sheath around axons are only partially understood. Leucine-rich glioma-inactivated (LGI) proteins play important roles in nervous system development and mutations in their genes have been associated with epilepsy and amyelination. Their function involves interactions with ADAM22 and ADAM23 cell surface receptors, possibly in apposing membranes, thus attenuating cellular interactions. LGI4-ADAM22 interactions are required for axonal sorting and myelination in the developing peripheral nervous system (PNS). Functional analysis revealed that, despite their high homology and affinity for ADAM22, LGI proteins are functionally distinct. To dissect the key residues in LGI proteins required for coordinating axonal sorting and myelination in the developing PNS, we adopted a phylogenetic and computational approach and demonstrate that the mechanism of action of LGI4 depends on a cluster of three amino acids on the outer surface of the LGI4 protein, thus providing a structural basis for the mechanistic differences in LGI protein function in nervous system development and evolution.
  • Teniel S. Ramikie, Rita Nyilas, Rebecca J. Bluett, Joyonna C. Gamble-George, Nolan D. Hartley, Ken Mackie, Masahiko Watanabe, Istvan Katona, Sachin Patel
    NEURON 81 (5) 1111 - 1125 0896-6273 2014/03 [Refereed][Not invited]
     
    The central amygdala (CeA) is a key structure at the limbic-motor interface regulating stress responses and emotional learning. Endocannabinoid (eCB) signaling is heavily implicated in the regulation of stress-response physiology and emotional learning processes; however, the role of eCBs in the modulation of synaptic efficacy in the CeA is not well understood. Here we describe the subcellular localization of CB1 cannabinoid receptors and eCB synthetic machinery at glutamatergic synapses in the CeA and find that CeA neurons exhibit multiple mechanistically and temporally distinct modes of postsynaptic eCB mobilization. These data identify a prominent role for eCBs in the modulation of excitatory drive to CeA neurons and provide insight into the mechanisms by which eCB signaling and exogenous cannabinoids could regulate stress responses and emotional learning.
  • Ming-Dong Zhang, Giuseppe Tortoriello, Brian Hsueh, Raju Tomer, Li Ye, Nicholas Mitsios, Lotta Borgius, Gunnar Grant, Ole Kiehn, Masahiko Watanabe, Mathias Uhlen, Jan Mulder, Karl Deisseroth, Tibor Harkany, Tomas G. M. Hokfelt
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 111 (12) E1149 - E1158 0027-8424 2014/03 [Refereed][Not invited]
     
    Neuronal calcium (Ca2+)-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca2+-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small-and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca2+-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca2+-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.
  • Carmen Ballesteros-Merino, Masahiko Watanabe, Ryuichi Shigemoto, Yugo Fukazawa, John P. Adelman, Rafael Lujan
    EUROPEAN JOURNAL OF NEUROSCIENCE 39 (6) 883 - 892 0953-816X 2014/03 [Refereed][Not invited]
     
    Small-conductance, Ca2+-activated K+ (SK) channels are expressed in the hippocampus where they regulate synaptic responses, plasticity, and learning and memory. To investigate the expression of SK3 (KCNN3) subunits, we determined the developmental profile and subcellular distribution of SK3 in the developing mouse hippocampus using western blots, immunohistochemistry and high-resolution immunoelectron microscopy. The results showed that SK3 expression increased during postnatal development, and that the localization of SK3 changed from being mainly associated with the endoplasmic reticulum and intracellular sites during the first postnatal week to being progressively concentrated in dendritic spines during later stages. In the adult, SK3 was localized mainly in postsynaptic compartments, both at extrasynaptic sites and along the postsynaptic density of excitatory synapses. Double labelling showed that SK3 co-localized with SK2 (KCNN2) and with N-methyl-D-aspartate receptors. Finally, quantitative analysis of SK3 density revealed two subcellular distribution patterns in different hippocampal layers, with SK3 being unevenly distributed in CA1 region of the hippocampus pyramidal cells and homogeneously distributed in dentate gyrus granule cells. Our results revealed a complex cell surface distribution of SK3-containing channels and a distinct developmental program that may influence different hippocampal functions.
  • Eszter Horvath, Stephen G. Woodhams, Rita Nyilas, Christopher M. Henstridge, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe, Istvan Katona
    EUROPEAN JOURNAL OF NEUROSCIENCE 39 (3) 419 - 434 0953-816X 2014/02 [Refereed][Not invited]
     
    Monoacylglycerol lipase (MGL) is a multifunctional serine hydrolase, which terminates anti-nociceptive endocannabinoid signaling and promotes pro-nociceptive prostaglandin signaling. Accordingly, both acute nociception and its sensitization in chronic pain models are prevented by systemic or focal spinal inhibition of MGL activity. Despite its analgesic potential, the neurobiological substrates of beneficial MGL blockade have remained unexplored. Therefore, we examined the regional, cellular and subcellular distribution of MGL in spinal circuits involved in nociceptive processing. All immunohistochemical findings obtained with light, confocal or electron microscopy were validated in MGL-knockout mice. Immunoperoxidase staining revealed a highly concentrated accumulation of MGL in the dorsal horn, especially in superficial layers. Further electron microscopic analysis uncovered that the majority of MGL-immunolabeling is found in axon terminals forming either asymmetric glutamatergic or symmetric -aminobutyric acid/glycinergic synapses in laminae I/IIo. In line with this presynaptic localization, analysis of double-immunofluorescence staining by confocal microscopy showed that MGL colocalizes with neurochemical markers of peptidergic and non-peptidergic nociceptive terminals, and also with markers of local excitatory or inhibitory interneurons. Interestingly, the ratio of MGL-immunolabeling was highest in calcitonin gene-related peptide-positive peptidergic primary afferents, and the staining intensity of nociceptive terminals was significantly reduced in MGL-knockout mice. These observations highlight the spinal nociceptor synapse as a potential anatomical site for the analgesic effects of MGL blockade. Moreover, the presence of MGL in additional terminal types raises the possibility that MGL may play distinct regulatory roles in synaptic endocannabinoid or prostaglandin signaling according to its different cellular locations in the dorsal horn pain circuitry.
  • Yoshiaki Ohtani, Mariko Miyata, Kouichi Hashimoto, Toshihide Tabata, Yasushi Kishimoto, Masahiro Fukaya, Daisuke Kase, Hidetoshi Kassai, Kazuki Nakao, Tatsumi Hirata, Masahiko Watanabe, Masanobu Kano, Atsu Aiba
    JOURNAL OF NEUROSCIENCE 34 (7) 2702 - 2712 0270-6474 2014/02 [Refereed][Not invited]
     
    The metabotropic glutamate receptor subtype 1(mGluR1, Grm1) in cerebellar Purkinje cells (PCs) is essential for motor coordination and motor learning. At the synaptic level, mGluR1 has a critical role in long-term synaptic depression (LTD) at parallel fiber (PF)-PC synapses, and in developmental elimination of climbing fiber (CF)-PC synapses. mGluR1a, a predominant splice variant in PCs, has a long carboxyl (C)-terminal domain that interacts with Homer scaffolding proteins. Cerebellar roles of the C-terminal domain at both synaptic and behavior levels remain poorly understood. To address this question, we introduced a short variant, mGluR1b, which lacks this domain into PCs of mGluR1-knock-out (KO) mice (mGluR1b-rescue mice). In mGluR1b-rescue mice, mGluR1b showed dispersed perisynaptic distribution in PC spines. Importantly, mGluR1b-rescue mice exhibited impairments in inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release, CF synapse elimination, LTD induction, and delay eyeblink conditioning: they showed normal transient receptor potential canonical(TRPC) currents and normal motor coordination. In contrast, PC-specific rescue of mGluR1a restored all cerebellar defects of mGluR1-KO mice. We conclude that the long C-terminal domain of mGluR1a is required for the proper perisynaptic targeting of mGluR1, IP3R-mediated Ca2+ release, CF synapse elimination, LTD, and motor learning, but not for TRPC currents and motor coordination.
  • Xiaojing Dai, Hirohide Iwasaki, Masahiko Watanabe, Shigeo Okabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 39 (4) 531 - 547 0953-816X 2014/02 [Refereed][Not invited]
  • Inoue R, Yoshihisa Y, Tojo Y, Okamura C, Yoshida Y, Kishimoto J, Luan X, Watanabe M, Mizuguchi M, Nabeshima Y, Hamase K, Matsunaga K, Shimizu T, Mori H
    The Journal of investigative dermatology 0022-202X 2014/01 [Refereed][Not invited]
  • Toshiharu Yasaka, Sheena Y. X. Tiong, Erika Polgar, Masahiko Watanabe, Eiichi Kumamoto, John S. Riddell, Andrew J. Todd
    MOLECULAR PAIN 10 1744-8069 2014/01 [Refereed][Not invited]
     
    Background: Lamina I projection neurons respond to painful stimuli, and some are also activated by touch or hair movement. Neuropathic pain resulting from peripheral nerve damage is often associated with tactile allodynia (touch-evoked pain), and this may result from increased responsiveness of lamina I projection neurons to non-noxious mechanical stimuli. It is thought that polysynaptic pathways involving excitatory interneurons can transmit tactile inputs to lamina I projection neurons, but that these are normally suppressed by inhibitory interneurons. Vertical cells in lamina II provide a potential route through which tactile stimuli can activate lamina I projection neurons, since their dendrites extend into the region where tactile afferents terminate, while their axons can innervate the projection cells. The aim of this study was to determine whether vertical cell dendrites were contacted by the central terminals of low-threshold mechanoreceptive primary afferents. Results: We initially demonstrated contacts between dendritic spines of vertical cells that had been recorded in spinal cord slices and axonal boutons containing the vesicular glutamate transporter 1 (VGLUT1), which is expressed by myelinated low-threshold mechanoreceptive afferents. To confirm that the VGLUT1 boutons included primary afferents, we then examined vertical cells recorded in rats that had received injections of cholera toxin B subunit (CTb) into the sciatic nerve. We found that over half of the VGLUT1 boutons contacting the vertical cells were CTb-immunoreactive, indicating that they were of primary afferent origin. Conclusions: These results show that vertical cell dendritic spines are frequently contacted by the central terminals of myelinated low-threshold mechanoreceptive afferents. Since dendritic spines are associated with excitatory synapses, it is likely that most of these contacts were synaptic. Vertical cells in lamina II are therefore a potential route through which tactile afferents can activate lamina I projection neurons, and this pathway could play a role in tactile allodynia.
  • Erika Polgar, Thomas C. P. Sardella, Sheena Y. X. Tiong, Samantha Locke, Masahiko Watanabe, Andrew J. Todd
    PAIN 154 (12) 2606 - 2615 0304-3959 2013/12 [Refereed][Not invited]
     
    In order to understand how nociceptive information is processed in the spinal dorsal horn we need to unravel the complex synaptic circuits involving interneurons, which constitute the vast majority of the neurons in laminae I-III. The main limitation has been the difficulty in defining functional populations among these cells. We have recently identified 4 non-overlapping classes of inhibitory interneuron, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) and parvalbumin, in the rat spinal cord. In this study we demonstrate that these form distinct functional populations that differ in terms of sst(2A) receptor expression and in their responses to painful stimulation. The sst(2A) receptor was expressed by nearly all of the nNOS-and galanin-containing inhibitory interneurons but by few of those with NPY and none of the parvalbumin cells. Many galanin-and NPY-containing cells exhibited phosphorylated extracellular signal-regulated kinases (pERK) after mechanical, thermal or chemical noxious stimuli, but very few nNOS-containing cells expressed pERK after any of these stimuli. However, many nNOS-positive inhibitory interneurons up-regulated Fos after noxious thermal stimulation or injection of formalin, but not after capsaicin injection. Parvalbumin cells did not express either activity-dependent marker following any of these stimuli. These results suggest that interneurons belonging to the NPY, nNOS and galanin populations are involved in attenuating pain, and for NPY and nNOS cells this is likely to result from direct inhibition of nociceptive projection neurons. They also suggest that the nociceptive inputs to the nNOS cells differ from those to the galanin and NPY populations. (C) 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
  • Ana Fajardo-Serrano, Nicole Wydeven, Daniele Young, Masahiko Watanabe, Ryuichi Shigemoto, Kirill A. Martemyanov, Kevin Wickman, Rafael Lujan
    HIPPOCAMPUS 23 (12) 1231 - 1245 1050-9631 2013/12 [Refereed][Not invited]
     
    In the hippocampus, signaling through G protein-coupled receptors is modulated by Regulators of G protein signaling (Rgs) proteins, which act to stimulate the rate of GTP hydrolysis, and consequently, G protein inactivation. The R7-Rgs subfamily selectively deactivates the G(i/o)-class of G subunits that mediate the action of several GPCRs. Here, we used co-immunoprecipitation, electrophysiology and immunoelectron microscopy techniques to investigate the formation of macromolecular complexes and spatial relationship of Rgs7/G5 complexes and its prototypical signaling partners, the GABA(B) receptor and Girk channel. Co-expression of recombinant GABA(B) receptors and Girk channels in combination with co-immunoprecipitation experiments established that the Rgs7/G5 forms complexes with GABA(B) receptors or Girk channels. Using electrophysiological experiments, we found that GABA(B)-Girk current deactivation kinetics was markedly faster in cells coexpressing Rgs7/G5. At the electron microscopic level, immunolabeling for Rgs7 and G5 proteins was found primarily in the dendritic layers of the hippocampus and showed similar distribution patterns. Immunoreactivity was mostly localized along the extrasynaptic plasma membrane of dendritic shafts and spines of pyramidal cells and, to a lesser extent, to that of presynaptic terminals. Quantitative analysis of immunogold particles for Rgs7 and G5 revealed an enrichment of the two proteins around excitatory synapses on dendritic spines, virtually identical to that of Girk2 and GABA(B1). These data support the existence of macromolecular complexes composed of GABA(B) receptor-G protein-Rgs7-Girk channels in which Rgs7 and G5 proteins may preferentialy modulate GABA(B) receptor signaling through the deactivation of Girk channels on dendritic spines. In contrast, Rgs7 and Girk2 were associated but mainly segregated from GABA(B1) in dendritic shafts, where Rgs7/G5 signaling complexes might modulate Girk-dependent signaling via a different metabotropic receptor(s). (c) 2013 Wiley Periodicals, Inc.
  • Yumi Watanabe, Norikazu Katayama, Kosei Takeuchi, Tetsuya Togano, Rieko Itoh, Michiko Sato, Maya Yamazaki, Manabu Abe, Toshiya Sato, Kanako Oda, Minesuke Yokoyama, Keizo Takao, Masahiro Fukaya, Tsuyoshi Miyakawa, Masahiko Watanabe, Kenji Sakimura, Toshiya Manabe, Michihiro Igarashi
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 (48) 34906 - 34919 0021-9258 2013/11 [Refereed][Not invited]
     
    Background: Roles of the syntaxin-1ACaMKII interaction are not physiologically understood in vivo.Results: A point mutation in syntaxin-1A caused abnormal plasticity, recycling, and behaviors in mice. Conclusion: The CaMKII/syntaxin-1A interaction is essential for maintenance of neuronal plasticity. Significance: Syntaxin-1A is involved in regulatory pathways in higher brain functions. Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.
  • L. Benjamin Hills, Amira Masri, Kotaro Konno, Wataru Kakegawa, Anh-Thu N. Lam, Elizabeth Lim-Melia, Nandini Chandy, R. Sean Hill, Jennifer N. Partlow, Muna Al-Saffar, Ramzi Nasir, Joan M. Stoler, A. James Barkovich, Masahiko Watanabe, Michisuke Yuzaki, Ganeshwaran H. Mochida
    NEUROLOGY 81 (16) 1378 - 1386 0028-3878 2013/10 [Not refereed][Not invited]
     
    Objective: To identify the genetic cause of a syndrome causing cerebellar ataxia and eye movement abnormalities. Methods: We identified 2 families with cerebellar ataxia, eye movement abnormalities, and global developmental delay. We performed genetic analyses including single nucleotide polymorphism genotyping, linkage analysis, array comparative genomic hybridization, quantitative PCR, and Sanger sequencing. We obtained eye movement recordings of mutant mice deficient for the ortholog of the identified candidate gene, and performed immunohistochemistry using human and mouse brain specimens. Results: All affected individuals had ataxia, eye movement abnormalities, most notably tonic upgaze, and delayed speech and cognitive development. Homozygosity mapping identified the disease locus on chromosome 4q. Within this region, a homozygous deletion of GRID2 exon 4 in the index family and compound heterozygous deletions involving GRID2 exon 2 in the second family were identified. Grid2-deficient mice showed larger spontaneous and random eye movements compared to wild-type mice. In developing mouse and human cerebella, GRID2 localized to the Purkinje cell dendritic spines. Brain MRI in 2 affected children showed progressive cerebellar atrophy, which was more severe than that of Grid2-deficient mice. Conclusions: Biallelic deletions of GRID2 lead to a syndrome of cerebellar ataxia and tonic upgaze in humans. The phenotypic resemblance and similarity in protein expression pattern between humans and mice suggest a conserved role for GRID2 in the synapse organization between parallel fibers and Purkinje cells. However, the progressive and severe cerebellar atrophy seen in the affected individuals could indicate an evolutionarily unique role for GRID2 in the human cerebellum.
  • Sonia Marco, Albert Giralt, Milos M. Petrovic, Mahmoud A. Pouladi, Rebeca Martínez-Turrillas, José Martínez-Hernández, Linda S. Kaltenbach, Jesús Torres-Peraza, Rona K. Graham, Masahiko Watanabe, Rafael Luján, Nobuki Nakanishi, Stuart A. Lipton, Donald C. Lo, Michael R. Hayden, Jordi Alberch, John F. Wesseling, Isabel Pérez-Otaño
    Nature Medicine 19 (8) 1030 - 1038 1078-8956 2013/08 [Refereed][Not invited]
     
    Huntington's disease is caused by an expanded polyglutamine repeat in the huntingtin protein (HTT), but the pathophysiological sequence of events that trigger synaptic failure and neuronal loss are not fully understood. Alterations in N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) have been implicated. Yet, it remains unclear how the HTT mutation affects NMDAR function, and direct evidence for a causative role is missing. Here we show that mutant HTT redirects an intracellular store of juvenile NMDARs containing GluN3A subunits to the surface of striatal neurons by sequestering and disrupting the subcellular localization of the endocytic adaptor PACSIN1, which is specific for GluN3A. Overexpressing GluN3A in wild-type mouse striatum mimicked the synapse loss observed in Huntington's disease mouse models, whereas genetic deletion of GluN3A prevented synapse degeneration, ameliorated motor and cognitive decline and reduced striatal atrophy and neuronal loss in the YAC128 Huntington's disease mouse model. Furthermore, GluN3A deletion corrected the abnormally enhanced NMDAR currents, which have been linked to cell death in Huntington's disease and other neurodegenerative conditions. Our findings reveal an early pathogenic role of GluN3A dysregulation in Huntington's disease and suggest that therapies targeting GluN3A or pathogenic HTT-PACSIN1 interactions might prevent or delay disease progression. © 2013 Nature America, Inc. All rights reserved.
  • Miki Hashizume, Taisuke Miyazaki, Kenji Sakimura, Masahiko Watanabe, Kazuo Kitamura, Masanobu Kano
    FRONTIERS IN NEURAL CIRCUITS 7 130  1662-5110 2013/08 [Refereed][Not invited]
     
    Cerebellar cortex has an elaborate rostrocaudal organization comprised of numerous microzones. Purkinje cells (PCs) in the same microzones how synchronous activity of complex spikes (CSs) evoked by excitatory inputs from climbing fibers (CFs) that arise from neurons in the inferior olive (IO). The synchronous CS activity is considered to depend on electrical coupling among IO neurons and anatomical organization of the olivo-cerebellar projection. To determine how the CF-PC wiring contributes to the formation of microzone, we examined the synchronous CS activities between neighboring PCs in the glutamate receptor delta 2 knockout (GluD2 KO) mouse in which exuberant surplus CFs make ectopic innervations onto distal dendrites of PCs. We performed in vivo two-photon calcium imaging for PC populations to detect CF inputs. Neighboring PCs in GluD2 KO mice showed higher synchrony of calcium transients than those in wild-type(control) mice. Moreover, the synchrony in GluD2 KO mice hardly declined with mediolateral separation between PCs up to similar to 200 mu m, which was in marked contrast to the fall off of the synchrony in control mice. The enhanced synchrony was only partially affected by the blockade of gap junctional coupling. On the other hand, transverse CF collaterals in GluD2 KO mice extended beyond the border of microzone and formed locally clustered ectopic synapses onto dendrites of neighboring PCs. Furthermore, PCs in GluD2 KO mice exhibited clustered firing (Cf), the characteristic CF response that was not found in PCs of wild type mice. Importantly, Cf was often associated with localized calcium transients in distal dendrites of PCs, which are likely to contribute to the enhanced synchrony of calcium signals in GluD2 KO mice. Thus, our results indicate that CF signals in GluD2 KO mice propagate across multiple microzones, and that proper formation of longitudinal olivo-cerebellar projection is essential for the spatio temporal organization of CS activity in the cerebellum.
  • Masanobu Kano, Hisako Nakayama, Kouichi Hashimoto, Kazuo Kitamura, Kenji Sakimura, Masahiko Watanabe
    JOURNAL OF PHYSIOLOGY-LONDON 591 (13) 3151 - 3158 0022-3751 2013/07 [Refereed][Not invited]
     
    Functional neural circuit formation during postnatal development involves massive elimination of early-formed redundant synapses and strengthening of necessary synaptic connections. In the cerebellum, one-to-one connection from a climbing fibre (CF) to a Purkinje cell (PC) is established through four distinct phases: (1) strengthening of a single CF among multiple CFs in each PC at postnatal age P3-P7 days, (2) translocation of a single strengthened CF to PC dendrites from around P9, (3) early-phase (P7 to around P11) and (4) late-phase (around P12-P17) elimination of weak CF synapses from PC somata. Mice with PC-selective deletion of the P/Q-type voltage-dependent Ca2+ channel (VDCC) exhibit severe defects in strengthening of single CFs, dendritic translocation of single CFs and CF elimination from P7. In contrast, mice with a mutation of a single allele for the GABA synthesizing enzyme GAD67 show selective impairment of CF elimination from P10. Electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto PC somata from putative basket cells influences CF-induced Ca2+ transients and regulates elimination of redundant CF synapses from PC somata at P10-P16. Thus, regulation of Ca2+ influx to PCs through VDCCs is crucial for the four phases of CF synapse elimination during postnatal development.
  • Natsuko Tsujino, Tomomi Tsunematsu, Motokazu Uchigashima, Kohtarou Konno, Akihiro Yamanaka, Kazuto Kobayashi, Masahiko Watanabe, Yoshimasa Koyama, Takeshi Sakurai
    PLOS ONE 8 (7) 1932-6203 2013/07 [Refereed][Not invited]
     
    Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.
  • Erik Keimpema, Alan Alpar, Fiona Howell, Katarzyna Malenczyk, Carl Hobbs, Yasmin L. Hurd, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano, Patrick Doherty, Tibor Harkany
    Scientific Reports 3 2093  2045-2322 2013/06/28 [Refereed][Not invited]
     
    Endocannabinoids are small signaling lipids, with 2-Arachidonoylglycerol (2-AG) implicated in modulating axonal growth and synaptic plasticity. The concept of short-range extracellular signaling by endocannabinoids is supported by the lack of trans-synaptic 2-AG signaling in mice lacking sn-1-diacylglycerol lipases (DAGLs), synthesizing 2-AG. Nevertheless, how far endocannabinoids can spread extracellularly to evoke physiological responses at CB1 cannabinoid receptors (CB1Rs) remains poorly understood. Here, we first show that cholinergic innervation of CA1 pyramidal cells of the hippocampus is sensitive to the genetic disruption of 2-AG signaling in DAGLa null mice. Next, we exploit a hybrid COS-7-cholinergic neuron co-culture system to demonstrate that heterologous DAGLa overexpression spherically excludes cholinergic growth cones from 2-AG-rich extracellular environments, and minimizes cell-cell contact in vitro. CB1R-mediated exclusion responses lasted 3 days, indicating sustained spherical 2-AG availability. Overall, these data suggest that extracellular 2-AG concentrations can be sufficient to activate CB1Rs along discrete spherical boundaries to modulate neuronal responsiveness.
  • Yasukazu Hozumi, Hirooki Matsui, Fumio Sakane, Masahiko Watanabe, Kaoru Goto
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 61 (6) 462 - 476 0022-1554 2013/06 [Refereed][Not invited]
     
    Recent studies have revealed that phosphoinositide (PI) signaling molecules are expressed in mammalian retinas, suggesting their importance in its signal transduction. We previously showed that diacylglycerol kinase (DGK) isozymes are expressed in distinct patterns in rat retina at the mRNA level. However, little is known about the nature and morphological aspects of DGKs in the retina. For this study, we performed immunohistochemical analyses to investigate in the retina the expression and localization of DGK isozymes at the protein level. Here, we show that both DGK beta and DGK iota localize in the outer plexiform layer, within which photoreceptor cells make contact with bipolar and horizontal cells. These isozymes exhibit distinct subcellular localization patterns: DGK iota localizes to the synaptic area of bipolar cells in a punctate manner, whereas DGK beta distributes diffusely in the subsynaptic and dendritic regions of bipolar and horizontal cells. However, punctate labeling for DGK epsilon is evident in the outer limiting membrane. DGK zeta and DGK alpha localize predominantly to the nucleus of ganglion cells. These findings show distinct expression and localization of DGK isozymes in the retina, suggesting a different role of each isozyme.
  • Naosuke Hoshina, Asami Tanimura, Miwako Yamasaki, Takeshi Inoue, Ryoji Fukabori, Teiko Kuroda, Kazumasa Yokoyama, Tohru Tezuka, Hiroshi Sagara, Shinji Hirano, Hiroshi Kiyonari, Masahiko Takada, Kazuto Kobayashi, Masahiko Watanabe, Masanobu Kano, Takanobu Nakazawa, Tadashi Yamamoto
    NEURON 78 (5) 839 - 854 0896-6273 2013/06 [Refereed][Not invited]
     
    Highly topographic organization of neural circuits exists for the regulation of various brain functions in corticobasal ganglia circuits. Although neural circuit-specific refinement during synapse development is essential for the execution of particular neural functions, the molecular and cellular mechanisms for synapse refinement are largely unknown. Here, we show that protocadherin 17 (PCDH17), one of the nonclustered delta 2-protocadherin family members, is enriched along corticobasal ganglia synapses in a zone-specific manner during synaptogenesis and regulates presynaptic assembly in these synapses. PCDH17 deficiency in mice causes facilitated presynaptic vesicle accumulation and enhanced synaptic transmission efficacy in corticobasal ganglia circuits. Furthermore, PCDH17(-/-) mice exhibit antidepressant-like phenotypes that are known to be regulated by corticobasal ganglia circuits. Our findings demonstrate a critical role for PCDH17 in the synaptic development of specific corticobasal ganglia circuits and suggest the involvement of PCDH17 in such circuits in depressive behaviors.
  • Pablo R. Brumovsky, Rebecca P. Seal, Kerstin H. Lundgren, Kim B. Seroogy, Masahiko Watanabe, G. F. Gebhart
    JOURNAL OF UROLOGY 189 (6) 2342 - 2349 0022-5347 2013/06 [Refereed][Not invited]
     
    Purpose: VGLUTs, which are essential for loading glutamate into synaptic vesicles, are present in various neuronal systems. However, to our knowledge the expression of VGLUTs in neurons innervating the bladder has not yet been analyzed. We studied VGLUT(1), VGLUT(2) and VGLUT(3) in mouse bladder neurons. Materials and Methods: We analyzed the expression of VGLUT(1), VGLUT(2) and calcitonin gene-related peptide by immunohistochemistry in the retrograde labeled primary afferent and autonomic neurons of BALB/c mice after injecting fast blue in the bladder wall. To study VGLUT(3) we traced the bladder of transgenic mice, in which VGLUT(3) is identified by enhanced green fluorescent protein detection. Results: Most bladder dorsal root ganglion neurons expressed VGLUT(2). A smaller percentage of neurons also expressed VGLUT(1) or VGLUT(3). Co-expression with calcitonin gene-related peptide was only observed for VGLUT(2). Occasional VGLUT(2) immunoreactive neurons were seen in the major pelvic ganglia. Abundant VGLUT(2) immunoreactive nerves were detected in the bladder dome and trigone, and the urethra. VGLUT(1) immunoreactive nerves were discretely present. Conclusions: We present what are to our knowledge novel data on VGLUT expression in sensory and autonomic neurons innervating the mouse bladder. The frequent association of VGLUT(2) and calcitonin gene-related peptide in sensory neurons suggests interactions between glutamatergic and peptidergic neurotransmissions, potentially influencing commonly perceived sensations in the bladder, such as discomfort and pain.
  • Yoshiaki Shinohara, Hajime Hirase, Masahiko Watanabe, Makoto Itakura, Masami Takahashi, Ryuichi Shigemoto
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 (24) 10045 - 10046 0027-8424 2013/06 [Refereed][Not invited]
  • Takayuki Yoshida, Masanobu Kano, Masahiko Watanabe, Mitsuhiro Yoshiok
    Japanese Journal of Neuropsychopharmacology 33 (3) 95 - 99 1340-2544 2013/06 [Refereed][Not invited]
     
    In the amygdala, it has been demonstrated that cannabinoid CB1 receptors are particularly enriched in GABAergic axon terminals and moderately expressed on glutamatergic fibers, and involved in the fear coping strategies. In this study, we found predominant neuronal projections of serotonergic, dopaminergic and cholinergic fibers in the basolateral amygdala (BA), and CB1 receptors were frequently localized on serotonergic axons but not dopaminergic, noradrenergic or cholinergic fibers. Furthermore, extracellular release of serotonin was significantly reduced by WIN55212-2, a CB agonist, whereas dopamine concentration was not altered, indicating presynaptic serotonin release is modulated by CB1 receptors. On the other hand, α-methyl-5-HT, 5-HT2 receptor agonist decreased the evoked IPSC and EPSC amplitude accompanied with enhancement of paired pulse ratio and induced inward currents from the patch-clamp recording BA pyramidal neurons these are parameters of presynaptic effect and postsynaptic localization of 5-HT2 receptors, respectively. This suppression of IPSC amplitude was completely blocked by MDL100907, a 5-HT2A antagonist, or AM251, a CB1 antagonist. These findings suggest that endocannabinoid is synthesized via activation of postsynaptic 5-HT2A receptors, and regulates not only inhibitory presynaptic GABA release but also local serotonergic transmission in the BA.
  • Dan Yan, Miwako Yamasaki, Christoph Straub, Masahiko Watanabe, Susumu Tomita
    NEURON 78 (4) 687 - 699 0896-6273 2013/05 [Refereed][Not invited]
     
    Glutamate is the most abundant excitatory neurotransmitter in the brain, and distinct classes of glutamate receptors coordinate synaptic transmission and spike generation upon various levels of neuronal activity. However, the mechanisms remain unclear. Here, we found that loss of synaptic AMPA receptors increased kainate receptor activity in cerebellar granule cells without changing NMDA receptors. The augmentation of kainate receptor-mediated currents in the absence of AMPA receptor activity is required for spike generation and is mediated by the increased expression of the GluK5 high-affinity kainate receptor subunit. Increase in GluK5 expression is sufficient to enhance kainate receptor activity by modulating receptor channel properties, but not localization. Furthermore, we demonstrate that the combined loss of the AMPA receptor auxiliary TARP gamma-2 subunit and the GluK5 subunit leads to early mouse lethality. Our findings reveal mechanisms mediated by distinct classes of postsynaptic glutamate receptors for the homeostatic maintenance of the neuronal activity.
  • Hiroyuki Sakagami, Osamu Katsumata, Yoshinobu Hara, Hideaki Tamaki, Masahiko Watanabe, Robert J. Harvey, Masahiro Fukaya
    Journal of Comparative Neurology 521 (4) 860 - 876 0021-9967 2013/03/01 [Refereed][Not invited]
     
    The BRAG/IQSEC is a family of guanine nucleotide exchange factors for ADP ribosylation factors, small GTPases that regulate membrane trafficking and actin cytoskeleton, and comprises three structurally related members (BRAG1-3) generated from different genes. In the mouse retina, BRAG1 (also known as IQSEC2) was previously shown to localize at synaptic ribbons of photoreceptor terminals and to form a protein complex with RIBEYE. In this study, we examined the immunohistochemical localization of BRAG2 (IQSEC1) and BRAG3 (IQSEC3) in the adult mouse retina at the light and electron microscopic levels. In the outer plexiform layer, BRAG2 showed a punctate distribution in intimate association with dystrophin and β-dystroglycan. Immunoelectron microscopic analysis revealed that BRAG2 localized at specific subcompartments of photoreceptor terminals in both rod spherules and cone pedicles. In the inner plexiform layer, immunolabeling for both BRAG2 and BRAG3 had a punctate appearance, suggestive of synaptic labeling. Double immunostaining demonstrated that BRAG2 colocalized preferentially with PSD-95 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptors (AMPARs). By contrast, BRAG3 colocalized with gephyrin and a subpopulation of inhibitory synapses expressing glycine receptors or γ-aminobutyric acid type A receptors (GABAARs). Immunoelectron microscopic analysis revealed that BRAG2 localized to postsynaptic processes at bipolar dyads, while BRAG3 localized to postsynaptic components at conventional synapses. These findings suggest that BRAG/IQSEC family members have key roles in the function and organization of distinct excitatory and inhibitory synapses in the retina. © 2012 Wiley Periodicals, Inc.
  • K. Mizukoshi, M. Sasaki, Y. Izumi, M. Miura, M. Watanabe, F. Amaya
    NEUROSCIENCE 234 77 - 87 0306-4522 2013/03 [Refereed][Not invited]
     
    Background The phosphorylation of p38 mitogen-activated protein kinase (MAPK) in the dorsal root ganglion (DRG) promotes primary afferent sensitization. The role of p38MAPK signaling in the DRG in the pathogenesis of plantar incision hyperalgesia has not been investigated. Results: Levels of phosphorylated p38MAPK (p-p38MAPK) obviously increased in the DRG after plantar incision. Unmyelinated and myelinated DRG neurons that express p-p38MAPK contained small to medium cell bodies, suggesting that p-p38MAPK expression is induced in neurons with C- and A delta-fibers. The p-p38MAPK inhibitors FR167653 or SB203580 inhibited incision-induced mechanical hypersensitivity and spontaneous pain behavior. The systemic administration of tumor necrosis factor-alpha (TNF-alpha) inhibitor prevented subsequent incision-induced activation of p38MAPK in the DRG and alleviated mechanical hypersensitivity after the incision. Conclusions: p38MAPK signaling in the DRG plays a crucial role in the development of primary afferent sensitization and pain behavior caused by plantar incision. (C) 2013 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Tomoko Sayano, Yuriko Kawakami, Wataru Kusada, Takeshi Suzuki, Yuki Kawano, Akihiro Watanabe, Kana Takashima, Yashiho Arimoto, Kayoko Esaki, Akira Wada, Fumiaki Yoshizawa, Masahiko Watanabe, Masahiro Okamoto, Yoshio Hirabayashi, Shigeki Furuya
    FEBS Journal 280 (6) 1502 - 1517 1742-464X 2013/03 [Refereed][Not invited]
     
    Targeted disruption in mice of the gene encoding d-3-phosphoglycerate dehydrogenase (Phgdh) results in embryonic lethality associated with a striking reduction in free l-serine and growth retardation including severe brain malformation. We previously observed a severe impairment in neurogenesis of the central nervous system of Phgdh knockout (KO) embryos and a reduction in the protein content of their brains. Although these findings suggest that l-serine deficiency links attenuation of mRNA translation to severe developmental malformation of the central nervous system, the underlying key molecular event remains unexplored. Here we demonstrate that mRNA of Eif4ebp1 encoding eukaryotic initiation factor 4 binding protein 1 and its protein, 4E-BP1, are markedly induced in the central nervous system of Phgdh KO embryos, whereas a modest induction is observed in the liver. The increase in 4E-BP1 was associated with a decrease in the cap initiation complex in the brain, as shown by lower levels of eukaryotic translation initiation factor 4G bound to eukaryotic translation initiation factor 4E (eIF4E) and increased eIF4E interaction with 4E-BP1 based on 7-methyl-GTP chromatography. eIF4E protein and polysomes were also diminished in Phgdh KO embryos. Induction of Eif4ebp1 mRNA and of 4E-BP1 was reproduced in mouse embryonic fibroblasts established from Phgdh KO embryos under the condition of l-serine deprivation. Induction of Eif4ebp1 mRNA was suppressed only when l-serine was supplemented in the culture medium, indicating that reduced l-serine availability regulates the induction of Eif4ebp1/4E-BP1. These data suggest that elevated levels of 4E-BP1 may be involved in a mechanism to arrest brain development in Phgdh KO embryos. © 2013 FEBS.
  • Issei Kawakita, Motokazu Uchigashima, Kohtarou Konno, Taisuke Miyazaki, Miwako Yamasaki, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 37 (4) 532 - 543 0953-816X 2013/02 [Refereed][Not invited]
     
    Postnatal expression of the type 2K+Cl cotransporter (KCC2) in neurons lowers the Cl equilibrium potential to values that are more negative than the resting potential, thereby converting the action of Cl-permeable GABAA and glycine receptors from excitatory to inhibitory. In the present study, we investigated the spatiotemporal expression of KCC2 in mouse cerebella, particularly focusing on Purkinje cells (PCs). First, we confirmed the fundamental expression profiles of KCC2 in the cerebellum, i.e. neuron-specific expression, somatodendritic distribution, and postnatal upregulation. We also found preferential recruitment to climbing fiber (CF) synapses during the second and third postnatal weeks, when perisomatic innervation in PCs switches from CFs to basket cell axons (BAs) and also when single winner CFs translocate from somata to dendrites. In parallel with this synaptic recruitment, the intracellular distribution shifted from a diffuse cytoplasmic to a predominantly cell surface pattern. In adult PCs, CF synapse-associated accumulation was obscured. Instead, significantly high expression was noted on the surface of PC dendrites in the superficial two-thirds of the molecular layer, in which stellate cells reside and project axons to innervate PC dendrites. Thus, the somatodendritic distribution in PCs is regulated in relation to particular inputs or input zones. During development, timed recruitment of KCC2 to CF synapses will augment inhibitory GABAergic actions by incoming BAs, promoting the CF-to-BA switchover in perisomatic PC innervation. In adulthood, enriched KCC2 expression at the stellate cell-targeting territory of PC dendrites might help in maintaining intracellular Cl homeostasis and the polarity of GABAA receptor-mediated responses upon sustained activity of this interneuron.
  • Dwi Wahyu Indriati, Naomi Kamasawa, Ko Matsui, Andrea L. Meredith, Masahiko Watanabe, Ryuichi Shigemoto
    JOURNAL OF NEUROSCIENCE 33 (8) 3668 - + 0270-6474 2013/02 [Refereed][Not invited]
     
    P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Ca(v)2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Ca(v)2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Ca(v)2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Ca(v)2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Ca(v)2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of similar to 40 nm. Calcium nanodomain created by the opening of Ca(v)2.1 channels likely activates the two channels that limit the extent of depolarization.
  • Taisuke Yoneda, Katsuro Kameyama, Kazusa Esumi, Yohei Daimyo, Masahiko Watanabe, Yoshio Hata
    PLoS ONE 8 (1) 1932-6203 2013/01/08 [Refereed][Not invited]
     
    The mammalian visual system exhibits significant experience-induced plasticity in the early postnatal period. While physiological studies have revealed the contribution of the CB1 cannabinoid receptor (CB1) to developmental plasticity in the primary visual cortex (V1), it remains unknown whether the expression and localization of CB1 is regulated during development or by visual experience. To explore a possible role of the endocannabinoid system in visual cortical plasticity, we examined the expression of CB1 in the visual cortex of mice. We found intense CB1 immunoreactivity in layers II/III and VI. CB1 mainly localized at vesicular GABA transporter-positive inhibitory nerve terminals. The amount of CB1 protein increased throughout development, and the specific laminar pattern of CB1 appeared at P20 and remained until adulthood. Dark rearing from birth to P30 decreased the amount of CB1 protein in V1 and altered the synaptic localization of CB1 in the deep layer. Dark rearing until P50, however, did not influence the expression of CB1. Brief monocular deprivation for 2 days upregulated the localization of CB1 at inhibitory nerve terminals in the deep layer. Taken together, the expression and the localization of CB1 are developmentally regulated, and both parameters are influenced by visual experience. © 2013 Yoneda et al.
  • Tie-Jun Sten Shi, Ming-Dong Zhang, Hugo Zeberg, Johanna Nilsson, Jacob Grunler, Su-Xing Liu, Qiong Xiang, Jonas Persson, Kaj J. Fried, Sergiu Bogdan Catrina, Masahiko Watanabe, Peter Arhem, Kerstin Brismar, Tomas G. M. Hokfelt
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 (2) 690 - 695 0027-8424 2013/01 [Refereed][Not invited]
     
    Diabetic peripheral neuropathy (DPN) is the most common complication in both type 1 and type 2 diabetes. Here we studied some phenotypic features of a well-established animal model of type 2 diabetes, the leptin receptor-deficient db(-)/db(-) mouse, and also the effect of long-term (6 mo) treatment with coenzyme Q10 (CoQ10), an endogenous antioxidant. Diabetic mice at 8 mo of age exhibited loss of sensation, hypoalgesia (an increase in mechanical threshold), and decreases in mechanical hyperalgesia, cold allodynia, and sciatic nerve conduction velocity. All these changes were virtually completely absent after the 6-mo, daily CoQ10 treatment in db(-)/db(-) mice when started at 7 wk of age. There was a 33% neuronal loss in the lumbar 5 dorsal root ganglia (DRGs) of the db(-)/db(-) mouse versus controls at 8 mo of age, which was significantly attenuated by CoQ10. There was no difference in neuron number in 5/6-wk-old mice between diabetic and control mice. We observed a strong down-regulation of phospholipase C (PLC) beta 3 in the DRGs of diabetic mice at 8 mo of age, a key molecule in pain signaling, and this effect was also blocked by the 6-mo CoQ10 treatment. Many of the phenotypic, neurochemical regulations encountered in lumbar DRGs in standard models of peripheral nerve injury were not observed in diabetic mice at 8 mo of age. These results suggest that reactive oxygen species and reduced PLC beta 3 expression may contribute to the sensory deficits in the late-stage diabetic db(-)/db(-) mouse, and that early long-term administration of the antioxidant CoQ10 may represent a promising therapeutic strategy for type 2 diabetes neuropathy.
  • Sam A. Booker, Anna Gross, Daniel Althof, Ryuichi Shigemoto, Bernhard Bettler, Michael Frotscher, Matthew Hearing, Kevin Wickman, Masahiko Watanabe, Ákos Kulik, Imre Vida
    Journal of Neuroscience 33 (18) 7961 - 7974 0270-6474 2013 [Refereed][Not invited]
     
    Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gammafrequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABAB receptors (GABABRs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABABRs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity forGABABRs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABABR-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABABR activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABAB IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABABRs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABABRs may shift the balance between perisomatic and dendritic inhibition. © 2013 the authors.
  • B. Wei, T. Kumada, T. Furukawa, K. Inoue, M. Watanabe, K. Sato, A. Fukuda
    NEUROSCIENCE 228 334 - 348 0306-4522 2013/01 [Refereed][Not invited]
     
    Although trigeminal neuropathic pain is one of the most common chronic pain syndromes, the etiology is still unknown. Here, a rat model was generated using chronic constrictive injury (CCI) with ligation of the infraorbital nerve to test the hypothesis that collapse of chloride homeostasis in trigeminal neurons causes impairment of gamma-aminobutyric acid-ergic (GABAergic) inhibition and induces trigeminal allodynia. Rats showed a reduction and increase in pain threshold and pain response scores, respectively, to mechanical stimulation, 1 and 3 weeks after CCI. In situ hybridization and immunohistochemical analysis showed that inward-directed Na+, K+-2Cl(-) cotransporter (NKCC1) mRNA and protein were upregulated in the small-sized and large-sized primary neurons in the injured side of the trigeminal ganglion and in the peripherin-positive terminal, respectively, for the first 2 weeks, while outward-directed K+-Cl- cotransporter (KCC2) mRNA and protein were downregulated in secondary relay neurons on the injured side of the spinal trigeminal nucleus caudalis (Sp5C). Optical imaging of evoked synaptic responses using a voltage-sensitive dye revealed that pre- and post-synaptic GABA actions were disinhibited and excitatory in the injured side, respectively, but inhibited in the sham-operated side of the Sp5C. This downregulation of KCC2 in the Sp5C may result in an excitatory switch by impairing postsynaptic GABA inhibition. GABA-mediated presynaptic disinhibition was attenuated by bumetanide, suggesting that NKCC1 upregulation in primary neurons may facilitate pain transmission by presynaptic GABAergic depolarization. Such Cl- homeostatic disruption resulting in perturbation of the inhibitory system possibly increases pain transmission, which may underlie the pathophysiology of trigeminal neuropathic pain. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Shinya Kawata, Kouichi Hashimoto, Maya Yamazaki, Taisuke Miyazaki, Miwako Yamasaki, Takayasu Mikuni, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    JOURNAL OF PHYSIOLOGICAL SCIENCES 63 S188 - S188 1880-6546 2013 [Refereed][Not invited]
  • Hiroki Shikanai, Takayuki Yoshida, Kohtarou Konno, Miwako Yamasaki, Takeshi Izumi, Yu Ohmura, Keiichi Shimamura, Masahiko Watanabe, Mitsuhiro Yoshioka
    JOURNAL OF PHARMACOLOGICAL SCIENCES 121 65P - 65P 1347-8613 2013 [Refereed][Not invited]
  • Toshika Ohkawa, Yuko Fukata, Miwako Yamasaki, Taisuke Miyazaki, Norihiko Yokoi, Hiroshi Takashima, Masahiko Watanabe, Osamu Watanabe, Masaki Fukata
    Journal of Neuroscience 33 (46) 18161 - 18174 0270-6474 2013 [Refereed][Not invited]
     
    More than 30 mutations in LGI1, a secreted neuronal protein, have been reported with autosomal dominant lateral temporal lobe epilepsy (ADLTE). Although LGI1 haploinsufficiency is thought to cause ADLTE, the underlying molecular mechanism that results in abnormal brain excitability remains mysterious. Here, we focused on a mode of action of LGI1 autoantibodies associated with limbic encephalitis (LE), which is one of acquired epileptic disorders characterized by subacute onset of amnesia and seizures.Wecomprehensively screened human sera from patients with immune-mediated neurological disorders for LGI1 autoantibodies, which also uncovered novel autoantibodies against six cell surface antigens including DCC, DPP10, and ADAM23. Our developed ELISA arrays revealed a specific role for LGI1 antibodies in LE and concomitant involvement of multiple antibodies, including LGI1 antibodies in neuromyotonia, a peripheral nerve disorder. LGI1 antibodies associated with LE specifically inhibited the ligand-receptor interaction between LGI1 and ADAM22/23 by targeting the EPTP repeat domain of LGI1 and reversibly reduced synaptic AMPA receptor clusters in rat hippocampal neurons. Furthermore, we found that disruption of LGI1-ADAM22 interaction by soluble extracellular domain ofADAM22was sufficient to reduce synapticAMPAreceptors in rat hippocampal neurons and that levels ofAMPAreceptor were greatly reduced in the hippocampal dentate gyrus in the epileptic LGI1 knock-out mouse. Therefore, either genetic or acquired loss of the LGI1-ADAM22 interaction reduces theAMPAreceptor function, causing epileptic disorders. These results suggest that by finely regulating the synapticAMPAreceptors, the LGI1-ADAM22 interaction maintains physiological brain excitability throughout life. © 2013 the authors.
  • Ageta-Ishihara N, Miyata T, Ohshima C, Watanabe M, Sato Y, Hamamura Y, Higashiyama T, Mazitschek R, Bito H, Kinoshita M
    Nature communications 4 2532 - 2532 2013 [Refereed][Not invited]
     
    Neurite growth requires two guanine nucleotide-binding protein polymers of tubulins and septins. However, whether and how those cytoskeletal systems are coordinated was unknown. Here we show that the acute knockdown or knockout of the pivotal septin subunit SEPT7 from cerebrocortical neurons impairs their interhemispheric and cerebrospinal axon projections and dendritogenesis in perinatal mice, when the microtubules are severely hyperacetylated. The resulting hyperstabilization and growth retardation of microtubules are demonstrated in vitro. The phenotypic similarity between SEPT7 depletion and the pharmacological inhibition of α-tubulin deacetylase HDAC6 reveals that HDAC6 requires SEPT7 not for its enzymatic activity, but to associate with acetylated α-tubulin. These and other findings indicate that septins provide a physical scaffold for HDAC6 to achieve efficient microtubule deacetylation, thereby negatively regulating microtubule stability to an optimal level for neuritogenesis. Our findings shed light on the mechanisms underlying the HDAC6-mediated coupling of the two ubiquitous cytoskeletal systems during neural development.
  • Rebeca Martinez-Turrillas, Elena Puerta, Dhrubajyoti Chowdhury, Sonia Marco, Masahiko Watanabe, Norberto Aguirre, Isabel Perez-Otano
    NEUROBIOLOGY OF DISEASE 48 (3) 290 - 298 0969-9961 2012/12 [Refereed][Not invited]
     
    Excitotoxicity due to excessive activation of glutamate receptors is a primary mediator of cell death in acute and chronic neurological disorders, and NMDA-type glutamate receptors (NMDARs) are thought to be involved. NMDARs assemble from heteromeric combinations of GluNl, GluN2 and GluN3 subunits, yielding a variety of receptor subtypes that differ in biophysical properties, signaling, and synaptic targeting. Inclusion of inhibitory GluN3 subunits reduces Ca2+ influx via NMDAR channels and alters their synaptic targeting, thus modifying the two hallmarks of NMDARs that are critical for their roles on neuronal death and survival. Here we evaluated the neuroprotective potential of GluN3A subunits by analyzing the susceptibility to striatal excitotoxic damage of transgenic mice overexpressing GluN3A. We found that mild GluN3A overexpression protected susceptible striatal neurons from lesions induced by the neurotoxin 3-nitropropionic acid (3-NP), an inhibitor of mitochondrial complex 11/succinate dehydrogenase. GluN3A-mediated neuroprotection was dose-dependent and correlated with the levels of transgenic GluN3A expressed by two different mice strains. Neuroprotection was associated with a potent reduction of the activation of calpain, a Ca2+-dependent protease, which was measured as a decrease in 3-NP-induced fodrin and STEP cleavage in GluN3A transgenic mice relative to controls. We further show that transgenic GluN3A subunits incorporate into extrasynaptic compartments in mouse striatum, suggesting that reductions of toxic calpain activation might be linked to inhibition by GluN3A of pathological extrasynaptic NMDAR activity. (c) 2012 Elsevier Inc. All rights reserved.
  • Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Kohtarou Konno, Miwako Yamasaki, Yuchio Yanagawa, Masabumi Minami, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 32 (50) 18035 - 18046 0270-6474 2012/12 [Refereed][Not invited]
     
    Dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) play crucial roles in motivational control of behaviors, and their activity is regulated directly or indirectly via GABAergic neurons by extrinsic afferents from various sources, including the bed nucleus of the stria terminalis ( BST). Here, the neurochemical composition of VTA-projecting BST neurons and their outputs to the VTA were studied in adult mouse brains. By combining retrograde tracing with fluorescence in situ hybridization for 67 kDa glutamate decarboxylase (GAD67) and vesicular glutamate transporters (VGluTs), VTA-targeting BST neurons were classified into GAD67-positive (GAD67(+))/VGluT3-negative (VGluT3(-)), GAD67(+)/VGluT3(+), and VGluT2(+) neurons, of which GAD67(+)/VGluT3(-)neurons constituted the majority (similar to 90%) of VTA-projecting BST neurons. GABAergic efferents from the BST formed symmetrical synapses on VTA neurons, which were mostly GABAergic neurons, and expressed GABA(A) receptor alpha 1 subunit on their synaptic and extrasynaptic membranes. In the VTA, VGluT3 was detected in terminals expressing vesicular inhibitory amino acid transporter ( VIAAT), plasmalemmal serotonin transporter, or neither. Of these, VIAAT(+)/VGluT3(+) terminals, which should include those from GAD67(+)/VGluT3(+) BST neurons, formed symmetrical synapses. When single axons from VGluT3(+) BST neurons were examined, almost all terminals were labeled for VIAAT, whereas VGluT3 was often absent from terminals with high VIAAT loads. VGluT2(+) terminals in the VTA exclusively formed asymmetrical synapses, which expressed AMPA receptors on postsynaptic membrane. Therefore, the major mode of the BST-VTA projection is GABAergic, and its activation is predicted to disinhibit VTA DAergic neurons. VGluT2(+) and VGluT3(+) BST neurons further supply additional projections, which may principally convey excitatory or inhibitory inputs, respectively, to the VTA.
  • Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Kohtarou Konno, Miwako Yamasaki, Yuchio Yanagawa, Masabumi Minami, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 32 (50) 18035 - 18046 0270-6474 2012/12 [Refereed][Not invited]
     
    Dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) play crucial roles in motivational control of behaviors, and their activity is regulated directly or indirectly via GABAergic neurons by extrinsic afferents from various sources, including the bed nucleus of the stria terminalis ( BST). Here, the neurochemical composition of VTA-projecting BST neurons and their outputs to the VTA were studied in adult mouse brains. By combining retrograde tracing with fluorescence in situ hybridization for 67 kDa glutamate decarboxylase (GAD67) and vesicular glutamate transporters (VGluTs), VTA-targeting BST neurons were classified into GAD67-positive (GAD67(+))/VGluT3-negative (VGluT3(-)), GAD67(+)/VGluT3(+), and VGluT2(+) neurons, of which GAD67(+)/VGluT3(-)neurons constituted the majority (similar to 90%) of VTA-projecting BST neurons. GABAergic efferents from the BST formed symmetrical synapses on VTA neurons, which were mostly GABAergic neurons, and expressed GABA(A) receptor alpha 1 subunit on their synaptic and extrasynaptic membranes. In the VTA, VGluT3 was detected in terminals expressing vesicular inhibitory amino acid transporter ( VIAAT), plasmalemmal serotonin transporter, or neither. Of these, VIAAT(+)/VGluT3(+) terminals, which should include those from GAD67(+)/VGluT3(+) BST neurons, formed symmetrical synapses. When single axons from VGluT3(+) BST neurons were examined, almost all terminals were labeled for VIAAT, whereas VGluT3 was often absent from terminals with high VIAAT loads. VGluT2(+) terminals in the VTA exclusively formed asymmetrical synapses, which expressed AMPA receptors on postsynaptic membrane. Therefore, the major mode of the BST-VTA projection is GABAergic, and its activation is predicted to disinhibit VTA DAergic neurons. VGluT2(+) and VGluT3(+) BST neurons further supply additional projections, which may principally convey excitatory or inhibitory inputs, respectively, to the VTA.
  • Aya Ito-Ishida, Taisuke Miyazaki, Eriko Miura, Keiko Matsuda, Masahiko Watanabe, Michisuke Yuzaki, Shigeo Okabe
    NEURON 76 (3) 549 - 564 0896-6273 2012/11 [Refereed][Not invited]
     
    Differentiation of pre- and postsynaptic sites is coordinated by reciprocal interaction across synaptic clefts. At parallel fiber (PF)-Purkinje cell (PC) synapses, dendritic spines are autonomously formed without PF influence. However, little is known about how presynaptic structural changes are induced and how they lead to differentiation of mature synapses. Here, we show that Cbln1 released from PFs induces dynamic structural changes in PFs by a mechanism that depends on postsynaptic glutamate receptor delta2 (GluD2) and presynaptic neurexin (Nrx). Time-lapse imaging in organotypic culture and ultrastructural analyses in vivo revealed that Nrx-Cbln1-GluD2 signaling induces PF protrusions that often formed circular structures and encapsulated PC spines. Such structural changes in PFs were associated with the accumulation of synaptic vesicles and GluD2, leading to formation of mature synapses. Thus, PF protrusions triggered by Nrx-Cbln1-GluD2 signaling may promote bidirectional maturation of PF-PC synapses by a positive feedback mechanism.
  • N. M. Derks, O. Pinter, W. Zuure, C. Ledent, M. Watanabe, C. S. Molnar, Y. Wei, E. W. Roubos, S. Wu, E. Hrabovszky, D. Zelena, T. Kozicz
    JOURNAL OF NEUROENDOCRINOLOGY 24 (11) 1447 - 1461 0953-8194 2012/11 [Refereed][Not invited]
     
    Neurones in the centrally projecting EdingerWestphal nucleus (EWcp) are the main site of urocortin 1 (Ucn1) synthesis in the mammalian brain, and are assumed to play a role in the stress response of the animal. Because endocannabinoid signalling has also been strongly implicated in stress, we hypothesised that endocannabinoids may modulate the functioning of the urocortinergic EWcp. First, using in situ hybridisation, we demonstrated cannabinoid receptor 1 (CB1R) mRNA expression in mouse EWcp-neurones that were Ucn1-negative. Dual- and triple-label immunocytochemistry revealed the presence of CB1R in several GABA-immunopositive fibres juxtaposed to EWcp-Ucn1 neurones. To test functional aspects of such an anatomical constellation, we compared acute (1 similar to h of restraint) and chronic (14 similar to days of chronic mild stress) stress-induced changes in wild-type (WT) and CB1R knockout (CB1R-KO) mice. Acute and especially chronic stress resulted in an increase in Ucn1 content of the EWcp, which was attenuated in CB1R-KO mice. CB1R-KO mice had higher basal and chronic stress-induced adrenocorticotrophin and corticosterone levels and were more anxious on the elevated plus-maze versus WT. Collectively, our results show for the first time EWcp-Ucn1 neurones are putatively innervated by endocannabinoid sensitive, inhibitory, GABAergic afferents. In addition, we provide novel evidence that the absence of the CB1 receptor alters the Ucn1 mRNA and peptide levels in EWcp neurones, concomitant with an augmented stress response and increased anxiety-like behaviour.
  • Hiroki Shikanai, Takayuki Yoshida, Kohtarou Konno, Miwako Yamasaki, Takeshi Izumi, Yu Ohmura, Masahiko Watanabe, Mitsuhiro Yoshioka
    JOURNAL OF NEUROSCIENCE 32 (41) 14415 - 14426 0270-6474 2012/10 [Refereed][Not invited]
     
    The serotonergic (5-HTergic) system arising from the dorsal raphe nucleus (DRN) is implicated in various physiological and behavioral processes, including stress responses. The DRN is comprised of several subnuclei, serving specific functions with distinct afferent and efferent connections. Furthermore, subsets of 5-HTergic neurons are known to coexpress other transmitters, including GABA, glutamate, or neuropeptides, thereby generating further heterogeneity. However, despite the growing evidence for functional variations among DRN subnuclei, relatively little is known about how they map onto neurochemical diversity of 5-HTergic neurons. In the present study, we characterized functional properties of GAD67-expressing 5-HTergic neurons (5-HT/GAD67 neurons) in the rat DRN, and compared with those of neurons expressing 5-HTergic molecules (5-HT neurons) or GAD67 alone. While 5-HT/GAD67 neurons were absent in the dorsomedial (DRD) or ventromedial (DRV) parts of the DRN, they were selectively distributed in the lateral wing of the DRN (DRL), constituting 12% of the total DRL neurons. They expressed plasmalemmal GABA transporter 1, but lacked vesicular inhibitory amino acid transporter. By using whole-cell patch-clamp recording, we found that 5-HT/GAD67 neurons had lower input resistance and firing frequency than 5-HT neurons. As revealed by c-Fos immunohistochemistry, neurons in the DRL, particularly 5-HT/GAD67 neurons, showed higher responsiveness to exposure to an open field arena than those in the DRD and DRV. By contrast, exposure to contextual fear conditioning stress showed no such regional differences. These findings indicate that 5-HT/GAD67 neurons constitute a unique neuronal population with distinctive neurochemical and electrophysiological properties and high responsiveness to innocuous stressor.
  • Shohei Oshima, Masahiko Watanabe
    EUROPEAN JOURNAL OF ORAL SCIENCES 120 (5) 386 - 394 0909-8836 2012/10 [Refereed][Not invited]
     
    Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase consisting of two subunits catalytic subunit A (CnA) and regulatory subunit B (CnB) and plays a critical role in transducing Ca2+ signals into cellular responses. In this study, we investigated the expression of calcineurin in the mouse developing tooth. In-situ hybridization detected mRNAs for the CnAa and CnA beta isoforms of CnA and for the CnB1 isoform of CnB in the upper molar tooth germ at embryonic day 15. Immunohistochemistry with antibodies specific for CnAa, CnA beta, and CnB1 showed strong immunoreactivity of these proteins in secretory-stage ameloblasts and in odontoblasts during dentin formation. CnA beta and CnB1 were strongly immunoreactive in ruffle-ended ameloblasts at the enamel-maturation stage. In ameloblasts and odontoblasts, we also noted different subcellular distributions of CnAa and CnA beta. From these data, temporal profiles of calcineurin expression appear to correlate with active mineralization in tooth development. Furthermore, the distinct subcellular distribution of the two CnA subunits may reflect their distinct substrates or responsive sites within single cells, thus contributing to the diversity of calcineurin-dependent cellular responses during active tooth mineralization.
  • Kayo Nishizawa, Ryoji Fukabori, Kana Okada, Nobuyuki Kai, Motokazu Uchigashima, Masahiko Watanabe, Akira Shiota, Masatsugu Ueda, Yuji Tsutsui, Kazuto Kobayashi
    JOURNAL OF NEUROSCIENCE 32 (39) 13421 - 13432 0270-6474 2012/09 [Refereed][Not invited]
     
    The dorsal striatum, which contains the dorsolateral striatum (DLS) and dorsomedial striatum (DMS), integrates the acquisition and implementation of instrumental learning in cooperation with the nucleus accumbens (NAc). The dorsal striatum regulates the basal ganglia circuitry through direct and indirect pathways. The mechanism by which these pathways mediate the learning processes of instrumental actions remains unclear. We investigated how the striatal indirect (striatopallidal) pathway arising from the DLS contributes to the performance of conditional discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D-2 receptor in the DLS of transgenic rats resulted in selective, efficient elimination of the striatopallidal pathway. This elimination impaired the accuracy of response selection in a two-choice reaction time task dependent on different auditory stimuli. The impaired response selection was elicited early in the test sessions and was gradually restored as the sessions continued. The restoration from the deficits in auditory discrimination was prevented by excitotoxic lesion of the NAc but not by that of the DMS. In addition, lesion of the DLS mimicked the behavioral consequence of the striatopallidal removal at the early stage of test sessions of discriminative performance. Our results demonstrate that the DLS-derived striatopallidal pathway plays an essential role in the execution of conditional discrimination, showing its contribution to the control of selection accuracy of learned motor responses. The results also suggest the presence of a mechanism that compensates for the learning deficits during the repetitive sessions, at least partly, demanding accumbal function.
  • Hidetaka Matsumoto, Koji Shibasaki, Motokazu Uchigashima, Amane Koizumi, Masashi Kurachi, Yasuhiro Moriwaki, Hidemi Misawa, Koichiro Kawashima, Masahiko Watanabe, Shoji Kishi, Yasuki Ishizaki
    PLOS ONE 7 (8) 1932-6203 2012/08 [Refereed][Not invited]
     
    It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that alpha 7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the alpha 7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through alpha 7 nAChRs in their microvilli.
  • Najma Baseer, Erika Polgar, Masahiko Watanabe, Takahiro Furuta, Takeshi Kaneko, Andrew J. Todd
    JOURNAL OF NEUROSCIENCE 32 (34) 11854 - 11863 0270-6474 2012/08 [Refereed][Not invited]
     
    Large projection neurons in lamina III of the rat spinal cord that express the neurokinin 1 receptor are densely innervated by peptidergic primary afferent nociceptors and more sparsely by low-threshold myelinated afferents. However, we know little about their input from other glutamatergic neurons. Here we show that these cells receive numerous contacts from nonprimary boutons that express the vesicular glutamate transporter 2 (VGLUT2), and form asymmetrical synapses on their dendrites and cell bodies. These synapses are significantly smaller than those formed by peptidergic afferents, but provide a substantial proportion of the glutamatergic synapses that the cells receive (over a third of those in laminae I-II and half of those in deeper laminae). Surprisingly, although the dynorphin precursor preprodynorphin (PPD) was only present in 4-7% of VGLUT2 boutons in laminae I-IV, it was found in 58% of the VGLUT2 boutons that contacted these cells. This indicates a highly selective targeting of the lamina III projection cells by glutamatergic neurons that express PPD, and these are likely to correspond to local neurons (interneurons and possibly projection cells). Since many PPD-expressing dorsal horn neurons respond to noxious stimulation, this suggests that the lamina III projection cells receive powerful monosynaptic and polysynaptic nociceptive input. Excitatory interneurons in the dorsal horn have been shown to possess I-A currents, which limit their excitability and can underlie a form of activity-dependent intrinsic plasticity. It is therefore likely that polysynaptic inputs to the lamina III projection neurons are recruited during the development of chronic pain states.
  • D. I. Hughes, S. Sikander, C. M. Kinnon, K. A. Boyle, M. Watanabe, R. J. Callister, B. A. Graham
    Journal of Physiology 590 (16) 3927 - 3951 0022-3751 2012/08 [Refereed][Not invited]
     
    Axo-axonic synapses on the central terminals of primary afferent fibres modulate sensory input and are the anatomical correlate of presynaptic inhibition. Although several classes of primary afferents are under such inhibitory control, the origin of these presynaptic inputs in the dorsal horn is unknown. Here, we characterize the neurochemical, anatomical and electrophysiological properties of parvalbumin (PV)-expressing cells in wild-type and transgenic mice where enhanced green fluorescent protein (eGFP) is expressed under the PV promoter. We show that most PV cells have either islet or central cell-like morphology, receive inputs from myelinated primary afferent fibres and are concentrated in laminae II inner and III. We also show that inhibitory PV terminals in lamina II inner selectively target the central terminals of myelinated afferents (∼80% of 935 PVeGFP boutons) and form axo-axonic synapses (∼75% of 71 synapses from PV boutons). Targeted whole-cell patch-clamp recordings from PVeGFP positive cells in laminae II and III showed action potential discharge was restricted to the tonic firing and initial bursting patterns (67% and 33% respectively n= 18), and virtually all express Ih subthreshold voltage-gated currents (94% n= 18). These neurons show higher rheobase current than non-eGFP cells but respond with high frequency action potential discharge upon activation. Together, our findings show that PV neurons in laminae II and III are a likely source of inhibitory presynaptic input on to myelinated primary afferents. Consequently PV cells are ideally placed to play an important role in the development of central sensitization and tactile allodynia. © 2012 The Authors. The Journal of Physiology © 2012 The Physiological Society.
  • Najma Baseer, Erika Polgar, Masahiko Watanabe, Takahiro Furuta, Takeshi Kaneko, Andrew J. Todd
    JOURNAL OF NEUROSCIENCE 32 (34) 11854 - 11863 0270-6474 2012/08 [Refereed][Not invited]
     
    Large projection neurons in lamina III of the rat spinal cord that express the neurokinin 1 receptor are densely innervated by peptidergic primary afferent nociceptors and more sparsely by low-threshold myelinated afferents. However, we know little about their input from other glutamatergic neurons. Here we show that these cells receive numerous contacts from nonprimary boutons that express the vesicular glutamate transporter 2 (VGLUT2), and form asymmetrical synapses on their dendrites and cell bodies. These synapses are significantly smaller than those formed by peptidergic afferents, but provide a substantial proportion of the glutamatergic synapses that the cells receive (over a third of those in laminae I-II and half of those in deeper laminae). Surprisingly, although the dynorphin precursor preprodynorphin (PPD) was only present in 4-7% of VGLUT2 boutons in laminae I-IV, it was found in 58% of the VGLUT2 boutons that contacted these cells. This indicates a highly selective targeting of the lamina III projection cells by glutamatergic neurons that express PPD, and these are likely to correspond to local neurons (interneurons and possibly projection cells). Since many PPD-expressing dorsal horn neurons respond to noxious stimulation, this suggests that the lamina III projection cells receive powerful monosynaptic and polysynaptic nociceptive input. Excitatory interneurons in the dorsal horn have been shown to possess I-A currents, which limit their excitability and can underlie a form of activity-dependent intrinsic plasticity. It is therefore likely that polysynaptic inputs to the lamina III projection neurons are recruited during the development of chronic pain states.
  • Asami Tanimura, Motokazu Uchigashima, Maya Yamazaki, Naofumi Uesaka, Takayasu Mikuni, Manabu Abe, Kouichi Hashimoto, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 (30) 12195 - 12200 0027-8424 2012/07 [Refereed][Not invited]
     
    The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic suppression. Although the mechanisms of 2AG production are well characterized, how 2-AG is degraded is less clearly understood. Here we found that expression of the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MGL) was highly heterogeneous in the cerebellum, being rich within parallel fiber (PF) terminals, weak in Bergman glia (BG), and absent in other synaptic terminals. Despite this highly selective MGL expression pattern, 2-AG-mediated retrograde suppression was significantly prolonged at not only PF-Purkinje cell (PC) synapses but also climbing fiber-PC synapses in granule cell-specific MGL knockout (MGL-KO) mice whose cerebellar MGL expression was confined to the BG. Virus-mediated expression of MGL into the BG of global MGL-KO mice significantly shortened 2-AG-mediated retrograde suppression at PF-PC synapses. Furthermore, contribution of MGL to termination of 2-AG signaling depended on the distance from MGL-rich PFs to inhibitory synaptic terminals. Thus, 2-AG is degraded in a synapse-type independent manner by MGL present in PFs and the BG. The results of the present study strongly suggest that MGL regulates 2-AG signaling rather broadly within a certain range of neural tissue, although MGL expression is heterogeneous and limited to a subset of nerve terminals and astrocytes.
  • Atsushi Iwakura, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 32 (27) 9438 - 9448 0270-6474 2012/07 [Refereed][Not invited]
     
    The axon initial segment (AIS) of cerebellar Purkinje cells (PCs) is embraced by ramified axons of GABAergic basket cells (BCs) called the pinceau formation. This unique structure has been assumed to be a device for the modulation of PC outputs through electrical and/or GABAergic inhibition. Electrical inhibition is supported by enriched potassium channels, absence of sodium channels, and developed septate-like junctions between BC axons. The neurochemical basis for GABAergic inhibition, however, has not been well investigated. Here we addressed this issue using C56BL/6 mice. First, we confirmed previous observations that typical synaptic contacts were rare and confined to proximal axonal portions, with the remaining portions being mostly covered by astrocytic processes. Then we examined the expression of molecules involved in GABAergic signaling, including GABA synthetic enzyme glutamic acid decarboxylase (GAD), vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT), cytomatrix active zone protein bassoon, GABA receptor GABA(A)R alpha 1, and cell adhesion molecule neuroligin-2. These molecules were recruited to form a functional assembly at perisomatic BC-PC synapses and along the AIS of hippocampal and neocortical pyramidal cells. GAD and VIAAT immunogold labeling was five times lower in the pinceau formation compared with perisomatic BC terminals and showed no accumulation toward the AIS. Moreover, bassoon, neuroligin-2, and GABA(A)R alpha 1 formed no detectable clusters along the ankyrin-G-positive AIS proper. These findings indicate that GABAergic signaling machinery is organized loosely and even incompletely in the pinceau formation. Together, BCs do not appear to exert GABAergic synaptic inhibition on the AIS, although the mode of action of the pinceau formation remains to be explored.
  • Noemi Holderith, Andrea Lorincz, Gergely Katona, Balazs Rozsa, Akos Kulik, Masahiko Watanabe, Zoltan Nusser
    NATURE NEUROSCIENCE 15 (7) 988 - 997 1097-6256 2012/07 [Refereed][Not invited]
     
    Cortical synapses have structural, molecular and functional heterogeneity; our knowledge regarding the relationship between their ultrastructural and functional parameters is still fragmented. Here we asked how the neurotransmitter release probability and presynaptic [Ca2+] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca2+ imaging derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active-zone area. Peak amplitude of [Ca2+] transients in single boutons also positively correlated with the active-zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated calcium channel subunit Cav2.1 and the presynaptic protein Rim 1/2 are confined to the active zone and their numbers scale linearly with the active-zone area. Gold particles labeling Cav2.1 were nonrandomly distributed in the active zones. Our results demonstrate that the numbers of several active-zone proteins, including presynaptic calcium channels, as well as the number of docked vesicles and the release probability, scale linearly with the active-zone area.
  • Laura C. Gregg, Kwang-Mook Jung, Jessica M. Spradley, Rita Nyilas, Richard L. Suplita, Andreas Zimmer, Masahiko Watanabe, Ken Mackie, Istvan Katona, Daniele Piomelli, Andrea G. Hohmann
    JOURNAL OF NEUROSCIENCE 32 (28) 9457 - 9468 0270-6474 2012/07 [Refereed][Not invited]
     
    Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here, we show that footshock stress produces antinociception in rats by activating type 5 metabotropic glutamate receptors (mGlu(5)) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu(5) in the dlPAG with DHPG [( S)-3,5-dihydroxyphenylglycine] triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB1 cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 [1,6-bis(cyclohexyloximinocarbonylamino)hexane] and (-)tetrahydrolipstatin (THL), which inhibit activity of DGL-alpha and DGL-beta isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-alpha colocalized with mGlu5 at postsynaptic sites of the dlPAG, whereas CB1 was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally mediated silencing of DGL-alpha, but not DGL-alpha, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu(5)-DGL-alpha cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.
  • Hiroshi Katoh, Keishi Yamashita, Mina Waraya, Ofer Margalit, Akira Ooki, Hideaki Tamaki, Hiroyuki Sakagami, Kenichi Kokubo, David Sidransky, Masahiko Watanabe
    NEOPLASIA 14 (7) 559 - + 1522-8002 2012/07 [Refereed][Not invited]
     
    Homeodomain-only protein X (HOPX)-beta promoter methylation was recently shown to be frequent in human cancers and was suggested as tumor suppressor gene in esophageal and gastric cancer. The aim of this study was to investigate the mechanistic roles of HOPX-beta promoter methylation and its clinical relevance in colorectal cancer (CRC). HOPX-beta promoter methylation was assessed in human CRC cell lines and 294 CRC tissues. HOPX mRNA and protein levels were measured in relation to HOPX-beta promoter methylation. The effects of forced HOPX expression on tumorigenesis were studied using in vitro and in vivo assays. The association between HOPX-beta promoter methylation and clinical relevance of CRC patients was determined. HOPX-beta promoter methylation is cancer-specific and frequently found in CRC cell lines and tissues, resulting in the down-regulation of HOPX mRNA and protein levels. In CRC cell lines, forced expression of HOPX suppressed proliferation, invasion, and anchorage-independent growth. DNA microarray analyses suggested critical downstream genes that are associated with cancer cell proliferation, invasion or angiogenesis. In a mouse xenograft model, HOPX inhibited tumorigenesis and angiogenesis. Finally, HOPX-beta promoter methylation was associated with worse prognosis of stage III CRC patients (hazard ratio = 1.40, P = .035) and also with poor differentiation (P = .014). In conclusion, HOPX-beta promoter methylation is a frequent and cancer-specific event in CRC progression. This epigenetic alteration may have clinical ramifications in the diagnosis and treatment of CRC patients.
  • Erik Hrabovszky, Csilla S. Molnar, Robert Nagy, Barbara Vida, Beata A. Borsay, Kalman Racz, Laszlo Herczeg, Masahiko Watanabe, Imre Kallo, Zsolt Liposits
    ENDOCRINOLOGY 153 (6) 2766 - 2776 0013-7227 2012/06 [Refereed][Not invited]
     
    Amino acid (aa) neurotransmitters in synaptic afferents to hypothalamic GnRH-I neurons are critically involved in the neuroendocrine control of reproduction. Although in rodents the major aa neurotransmitter in these afferents is gamma-aminobutyric acid (GABA), glutamatergic axons also innervate GnRH neurons directly. Our aim with the present study was to address the relative contribution of GABAergic and glutamatergic axons to the afferent control of human GnRH neurons. Formalin-fixed hypothalamic samples were obtained from adult male individuals (n = 8) at autopsies, and their coronal sections processed for dual-label immunohistochemical studies. GABAergic axons were labeled with vesicular inhibitory aa transporter antibodies, whereas glutamatergic axons were detected with antisera against the major vesicular glutamate transporter (VGLUT) isoforms, VGLUT1 and VGLUT2. The relative incidences of GABAergic and glutamatergic axonal appositions to GnRH-immunoreactive neurons were compared quantitatively in two regions, the infundibular and paraventricular nuclei. Results showed that GABAergic axons established the most frequently encountered type of axo-somatic apposition. Glutamatergic contacts occurred in significantly lower numbers, with similar contributions by their VGLUT1 and VGLUT2 subclasses. The innervation pattern was different on GnRH dendrites where the combined incidence of glutamatergic (VGLUT1 + VGLUT2) contacts slightly exceeded that of the GABAergic appositions. We conclude that GABA represents the major aa neurotransmitter in axo-somatic afferents to human GnRH neurons, whereas glutamatergic inputs occur somewhat more frequently than GABAergic inputs on GnRH dendrites. Unlike in rats, the GnRH system of the human receives innervation from the VGLUT1, in addition to the VGLUT2, subclass of glutamatergic neurons. (Endocrinology 153: 2766-2776, 2012)
  • Carmen Ballesteros-Merino, Mike Lin, Wendy W. Wu, Clotilde Ferrandiz-Huertas, Maria J. Cabanero, Masahiko Watanabe, Yugo Fukazawa, Ryuichi Shigemoto, James Maylie, John P. Adelman, Rafael Lujan
    HIPPOCAMPUS 22 (6) 1467 - 1480 1050-9631 2012/06 [Refereed][Not invited]
     
    We investigated the temporal and spatial expression of SK2 in the developing mouse hippocampus using molecular and biochemical techniques, quantitative immunogold electron microscopy, and electrophysiology. The mRNA encoding SK2 was expressed in the developing and adult hippocampus. Western blotting and immunohistochemistry showed that SK2 protein increased with age. This was accompanied by a shift in subcellular localization. Early in development (P5), SK2 was predominantly localized to the endoplasmic reticulum in the pyramidal cell layer. But by P30 SK2 was almost exclusively expressed in the dendrites and spines. The level of SK2 at the postsynaptic density (PSD) also increased during development. In the adult, SK2 expression on the spine plasma membrane showed a proximal-to-distal gradient. Consistent with this redistribution and gradient of SK2, the selective SK channel blocker apamin increased evoked excitatory postsynaptic potentials (EPSPs) only in CA1 pyramidal neurons from mice older than P15. However, the effect of apamin on EPSPs was not different between synapses in proximal or distal stratum radiatum or stratum lacunosum-moleculare in adult. These results show a developmental increase and gradient in SK2-containing channel surface expression that underlie their influence on neurotransmission, and that may contribute to increased memory acquisition during early development. 2011 Wiley Periodicals, Inc.
  • Yuichi Takeuchi, Miwako Yamasaki, Yasuyuki Nagumo, Keiji Imoto, Masahiko Watanabe, Mariko Miyata
    JOURNAL OF NEUROSCIENCE 32 (20) 6917 - 6930 0270-6474 2012/05 [Refereed][Not invited]
     
    The remodeling of neural circuitry and changes in synaptic efficacy after peripheral sensory nerve injury are considered the basis for functional reorganization in the brain, including changes in receptive fields. However, when or how the remodeling occurs is largely unknown. Here we show the rapid rewiring of afferent fibers in the mature ventral posteromedial thalamic nucleus of mice after transection of the peripheral whisker sensory nerve, using the whole-cell voltage-clamp technique. Transection induced the recruitment of afferent fibers to a thalamic relay neuron within 5-6 d of injury. The rewiring was pathway specific, but not sensory experience dependent or peripheral nerve activity dependent. The newly recruited fibers mediated small EPSCs, and postsynaptic GluA2-containing AMPA receptors were selectively upregulated at the new synapses. This rapid and pathway-specific remodeling of thalamic circuitry may be an initial step in the massive axonal reorganization at supraspinal levels, which occurs months or years after peripheral sensory nerve injury.
  • Csaba Cserep, Eszter Szabadits, Andras Szonyi, Masahiko Watanabe, Tamas F. Freund, Gabor Nyiri
    PLOS ONE 7 (5) 1932-6203 2012/05 [Refereed][Not invited]
     
    GABA (gamma-aminobutyric-acid), the main inhibitory neurotransmitter in the adult brain, exerts depolarizing (excitatory) actions during development and this GABAergic depolarization cooperates with NMDARs (N-methyl-D-aspartate receptors) to drive spontaneous synchronous activity (SSA) that is fundamentally important for developing neuronal networks. Although GABAergic depolarization is known to assist in the activation of NMDARs during development, the subcellular localization of NMDARs relative to GABAergic synapses is still unknown. Here, we investigated the subcellular distribution of NMDARs in association with GABAergic synapses at the developmental stage when SSA is most prominent in mice. Using multiple immunofluorescent labeling and confocal laser-scanning microscopy in the developing mouse hippocampus, we found that NMDARs were associated with both glutamatergic and GABAergic synapses at postnatal day 6-7 and we observed a direct colocalization of GABA(A)- and NMDA-receptor labeling in GABAergic synapses. Electron microscopy of preembedding immunogold-immunoperoxidase reactions confirmed that GluN1, GluN2A and GluN2B NMDAR subunits were all expressed in glutamatergic and GABAergic synapses postsynaptically. Finally, quantitative post-embedding immunogold labeling revealed that the density of NMDARs was 3 times higher in glutamatergic than in GABAergic synapses. Since GABAergic synapses were larger, there was little difference in the total number of NMDA receptors in the two types of synapses. In addition, receptor density in synapses was substantially higher than extrasynaptically. These data can provide the neuroanatomical basis of a new interpretation of previous physiological data regarding the GABA(A)R-NMDAR cooperation during early development. We suggest that during SSA, synaptic GABA(A)R-mediated depolarization assists NMDAR activation right inside GABAergic synapses and this effective spatial cooperation of receptors and local change of membrane potential will reach developing glutamatergic synapses with a higher probability and efficiency even further away on the dendrites. This additional level of cooperation that operates within the depolarizing GABAergic synapse, may also allow its own modification triggered by Ca2+-influx through the NMDA receptors.
  • Hiroaki Yaguchi, Fumihiko Okumura, Hidehisa Takahashi, Takahiro Kano, Hiroyuki Kameda, Motokazu Uchigashima, Shinya Tanaka, Masahiko Watanabe, Hidenao Sasaki, Shigetsugu Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 287 (15) 12050 - 12059 0021-9258 2012/04 [Refereed][Not invited]
     
    Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis.
  • Hisako Nakayama, Taisuke Miyazaki, Kazuo Kitamura, Kouichi Hashimoto, Yuchio Yanagawa, Kunihiko Obata, Kenji Sakimura, Masahiko Watanabe, Masanobu Kano
    NEURON 74 (2) 384 - 396 0896-6273 2012/04 [Refereed][Not invited]
     
    Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABAA receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16.
  • Daiki D. Horikawa, Ayami Yamaguchi, Tetsuya Sakashita, Daisuke Tanaka, Nobuyuki Hamada, Fumiko Yukuhiro, Hirokazu Kuwahara, Takekazu Kunieda, Masahiko Watanabe, Yuichi Nakahara, Seiichi Wada, Tomoo Funayama, Chihiro Katagiri, Seigo Higashi, Shin-Ichi Yokobori, Mikinori Kuwabara, Lynn J. Rothschild, Takashi Okuda, Hirofumi Hashimoto, Yasuhiko Kobayashi
    ASTROBIOLOGY 12 (4) 283 - 289 1531-1074 2012/04 [Refereed][Not invited]
     
    Tardigrades are tiny (less than 1 mm in length) invertebrate animals that have the potential to survive travel to other planets because of their tolerance to extreme environmental conditions by means of a dry ametabolic state called anhydrobiosis. While the tolerance of adult tardigrades to extreme environments has been reported, there are few reports on the tolerance of their eggs. We examined the ability of hydrated and anhydrobiotic eggs of the tardigrade Ramazzottius varieornatus to hatch after exposure to ionizing irradiation (helium ions), extremely low and high temperatures, and high vacuum. We previously reported that there was a similar pattern of tolerance against ionizing radiation between hydrated and anhydrobiotic adults. In contrast, anhydrobiotic eggs (50% lethal dose; 1690 Gy) were substantially more radioresistant than hydrated ones (50% lethal dose; 509 Gy). Anhydrobiotic eggs also have a broader temperature resistance compared with hydrated ones. Over 70% of the anhydrobiotic eggs treated at either -196 degrees C or +50 degrees C hatched successfully, but all the hydrated eggs failed to hatch. After exposure to high-vacuum conditions (5.3 x 10(-4) Pa to 6.2 x 10(-5) Pa), the hatchability of the anhydrobiotic eggs was comparable to that of untreated control eggs.
  • Claire L. Padgett, Arnaud L. Lalive, Kelly R. Tan, Miho Terunuma, Michaelanne B. Munoz, Menelas N. Pangalos, Jose Martinez-Hernandez, Masahiko Watanabe, Stephen J. Moss, Rafael Lujan, Christian Luescher, Paul A. Slesinger
    NEURON 73 (5) 978 - 989 0896-6273 2012/03 [Refereed][Not invited]
     
    Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABABR-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.
  • Ryoji Fukabori, Kana Okada, Kayo Nishizawa, Nobuyuki Kai, Kenta Kobayashi, Motokazu Uchigashima, Masahiko Watanabe, Yuji Tsutsui, Kazuto Kobayashi
    EUROPEAN JOURNAL OF NEUROSCIENCE 35 (5) 784 - 797 0953-816X 2012/03 [Refereed][Not invited]
     
    The dorsal striatum in the basal ganglia circuitry is a principal structure that mediates the acquisition and performance of instrumental learning. The projections from the dorsal striatum are composed of two subpopulations of medium spiny neurons that constitute the direct and indirect pathways. The mechanism by which these striatal projections control the learning processes of instrumental actions remains unknown. We addressed the behavioral role of the striatal direct (striatonigral) pathway in the performance of visual discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D1 receptor in mice resulted in a moderate level of elimination of the striatonigral pathway. Targeting of the neural pathway from the whole region of the dorsal striatum lengthened the response time but did not affect the accuracy of response selection in a two-choice reaction time task dependent on light stimulus. This lengthened motor response was induced early in the test sessions and was gradually restored to normal levels during repetitive sessions. In addition, subregion-specific pathway targeting revealed that the delay in learned motor response was generated by the elimination of the striatonigral pathway arising from the dorsomedial striatum but not from the dorsolateral striatum. Our findings indicate that the striatonigral pathway, in particular from the dorsomedial striatum, contributes to the regulation of response time in the execution of visual discrimination. The restoration of motor response deficits during repetitive sessions suggests the presence of a mechanism by which the response facilitation is acquired through continuation of learning despite the removal of the striatonigral pathway.
  • Valentina Chiodi, Motokazu Uchigashima, Sarah Beggiato, Antonella Ferrante, Monica Armida, Alberto Martire, Rosa Luisa Potenza, Luca Ferraro, Sergio Tanganelli, Masahiko Watanabe, Maria Rosaria Domenici, Patrizia Popoli
    NEUROBIOLOGY OF DISEASE 45 (3) 983 - 991 0969-9961 2012/03 [Refereed][Not invited]
     
    Cannabinoid CB1 receptors (CB1Rs) are known to be downregulated in patients and in animal models of Huntington's disease (HD). However, the functional meaning of this reduction, if any, is still unclear. Here, the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN) were investigated on striatal synaptic transmission and on glutamate and GABA release in symptomatic R6/2 mice, a genetic model of HD. The expression levels of CB1Rs in glutamatergic and GABAergic synapses were also evaluated. We found that in R6/2 mice, WIN effects on synaptic transmission and glutamate release were significantly increased with respect to wild type mice. On the contrary, a decrease in WIN-induced reduction of GABA release was found in R6/2 versus WT mice. The expression of CB1Rs in GABAergic neurons was drastically reduced, while CB1Rs levels in glutamatergic neurons were unchanged. These results demonstrate that the expression and functionality of CB1Rs are differentially affected in GABAergic and glutamatergic neurons in R6/2 mice. As a result, the balance between CB1Rs expressed by the two neuronal populations and, thus, the net effect of CB1R stimulation, is profoundly altered in HD mice. (C) 2011 Elsevier Inc. All rights reserved.
  • The interaction of the cannabinoidergic and serotonergic system modulating synaptic transmission in the basal amygdala
    Yoshida, T, Izumi, T, Ohmura, Y, Watanabe, M, Yoshioka, M
    Journal of Pharmacological Sciences 118 179P - 179P 1347-8613 2012 [Refereed][Not invited]
  • Inositol 1,4,5-trisphosphate signaling maintains the activity of glutamate uptake in Bergmann glia
    Okubo, Y, Mashimo, M, Yamazawa, T, Yamasaki, M, Watanabe, M, Murayama, T, Lino, M
    Journal of Pharmacological Sciences 118 102P - 102P 1347-8613 2012 [Refereed][Not invited]
  • Taisuke Miyazaki, Miwako Yamasaki, Kouichi Hashimoto, Maya Yamazaki, Manabu Abe, Hiroshi Usui, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 32 (4) 1311 - 1328 0270-6474 2012/01 [Refereed][Not invited]
     
    In the adult cerebellum, each Purkinje cell (PC) is innervated by a single climbing fiber (CF) in proximal dendrites and 10(5)-10(6) parallel fibers (PFs) in distal dendrites. This organized wiring is established postnatally through heterosynaptic competition between PFs and CFs and homosynaptic competition among multiple CFs. Using PC-specific Ca(v)2.1 knock-out mice (PC-Ca(v)2.1 KO mice), we have demonstrated recently that postsynaptic Ca(v)2.1 plays a key role in the homosynaptic competition by promoting functional strengthening and dendritic translocation of single "winner" CFs. Here, we report that Ca(v)2.1 in PCs, but not in granule cells, is also essential for the heterosynaptic competition. In PC-Ca(v)2.1 KO mice, the extent of CF territory was limited to the soma and basal dendrites, whereas PF territory was expanded reciprocally. Consequently, the proximal somatodendritic domain of PCs displayed hyperspiny transformation and fell into chaotic innervation by multiple CFs and numerous PFs. PC-Ca(v)2.1 KO mice also displayed patterned degeneration of PCs, which occurred preferentially in aldolase C/zebrin II-negative cerebellar compartments. Furthermore, the mutually complementary expression of phospholipase C beta 3 (PLC beta 3) and PLC beta 4 was altered such that their normally sharp boundary was blurred in the PCs of PC-Cav2.1 KO mice. This blurring was caused by an impaired posttranscriptional downregulation of PLC beta 3 in PLC beta 4-dominant PCs during the early postnatal period. A similar alteration was noted in the banded expression of the glutamate transporter EAAT4 in PC-Cav2.1 KO mice. Therefore, Cav2.1 in PCs is essential for competitive synaptic wiring, cell survival, and the establishment of precise boundaries and reciprocity of biochemical compartments in PCs.
  • Sho Kakizawa, Toshiko Yamazawa, Yili Chen, Akihiro Ito, Takashi Murayama, Hideto Oyamada, Nagomi Kurebayashi, Osamu Sato, Masahiko Watanabe, Nozomu Mori, Katsuji Oguchi, Takashi Sakurai, Hiroshi Takeshima, Nobuhito Saito, Masamitsu Iino
    EMBO JOURNAL 31 (2) 417 - 428 0261-4189 2012/01 [Refereed][Not invited]
     
    Mobilization of intracellular Ca2+ stores regulates a multitude of cellular functions, but the role of intracellular Ca2+ release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca2+ release from intracellular stores of central neurons, and thereby promote prolonged Ca2+ signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca2+ release. NO-induced Ca2+ release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain. The EMBO Journal (2012) 31, 417-428. doi:10.1038/emboj.2011.386; Published online 28 October 2011
  • Duane Allen, Shin Nakayama, Masayuki Kuroiwa, Takaaki Nakano, Julie Palmateer, Yasuharu Kosaka, Carmen Ballesteros, Masahiko Watanabe, Chris T. Bond, Rafael Lujan, James Maylie, John P. Adelman, Paco S. Herson
    JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 31 (12) 2302 - 2312 0271-678X 2011/12 [Refereed][Not invited]
     
    In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca2+-activated K+ channels type 2 (SK2 channels) provides a negative feedback on N-methyl-D-aspartate receptors (NMDARs), reestablishing Mg2+ block that reduces Ca2+ influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2302-2312; doi:10.1038/jcbfm.2011.90; published online 29 June 2011
  • Laura Fernandez-Alacid, Masahiko Watanabe, Elek Molnar, Kevin Wickman, Rafael Lujan
    EUROPEAN JOURNAL OF NEUROSCIENCE 34 (11) 1724 - 1736 0953-816X 2011/12 [Refereed][Not invited]
     
    G protein-gated inwardly-rectifying K+ (GIRK/family 3 of inwardly-rectifying K+) channels are coupled to neurotransmitter action and can play important roles in modulating neuronal excitability. We investigated the temporal and spatial expression of GIRK1, GIRK2 and GIRK3 subunits in the developing and adult brain of mice and rats using biochemical, immunohistochemical and immunoelectron microscopic techniques. At all ages analysed, the overall distribution patterns of GIRK1-3 were very similar, with high expression levels in the neocortex, cerebellum, hippocampus and thalamus. Focusing on the hippocampus, histoblotting and immunohistochemistry showed that GIRK1-3 protein levels increased with age, and this was accompanied by a shift in the subcellular localization of the subunits. Early in development (postnatal day 5), GIRK subunits were predominantly localized to the endoplasmic reticulum in the pyramidal cells, but by postnatal day 60 they were mostly found along the plasma membrane. During development, GIRK1 and GIRK2 were found primarily at postsynaptic sites, whereas GIRK3 was predominantly detected at presynaptic sites. In addition, GIRK1 and GIRK2 expression on the spine plasma membrane showed identical proximal-to-distal gradients that differed from GIRK3 distribution. Furthermore, although GIRK1 was never found within the postsynaptic density (PSD), the level of GIRK2 in the PSD progressively increased and GIRK3 did not change in the PSD during development. Together, these findings shed new light on the developmental regulation and subcellular diversity of neuronal GIRK channels, and support the contention that distinct subpopulations of GIRK channels exert separable influences on neuronal excitability. The ability to selectively target specific subpopulations of GIRK channels may prove effective in the treatment of disorders of excitability.
  • Duane Allen, Shin Nakayama, Masayuki Kuroiwa, Takaaki Nakano, Julie Palmateer, Yasuharu Kosaka, Carmen Ballesteros, Masahiko Watanabe, Chris T. Bond, Rafael Lujan, James Maylie, John P. Adelman, Paco S. Herson
    JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 31 (12) 2302 - 2312 0271-678X 2011/12 [Refereed][Not invited]
     
    In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca2+-activated K+ channels type 2 (SK2 channels) provides a negative feedback on N-methyl-D-aspartate receptors (NMDARs), reestablishing Mg2+ block that reduces Ca2+ influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2302-2312; doi:10.1038/jcbfm.2011.90; published online 29 June 2011
  • Masahiko Watanabe, Masanobu Kano
    EUROPEAN JOURNAL OF NEUROSCIENCE 34 (10) 1697 - 1710 0953-816X 2011/11 [Refereed][Not invited]
     
    Innervation of Purkinje cells (PCs) by multiple climbing fibers (CFs) is refined into mono-innervation during the first three postnatal weeks of rodents lives. In this review article, we will integrate the current knowledge on developmental process and mechanisms of CF synapse elimination. In the creeper stage of CF innervation (postnatal day 0 (P0)similar to), CFs creep among PC somata to form transient synapses on immature dendrites. In the pericellular nest stage (P5 similar to), CFs densely surround and innervate PC somata. CF innervation is then displaced to the apical portion of PC somata in the capuchon stage (P9 similar to), and translocate to dendrites in the dendritic (P12 similar to) stage. Along with the developmental changes in CF wiring, functional and morphological distinctions become larger among CF inputs. PCs are initially innervated by more than five CFs with similar strengths (similar to P3). During P37 only a single CF is selectively strengthened (functional differentiation), and it undergoes dendritic translocation from P9 on (dendritic translocation). Following the functional differentiation, perisomatic CF synapses are eliminated nonselectively; this proceeds in two distinct phases. The early phase (P711) is conducted independently of parallel fiber (PF)PC synapse formation, while the late phase (P1217) critically depends on it. The P/Q-type voltage-dependent Ca2+ channel in PCs triggers selective strengthening of single CF inputs, promotes dendritic translocation of the strengthened CFs, and drives the early phase of CF synapse elimination. In contrast, the late phase is mediated by the mGluR1GaqPLC beta 4PKC signaling cascade in PCs driven at PFPC synapses, whose structural connectivity is stabilized and maintained by the GluRd2Cbln1neurexin system.
  • Shigeki Kato, Masahito Kuramochi, Kenta Kobayashi, Ryoji Fukabori, Kana Okada, Motokazu Uchigashima, Masahiko Watanabe, Yuji Tsutsui, Kazuto Kobayashi
    JOURNAL OF NEUROSCIENCE 31 (47) 17169 - 17179 0270-6474 2011/11 [Refereed][Not invited]
     
    The dorsal striatum receives converging excitatory inputs from diverse brain regions, including the cerebral cortex and the intralaminar/midline thalamic nuclei, and mediates learning processes contributing to instrumental motor actions. However, the roles of each striatal input pathway in these learning processes remain uncertain. We developed a novel strategy to target specific neural pathways and applied this strategy for studying behavioral roles of the pathway originating from the parafascicular nucleus (PF) and projecting to the dorsolateral striatum. A highly efficient retrograde gene transfer vector encoding the recombinant immunotoxin (IT) receptor was injected into the dorsolateral striatum in mice to express the receptor in neurons innervating the striatum. IT treatment into the PF of the vector-injected animals caused a selective elimination of neurons of the PF-derived thalamostriatal pathway. The elimination of this pathway impaired the response selection accuracy and delayed the motor response in the acquisition of a visual cue-dependent discrimination task. When the pathway elimination was induced after learning acquisition, it disturbed the response accuracy in the task performance with no apparent change in the response time. The elimination did not influence spontaneous locomotion, methamphetamine-induced hyperactivity, and motor skill learning that demand the function of the dorsal striatum. These results demonstrate that thalamostriatal projection derived from the PF plays essential roles in the acquisition and execution of discrimination learning in response to sensory stimulus. The temporal difference in the pathway requirement for visual discrimination suggests a stage-specific role of thalamostriatal pathway in the modulation of response time of learned motor actions.
  • Maria R. Karlocai, Kinga Toth, Masahiko Watanabe, Catherine Ledent, Gabor Juhasz, Tamas F. Freund, Zsofia Magloczky
    PLOS ONE 6 (11) 1932-6203 2011/11 [Refereed][Not invited]
     
    The endocannabinoid system plays a central role in retrograde synaptic communication and may control the spread of activity in an epileptic network. Using the pilocarpine model of temporal lobe epilepsy we examined the expression pattern of the Type 1 cannabinoid receptor (CB1-R) in the hippocampi of CD1 mice at survival times of 2 hours, 1 day, 3 days and 2 months (acute, latent and chronic phases). Based on the behavioral signs of the acute seizures, animals were classified as "weakly'' or "strongly'' epileptic using the modified Racine scale. Mice of the weak group had mild seizures, whereas seizures in the strong group were frequent with intense motor symptoms and the majority of these animals developed sclerosis in the chronic phase. In control samples the most intense staining of CB1-R-positive fibers was found in the molecular layer of the dentate gyrus and in str. pyramidale of the cornu Ammonis. In weak animals no significant changes were seen at any survival time compared to controls. In strong animals, however, in the acute phase, a massive reduction in CB1-R-stained terminals occurred in the hippocampus. In the latent phase CB1-R immunoreactivity gradually recovered. In the chronic phase, CB1-immunostaining in sclerotic samples was stronger throughout the hippocampus. Quantitative electron microscopic analysis showed an increase in the number of CB1-R-positive terminals in the dentate gyrus. Moreover, the number of immunogold particles significantly increased in GABAergic terminals. Our results suggest a proconvulsive downregulation of CB1 receptors in the acute phase most probably due to receptor internalization, followed by compensatory upregulation and sprouting in the chronic phase of epilepsy. In conclusion, the changes in CB1 receptor expression pattern revealed in this study are associated with the severity of hippocampal injury initiated by acute seizures that ultimately leads to sclerosis in the vulnerable regions in the chronic phase.
  • Ryoichi Ichikawa, Miwako Yamasaki, Taisuke Miyazaki, Kohtarou Konno, Kouichi Hashimoto, Haruyuki Tatsumi, Yoshiro Inoue, Masanobu Kano, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 31 (47) 16916 - 16927 0270-6474 2011/11 [Refereed][Not invited]
     
    In early postnatal development, perisomatic innervation of cerebellar Purkinje cells (PCs) switches from glutamatergic climbing fibers (CFs) to GABAergic basket cell fibers (BFs). Here we examined the switching process in C57BL/6 mice. At postnatal day 7 (P7), most perisomatic synapses were formed by CFs on to somatic spines. The density of CF-spine synapses peaked at P9, when pericellular nest around PCs by CFs was most developed, and CF-spine synapses constituted 88% of the total perisomatic synapses. Thereafter, CF-spine synapses dropped to 63% at P12, 6% at P15, and <1% at P20, whereas BF synapses increased reciprocally. During the switching period, a substantial number of BF synapses existed as BF-spine synapses (37% of the total perisomatic synapses at P15), and free spines surrounded by BFs or Bergmann glia also emerged. By P20, BF-spine synapses and free spines virtually disappeared, and BF-soma synapses became predominant (88%), thus attaining the adult pattern of perisomatic innervation. Parallel with the presynaptic switching, postsynaptic receptor phenotype also switched from glutamatergic to GABAergic. In the active switching period, particularly at P12, fragmental clusters of AMPA-type glutamate receptor were juxtaposed with those of GABA(A) receptor. When examined with serial ultrathin sections, immunogold labeling for glutamate and GABA(A) receptors was often clustered beneath single BF terminals. These results suggest that a considerable fraction of somatic spines is succeeded from CFs to BFs and Bergmann glia in the early postnatal period, and that the switching of postsynaptic receptor phenotypes mainly proceeds under the coverage of BF terminals.
  • Pablo R. Brumovsky, David R. Robinson, Jun-Ho La, Kim B. Seroogy, Kerstin H. Lundgren, Kathryn M. Albers, Michael E. Kiyatkin, Rebecca P. Seal, Robert H. Edwards, Masahiko Watanabe, Tomas Hokfelt, G. F. Gebhart
    JOURNAL OF COMPARATIVE NEUROLOGY 519 (16) 3346 - 3366 0021-9967 2011/11 [Refereed][Not invited]
     
    Vesicular glutamate transporters (VGLUTs) have been extensively studied in various neuronal systems, but their expression in visceral sensory and autonomic neurons remains to be analyzed in detail. Here we studied VGLUTs type 1 and 2 (VGLUT(1) and VGLUT(2), respectively) in neurons innervating the mouse colorectum. Lumbosacral and thoracolumbar dorsal root ganglion (DRG), lumbar sympathetic chain (LSC), and major pelvic ganglion (MPG) neurons innervating the colorectum of BALB/C mice were retrogradely traced with Fast Blue, dissected, and processed for immunohistochemistry. Tissue from additional naive mice was included. Previously characterized antibodies against VGLUT(1), VGLUT(2), and calcitonin gene-related peptide (CGRP) were used. Riboprobe in situ hybridization, using probes against VGLUT(1) and VGLUT(2), was also performed. Most colorectal DRG neurons expressed VGLUT(2) and often colocalized with CGRP. A smaller percentage of neurons expressed VGLUT(1). VGLUT(2)-immunoreactive (IR) neurons in the MPG were rare. Abundant VGLUT(2)-IR nerves were detected in all layers of the colorectum; VGLUT(1)-IR nerves were sparse. A subpopulation of myenteric plexus neurons expressed VGLUT2 protein and mRNA, but VGLUT1 mRNA was undetectable. In conclusion, we show 1) that most colorectal DRG neurons express VGLUT(2), and to a lesser extent, VGLUT(1); 2) abundance of VGLUT2-IR fibers innervating colorectum; and 3) a subpopulation of myenteric plexus neurons expressing VGLUT(2). Altogether, our data suggests a role for VGLUT(2) in colorectal glutamatergic neurotransmission, potentially influencing colorectal sensitivity and motility. J. Comp. Neurol. 519:3346-3366, 2011. (C) 2011 Wiley-Liss, Inc.
  • T. C. P. Sardella, E. Polgar, M. Watanabe, A. J. Todd
    NEUROSCIENCE 192 708 - 720 0306-4522 2011/09 [Refereed][Not invited]
     
    Nitric oxide produced by neuronal nitric oxide synthase (nNOS) in the spinal cord is required for development of hyperalgesia in inflammatory and neuropathic pain states. nNOS is expressed by some dorsal horn neurons, and an early study that used a histochemical method to identify these cells suggested that they were mainly inhibitory interneurons. We have carried out a quantitative analysis of nNOS-immunoreactivity in laminae I-III of the rat dorsal horn, to determine the proportion of inhibitory and excitatory neurons and axonal boutons that express the protein. nNOS was present in similar to 5% of neurons in laminae I and III, and 18% of those in lamina II. Although most cells with strong nNOS immunostaining were GABA-immunoreactive, two-thirds of the nNOS-positive cells in lamina II and half of those in lamina III were not GABAergic, and some of these expressed protein kinase C gamma (PKC gamma). We estimate that nNOS is present in 17-19% of the inhibitory interneurons in laminae I-II, and 6% of those in lamina III. However, our results suggest that nNOS is also expressed at a relatively low level by a significant proportion (similar to 17%) of excitatory interneurons in lamina II. nNOS was seldom seen in boutons that contained vesicular glutamate transporter 2, which is expressed by excitatory interneurons, but was co-localised with the vesicular GABA transporter (VGAT, a marker for GABAergic and glycinergic axons). nNOS was detected in 13% of VGAT boutons in lamina I and in 7-8% of those in laminae II-III. However, it was only found in 2-4% of the VGAT boutons that were presynaptic to PKC gamma-expressing interneurons in this region. These results indicate that nNOS is more widely expressed than previously thought, being present in both inhibitory and excitatory neurons. They provide further evidence that axons of neurochemically defined populations of inhibitory interneuron are selective in their post-synaptic targets. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Thomas C. P. Sardella, Erika Polgar, Francesca Garzillo, Takahiro Furuta, Takeshi Kaneko, Masahiko Watanabe, Andrew J. Todd
    MOLECULAR PAIN 7 1744-8069 2011/09 [Refereed][Not invited]
     
    Background: The opioid peptide dynorphin is expressed by certain neurons in the superficial dorsal horn of the spinal cord, but little is known about the types of cell that contain dynorphin. In this study, we have used an antibody against the dynorphin precursor preprodynorphin (PPD), to reveal the cell bodies and axons of dynorphin-expressing neurons in the rat spinal cord. The main aims were to estimate the proportion of neurons in each of laminae I-III that express dynorphin and to determine whether they are excitatory or inhibitory neurons. Results: PPD-immunoreactive cells were concentrated in lamina I and the outer part of lamina II (IIo), where they constituted 17% and 8%, respectively, of all neurons. Around half of those in lamina I and 80% of those in lamina II were GABA-immunoreactive. We have previously identified four non-overlapping neurochemical populations of inhibitory interneurons in this region, defined by the presence of neuropeptide Y, galanin, parvalbumin and neuronal nitric oxide synthase. PPD co-localised extensively with galanin in both cell bodies and axons, but rarely or not at all with the other three markers. PPD was present in around 4% of GABAergic boutons (identified by the presence of the vesicular GABA transporter) in laminae I-II. Conclusions: These results show that most dynorphin-expressing cells in the superficial dorsal horn are inhibitory interneurons, and that they largely correspond to the population that is defined by the presence of galanin. We estimate that dynorphin is present in similar to 32% of inhibitory interneurons in lamina I and 11% of those in lamina II. Since the proportion of GABAergic boutons that contain PPD in these laminae was considerably lower than this, our findings suggest that these neurons may generate relatively small axonal arborisations.
  • Mari Hondo, Naoki Furutani, Miwako Yamasaki, Masahiko Watanabe, Takeshi Sakurai
    PLOS ONE 6 (9) 1932-6203 2011/09 [Refereed][Not invited]
     
    Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation.
  • Mari Hondo, Naoki Furutani, Miwako Yamasaki, Masahiko Watanabe, Takeshi Sakurai
    PLOS ONE 6 (9) 1932-6203 2011/09 [Refereed][Not invited]
     
    Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation.
  • Pablo R. Brumovsky, Kim B. Seroogy, Kerstin H. Lundgren, Masahiko Watanabe, Tomas Hokfelt, G. F. Gebhart
    EXPERIMENTAL NEUROLOGY 230 (2) 258 - 272 0014-4886 2011/08 [Refereed][Not invited]
     
    Glutamate is the main excitatory neurotransmitter in the nervous system, including in primary afferent neurons. However, to date a glutamatergic phenotype of autonomic neurons has not been described. Therefore, we explored the expression of vesicular glutamate transporter (VGLUT) types 1,2 and 3 in lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) of naive BALB/C mice, as well as after pelvic nerve axotomy (PNA), using immunohistochemistry and in situ hybridization. Colocalization with activating transcription factor-3 (ATF-3), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT) and calcitonin gene-related peptide was also examined. Sham-PNA, sciatic nerve axotomy (SNA) or naive mice were included. In naive mice, VGLUT(2)-like immunoreactivity (LI) was only detected in fibers and varicosities in LSC and MPG; no ATF-3-immunoreactive (IR) neurons were visible. In contrast, PNA induced upregulation of VGLUT(2) protein and transcript, as well as of ATF-3-LI in subpopulations of LSC neurons. Interestingly, VGLUT(2)-IR LSC neurons coexpressed ATF-3, and often lacked the noradrenergic marker TH. SNA only increased VGLUT(2) protein and transcript in scattered LSC neurons. Neither PNA nor SNA upregulated VGLUT(2) in MPG neurons. We also found perineuronal baskets immunoreactive either for VGLUT(2) or the acetylcholinergic marker VAChT in non-PNA MPGs, usually around TH-IR neurons. VGLUT(1)-LI was restricted to some varicosities in MPGs, was absent in LSCs, and remained largely unaffected by PNA or SNA. This was confirmed by the lack of expression of VGLUT(1) or VGLUT(3) mRNAs in LSCs, even after PNA or SNA. Taken together, axotomy of visceral and non-visceral nerves results in a glutamatergic phenotype of some LSC neurons. In addition, we show previously non-described MPG perineuronal glutamatergic baskets. (C) 2011 Elsevier Inc. All rights reserved.
  • Pablo R. Brumovsky, Kim B. Seroogy, Kerstin H. Lundgren, Masahiko Watanabe, Tomas Hokfelt, G. F. Gebhart
    EXPERIMENTAL NEUROLOGY 230 (2) 258 - 272 0014-4886 2011/08 [Refereed][Not invited]
     
    Glutamate is the main excitatory neurotransmitter in the nervous system, including in primary afferent neurons. However, to date a glutamatergic phenotype of autonomic neurons has not been described. Therefore, we explored the expression of vesicular glutamate transporter (VGLUT) types 1,2 and 3 in lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) of naive BALB/C mice, as well as after pelvic nerve axotomy (PNA), using immunohistochemistry and in situ hybridization. Colocalization with activating transcription factor-3 (ATF-3), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT) and calcitonin gene-related peptide was also examined. Sham-PNA, sciatic nerve axotomy (SNA) or naive mice were included. In naive mice, VGLUT(2)-like immunoreactivity (LI) was only detected in fibers and varicosities in LSC and MPG; no ATF-3-immunoreactive (IR) neurons were visible. In contrast, PNA induced upregulation of VGLUT(2) protein and transcript, as well as of ATF-3-LI in subpopulations of LSC neurons. Interestingly, VGLUT(2)-IR LSC neurons coexpressed ATF-3, and often lacked the noradrenergic marker TH. SNA only increased VGLUT(2) protein and transcript in scattered LSC neurons. Neither PNA nor SNA upregulated VGLUT(2) in MPG neurons. We also found perineuronal baskets immunoreactive either for VGLUT(2) or the acetylcholinergic marker VAChT in non-PNA MPGs, usually around TH-IR neurons. VGLUT(1)-LI was restricted to some varicosities in MPGs, was absent in LSCs, and remained largely unaffected by PNA or SNA. This was confirmed by the lack of expression of VGLUT(1) or VGLUT(3) mRNAs in LSCs, even after PNA or SNA. Taken together, axotomy of visceral and non-visceral nerves results in a glutamatergic phenotype of some LSC neurons. In addition, we show previously non-described MPG perineuronal glutamatergic baskets. (C) 2011 Elsevier Inc. All rights reserved.
  • Devinder Arora, Matthew Hearing, Desirae M. Haluk, Kelsey Mirkovic, Ana Fajardo-Serrano, Martin W. Wessendorf, Masahiko Watanabe, Rafael Lujan, Kevin Wickman
    JOURNAL OF NEUROSCIENCE 31 (34) 12251 - 12257 0270-6474 2011/08 [Refereed][Not invited]
     
    Enhanced glutamatergic neurotransmission in dopamine (DA) neurons of the ventral tegmental area (VTA), triggered by a single cocaine injection, represents an early adaptation linked to the more enduring effects of abused drugs that characterize addiction. Here, we examined the impact of in vivo cocaine exposure on metabotropic inhibitory signaling involving G-protein-gated inwardly rectifying K+ (Girk) channels in VTA DA neurons. Somatodendritic Girk currents evoked by the GABA(B) receptor (GABA(B)R) agonist baclofen were diminished in a dose-dependent manner in mice given a single cocaine injection. This adaptation persisted for 3-4 d, was specific for DA neurons of the VTA, and occurred in parallel with an increase in spontaneous glutamatergic neurotransmission. No additional suppression of GABA(B)R-Girk signaling was observed following repeated cocaine administration. While total Girk2 and GABA(B)R1 mRNA and protein levels were unaltered by cocaine exposure in VTA DA neurons, the cocaine-induced decrease in GABA(B)R-Girk signaling correlated with a reduction in Girk2-containing channels at the plasma membrane in VTA DA neurons. Systemic pretreatment with sulpiride, but not SCH23390 (7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol), prevented the cocaine-induced suppression of GABA(B)R-Girk signaling, implicating D-2/3 DA receptor activation in this adaptation. The acute cocaine-induced weakening of somatodendritic Girk signaling complements the previously demonstrated cocaine-induced strengthening of glutamatergic neurotransmission, likely contributing to enhanced output of VTA DA neurons during the early stages of addiction.
  • Christoph Straub, David L. Hunt, Miwako Yamasaki, Kwang S. Kim, Masahiko Watanabe, Pablo E. Castillo, Susumu Tomita
    NATURE NEUROSCIENCE 14 (7) 866 - U83 1097-6256 2011/07 [Refereed][Not invited]
     
    Ionotropic glutamate receptors principally mediate fast excitatory transmission in the brain. Among the three classes of ionotropic glutamate receptors, kainate receptors (KARs) have a unique brain distribution, which has been historically defined by (3)H-radiolabeled kainate binding. Compared with recombinant KARs expressed in heterologous cells, synaptic KARs exhibit characteristically slow rise-time and decay kinetics. However, the mechanisms responsible for these distinct KAR properties remain unclear. We found that both the high-affinity binding pattern in the mouse brain and the channel properties of native KARs are determined by the KAR auxiliary subunit Neto1. Through modulation of agonist binding affinity and off-kinetics of KARs, but not trafficking of KARs, Neto1 determined both the KAR high-affinity binding pattern and the distinctively slow kinetics of postsynaptic KARs. By regulating KAR excitatory postsynaptic current kinetics, Neto1 can control synaptic temporal summation, spike generation and fidelity.
  • Yamato Hida, Masahiro Fukaya, Akari Hagiwara, Maki Deguchi-Tawarada, Toshinori Yoshioka, Isao Kitajima, Eiji Inoue, Masahiko Watanabe, Toshihisa Ohtsuka
    JOURNAL OF BIOCHEMISTRY 149 (6) 693 - 700 0021-924X 2011/06 [Refereed][Not invited]
     
    The planar cell polarity (PCP) protein, Prickle (Pk), is conserved in invertebrates and vertebrates, and regulates cellular morphogenesis and movement. Vertebrate Pk consists of at least two family members, Pk1 and Pk2, both of which are expressed in the brain; however, their localization and function at synapses remain elusive. Here, we show that Pk2 is expressed mainly in the adult brain and is tightly associated with the postsynaptic density (PSD) fraction obtained by subcellular fractionation. In primary cultured rat hippocampal neurons, Pk2 is colocalized with PSD-95 and synaptophysin at synapses. Moreover, immunoelectron microcopy shows that Pk2 is localized at the PSD of asymmetric synapses in the hippocampal CA1 region. Biochemical assays identified that Pk2 forms a complex with PSD proteins including PSD-95 and NMDA receptor subunits via the direct binding to the C-terminal guanylate kinase domain of PSD-95. These results indicate that Pk2 is a novel PSD protein that interacts with PSD-95 and NMDA receptors through complex formations in the brain.
  • Duane Allen, Chris T. Bond, Rafael Lujan, Carmen Ballesteros-Merino, Mike T. Lin, Kang Wang, Nathan Klett, Masahiko Watanabe, Ryuichi Shigemoto, Robert W. Stackman, James Maylie, John P. Adelman
    NATURE NEUROSCIENCE 14 (6) 744 - U344 1097-6256 2011/06 [Refereed][Not invited]
     
    SK2-containing channels are expressed in the postsynaptic density (PSD) of dendritic spines on mouse hippocampal area CA1 pyramidal neurons and influence synaptic responses, plasticity and learning. The Sk2 gene (also known as Kcnn2) encodes two isoforms that differ only in the length of their N-terminal domains. SK2-long (SK2-L) and SK2-short (SK2-S) are coexpressed in CA1 pyramidal neurons and likely form heteromeric channels. In mice lacking SK2-L (SK2-S only mice), SK2-S-containing channels were expressed in the extrasynaptic membrane, but were excluded from the PSD. The SK channel contribution to excitatory postsynaptic potentials was absent in SK2-S only mice and was restored by SK2-L re-expression. Blocking SK channels increased the amount of long-term potentiation induced in area CA1 in slices from wild-type mice but had no effect in slices from SK2-S only mice. Furthermore, SK2-S only mice outperformed wild-type mice in the novel object recognition task. These results indicate that SK2-L directs synaptic SK2-containing channel expression and is important for normal synaptic signaling, plasticity and learning.
  • Kouichi Hashimoto, Mika Tsujita, Taisuke Miyazaki, Kazuo Kitamura, Maya Yamazaki, Hee-Sup Shin, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 (24) 9987 - 9992 0027-8424 2011/06 [Refereed][Not invited]
     
    Neural circuits are initially redundant but rearranged through activity-dependent synapse elimination during postnatal development. This process is crucial for shaping mature neural circuits and for proper brain function. At birth, Purkinje cells (PCs) in the cerebellum are innervated by multiple climbing fibers (CFs) with similar synaptic strengths. During postnatal development, a single CF is selectively strengthened in each PC through synaptic competition, the strengthened single CF undergoes translocation to a PC dendrite, and massive elimination of redundant CF synapses follows. To investigate the cellular mechanisms of this activity-dependent synaptic refinement, we generated mice with PC-selective deletion of the Ca(v)2.1 P/Q-type Ca2+ channel, the major voltage-dependent Ca2+ channel in PCs. In the PC-selective Ca(v)2.1 knockout mice, Ca2+ transients induced by spontaneous CF inputs are markedly reduced in PCs in vivo. Not a single but multiple CFs were equally strengthened in each PC from postnatal day 5 (P5) to P8, multiple CFs underwent translocation to PC dendrites, and subsequent synapse elimination until around P12 was severely impaired. Thus, P/Q-type Ca2+ channels in postsynaptic PCs mediate synaptic competition among multiple CFs and trigger synapse elimination in developing cerebellum.
  • Yamato Hida, Masahiro Fukaya, Akari Hagiwara, Maki Deguchi-Tawarada, Toshinori Yoshioka, Isao Kitajima, Eiji Inoue, Masahiko Watanabe, Toshihisa Ohtsuka
    JOURNAL OF BIOCHEMISTRY 149 (6) 693 - 700 0021-924X 2011/06 [Refereed][Not invited]
     
    The planar cell polarity (PCP) protein, Prickle (Pk), is conserved in invertebrates and vertebrates, and regulates cellular morphogenesis and movement. Vertebrate Pk consists of at least two family members, Pk1 and Pk2, both of which are expressed in the brain; however, their localization and function at synapses remain elusive. Here, we show that Pk2 is expressed mainly in the adult brain and is tightly associated with the postsynaptic density (PSD) fraction obtained by subcellular fractionation. In primary cultured rat hippocampal neurons, Pk2 is colocalized with PSD-95 and synaptophysin at synapses. Moreover, immunoelectron microcopy shows that Pk2 is localized at the PSD of asymmetric synapses in the hippocampal CA1 region. Biochemical assays identified that Pk2 forms a complex with PSD proteins including PSD-95 and NMDA receptor subunits via the direct binding to the C-terminal guanylate kinase domain of PSD-95. These results indicate that Pk2 is a novel PSD protein that interacts with PSD-95 and NMDA receptors through complex formations in the brain.
  • Kouichi Hashimoto, Mika Tsujita, Taisuke Miyazaki, Kazuo Kitamura, Maya Yamazaki, Hee-Sup Shin, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 (24) 9987 - 9992 0027-8424 2011/06 [Refereed][Not invited]
     
    Neural circuits are initially redundant but rearranged through activity-dependent synapse elimination during postnatal development. This process is crucial for shaping mature neural circuits and for proper brain function. At birth, Purkinje cells (PCs) in the cerebellum are innervated by multiple climbing fibers (CFs) with similar synaptic strengths. During postnatal development, a single CF is selectively strengthened in each PC through synaptic competition, the strengthened single CF undergoes translocation to a PC dendrite, and massive elimination of redundant CF synapses follows. To investigate the cellular mechanisms of this activity-dependent synaptic refinement, we generated mice with PC-selective deletion of the Ca(v)2.1 P/Q-type Ca2+ channel, the major voltage-dependent Ca2+ channel in PCs. In the PC-selective Ca(v)2.1 knockout mice, Ca2+ transients induced by spontaneous CF inputs are markedly reduced in PCs in vivo. Not a single but multiple CFs were equally strengthened in each PC from postnatal day 5 (P5) to P8, multiple CFs underwent translocation to PC dendrites, and subsequent synapse elimination until around P12 was severely impaired. Thus, P/Q-type Ca2+ channels in postsynaptic PCs mediate synaptic competition among multiple CFs and trigger synapse elimination in developing cerebellum.
  • Takeshi Izumi, Shuken Boku, Weng Shinmin, Takeshi Inoue, Kotaro Konno, Taku Yamaguchi, Takayuki Yoshida, Machiko Matsumoto, Masahiko Watanabe, Tsukasa Koyama, Mitsuhiro Yoshioka
    JOURNAL OF NEUROSCIENCE RESEARCH 89 (5) 773 - 790 0360-4012 2011/05 [Refereed][Not invited]
     
    The amygdala is one of the crucial brain structures for conditioned fear, in which conditioned stimuli are received by the basolateral nucleus of the amygdala (BLA), inducing a fear reaction via the central nucleus of the amygdala (CeA). Whereas BLA sends glutamatergic projections into CeA, the intercalated nucleus of the amygdala (ITC) sends GABAergic projections into CeA, which is doubly regulated by BLA and ITC. In the present study, we investigated the characteristics of the neural cells activated by retrieval of conditioned fear in BLA and ITC using immunohistochemistry, in situ hybridization, and Western blot analysis of transcription factors and neural cell markers. Because most conditioned fear-induced c-Fos-positive cells in BLA were glutaminase positive and 67-kDa isomer of glutamic acid decarboxylase (GAD67) negative, these cells are speculated to be glutamatergic. Seventy-eight percent of the phosphorylated CREB (pCREB)positive cells were glutaminase double positive and 13% of the pCREB-positive cells were GAD67 double positive, indicating that many of the conditioned fear-induced pCREB-positive cells in BLA were glutamatergic, but at least some of the pCREB-positive cells were GABAergic. These results suggested that CREB phosphorylation was increased both in glutamatergic and in GABAergic neurons, but c-Fos expression was increased mainly in glutamatergic neurons in BLA. CREB phosphorylation but not c-Fos expression in ITC was specifically increased by retrieval of conditioned fear. It is therefore speculated that ITC GABAergic neurons were activated by retrieval of conditioned fear and that transcription factors other than c-Fos were relevant to the activation. (C) 2011 Wiley-Liss, Inc.
  • Davor Stanic, Jan Mulder, Masahiko Watanabe, Tomas Hokfelt
    JOURNAL OF COMPARATIVE NEUROLOGY 519 (7) 1219 - 1257 0021-9967 2011/05 [Refereed][Not invited]
     
    Neuropeptide Y (NPY) is widely expressed in the brain and its biological effects are mediated through a variety of receptors. We examined, using immunohistochemistry, expression of the Y2 receptor (R) protein in the adult mouse brain and its association with NPY and the Y1R, as well as a range of additional neurotransmitters and signaling-related molecules, which previously have not been defined. Our main focus was on the hippocampal formation (HiFo), amygdaloid complex, and hypothalamus, considering the known functions of NPY and the wide expression of NPY, Y1R, and Y2R in these regions. Y2R-like immunoreactivity (-LI) was distributed in nerve fibers/terminal endings throughout the brain axis, without apparent colocalization with NPY or the Y1R. Occasional coexistence between NPY- and Y1R-LI was found in the HiFo. Following colchicine treatment, Y2R-LI accumulated in cell bodies that coexpressed gamma-aminobutyric acid (GABA) in a population of cells in the amygdaloid complex and lateral septal nucleus, but not in the HiFo. Instead, Y2R-positive nerve terminals appeared to surround GABA-immunoreactive (ir) cells in the HiFo and other neuronal populations, e. g., NPY-ir cells in HiFo and tyrosine hydroxylase-ir cells in the hypothalamus. In the HiFo, Y2R-ir mossy fibers coexpressed GABA, glutamic acid decarboxylase 67 and calbindin, and Y2R-LI was found in the same fibers that contained the presynaptic metabotropic glutamate receptor 2, but not together with any of the three vesicular glutamate transporters. Our findings provide further support that Y2R is mostly presynaptic, and that Y2Rs thus have a modulatory role in mediating presynaptic neurotransmitter release. J. Comp. Neurol. 519: 1219-1257, 2011. (C) 2011 Wiley-Liss, Inc.
  • Shunya Suzuki, Keiichi Tamai, Masahiko Watanabe, Masanao Kyuuma, Masao Ono, Kazuo Sugamura, Nobuyuki Tanaka
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 408 (4) 582 - 588 0006-291X 2011/05 [Refereed][Not invited]
     
    Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. AMSH (associated molecule with the SH3 domain of STAM) is a deubiquitination enzyme that participates in the endosomal sorting of several cell-surface molecules. AMSH impairment results in missorted ubiquitinated cargoes in vitro and severe neurodegeneration in vivo, but it is not known how AMSH deficiency causes neuronal damage in the brain. Here, we demonstrate that AMSH(-1-) mice developed ubiquitinated protein accumulations as early as embryonic day 10 (E10), and that severe deposits were present in the brain at postnatal day 8 (P8) and P18. Interestingly, TDP-43 was found to accumulate and colocalize with glial marker-positive cells in the brain. Glutamate receptor and p62 accumulations were also found; these molecules colocalized with ubiquitinated aggregates in the brain. These data suggest that AMSH plays an important role in degrading ubiquitinated proteins and glutamate receptors in vivo. AMSH(-1-) mice provide an animal model for neurodegenerative diseases, which are commonly characterized by the generation of proteinaceous aggregates. (C) 2011 Elsevier Inc. All rights reserved.
  • Motokazu Uchigashima, Maya Yamazaki, Miwako Yamasaki, Asami Tanimura, Kenji Sakimura, Masanobu Kano, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 31 (21) 7700 - 7714 0270-6474 2011/05 [Refereed][Not invited]
     
    2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of neurotransmission in the brain. In the present study, we investigated the 2-AG signaling system at mossy cell (MC)-granule cell (GC) synapses in the mouse dentate gyrus, an excitatory recurrent circuit where endocannabinoids are thought to suppress epileptogenesis. First, we showed by electrophysiology that 2-AG produced by diacylglycerol lipase alpha (DGL alpha) mediated both depolarization-induced suppression of excitation and its enhancement by group I metabotropic glutamate receptor activation at MC-GC synapses, as they were abolished in DGL alpha-knock-out mice. Immunohistochemistry revealed that DGL alpha was enriched in the neck portion of GC spines forming synapses with MC terminals, whereas cannabinoid CB(1) receptors accumulated in the terminal portion of MC axons. On the other hand, the major 2-AG-degrading enzyme, monoacylglycerol lipase (MGL), was absent at MC-GC synapses but was expressed in astrocytes and some inhibitory terminals. Serial electron microscopy clarified that a given GC spine was innervated by a single MC terminal and also contacted nonsynaptically by other MC terminals making synapses with other GC spines in the neighborhood. MGL-expressing elements, however, poorly covered GC spines, amounting to 17% of the total surface of GC spines by astrocytes and 4% by inhibitory terminals. Our findings provide a basis for 2-AG-mediated retrograde suppression of MC-GC synaptic transmission and also suggest that 2-AG released from activated GC spines is readily accessible to nearby MC-GC synapses by escaping from enzymatic degradation. This molecular-anatomical configuration will contribute to adjust network activity in the dentate gyrus after enhanced excitation.
  • Motokazu Uchigashima, Maya Yamazaki, Miwako Yamasaki, Asami Tanimura, Kenji Sakimura, Masanobu Kano, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 31 (21) 7700 - 7714 0270-6474 2011/05 [Refereed][Not invited]
     
    2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of neurotransmission in the brain. In the present study, we investigated the 2-AG signaling system at mossy cell (MC)-granule cell (GC) synapses in the mouse dentate gyrus, an excitatory recurrent circuit where endocannabinoids are thought to suppress epileptogenesis. First, we showed by electrophysiology that 2-AG produced by diacylglycerol lipase alpha (DGL alpha) mediated both depolarization-induced suppression of excitation and its enhancement by group I metabotropic glutamate receptor activation at MC-GC synapses, as they were abolished in DGL alpha-knock-out mice. Immunohistochemistry revealed that DGL alpha was enriched in the neck portion of GC spines forming synapses with MC terminals, whereas cannabinoid CB(1) receptors accumulated in the terminal portion of MC axons. On the other hand, the major 2-AG-degrading enzyme, monoacylglycerol lipase (MGL), was absent at MC-GC synapses but was expressed in astrocytes and some inhibitory terminals. Serial electron microscopy clarified that a given GC spine was innervated by a single MC terminal and also contacted nonsynaptically by other MC terminals making synapses with other GC spines in the neighborhood. MGL-expressing elements, however, poorly covered GC spines, amounting to 17% of the total surface of GC spines by astrocytes and 4% by inhibitory terminals. Our findings provide a basis for 2-AG-mediated retrograde suppression of MC-GC synaptic transmission and also suggest that 2-AG released from activated GC spines is readily accessible to nearby MC-GC synapses by escaping from enzymatic degradation. This molecular-anatomical configuration will contribute to adjust network activity in the dentate gyrus after enhanced excitation.
  • Sheena Y. X. Tiong, Erika Polgar, Josie C. van Kralingen, Masahiko Watanabe, Andrew J. Todd
    MOLECULAR PAIN 7 1744-8069 2011/05 [Refereed][Not invited]
     
    Background: Inhibitory interneurons constitute 30-40% of neurons in laminae I-III and have an important anti-nociceptive role. However, because of the difficulty in classifying them we know little about their organisation. Previous studies have identified 3 non-overlapping groups of inhibitory interneuron, which contain neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin, and have shown that these differ in postsynaptic targets. Some inhibitory interneurons contain galanin and the first aim of this study was to determine whether these form a different population from those containing NPY, nNOS or parvalbumin. We also estimated the proportion of neurons and GABAergic axons that contain galanin in laminae I-III. Results: Galanin cells were concentrated in laminae I-IIo, with few in laminae IIi-III. Galanin showed minimal co-localisation with NPY, nNOS or parvalbumin in laminae I-II, but most galanin-containing cells in lamina III were nNOS-positive. Galanin cells constituted similar to 7%, 3% and 2% of all neurons in laminae I, II and III, and we estimate that this corresponds to 26%, 10% and 5% of the GABAergic neurons in these laminae. However, galanin was only found in similar to 6% of GABAergic boutons in laminae I-IIo, and similar to 1% of those in laminae IIi-III. Conclusions: These results show that galanin, NPY, nNOS and parvalbumin can be used to define four distinct neurochemical populations of inhibitory interneurons. Together with results of a recent study, they suggest that the galanin and NPY populations account for around half of the inhibitory interneurons in lamina I and a quarter of those in lamina II.
  • Shunya Suzuki, Keiichi Tamai, Masahiko Watanabe, Masanao Kyuuma, Masao Ono, Kazuo Sugamura, Nobuyuki Tanaka
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 408 (4) 582 - 588 0006-291X 2011/05 [Refereed][Not invited]
     
    Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. AMSH (associated molecule with the SH3 domain of STAM) is a deubiquitination enzyme that participates in the endosomal sorting of several cell-surface molecules. AMSH impairment results in missorted ubiquitinated cargoes in vitro and severe neurodegeneration in vivo, but it is not known how AMSH deficiency causes neuronal damage in the brain. Here, we demonstrate that AMSH(-1-) mice developed ubiquitinated protein accumulations as early as embryonic day 10 (E10), and that severe deposits were present in the brain at postnatal day 8 (P8) and P18. Interestingly, TDP-43 was found to accumulate and colocalize with glial marker-positive cells in the brain. Glutamate receptor and p62 accumulations were also found; these molecules colocalized with ubiquitinated aggregates in the brain. These data suggest that AMSH plays an important role in degrading ubiquitinated proteins and glutamate receptors in vivo. AMSH(-1-) mice provide an animal model for neurodegenerative diseases, which are commonly characterized by the generation of proteinaceous aggregates. (C) 2011 Elsevier Inc. All rights reserved.
  • Davor Stanic, Jan Mulder, Masahiko Watanabe, Tomas Hokfelt
    JOURNAL OF COMPARATIVE NEUROLOGY 519 (7) 1219 - 1257 0021-9967 2011/05 [Refereed][Not invited]
     
    Neuropeptide Y (NPY) is widely expressed in the brain and its biological effects are mediated through a variety of receptors. We examined, using immunohistochemistry, expression of the Y2 receptor (R) protein in the adult mouse brain and its association with NPY and the Y1R, as well as a range of additional neurotransmitters and signaling-related molecules, which previously have not been defined. Our main focus was on the hippocampal formation (HiFo), amygdaloid complex, and hypothalamus, considering the known functions of NPY and the wide expression of NPY, Y1R, and Y2R in these regions. Y2R-like immunoreactivity (-LI) was distributed in nerve fibers/terminal endings throughout the brain axis, without apparent colocalization with NPY or the Y1R. Occasional coexistence between NPY- and Y1R-LI was found in the HiFo. Following colchicine treatment, Y2R-LI accumulated in cell bodies that coexpressed gamma-aminobutyric acid (GABA) in a population of cells in the amygdaloid complex and lateral septal nucleus, but not in the HiFo. Instead, Y2R-positive nerve terminals appeared to surround GABA-immunoreactive (ir) cells in the HiFo and other neuronal populations, e. g., NPY-ir cells in HiFo and tyrosine hydroxylase-ir cells in the hypothalamus. In the HiFo, Y2R-ir mossy fibers coexpressed GABA, glutamic acid decarboxylase 67 and calbindin, and Y2R-LI was found in the same fibers that contained the presynaptic metabotropic glutamate receptor 2, but not together with any of the three vesicular glutamate transporters. Our findings provide further support that Y2R is mostly presynaptic, and that Y2Rs thus have a modulatory role in mediating presynaptic neurotransmitter release. J. Comp. Neurol. 519: 1219-1257, 2011. (C) 2011 Wiley-Liss, Inc.
  • Takeshi Izumi, Shuken Boku, Weng Shinmin, Takeshi Inoue, Kotaro Konno, Taku Yamaguchi, Takayuki Yoshida, Machiko Matsumoto, Masahiko Watanabe, Tsukasa Koyama, Mitsuhiro Yoshioka
    JOURNAL OF NEUROSCIENCE RESEARCH 89 (5) 773 - 790 0360-4012 2011/05 [Refereed][Not invited]
     
    The amygdala is one of the crucial brain structures for conditioned fear, in which conditioned stimuli are received by the basolateral nucleus of the amygdala (BLA), inducing a fear reaction via the central nucleus of the amygdala (CeA). Whereas BLA sends glutamatergic projections into CeA, the intercalated nucleus of the amygdala (ITC) sends GABAergic projections into CeA, which is doubly regulated by BLA and ITC. In the present study, we investigated the characteristics of the neural cells activated by retrieval of conditioned fear in BLA and ITC using immunohistochemistry, in situ hybridization, and Western blot analysis of transcription factors and neural cell markers. Because most conditioned fear-induced c-Fos-positive cells in BLA were glutaminase positive and 67-kDa isomer of glutamic acid decarboxylase (GAD67) negative, these cells are speculated to be glutamatergic. Seventy-eight percent of the phosphorylated CREB (pCREB)positive cells were glutaminase double positive and 13% of the pCREB-positive cells were GAD67 double positive, indicating that many of the conditioned fear-induced pCREB-positive cells in BLA were glutamatergic, but at least some of the pCREB-positive cells were GABAergic. These results suggested that CREB phosphorylation was increased both in glutamatergic and in GABAergic neurons, but c-Fos expression was increased mainly in glutamatergic neurons in BLA. CREB phosphorylation but not c-Fos expression in ITC was specifically increased by retrieval of conditioned fear. It is therefore speculated that ITC GABAergic neurons were activated by retrieval of conditioned fear and that transcription factors other than c-Fos were relevant to the activation. (C) 2011 Wiley-Liss, Inc.
  • Erika Polgar, Thomas C. P. Sardella, Masahiko Watanabe, Andrew J. Todd
    JOURNAL OF COMPARATIVE NEUROLOGY 519 (6) 1007 - 1023 0021-9967 2011/04 [Refereed][Not invited]
     
    Between 25-40% of neurons in laminae I-III are GABAergic, and some of these express neuropeptide Y (NPY). We previously reported that NPY-immunoreactive axons form numerous synapses on lamina III projection neurons that possess the neurokinin 1 receptor (NK1r). The aims of this study were to determine the proportion of neurons and GABAergic boutons in this region that contain NPY, and to look for evidence that they selectively innervate different neuronal populations. We found that 4-6% of neurons in laminae I-III were NPY-immunoreactive and based on the proportions of neurons that are GABAergic, we estimate that NPY is expressed by 18% of inhibitory interneurons in laminae I-II and 9% of those in lamina III. GABAergic boutons were identified by the presence of the vesicular GABA transporter (VGAT) and NPY was found in 13-15% of VGAT-immunoreactive boutons in laminae I-II, and 5% of those in lamina III. For both the lamina III NK1r-immunoreactive projection neurons and protein kinase Cc (PKC gamma)-immunoreactive interneurons in lamina II, we found that around one-third of the VGAT boutons that contacted them were NPY-immunoreactive. However, based on differences in the sizes of these boutons and the strength of their NPY-immunoreactivity, we conclude that these originate from different populations of interneurons. Only 6% of VGAT boutons presynaptic to large lamina I projection neurons that lacked NK1rs contained NPY. These results show that NPY-containing neurons make up a considerable proportion of the inhibitory interneurons in laminae I-III, and that their axons preferentially target certain classes of dorsal horn neuron. J. Comp. Neurol. 519: 1007-1023, 2011. (C) 2010 Wiley-Liss, Inc.
  • Jan Mulder, Misha Zilberter, Susana J. Pasquare, Alan Alpar, Gunnar Schulte, Samira G. Ferreira, Attila Koefalvi, Ana M. Martin-Moreno, Erik Keimpema, Heikki Tanila, Masahiko Watanabe, Ken Mackie, Tibor Hortobagyi, Maria L. de Ceballos, Tibor Harkany
    BRAIN 134 1041 - 1060 0006-8950 2011/04 [Refereed][Not invited]
     
    Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB(1) cannabinoid receptors. Cognitive decline in Alzheimer's disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimer's disease. Comparative protein profiling and quantitative morphometry showed that overall CB(1) cannabinoid receptor protein levels in the hippocampi of patients with Alzheimer's disease remain unchanged relative to age-matched controls, and CB(1) cannabinoid receptor-positive presynapses engulf amyloid-beta-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase alpha and beta isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimer's (Braak stage VI), with ectopic sn-1-diacylglycerol lipase beta expression found in microglia accumulating near senile plaques and apposing CB(1) cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase alpha/beta-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimer's disease (Braak stage III). However, Alzheimer's pathology differentially impacts serine hydrolase alpha/beta-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase alpha/beta-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB(1) cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimer's tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimer's disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-beta. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimer's disease.
  • Eszter Szabadits, Csaba Cserep, Andras Szonyi, Yugo Fukazawa, Ryuichi Shigemoto, Masahiko Watanabe, Shigeyoshi Itohara, Tamas F. Freund, Gabor Nyiri
    JOURNAL OF NEUROSCIENCE 31 (16) 5893 - 5904 0270-6474 2011/04 [Refereed][Not invited]
     
    GABAergic inhibition plays a central role in the control of pyramidal cell ensemble activities; thus, any signaling mechanism that regulates inhibition is able to fine-tune network patterns. Here, we provide evidence that the retrograde nitric oxide (NO)-cGMP cascade triggered by NMDA receptor(NMDAR) activation plays a role in the control of hippocampal GABAergic transmission in mice. GABAergic synapses express neuronal nitric oxide synthase (nNOS) postsynaptically and NO receptors (NO-sensitive guanylyl cyclase) in the presynaptic terminals. We hypothesized that-similar to glutamatergic synapses-the Ca2+ transients required to activate nNOS were provided by NMDA receptor activation. Indeed, administration of 5 mu M NMDA induced a robust nNOS-dependent cGMP production in GABAergic terminals, selectively in the CA1 and CA3c areas. Furthermore, using preembedding, postembedding, and SDS-digested freeze-fracture replica immunogold labeling, we provided quantitative immunocytochemical evidence that NMDAR subunits GluN1, GluN2A, and GluN2B were present in most somatic GABAergic synapses postsynaptically. These data indicate that NMDARs can modulate hippocampal GABAergic inhibition via NO-cGMP signaling in an activity-dependent manner and that this effect is subregion specific in the mouse hippocampus.
  • Eszter Szabadits, Csaba Cserep, Andras Szonyi, Yugo Fukazawa, Ryuichi Shigemoto, Masahiko Watanabe, Shigeyoshi Itohara, Tamas F. Freund, Gabor Nyiri
    JOURNAL OF NEUROSCIENCE 31 (16) 5893 - 5904 0270-6474 2011/04 [Refereed][Not invited]
     
    GABAergic inhibition plays a central role in the control of pyramidal cell ensemble activities; thus, any signaling mechanism that regulates inhibition is able to fine-tune network patterns. Here, we provide evidence that the retrograde nitric oxide (NO)-cGMP cascade triggered by NMDA receptor(NMDAR) activation plays a role in the control of hippocampal GABAergic transmission in mice. GABAergic synapses express neuronal nitric oxide synthase (nNOS) postsynaptically and NO receptors (NO-sensitive guanylyl cyclase) in the presynaptic terminals. We hypothesized that-similar to glutamatergic synapses-the Ca2+ transients required to activate nNOS were provided by NMDA receptor activation. Indeed, administration of 5 mu M NMDA induced a robust nNOS-dependent cGMP production in GABAergic terminals, selectively in the CA1 and CA3c areas. Furthermore, using preembedding, postembedding, and SDS-digested freeze-fracture replica immunogold labeling, we provided quantitative immunocytochemical evidence that NMDAR subunits GluN1, GluN2A, and GluN2B were present in most somatic GABAergic synapses postsynaptically. These data indicate that NMDARs can modulate hippocampal GABAergic inhibition via NO-cGMP signaling in an activity-dependent manner and that this effect is subregion specific in the mouse hippocampus.
  • Erika Polgar, Thomas C. P. Sardella, Masahiko Watanabe, Andrew J. Todd
    JOURNAL OF COMPARATIVE NEUROLOGY 519 (6) 1007 - 1023 0021-9967 2011/04 [Refereed][Not invited]
     
    Between 25-40% of neurons in laminae I-III are GABAergic, and some of these express neuropeptide Y (NPY). We previously reported that NPY-immunoreactive axons form numerous synapses on lamina III projection neurons that possess the neurokinin 1 receptor (NK1r). The aims of this study were to determine the proportion of neurons and GABAergic boutons in this region that contain NPY, and to look for evidence that they selectively innervate different neuronal populations. We found that 4-6% of neurons in laminae I-III were NPY-immunoreactive and based on the proportions of neurons that are GABAergic, we estimate that NPY is expressed by 18% of inhibitory interneurons in laminae I-II and 9% of those in lamina III. GABAergic boutons were identified by the presence of the vesicular GABA transporter (VGAT) and NPY was found in 13-15% of VGAT-immunoreactive boutons in laminae I-II, and 5% of those in lamina III. For both the lamina III NK1r-immunoreactive projection neurons and protein kinase Cc (PKC gamma)-immunoreactive interneurons in lamina II, we found that around one-third of the VGAT boutons that contacted them were NPY-immunoreactive. However, based on differences in the sizes of these boutons and the strength of their NPY-immunoreactivity, we conclude that these originate from different populations of interneurons. Only 6% of VGAT boutons presynaptic to large lamina I projection neurons that lacked NK1rs contained NPY. These results show that NPY-containing neurons make up a considerable proportion of the inhibitory interneurons in laminae I-III, and that their axons preferentially target certain classes of dorsal horn neuron. J. Comp. Neurol. 519: 1007-1023, 2011. (C) 2010 Wiley-Liss, Inc.
  • Jan Mulder, Misha Zilberter, Susana J. Pasquare, Alan Alpar, Gunnar Schulte, Samira G. Ferreira, Attila Koefalvi, Ana M. Martin-Moreno, Erik Keimpema, Heikki Tanila, Masahiko Watanabe, Ken Mackie, Tibor Hortobagyi, Maria L. de Ceballos, Tibor Harkany
    BRAIN 134 1041 - 1060 0006-8950 2011/04 [Refereed][Not invited]
     
    Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB(1) cannabinoid receptors. Cognitive decline in Alzheimer's disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimer's disease. Comparative protein profiling and quantitative morphometry showed that overall CB(1) cannabinoid receptor protein levels in the hippocampi of patients with Alzheimer's disease remain unchanged relative to age-matched controls, and CB(1) cannabinoid receptor-positive presynapses engulf amyloid-beta-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase alpha and beta isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimer's (Braak stage VI), with ectopic sn-1-diacylglycerol lipase beta expression found in microglia accumulating near senile plaques and apposing CB(1) cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase alpha/beta-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimer's disease (Braak stage III). However, Alzheimer's pathology differentially impacts serine hydrolase alpha/beta-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase alpha/beta-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB(1) cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimer's tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimer's disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-beta. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimer's disease.
  • Taisuke Miyazaki, Masahiko Watanabe
    ANATOMICAL SCIENCE INTERNATIONAL 1 86 (1) 10 - 18 1447-6959 2011/03 [Refereed][Not invited]
     
    In the adult cerebellum, a single climbing fiber (CF) innervates proximal dendrites of Purkinje cells (PCs). This monoinnervation is established by the developmental elimination of surplus CFs through homosynaptic competition among multiply innervating CFs and heterosynaptic competition between CFs and parallel fibers, i.e., granule cell axons innervating distal PC dendrites. Although the developmental process of CF monoinnervation and defects in it in mutant and experimental animal models have been extensively studied by electrophysiological techniques, for quite some time this subject was poorly understood from a morphological perspective due to a lack of neuroanatomical methods that could distinguish CFs with different neuronal origins. Soon after the identification of type 2 vesicular glutamate transporter (VGluT2) that selectively detects CF terminals in the molecular layer, we developed a novel method of combined anterograde tracer labeling and VGluT2 immunohistochemistry. This method enables us to identify the mode (mono vs. multiple) of CF innervation and the site of multiple innervation. Since then, we have applied this method to various kinds of gene-manipulated mice manifesting ataxia and other cerebellar phenotypes. In this review, we summarize experimental procedures for the combined tracer/VGluT2 labeling method, and then introduce what we have learned by applying this method in studies on the role of GluR delta 2 and Ca(v)2.1 in CF monoinnervation. This method has provided informative anatomical correlates to electrophysiological data and vice versa, and will extend our knowledge of the molecular and cellular mechanisms for the development, plasticity, degeneration, and repair of the CF-PC projection system.
  • Tayo Katano, Takanobu Nakazawa, Terumasa Nakatsuka, Masahiko Watanabe, Tadashi Yamamoto, Seiji Ito
    NEUROPHARMACOLOGY 60 (4) 609 - 616 0028-3908 2011/03 [Refereed][Not invited]
     
    Previously we demonstrated that phosphorylation of NR2B subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor at Tyr1472 is increased in a neuropathic-pain model and that this phosphorylation is required for the maintenance of neuropathic pain by L5-spinal nerve transection. We obtained these results by using a selective NR2B antagonist and mice deficient in Fyn, which is an Src-family tyrosine protein kinase. However, how Tyr1472 phosphorylation of NR2B is involved in the maintenance of neuropathic pain was unclear. Here, we demonstrated that neuropathic pain was markedly attenuated in the spared nerve injury model of mice with a knock-in mutation of the Tyr1472 site to phenylalanine of NR2B (Y1472F-KI). While phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) at its Thr286 and that of the GluR1 subunit of the AMPA receptor at its Ser831 was enhanced in the spinal dorsal horn after spared nerve injury in wild-type mice, such phosphorylation was markedly impaired in Y1472F-KI mice. Inhibition of CaMKII by intrathecal injection of KN93, an inhibitor of CaMKII, reduced mechanical allodynia and phosphorylation of CaMKII at its Thr286 and that of GluR1 at its Ser831 in the spinal cord 7 days after spared nerve injury. These results demonstrate that the phosphorylation of CaMKII and GluR1 occurs downstream of the Tyr1472 phosphorylation of NR2B subunits in the spinal cord and give the first suggestion that activation of CaMKII and GluR1-AMPA receptors may be involved in mechanical allodynia caused by peripheral nerve injury. (C) 2011 Elsevier Ltd. All rights reserved.
  • Masahiro Fukaya, Akifumi Kamata, Yoshinobu Hara, Hideaki Tamaki, Osamu Katsumata, Naoki Ito, Shin'ichi Takeda, Yutaka Hata, Tatsuo Suzuki, Masahiko Watanabe, Robert J. Harvey, Hiroyuki Sakagami
    JOURNAL OF NEUROCHEMISTRY 116 (6) 1122 - 1137 0022-3042 2011/03 [Refereed][Not invited]
     
    P>SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both beta-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.
  • Rylan S. Larsen, Rebekah J. Corlew, Maile A. Henson, Adam C. Roberts, Masayoshi Mishina, Masahiko Watanabe, Stuart A. Lipton, Nobuki Nakanishi, Isabel Perez-Otano, Richard J. Weinberg, Benjamin D. Philpot
    NATURE NEUROSCIENCE 14 (3) 338 - 344 1097-6256 2011/03 [Refereed][Not invited]
     
    Recent evidence suggests that presynaptic-acting NMDA receptors (preNMDARs) are important for neocortical synaptic transmission and plasticity. We found that unique properties of the NR3A subunit enable preNMDARs to enhance spontaneous and evoked glutamate release and that NR3A is required for spike timing-dependent long-term depression in the juvenile mouse visual cortex. In the mature cortex, NR2B-containing preNMDARs enhanced neurotransmission in the absence of magnesium, indicating that presynaptic NMDARs may function under depolarizing conditions throughout life. Our findings indicate that NR3A relieves preNMDARs from the dual-activation requirement of ligand-binding and depolarization; the developmental removal of NR3A limits preNMDAR functionality by restoring this associative property.
  • Jae-Yeol Joo, Sung-Jin Lee, Takeshi Uemura, Tomoyuki Yoshida, Misato Yasumura, Masahiko Watanabe, Masayoshi Mishina
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 406 (4) 627 - 632 0006-291X 2011/03 [Refereed][Not invited]
     
    Trans-synaptic interaction of postsynaptic glutamate receptor 62 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates synapse formation in the cerebellum [T. Uemura, S.J. Lee, M. Yasumura, T. Takeuchi, T. Yoshida, M. Ra, R. Taguchi, K. Sakimura, M. Mishina, Cell 141 (2010) 1068-1079]. This finding raises a question whether other Cbln family members interact with NRXNs to regulate synapse formation in the forebrain. Here, we showed that Cbln1 and Cbln2 induced presynaptic differentiation of cultured cortical neurons, while Cbln4 exhibited little activity. When compared with neuroligin 1, Cbln1 and Cbln2 induced preferentially inhibitory presynaptic differentiation rather than excitatory one in cortical cultures. The synaptogenic activities of Cbln1 and Cbln2 were suppressed by the addition of the extracellular domain of NRXN1 beta to the cortical neuron cultures. Consistently, Cbln1 and Cbln2 showed robust binding activities to NRXN1 alpha and three beta-NRXNs, while only weak interactions were observed between Cbln4 and NRXNs. The interactions of Cbln1, Cbln2 and Cbln4 were selective for NRXN variants containing splice segment (S) 4. Affinities for NRXNs estimated by surface plasmon resonance analysis were variable among Cbln subtypes. Cbln1 showed higher affinities to NRXNs than Cbln2, while the binding ability of Cbln4 was much lower than those of Cbln1 and Cbln2. The affinities of Cbln1 and Cbln2 were comparable between NRXN1 alpha and NRXN1 beta, but those for NRXN2 beta and NRXN3 beta were lower. These results suggest that Cbln subtypes exert synaptogenic activities in cortical neurons by differentially interacting with NRXN variants containing S4. (C) 2011 Elsevier Inc. All rights reserved.
  • Miwako Yamasaki, Taisuke Miyazaki, Hirotsugu Azechi, Manabu Abe, Rie Natsume, Teruki Hagiwara, Atsu Aiba, Masayoshi Mishina, Kenji Sakimura, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 9 31 (9) 3362 - 3374 0270-6474 2011/03 [Refereed][Not invited]
     
    The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor delta 2 (GluR delta 2 or G1uD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRS2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluR delta 2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluR delta 2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluR52. These results suggest that GluR delta 2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.
  • Miwako Yamasaki, Taisuke Miyazaki, Hirotsugu Azechi, Manabu Abe, Rie Natsume, Teruki Hagiwara, Atsu Aiba, Masayoshi Mishina, Kenji Sakimura, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 31 (9) 3362 - 3374 0270-6474 2011/03 [Refereed][Not invited]
     
    The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor delta 2 (GluR delta 2 or G1uD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRS2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluR delta 2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluR delta 2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluR52. These results suggest that GluR delta 2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.
  • H. Yamamoto, Y. Takamatsu, K. Imai, E. Kamegaya, Y. Hagino, M. Watanabe, T. Yamamoto, I. Sora, H. Koga, K. Ikeda
    CURRENT NEUROPHARMACOLOGY 9 (1) 73 - 78 1570-159X 2011/03 [Refereed][Not invited]
     
    Previously, we found fluoxetine reduces methamphetamine preference in mice. However, effects of fluoxetine on developed methamphetamine preference and on methamphetamine induced gene expression changes have been largely unknown. The present study investigates effects of post-treatment with fluoxetine on methamphetamine dependence and on gene expressions after long-term withdrawal in mice. First, we examined whether chronic post-treatment with fluoxetine attenuated methamphetamine-conditioned place preference. Next, we examined the changes in gene expression levels after long-term withdrawal (with saline or fluoxetine treatment) following chronic methamphetamine treatment. Using mRNA from the pooled frontal cortices of 10 mice per group, gene expression analyses were performed using a custom-developed cDNA array and a real-time quantitative reverse transcription-PCR. Chronic post-treatments with fluoxetine abolished the conditioned place preference developed by methamphetamine administrations. Even after long-term withdrawal from repeated methamphetamine administration, -opioid receptor (MOP) gene expression was significantly reduced in the frontal cortex. The reduced MOP gene expression in the frontal cortex was restored by chronic administration with fluoxetine. These changes were confirmed by Western blot analyses. These findings suggest that the chronic post-treatments with fluoxetine might be effective for restoring the reduction of MOP levels in the frontal cortex following long-term abstinence from methamphetamine.
  • Taisuke Miyazaki, Masahiko Watanabe
    ANATOMICAL SCIENCE INTERNATIONAL 86 (1) 10 - 18 1447-6959 2011/03 [Refereed][Not invited]
     
    In the adult cerebellum, a single climbing fiber (CF) innervates proximal dendrites of Purkinje cells (PCs). This monoinnervation is established by the developmental elimination of surplus CFs through homosynaptic competition among multiply innervating CFs and heterosynaptic competition between CFs and parallel fibers, i.e., granule cell axons innervating distal PC dendrites. Although the developmental process of CF monoinnervation and defects in it in mutant and experimental animal models have been extensively studied by electrophysiological techniques, for quite some time this subject was poorly understood from a morphological perspective due to a lack of neuroanatomical methods that could distinguish CFs with different neuronal origins. Soon after the identification of type 2 vesicular glutamate transporter (VGluT2) that selectively detects CF terminals in the molecular layer, we developed a novel method of combined anterograde tracer labeling and VGluT2 immunohistochemistry. This method enables us to identify the mode (mono vs. multiple) of CF innervation and the site of multiple innervation. Since then, we have applied this method to various kinds of gene-manipulated mice manifesting ataxia and other cerebellar phenotypes. In this review, we summarize experimental procedures for the combined tracer/VGluT2 labeling method, and then introduce what we have learned by applying this method in studies on the role of GluR delta 2 and Ca(v)2.1 in CF monoinnervation. This method has provided informative anatomical correlates to electrophysiological data and vice versa, and will extend our knowledge of the molecular and cellular mechanisms for the development, plasticity, degeneration, and repair of the CF-PC projection system.
  • Rylan S. Larsen, Rebekah J. Corlew, Maile A. Henson, Adam C. Roberts, Masayoshi Mishina, Masahiko Watanabe, Stuart A. Lipton, Nobuki Nakanishi, Isabel Perez-Otano, Richard J. Weinberg, Benjamin D. Philpot
    NATURE NEUROSCIENCE 14 (3) 338 - 344 1097-6256 2011/03 [Refereed][Not invited]
     
    Recent evidence suggests that presynaptic-acting NMDA receptors (preNMDARs) are important for neocortical synaptic transmission and plasticity. We found that unique properties of the NR3A subunit enable preNMDARs to enhance spontaneous and evoked glutamate release and that NR3A is required for spike timing-dependent long-term depression in the juvenile mouse visual cortex. In the mature cortex, NR2B-containing preNMDARs enhanced neurotransmission in the absence of magnesium, indicating that presynaptic NMDARs may function under depolarizing conditions throughout life. Our findings indicate that NR3A relieves preNMDARs from the dual-activation requirement of ligand-binding and depolarization; the developmental removal of NR3A limits preNMDAR functionality by restoring this associative property.
  • Tayo Katano, Takanobu Nakazawa, Terumasa Nakatsuka, Masahiko Watanabe, Tadashi Yamamoto, Seiji Ito
    NEUROPHARMACOLOGY 60 (4) 609 - 616 0028-3908 2011/03 [Refereed][Not invited]
     
    Previously we demonstrated that phosphorylation of NR2B subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor at Tyr1472 is increased in a neuropathic-pain model and that this phosphorylation is required for the maintenance of neuropathic pain by L5-spinal nerve transection. We obtained these results by using a selective NR2B antagonist and mice deficient in Fyn, which is an Src-family tyrosine protein kinase. However, how Tyr1472 phosphorylation of NR2B is involved in the maintenance of neuropathic pain was unclear. Here, we demonstrated that neuropathic pain was markedly attenuated in the spared nerve injury model of mice with a knock-in mutation of the Tyr1472 site to phenylalanine of NR2B (Y1472F-KI). While phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) at its Thr286 and that of the GluR1 subunit of the AMPA receptor at its Ser831 was enhanced in the spinal dorsal horn after spared nerve injury in wild-type mice, such phosphorylation was markedly impaired in Y1472F-KI mice. Inhibition of CaMKII by intrathecal injection of KN93, an inhibitor of CaMKII, reduced mechanical allodynia and phosphorylation of CaMKII at its Thr286 and that of GluR1 at its Ser831 in the spinal cord 7 days after spared nerve injury. These results demonstrate that the phosphorylation of CaMKII and GluR1 occurs downstream of the Tyr1472 phosphorylation of NR2B subunits in the spinal cord and give the first suggestion that activation of CaMKII and GluR1-AMPA receptors may be involved in mechanical allodynia caused by peripheral nerve injury. (C) 2011 Elsevier Ltd. All rights reserved.
  • Shinya Kasai, Hideko Yamamoto, Etsuko Kamegaya, George R. Uhl, Ichiro Sora, Masahiko Watanabe, Kazutaka Ikeda
    CURRENT NEUROPHARMACOLOGY 9 (1) 219 - 222 1570-159X 2011/03 [Refereed][Not invited]
     
    Increasing evidence suggests that mu opioid receptor (MOP) expression is altered during the development of and withdrawal from substance dependence. Although anti-MOP antibodies have been hypothesized to be useful for estimating MOP expression levels, inconsistent MOP molecular weights (MWs) have been reported in studies using anti-MOP antibodies. In the present study, we generated a new anti-MOP antibody (N38) against the 1-38 amino acid sequence of the mouse MOP N-terminus and conducted Western blot analysis with wildtype and MOP knockout brain lysates to determine the MWs of intrinsic MOP. The N38 antibody detected migrating bands with relative MWs of 60-67 kDa in the plasma membrane fraction isolated from wildtype brain, but not from the MOP knockout brain. These migrating bands exhibited semi-linear density in the range of 3-30 mu g membrane proteins/lane. The N38 antibody may be useful for quantitatively detecting MOP.
  • Jae-Yeol Joo, Sung-Jin Lee, Takeshi Uemura, Tomoyuki Yoshida, Misato Yasumura, Masahiko Watanabe, Masayoshi Mishina
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 406 (4) 627 - 632 0006-291X 2011/03 [Refereed][Not invited]
     
    Trans-synaptic interaction of postsynaptic glutamate receptor 62 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates synapse formation in the cerebellum [T. Uemura, S.J. Lee, M. Yasumura, T. Takeuchi, T. Yoshida, M. Ra, R. Taguchi, K. Sakimura, M. Mishina, Cell 141 (2010) 1068-1079]. This finding raises a question whether other Cbln family members interact with NRXNs to regulate synapse formation in the forebrain. Here, we showed that Cbln1 and Cbln2 induced presynaptic differentiation of cultured cortical neurons, while Cbln4 exhibited little activity. When compared with neuroligin 1, Cbln1 and Cbln2 induced preferentially inhibitory presynaptic differentiation rather than excitatory one in cortical cultures. The synaptogenic activities of Cbln1 and Cbln2 were suppressed by the addition of the extracellular domain of NRXN1 beta to the cortical neuron cultures. Consistently, Cbln1 and Cbln2 showed robust binding activities to NRXN1 alpha and three beta-NRXNs, while only weak interactions were observed between Cbln4 and NRXNs. The interactions of Cbln1, Cbln2 and Cbln4 were selective for NRXN variants containing splice segment (S) 4. Affinities for NRXNs estimated by surface plasmon resonance analysis were variable among Cbln subtypes. Cbln1 showed higher affinities to NRXNs than Cbln2, while the binding ability of Cbln4 was much lower than those of Cbln1 and Cbln2. The affinities of Cbln1 and Cbln2 were comparable between NRXN1 alpha and NRXN1 beta, but those for NRXN2 beta and NRXN3 beta were lower. These results suggest that Cbln subtypes exert synaptogenic activities in cortical neurons by differentially interacting with NRXN variants containing S4. (C) 2011 Elsevier Inc. All rights reserved.
  • Masahiro Fukaya, Akifumi Kamata, Yoshinobu Hara, Hideaki Tamaki, Osamu Katsumata, Naoki Ito, Shin'ichi Takeda, Yutaka Hata, Tatsuo Suzuki, Masahiko Watanabe, Robert J. Harvey, Hiroyuki Sakagami
    JOURNAL OF NEUROCHEMISTRY 116 (6) 1122 - 1137 0022-3042 2011/03 [Refereed][Not invited]
     
    P>SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both beta-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.
  • Kenta Kobayashi, Tomoyuki Masuda, Masanori Takahashi, Jun-ichi Miyazaki, Masahiro Nakagawa, Motokazu Uchigashima, Masahiko Watanabe, Hiroyuki Yaginuma, Noriko Osumi, Kozo Kaibuchi, Kazuto Kobayashi
    EUROPEAN JOURNAL OF NEUROSCIENCE 33 (4) 612 - 621 0953-816X 2011/02 [Refereed][Not invited]
     
    Cranial motor neurons, which are divided into somatic motor (SM), branchiomotor (BM) and visceral motor (VM) neurons, form distinct axonal trajectories to innervate their synapse targets. Rho GTPase regulates various neuronal functions through one of the major effector proteins, Rho-kinase. Here, we addressed the in vivo role of the Rho/Rho-kinase signaling pathway in axon patterning of cranial motor neurons. We performed conditional expression of a dominant-negative mutant for RhoA or Rho-kinase in transgenic mice by using the Cre-loxP system to suppress the activity of these molecules in developing cranial motor neurons. Blockade of the Rho/Rho-kinase signaling pathway caused defects in the patterning of SM axons but not in that of BM/VM axons, in which defects were accompanied by reduced muscle innervation and reduced synapse formation by SM neurons. In addition, blockade of the signaling pathway shifted the trajectory of growing SM axons in explant cultures, whereas it did not appear to affect the rate of spontaneous axonal outgrowth. These results indicate that the Rho/Rho-kinase signaling pathway plays an essential role in the axon patterning of cranial SM neurons during development.
  • Takayuki Yoshida, Motokazu Uchigashima, Miwako Yamasaki, Istvan Katona, Maya Yamazaki, Kenji Sakimura, Masanobu Kano, Mitsuhiro Yoshioka, Masahiko Watanabe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 (7) 3059 - 3064 0027-8424 2011/02 [Refereed][Not invited]
     
    2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of synaptic transmission in the brain. 2-AG is synthesized in activated postsynaptic neurons by sn-1-specific diacylglycerol lipase (DGL), binds to presynaptic cannabinoid CB(1) receptors, suppresses neurotransmitter release, and is degraded mainly by monoacylglycerol lipase (MGL). In the basolateral amygdala complex, it has been demonstrated that CB(1) is particularly enriched in axon terminals of cholecystokinin (CCK)-positive GABAergic interneurons, induces short-and long-term depression at inhibitory synapses, and is involved in extinction of fear memory. Here, we clarified a unique molecular convergence of DGL alpha, CB(1), and MGL at specific inhibitory synapses in the basal nucleus (BA), but not lateral nucleus, of the basolateral amygdala. The synapses, termed invaginating synapses, consisted of conventional symmetrical contact and unique perisynaptic invagination of nerve terminals into perikarya. At invaginating synapses, DGL alpha was preferentially recruited to concave somatic membrane of postsynaptic pyramidal neurons, whereas invaginating presynaptic terminals highly expressed CB(1), MGL, and CCK. No such molecular convergence was seen for flat perisomatic synapses made by parvalbumin-positive interneurons. On the other hand, DGL alpha and CB(1) were expressed weakly at axospinous excitatory synapses. Consistent with these morphological data, thresholds for DGL alpha-mediated depolarization-induced retrograde suppression were much lower for inhibitory synapses than for excitatory synapses in BA pyramidal neurons. Moreover, depolarization-induced suppression was readily saturated for inhibition, but never for excitation. These findings suggest that perisomatic inhibition by invaginating synapses is a key target of 2-AG-mediated control of the excitability of BA pyramidal neurons.
  • A. Ludanyi, S. -J. Hu, M. Yamazaki, A. Tanimura, D. Piomelli, M. Watanabe, M. Kano, K. Sakimura, Z. Magloczky, K. Mackie, T. F. Freund, I. Katona
    NEUROSCIENCE 174 50 - 63 0306-4522 2011/02 [Refereed][Not invited]
     
    Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders. Their medical significance may be best explained by the emerging concept that endocannabinoids are essential modulators of synaptic transmission throughout the central nervous system. However, the precise molecular architecture of the endocannabinoid signaling machinery in the human brain remains elusive. To address this issue, we investigated the synaptic distribution of metabolic enzymes for the most abundant endocannabinoid molecule, 2-arachidonoylglycerol (2-AG), in the postmortem human hippocampus. Immunostaining for diacylglycerol lipase-alpha (DGL-alpha), the main synthesizing enzyme of 2-AG, resulted in a laminar pattern corresponding to the termination zones of glutamatergic pathways. The highest density of DGL-alpha-immunostaining was observed in strata radiatum and oriens of the cornu ammonis and in the inner third of stratum moleculare of the dentate gyrus. At higher magnification, DGL-alpha-immunopositive puncta were distributed throughout the neuropil outlining the immunonegative main dendrites of pyramidal and granule cells. Electron microscopic analysis revealed that this pattern was due to the accumulation of DGL-alpha in dendritic spine heads. Similar DGL-alpha-immunostaining pattern was also found in hippocampi of wild-type, but not of DGL-alpha knockout mice. Using two independent antibodies developed against monoacylglycerol lipase (MGL), the predominant enzyme inactivating 2-AG, immunostaining also revealed a laminar and punctate staining pattern. However, as observed previously in rodent hippocampus, MGL was enriched in axon terminals instead of postsynaptic structures at the ultrastructural level. Taken together, these findings demonstrate the post- and presynaptic segregation of primary enzymes responsible for synthesis and elimination of 2-AG, respectively, in the human hippocampus. Thus, molecular architecture of the endocannabinoid signaling machinery supports retrograde regulation of synaptic activity, and its similar blueprint in rodents and humans further indicates that 2-AG's physiological role as a negative feed-back signal is an evolutionarily conserved feature of excitatory synapses. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Taisuke Miyazaki, Miwako Yamasaki, Motokazu Uchigashima, Ayano Matsushima, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 33 (1) 82 - 94 0953-816X 2011/01 [Refereed][Not invited]
     
    Secretogranin II (SgII), or chromogranin C, is thought to participate in the sorting and packaging of peptide hormones and neuropeptides into secretory granules and large dense-core vesicle (LDCVs), and also functions as a precursor of neuropeptide secretoneurin. Although SgII is widely distributed in the brain and is predominantly localized at terminals of mossy fibers in the hippocampus and cerebellum and climbing fibers in the cerebellum, its cellular expression and ultrastructural localization remain largely unknown. In the present study, we addressed this issue in the adult mouse brain by multiple-labeling fluorescence in situ hybridization and immunofluorescence and by preembedding and postembedding immunoelectron microscopies. SgII was expressed in various neurons, distributed as either tiny puncta or coarse aggregates in the neuropil, and intensely accumulated in perikarya of particular neurons, such as parvalbumin-positive interneurons and mossy cells in the hippocampus and Purkinje cells in the cerebellum. Coarse aggregates were typical of terminals of mossy fibers and climbing fibers. In these terminals, numerous immunogold particles were clustered on individual LDCVs, and one or two particles also fell within small synaptic vesicle-accumulating portions. SgII was further detected as tiny puncta in neural elements lacking LDCVs, such as parallel fibers of cerebellar granule cells, somatodendritic elements of various neurons and Bergmann glia. Thus, SgII is present in LDCV and non-LDCV compartments of various neural cells. The wide subcellular localization of SgII may reflect diverse release sites of neuropeptides and secretorneurin, or suggests its role in the sorting and packaging of molecules other than neuropeptides in non-LDCV compartments.
  • Taisuke Miyazaki, Miwako Yamasaki, Motokazu Uchigashima, Ayano Matsushima, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 33 (1) 82 - 94 0953-816X 2011/01 [Refereed][Not invited]
     
    Secretogranin II (SgII), or chromogranin C, is thought to participate in the sorting and packaging of peptide hormones and neuropeptides into secretory granules and large dense-core vesicle (LDCVs), and also functions as a precursor of neuropeptide secretoneurin. Although SgII is widely distributed in the brain and is predominantly localized at terminals of mossy fibers in the hippocampus and cerebellum and climbing fibers in the cerebellum, its cellular expression and ultrastructural localization remain largely unknown. In the present study, we addressed this issue in the adult mouse brain by multiple-labeling fluorescence in situ hybridization and immunofluorescence and by preembedding and postembedding immunoelectron microscopies. SgII was expressed in various neurons, distributed as either tiny puncta or coarse aggregates in the neuropil, and intensely accumulated in perikarya of particular neurons, such as parvalbumin-positive interneurons and mossy cells in the hippocampus and Purkinje cells in the cerebellum. Coarse aggregates were typical of terminals of mossy fibers and climbing fibers. In these terminals, numerous immunogold particles were clustered on individual LDCVs, and one or two particles also fell within small synaptic vesicle-accumulating portions. SgII was further detected as tiny puncta in neural elements lacking LDCVs, such as parallel fibers of cerebellar granule cells, somatodendritic elements of various neurons and Bergmann glia. Thus, SgII is present in LDCV and non-LDCV compartments of various neural cells. The wide subcellular localization of SgII may reflect diverse release sites of neuropeptides and secretorneurin, or suggests its role in the sorting and packaging of molecules other than neuropeptides in non-LDCV compartments.
  • Yohei Okubo, Masato Mashimo, Toshiko Yamazawa, Miwako Yamasaki, Masahiko Watanabe, Toshihiko Murayama, Masamitsu Iino
    NEUROSCIENCE RESEARCH 71 E327 - E327 0168-0102 2011 [Refereed][Not invited]
  • Takayuki Yoshida, Motokazu Uchigashima, Miwako Yamasaki, Istvan Katona, Maya Yamazaki, Kenji Sakimura, Masanobu Kano, Mitsuhiro Yoshioka, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E93 - E94 0168-0102 2011 [Refereed][Not invited]
  • Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Masabumi Minami, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E322 - E322 0168-0102 2011 [Refereed][Not invited]
  • Taisuke Miyazaki, Miwako Yamasaki, Kouichi Hashimoto, Keiko Shimamoto, Kazuhisa Kohda, Michisuke Yuzaki, Kohichi Tanaka, Masanobu Kano, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E63 - E63 0168-0102 2011 [Refereed][Not invited]
  • Mari Hondo, Naoki Furutani, Miwako Yamasaki, Masahiko Watanabe, Takeshi Sakurai
    NEUROSCIENCE RESEARCH 71 E170 - E170 0168-0102 2011 [Refereed][Not invited]
  • Kohtarou Konno, Miwako Yamasaki, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E213 - E213 0168-0102 2011 [Refereed][Not invited]
  • Ryoichi Ichikawa, Miwako Yamasaki, Taisuke Miyazaki, Haruyuki Tatsumi, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E215 - E215 0168-0102 2011 [Refereed][Not invited]
  • Miwako Yamasaki, Taisuke Miyazaki, Hirotsugu Azechi, Manabu Abe, Rie Natsume, Teruki Hagiwara, Atsu Aiba, Masayoshi Mishina, Kenji Sakimura, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E93 - E93 0168-0102 2011 [Refereed][Not invited]
  • Izumi Watanabe, Hirotsugu Azechi, Kaori Akashi, Maya Yamazaki, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura
    NEUROSCIENCE RESEARCH 71 E315 - E316 0168-0102 2011 [Refereed][Not invited]
  • Imre Farkas, Imre Kallo, Levente Deli, Barbara Vida, Erik Hrabovszky, Csaba Fekete, Suzanne M. Moenter, Masahiko Watanabe, Zsolt Liposits
    ENDOCRINOLOGY 151 (12) 5818 - 5829 0013-7227 2010/12 [Refereed][Not invited]
     
    Cannabinoids suppress fertility via reducing hypothalamic GnRH output. gamma-Aminobutyric acid (GABA)(A) receptor (GABA(A)-R)-mediated transmission is a major input to GnRH cells that can be excitatory. We hypothesized that cannabinoids act via inhibiting GABAergic input. We performed loose-patch electrophysiological studies of acute slices from adult male GnRH-green fluorescent protein transgenic mice. Bath application of type 1 cannabinoid receptor (CB1) agonist WIN55,212 decreased GnRH neuron firing rate. This action was detectable in presence of the glutamate receptor antagonist kynurenic acid but disappeared when bicuculline was also present, indicating GABA(A)-R involvement. In immunocytochemical experiments, CB1-immunoreactive axons formed contacts with GnRH neurons and a subset established symmetric synapses characteristic of GABAergic neurotransmission. Functional studies were continued with whole-cell patch-clamp electrophysiology in presence of tetrodotoxin. WIN55,212 decreased the frequency of GABA(A)-R-mediated miniature postsynaptic currents (mPSCs) (reflecting spontaneous vesicle fusion), which was prevented with the CB1 antagonist AM251, indicating collectively that activation of presynaptic CB1 inhibits GABA release. AM251 alone increased mPSC frequency, providing evidence that endocannabinoids tonically inhibit GABA(A)-R drive onto GnRH neurons. Increased mPSC frequency was absent when diacylglycerol lipase was blocked intracellularly with tetrahydrolipstatin, showing that tonic inhibition is caused by 2-arachidonoylglycerol production of GnRH neurons. CdCl2 in extracellular solution can maintain both action potentials and spontaneous vesicle fusion. Under these conditions, when endocannabinoid-mediated blockade of spontaneous vesicle fusion was blocked with AM251, GnRH neuron firing increased, revealing an endogenous endocannabinoid brake on GnRH neuron firing. Retrograde endocannabinoid signaling may represent an important mechanism under physiological and pathological conditions whereby GnRH neurons regulate their excitatory GABAergic inputs. (Endocrinology 151:5818-5829,2010)
  • Jung Hoon Yang, Akira Wada, Kazuyuki Yoshida, Yurika Miyoshi, Tomoko Sayano, Kayoko Esaki, Masami O. Kinoshita, Shozo Tomonaga, Norihiro Azuma, Masahiko Watanabe, Kenji Hamase, Kiyoshi Zaitsu, Takeo Machida, Albee Messing, Shigeyoshi Itohara, Yoshio Hirabayashi, Shigeki Furuya
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 (53) 41380 - 41390 0021-9258 2010/12 [Refereed][Not invited]
     
    In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L-and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus.
  • Jung Hoon Yang, Akira Wada, Kazuyuki Yoshida, Yurika Miyoshi, Tomoko Sayano, Kayoko Esaki, Masami O. Kinoshita, Shozo Tomonaga, Norihiro Azuma, Masahiko Watanabe, Kenji Hamase, Kiyoshi Zaitsu, Takeo Machida, Albee Messing, Shigeyoshi Itohara, Yoshio Hirabayashi, Shigeki Furuya
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 (53) 41380 - 41390 0021-9258 2010/12 [Refereed][Not invited]
     
    In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L-and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus.
  • Imre Farkas, Imre Kallo, Levente Deli, Barbara Vida, Erik Hrabovszky, Csaba Fekete, Suzanne M. Moenter, Masahiko Watanabe, Zsolt Liposits
    ENDOCRINOLOGY 151 (12) 5818 - 5829 0013-7227 2010/12 [Refereed][Not invited]
     
    Cannabinoids suppress fertility via reducing hypothalamic GnRH output. gamma-Aminobutyric acid (GABA)(A) receptor (GABA(A)-R)-mediated transmission is a major input to GnRH cells that can be excitatory. We hypothesized that cannabinoids act via inhibiting GABAergic input. We performed loose-patch electrophysiological studies of acute slices from adult male GnRH-green fluorescent protein transgenic mice. Bath application of type 1 cannabinoid receptor (CB1) agonist WIN55,212 decreased GnRH neuron firing rate. This action was detectable in presence of the glutamate receptor antagonist kynurenic acid but disappeared when bicuculline was also present, indicating GABA(A)-R involvement. In immunocytochemical experiments, CB1-immunoreactive axons formed contacts with GnRH neurons and a subset established symmetric synapses characteristic of GABAergic neurotransmission. Functional studies were continued with whole-cell patch-clamp electrophysiology in presence of tetrodotoxin. WIN55,212 decreased the frequency of GABA(A)-R-mediated miniature postsynaptic currents (mPSCs) (reflecting spontaneous vesicle fusion), which was prevented with the CB1 antagonist AM251, indicating collectively that activation of presynaptic CB1 inhibits GABA release. AM251 alone increased mPSC frequency, providing evidence that endocannabinoids tonically inhibit GABA(A)-R drive onto GnRH neurons. Increased mPSC frequency was absent when diacylglycerol lipase was blocked intracellularly with tetrahydrolipstatin, showing that tonic inhibition is caused by 2-arachidonoylglycerol production of GnRH neurons. CdCl2 in extracellular solution can maintain both action potentials and spontaneous vesicle fusion. Under these conditions, when endocannabinoid-mediated blockade of spontaneous vesicle fusion was blocked with AM251, GnRH neuron firing increased, revealing an endogenous endocannabinoid brake on GnRH neuron firing. Retrograde endocannabinoid signaling may represent an important mechanism under physiological and pathological conditions whereby GnRH neurons regulate their excitatory GABAergic inputs. (Endocrinology 151:5818-5829,2010)
  • Laxmi Kumar Parajuli, Yugo Fukazawa, Masahiko Watanabe, Ryuichi Shigemoto
    JOURNAL OF COMPARATIVE NEUROLOGY 21 518 (21) 4362 - 4374 0021-9967 2010/11 [Refereed][Not invited]
     
    T-type calcium channels play a pivotal role in regulating neural membrane excitability in the nervous system. However, the precise subcellular distributions of T-type channel subunits and their implication for membrane excitability are not well understood. Here we investigated the subcellular distribution of the alpha 1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the alpha 1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the alpha 1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited a gradient such that the labeling density was higher in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane-associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about 3-fold higher than that of the interneurons. These results provide ultrastructural evidence for cell-type-specific expression levels and for uniform expression density of the alpha 1G subunit over the plasma membrane of dLGN cells. J. Comp. Neurol. 518: 4362-4374, 2010. (C) 2010 Wiley-Liss, Inc.
  • Masato Mashimo, Yohei Okubo, Toshiko Yamazawa, Miwako Yamasaki, Masahiko Watanabe, Toshihiko Murayama, Masamitsu Iino
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 (10) 1668 - 1677 0953-816X 2010/11 [Refereed][Not invited]
     
    The maintenance of synaptic functions is essential for neuronal information processing in the adult brain. Astrocytes express glutamate transporters that rapidly remove glutamate from the extracellular space and they play a critical role in the precise operation of glutamatergic transmission. However, how the glutamate clearance function of astrocytes is maintained remains elusive. Here, we describe a maintenance mechanism for the glutamate uptake capacity of Bergmann glial cells (BGs) in the cerebellum. When inositol 1,4,5-trisphosphate (IP(3)) signaling was chronically and selectively inhibited in BGs in vivo, the retention time of glutamate around parallel fiber-Purkinje cell synapses was increased. Under these conditions, a decrease in the level of the glutamate/aspartate transporter (GLAST) in BGs was observed. The same effects were observed after chronic in vivo inhibition of purinergic P2 receptors in the cerebellar cortex. These results suggest that the IP(3) signaling cascade is involved in regulating GLAST levels in BGs to maintain glutamate clearance in the mature cerebellum.
  • Taisuke Miyazaki, Miwako Yamasaki, Tomonori Takeuchi, Kenji Sakimura, Masayoshi Mishina, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 45 30 (45) 15196 - 15209 0270-6474 2010/11 [Refereed][Not invited]
     
    Glutamate receptor GluR delta 2 is exclusively expressed in Purkinje cells (PCs) from early development and plays key roles in parallel fiber (PF) synapse formation, elimination of surplus climbing fibers (CFs), long-term depression, motor coordination, and motor learning. To address its role in adulthood, we previously developed a mouse model of drug-induced GluR delta 2 ablation in adult PCs (Takeuchi et al., 2005). In that study, we demonstrated an essential role to maintain the connectivity of PF-PC synapses, based on the observation that both mismatching of presynaptic and postsynaptic specializations and disconnection of PF-PC synapses are progressively increased after GluR delta 2 ablation. Here, we pursued its role for CF wiring in adult cerebellum. In parallel with the disconnection of PF-PC synapses, ascending CF branches exhibited distal extension to innervate distal dendrites of the target and neighboring PCs. Furthermore, transverse CF branches, a short motile collateral rarely forming synapses in wild-type animals, displayed aberrant mediolateral extension to innervate distal dendrites of neighboring and remote PCs. Consequently, many PCs were wired by single main CF and other surplus CFs innervating a small part of distal dendrites. Electrophysiological recording further revealed that surplus CF-EPSCs characterized with slow rise time and small amplitude emerged after GluR delta 2 ablation, and increased progressively both in number and amplitude. Therefore, GluR delta 2 is essential for maintaining CF monoinnervation in adult cerebellum by suppressing aberrant invasion of CF branches to the territory of PF innervation. Thus, GluR delta 2 fuels heterosynaptic competition and gives PFs the competitive advantages over CFs throughout the animal's life.
  • Laxmi Kumar Parajuli, Yugo Fukazawa, Masahiko Watanabe, Ryuichi Shigemoto
    JOURNAL OF COMPARATIVE NEUROLOGY 518 (21) 4362 - 4374 0021-9967 2010/11 [Refereed][Not invited]
     
    T-type calcium channels play a pivotal role in regulating neural membrane excitability in the nervous system. However, the precise subcellular distributions of T-type channel subunits and their implication for membrane excitability are not well understood. Here we investigated the subcellular distribution of the alpha 1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the alpha 1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the alpha 1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited a gradient such that the labeling density was higher in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane-associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about 3-fold higher than that of the interneurons. These results provide ultrastructural evidence for cell-type-specific expression levels and for uniform expression density of the alpha 1G subunit over the plasma membrane of dLGN cells. J. Comp. Neurol. 518: 4362-4374, 2010. (C) 2010 Wiley-Liss, Inc.
  • Taisuke Miyazaki, Miwako Yamasaki, Tomonori Takeuchi, Kenji Sakimura, Masayoshi Mishina, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 30 (45) 15196 - 15209 0270-6474 2010/11 [Refereed][Not invited]
     
    Glutamate receptor GluR delta 2 is exclusively expressed in Purkinje cells (PCs) from early development and plays key roles in parallel fiber (PF) synapse formation, elimination of surplus climbing fibers (CFs), long-term depression, motor coordination, and motor learning. To address its role in adulthood, we previously developed a mouse model of drug-induced GluR delta 2 ablation in adult PCs (Takeuchi et al., 2005). In that study, we demonstrated an essential role to maintain the connectivity of PF-PC synapses, based on the observation that both mismatching of presynaptic and postsynaptic specializations and disconnection of PF-PC synapses are progressively increased after GluR delta 2 ablation. Here, we pursued its role for CF wiring in adult cerebellum. In parallel with the disconnection of PF-PC synapses, ascending CF branches exhibited distal extension to innervate distal dendrites of the target and neighboring PCs. Furthermore, transverse CF branches, a short motile collateral rarely forming synapses in wild-type animals, displayed aberrant mediolateral extension to innervate distal dendrites of neighboring and remote PCs. Consequently, many PCs were wired by single main CF and other surplus CFs innervating a small part of distal dendrites. Electrophysiological recording further revealed that surplus CF-EPSCs characterized with slow rise time and small amplitude emerged after GluR delta 2 ablation, and increased progressively both in number and amplitude. Therefore, GluR delta 2 is essential for maintaining CF monoinnervation in adult cerebellum by suppressing aberrant invasion of CF branches to the territory of PF innervation. Thus, GluR delta 2 fuels heterosynaptic competition and gives PFs the competitive advantages over CFs throughout the animal's life.
  • Masato Mashimo, Yohei Okubo, Toshiko Yamazawa, Miwako Yamasaki, Masahiko Watanabe, Toshihiko Murayama, Masamitsu Iino
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 (10) 1668 - 1677 0953-816X 2010/11 [Refereed][Not invited]
     
    The maintenance of synaptic functions is essential for neuronal information processing in the adult brain. Astrocytes express glutamate transporters that rapidly remove glutamate from the extracellular space and they play a critical role in the precise operation of glutamatergic transmission. However, how the glutamate clearance function of astrocytes is maintained remains elusive. Here, we describe a maintenance mechanism for the glutamate uptake capacity of Bergmann glial cells (BGs) in the cerebellum. When inositol 1,4,5-trisphosphate (IP(3)) signaling was chronically and selectively inhibited in BGs in vivo, the retention time of glutamate around parallel fiber-Purkinje cell synapses was increased. Under these conditions, a decrease in the level of the glutamate/aspartate transporter (GLAST) in BGs was observed. The same effects were observed after chronic in vivo inhibition of purinergic P2 receptors in the cerebellar cortex. These results suggest that the IP(3) signaling cascade is involved in regulating GLAST levels in BGs to maintain glutamate clearance in the mature cerebellum.
  • Francisco Ciruela, Victor Fernandez-Duenas, Kristoffer Sahlholm, Laura Fernandez-Alacid, Joel C. Nicolau, Masahiko Watanabe, Rafael Lujan
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 (8) 1265 - 1277 0953-816X 2010/10 [Refereed][Not invited]
     
    The stimulation of inhibitory neurotransmitter receptors, such as gamma-aminobutyric acid type B (GABA(B)) receptors, activates G protein-gated inwardly-rectifying K+ (GIRK) channels, which influence membrane excitability. There is now evidence suggesting that G protein-coupled receptors and G protein-gated inwardly-rectifying K+ [GIRK/family 3 of inwardly-rectifying K+ (Kir3)] channels do not diffuse freely within the plasma membrane, but instead there are direct protein-protein interactions between them. Here, we used bioluminescence resonance energy transfer, co-immunoprecipitation, confocal and electron microscopy techniques to investigate the oligomerization of GABA(B) receptors with GIRK channels containing the GIRK3 subunit, whose contribution to functional channels is still unresolved. Co-expression of GABA(B) receptors and GIRK channels in human embryonic kidney-293 cells in combination with co-immunoprecipitation experiments established that the metabotropic receptor forms stable complexes with GIRK channels. Using bioluminescence resonance energy transfer, we have shown that, in living cells under physiological conditions, GABA(B) receptors interact directly with GIRK1/GIRK3 heterotetramers. In addition, we have provided evidence that the receptor-effector complexes are also found in vivo and identified that the cerebellar granule cells are one neuron population where the interaction probably takes place. Altogether, our data show that signalling complexes containing GABA(B) receptors and GIRK channels are formed shortly after biosynthesis, probably in the endoplasmic reticulum and/or endoplasmic reticulum/Golgi apparatus complex, suggesting that this might be a general feature of receptor-effector ion channel signal transduction and supporting a channel-forming role for the GIRK3 subunit.
  • Chihiro Takasaki, Miwako Yamasaki, Motokazu Uchigashima, Kohtarou Konno, Yuchio Yanagawa, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 (8) 1326 - 1336 0953-816X 2010/10 [Refereed][Not invited]
     
    Neuronal cell bodies are associated with glial cells collectively referred to as perineuronal satellite cells. One such satellite cell is the perineuronal oligodendrocyte, which is unmyelinating oligodendrocytes attaching to large neurons in various neural regions. However, little is known about their cellular characteristics and function. In this study, we identified perineuronal oligodendrocytes as 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive cells attaching to neuronal perikarya immunostained for microtubule-associated protein 2, and examined their cytochemical and cytological properties in the mouse cerebral cortex. 2',3'-Cyclic nucleotide 3'-phosphodiesterase-positive perineuronal oligodendrocytes were immunonegative to representative glial markers for astrocytes (brain-type lipid binding protein and glial fibrillary acidic protein), microglia (Iba-1) and NG2+ glia. However, almost all perineuronal oligodendrocytes expressed glia-specific or glia-enriched metabolic enzymes, i.e. the creatine synthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase and l-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase. As to molecules participating in the glutamate-glutamine cycle, none of the perineuronal oligodendrocytes expressed the plasmalemmal glutamate transporters GLAST and GLT-1, although nearly half of the perineuronal oligodendrocytes were immunopositive for glutamine synthetase. Cytologically, perineuronal oligodendrocytes were mainly distributed in deep cortical layers (layers IV-VI), and attached directly and tightly to neuronal cell bodies, making a long concave impression to the contacting neurons. Interestingly, they attached more to glutamatergic principal neurons than to GABAergic interneurons, and this became evident at postnatal day 14, when the cerebral cortex develops and maturates. These cytochemical and cytological properties suggest that perineuronal oligodendrocytes are so differentiated as to fulfill metabolic support to the associating principal cortical neurons, rather than to regulate their synaptic transmission.
  • Erik Keimpema, Klaudia Barabas, Yury M. Morozov, Giuseppe Tortoriello, Masaaki Torii, Gary Cameron, Yuchio Yanagawa, Masahiko Watanabe, Ken Mackie, Tibor Harkany
    JOURNAL OF NEUROSCIENCE 30 (42) 13992 - 14007 0270-6474 2010/10 [Refereed][Not invited]
     
    Endocannabinoids, particularly 2-arachidonoyl glycerol (2-AG), impact the directional turning and motility of a developing axon by activating CB1 cannabinoid receptors (CB(1)Rs) in its growth cone. Recent findings posit that sn-1-diacylglycerol lipases (DAGL alpha/beta) synthesize 2-AG in the motile axon segment of developing pyramidal cells. Coincident axonal targeting of CB(1)Rs and DAGLs prompts the hypothesis that autocrine 2-AG signaling facilitates axonal outgrowth. However, DAGLs alone are insufficient to account for the spatial specificity and dynamics of 2-AG signaling. Therefore, we hypothesized that local 2-AG degradation by monoacylglycerol lipase (MGL) must play a role. We determined how subcellular recruitment of MGL is temporally and spatially restricted to establish the signaling competence of 2-AG during axonal growth. MGL is expressed in central and peripheral axons of the fetal nervous system by embryonic day 12.5. MGL coexists with DAGL alpha and CB(1)Rs in corticofugal axons of pyramidal cells. Here, MGL and DAGL alpha undergo differential axonal targeting with MGL being excluded from the motile neurite tip. Thus, spatially confined MGL activity generates a 2-AG-sensing microdomain and configures 2-AG signaling to promote axonal growth. Once synaptogenesis commences, MGL disperses in stationary growth cones. The axonal polarity of MGL is maintained by differential proteasomal degradation because inhibiting the ubiquitin proteasome system also induces axonal MGL redistribution. Because MGL inactivation drives a CB1R-dependent axonal growth response, we conclude that 2-AG may act as a focal protrusive signal for developing neurons and whose regulated metabolism is critical for attaining correct axonal complexity.
  • Chihiro Takasaki, Miwako Yamasaki, Motokazu Uchigashima, Kohtarou Konno, Yuchio Yanagawa, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 (8) 1326 - 1336 0953-816X 2010/10 [Refereed][Not invited]
     
    Neuronal cell bodies are associated with glial cells collectively referred to as perineuronal satellite cells. One such satellite cell is the perineuronal oligodendrocyte, which is unmyelinating oligodendrocytes attaching to large neurons in various neural regions. However, little is known about their cellular characteristics and function. In this study, we identified perineuronal oligodendrocytes as 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive cells attaching to neuronal perikarya immunostained for microtubule-associated protein 2, and examined their cytochemical and cytological properties in the mouse cerebral cortex. 2',3'-Cyclic nucleotide 3'-phosphodiesterase-positive perineuronal oligodendrocytes were immunonegative to representative glial markers for astrocytes (brain-type lipid binding protein and glial fibrillary acidic protein), microglia (Iba-1) and NG2+ glia. However, almost all perineuronal oligodendrocytes expressed glia-specific or glia-enriched metabolic enzymes, i.e. the creatine synthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase and l-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase. As to molecules participating in the glutamate-glutamine cycle, none of the perineuronal oligodendrocytes expressed the plasmalemmal glutamate transporters GLAST and GLT-1, although nearly half of the perineuronal oligodendrocytes were immunopositive for glutamine synthetase. Cytologically, perineuronal oligodendrocytes were mainly distributed in deep cortical layers (layers IV-VI), and attached directly and tightly to neuronal cell bodies, making a long concave impression to the contacting neurons. Interestingly, they attached more to glutamatergic principal neurons than to GABAergic interneurons, and this became evident at postnatal day 14, when the cerebral cortex develops and maturates. These cytochemical and cytological properties suggest that perineuronal oligodendrocytes are so differentiated as to fulfill metabolic support to the associating principal cortical neurons, rather than to regulate their synaptic transmission.
  • Erik Keimpema, Klaudia Barabas, Yury M. Morozov, Giuseppe Tortoriello, Masaaki Torii, Gary Cameron, Yuchio Yanagawa, Masahiko Watanabe, Ken Mackie, Tibor Harkany
    JOURNAL OF NEUROSCIENCE 30 (42) 13992 - 14007 0270-6474 2010/10 [Refereed][Not invited]
     
    Endocannabinoids, particularly 2-arachidonoyl glycerol (2-AG), impact the directional turning and motility of a developing axon by activating CB1 cannabinoid receptors (CB(1)Rs) in its growth cone. Recent findings posit that sn-1-diacylglycerol lipases (DAGL alpha/beta) synthesize 2-AG in the motile axon segment of developing pyramidal cells. Coincident axonal targeting of CB(1)Rs and DAGLs prompts the hypothesis that autocrine 2-AG signaling facilitates axonal outgrowth. However, DAGLs alone are insufficient to account for the spatial specificity and dynamics of 2-AG signaling. Therefore, we hypothesized that local 2-AG degradation by monoacylglycerol lipase (MGL) must play a role. We determined how subcellular recruitment of MGL is temporally and spatially restricted to establish the signaling competence of 2-AG during axonal growth. MGL is expressed in central and peripheral axons of the fetal nervous system by embryonic day 12.5. MGL coexists with DAGL alpha and CB(1)Rs in corticofugal axons of pyramidal cells. Here, MGL and DAGL alpha undergo differential axonal targeting with MGL being excluded from the motile neurite tip. Thus, spatially confined MGL activity generates a 2-AG-sensing microdomain and configures 2-AG signaling to promote axonal growth. Once synaptogenesis commences, MGL disperses in stationary growth cones. The axonal polarity of MGL is maintained by differential proteasomal degradation because inhibiting the ubiquitin proteasome system also induces axonal MGL redistribution. Because MGL inactivation drives a CB1R-dependent axonal growth response, we conclude that 2-AG may act as a focal protrusive signal for developing neurons and whose regulated metabolism is critical for attaining correct axonal complexity.
  • Francisco Ciruela, Victor Fernandez-Duenas, Kristoffer Sahlholm, Laura Fernandez-Alacid, Joel C. Nicolau, Masahiko Watanabe, Rafael Lujan
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 (8) 1265 - 1277 0953-816X 2010/10 [Refereed][Not invited]
     
    The stimulation of inhibitory neurotransmitter receptors, such as gamma-aminobutyric acid type B (GABA(B)) receptors, activates G protein-gated inwardly-rectifying K+ (GIRK) channels, which influence membrane excitability. There is now evidence suggesting that G protein-coupled receptors and G protein-gated inwardly-rectifying K+ [GIRK/family 3 of inwardly-rectifying K+ (Kir3)] channels do not diffuse freely within the plasma membrane, but instead there are direct protein-protein interactions between them. Here, we used bioluminescence resonance energy transfer, co-immunoprecipitation, confocal and electron microscopy techniques to investigate the oligomerization of GABA(B) receptors with GIRK channels containing the GIRK3 subunit, whose contribution to functional channels is still unresolved. Co-expression of GABA(B) receptors and GIRK channels in human embryonic kidney-293 cells in combination with co-immunoprecipitation experiments established that the metabotropic receptor forms stable complexes with GIRK channels. Using bioluminescence resonance energy transfer, we have shown that, in living cells under physiological conditions, GABA(B) receptors interact directly with GIRK1/GIRK3 heterotetramers. In addition, we have provided evidence that the receptor-effector complexes are also found in vivo and identified that the cerebellar granule cells are one neuron population where the interaction probably takes place. Altogether, our data show that signalling complexes containing GABA(B) receptors and GIRK channels are formed shortly after biosynthesis, probably in the endoplasmic reticulum and/or endoplasmic reticulum/Golgi apparatus complex, suggesting that this might be a general feature of receptor-effector ion channel signal transduction and supporting a channel-forming role for the GIRK3 subunit.
  • Shinji Matsumura, Shunji Kunori, Tamaki Mabuchi, Tayo Katano, Takanobu Nakazawa, Tetsuya Abe, Masahiko Watanabe, Tadashi Yamamoto, Emiko Okuda-Ashitaka, Seiji Ito
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 (5) 798 - 810 0953-816X 2010/09 [Refereed][Not invited]
     
    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of long-term potentiation (LTP), which can be triggered by N-methyl-d-aspartate (NMDA) receptor-mediated Ca2+ influx. We previously demonstrated that Fyn kinase-mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F-KI mice with a knock-in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F-KI mice among the major phosphorylation sites. There was no difference in the Ca2+ response to glutamate and sensitivity to NMDA receptor antagonists between naive wild-type and Y1472F-KI mice, and the Ca2+ response to glutamate was attenuated in the Y1472F-KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F-KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase C gamma at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild-type and Y1472F-KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor-mediated CaMKII signaling in Y1472F-KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain.
  • Mike T. Lin, Rafael Lujan, Masahiko Watanabe, Matthew Frerking, James Maylie, John P. Adelman
    JOURNAL OF NEUROSCIENCE 30 (35) 11726 - 11734 0270-6474 2010/09 [Refereed][Not invited]
     
    Small conductance Ca(2+)-activated K(+) type 2 (SK2) channels are expressed in the postsynaptic density of CA1 neurons where they are activated by synaptically evoked Ca(2+) influx to limit the size of EPSPs and spine Ca(2+) transients. At Schaffer collateral synapses, the induction of long-term potentiation (LTP) increases the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated contribution to synaptic transmission and decreases the synaptic SK2 channel contribution through protein kinase A-dependent channel endocytosis. Using a combination of electrophysiology and immunoelectron microscopy in mice, the relationship between the dynamics of spine SK2 channels and AMPARs was investigated. Unlike AMPARs, synaptic SK2 channels under basal conditions do not rapidly recycle. Furthermore, SK2 channels occupy a distinct population of endosomes separate from AMPARs. However, blocking vesicular exocytosis or the delivery of synaptic GluA1-containing AMPARs during the induction of LTP blocks SK2 channel endocytosis. By similar to 2 h after the induction of LTP, synaptic SK2 channel expression and function are restored. Thus, LTP-dependent endocytosis of SK2 is coupled to LTP-dependent AMPA exocytosis, and the similar to 2 h window after the induction of LTP during which synaptic SK2 activity is absent may be important for consolidating the later phases of LTP.
  • Shinji Matsumura, Shunji Kunori, Tamaki Mabuchi, Tayo Katano, Takanobu Nakazawa, Tetsuya Abe, Masahiko Watanabe, Tadashi Yamamoto, Emiko Okuda-Ashitaka, Seiji Ito
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 (5) 798 - 810 0953-816X 2010/09 [Refereed][Not invited]
     
    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of long-term potentiation (LTP), which can be triggered by N-methyl-d-aspartate (NMDA) receptor-mediated Ca2+ influx. We previously demonstrated that Fyn kinase-mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F-KI mice with a knock-in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F-KI mice among the major phosphorylation sites. There was no difference in the Ca2+ response to glutamate and sensitivity to NMDA receptor antagonists between naive wild-type and Y1472F-KI mice, and the Ca2+ response to glutamate was attenuated in the Y1472F-KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F-KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase C gamma at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild-type and Y1472F-KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor-mediated CaMKII signaling in Y1472F-KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain.
  • Mike T. Lin, Rafael Lujan, Masahiko Watanabe, Matthew Frerking, James Maylie, John P. Adelman
    JOURNAL OF NEUROSCIENCE 30 (35) 11726 - 11734 0270-6474 2010/09 [Refereed][Not invited]
     
    Small conductance Ca(2+)-activated K(+) type 2 (SK2) channels are expressed in the postsynaptic density of CA1 neurons where they are activated by synaptically evoked Ca(2+) influx to limit the size of EPSPs and spine Ca(2+) transients. At Schaffer collateral synapses, the induction of long-term potentiation (LTP) increases the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated contribution to synaptic transmission and decreases the synaptic SK2 channel contribution through protein kinase A-dependent channel endocytosis. Using a combination of electrophysiology and immunoelectron microscopy in mice, the relationship between the dynamics of spine SK2 channels and AMPARs was investigated. Unlike AMPARs, synaptic SK2 channels under basal conditions do not rapidly recycle. Furthermore, SK2 channels occupy a distinct population of endosomes separate from AMPARs. However, blocking vesicular exocytosis or the delivery of synaptic GluA1-containing AMPARs during the induction of LTP blocks SK2 channel endocytosis. By similar to 2 h after the induction of LTP, synaptic SK2 channel expression and function are restored. Thus, LTP-dependent endocytosis of SK2 is coupled to LTP-dependent AMPA exocytosis, and the similar to 2 h window after the induction of LTP during which synaptic SK2 activity is absent may be important for consolidating the later phases of LTP.
  • Clementina M. van Rijn, Silvana Gaetani, Ines Santolini, Aleksandra Badura, Aleksandra Gabova, Jin Fu, Masashiko Watanabe, Vincenzo Cuomo, Gilles van Luijtelaar, Ferdinando Nicoletti, Richard T. Ngomba
    EPILEPSIA 51 (8) 1511 - 1521 0013-9580 2010/08 [Refereed][Not invited]
     
    P>Purpose: Genetically epileptic WAG/Rij rats develop spontaneous absence-like seizures after 3 months of age. We used WAG/Rij rats to examine whether absence seizures are associated with changes in the expression of type-1 cannabinoid (CB1) receptors. Methods: Receptor expression was examined by in situ hybridization and western blot analysis in various brain regions of "presymptomatic" 2-month old and "symptomatic" 8-month-old WAG/Rij rats relative to age-matched nonepileptic control rats. Furthermore, we examined whether pharmacologic activation of CB1 receptor affects absence seizures. We recorded spontaneous spike-wave discharges (SWDs) in 8-month old WAG/Rij rats systemically injected with the potent CB1 receptor agonist, R(+)WIN55,212-2 (3-12 mg/kg, s.c.), given alone or combined with the CB1 receptor antagonist/inverse agonist, AM251 (12 mg/kg, s.c.). Results: Data showed a reduction of CB1 receptor mRNA and protein levels in the reticular thalamic nucleus, and a reduction in CB1 receptor protein levels in ventral basal thalamic nuclei of 8-month-old WAG/Rij rats, as compared with age-matched ACI control rats. In vivo, R(+)WIN55,212-2 caused a dose-dependent reduction in the frequency of SWDs in the first 3 h after the injection. This was followed by a late increase in the mean SWD duration, which suggests a biphasic modulation of SWDs by CB1 receptor agonists. Both effects were reversed or attenuated when R(+)WIN55,212-2 was combined with AM251. Discussion: These data indicate that the development of absence seizures is associated with plastic modifications of CB1 receptors within the thalamic-cortical-thalamic network, and raise the interesting possibility that CB1 receptors are targeted by novel antiabsence drugs.
  • Hassan Marzban, Seung-Hyuk Chung, Maryam Kherad Pezhouh, Hans Feirabend, Masahiko Watanabe, Jan Voogd, Richard Hawkes
    JOURNAL OF COMPARATIVE NEUROLOGY 518 (12) 2221 - 2239 0021-9967 2010/06 [Refereed][Not invited]
     
    The The chick is a well-understood developmental model of cerebellar pattern formation,but we know much less about the patterning of the adult chicken cerebellum. Therefore an expression study of two Purkinje cell stripe antigens zebrin II/aldolase C and phospholipase C beta 4 (PLC beta 4)-has been carried out in the adult chicken (Gallus domesticus). The mammalian cerebellar cortex is built around transverse expression domains ("transverse zones"), each of which is further subdivided into parasagittally oriented stripes. The results from the adult chicken reveal a similar pattern. Five distinct transverse domains were identified. In the anterior lobe a uniformly zebrin II-immunopositive/PLC beta 4-immunonegative lingular zone (LZ; lobule I) and a striped anterior zone (AZ; lobules II-Via) were distinguished. A central zone (CZ; similar to lobules Vla-VIIIa,b) and a posterior zone (PZ; similar to lobules VIIIa,b-IXc,d) were distinguished in the posterior lobe. Finally, the nodular zone (NZ; lobule X) is uniformly zebrin II-immunoreactive and is innervated by vestibular mossy fibers. Lobule IXc,d is considered as a transitional region between the PZ and the NZ, because the vestibular mossy fiber projection extends into these lobules and because they receive optokinetic mossy and climbing fiber input. It is proposed that the zebrin II-immunonegative P3- stripe corresponds to the lateral vermal B zone of the mammalian cerebellum and that the border between the avian homologs of the mammalian vermis and hemispheres is located immediately lateral to P3-. Thus, there seem to be transverse zones in chicken that are plausible homologs of those identified in mammals, together with an LZ that is characteristic of birds. J. Comp. Neurol. 518:2221-2239, 2010. (C) 2010 Wiley-Liss, Inc.
  • Maya Yamazaki, Masahiro Fukaya, Kouichi Hashimoto, Miwako Yamasaki, Mika Tsujita, Makoto Itakura, Manabu Abe, Rie Natsume, Masami Takahashi, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 31 (12) 2204 - 2220 0953-816X 2010/06 [Refereed][Not invited]
     
    The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, gamma-2 and gamma-7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, gamma-2 and gamma-7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in gamma-2-knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in gamma-7-KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in gamma-2/gamma-7 double-KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in gamma-2-KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber-Purkinje cell synapses, and AMPA receptor-mediated currents at climbing fiber-Purkinje cell synapses were all reduced to approximately 10% of the wild-type levels in DKO mice. On the other hand, the reduction in GluA4 in gamma-7-KO granular layer reflected its loss at mossy fiber-granule cell synapses, whereas that of GluA1 and GluA4 in gamma-7-KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, gamma-2 and gamma-7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression.
  • Takuma Kitanishi, Jun Sakai, Shinichi Kojima, Yoshito Saitoh, Kaoru Inokuchi, Masahiro Fukaya, Masahiko Watanabe, Norio Matsuki, Maki K Yamada
    Genes to cells : devoted to molecular & cellular mechanisms 15 (7) 737 - 47 1356-9597 2010/06 [Refereed][Not invited]
     
    Actin reorganization in dendritic spines is hypothesized to underlie neuronal plasticity. Actin-related proteins, therefore, might serve as useful markers of plastic changes in dendritic spines. Here, we utilized memory deficits induced by fimbria-fornix transection (FFT) in rats as a dementia model to screen candidate memory-associated molecules by using a two-dimensional gel method. Comparison of protein profiles between the transected and control sides of hippocampi after unilateral FFT revealed a reduction in the F-actin capping protein (CapZ) signal on the FFT side. Subsequent immunostaining of brain sections and cultured hippocampal neurons revealed that CapZ localized in dendritic spines and the signal intensity in each spine varied widely. The CapZ content decreased after suppression of neuronal firing by tetrodotoxin treatment in cultured neurons, indicating rapid and activity-dependent regulation of CapZ accumulation in spines. To test input specificity of CapZ accumulation in vivo, we delivered high-frequency stimuli to the medial perforant path unilaterally in awake rats. This path selectively inputs to the middle molecular layer of the dentate gyrus, where CapZ immunoreactivity increased. We conclude that activity-dependent, synapse-specific regulation of CapZ redistribution might be important in both maintenance and remodeling of synaptic connections in neurons receiving specific spatial and temporal patterns of inputs.
  • Maya Yamazaki, Masahiro Fukaya, Kouichi Hashimoto, Miwako Yamasaki, Mika Tsujita, Makoto Itakura, Manabu Abe, Rie Natsume, Masami Takahashi, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 31 (12) 2204 - 2220 0953-816X 2010/06 [Refereed][Not invited]
     
    The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, gamma-2 and gamma-7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, gamma-2 and gamma-7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in gamma-2-knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in gamma-7-KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in gamma-2/gamma-7 double-KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in gamma-2-KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber-Purkinje cell synapses, and AMPA receptor-mediated currents at climbing fiber-Purkinje cell synapses were all reduced to approximately 10% of the wild-type levels in DKO mice. On the other hand, the reduction in GluA4 in gamma-7-KO granular layer reflected its loss at mossy fiber-granule cell synapses, whereas that of GluA1 and GluA4 in gamma-7-KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, gamma-2 and gamma-7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression.
  • Hassan Marzban, Seung-Hyuk Chung, Maryam Kherad Pezhouh, Hans Feirabend, Masahiko Watanabe, Jan Voogd, Richard Hawkes
    JOURNAL OF COMPARATIVE NEUROLOGY 518 (12) 2221 - 2239 0021-9967 2010/06 [Refereed][Not invited]
     
    The The chick is a well-understood developmental model of cerebellar pattern formation,but we know much less about the patterning of the adult chicken cerebellum. Therefore an expression study of two Purkinje cell stripe antigens zebrin II/aldolase C and phospholipase C beta 4 (PLC beta 4)-has been carried out in the adult chicken (Gallus domesticus). The mammalian cerebellar cortex is built around transverse expression domains ("transverse zones"), each of which is further subdivided into parasagittally oriented stripes. The results from the adult chicken reveal a similar pattern. Five distinct transverse domains were identified. In the anterior lobe a uniformly zebrin II-immunopositive/PLC beta 4-immunonegative lingular zone (LZ; lobule I) and a striped anterior zone (AZ; lobules II-Via) were distinguished. A central zone (CZ; similar to lobules Vla-VIIIa,b) and a posterior zone (PZ; similar to lobules VIIIa,b-IXc,d) were distinguished in the posterior lobe. Finally, the nodular zone (NZ; lobule X) is uniformly zebrin II-immunoreactive and is innervated by vestibular mossy fibers. Lobule IXc,d is considered as a transitional region between the PZ and the NZ, because the vestibular mossy fiber projection extends into these lobules and because they receive optokinetic mossy and climbing fiber input. It is proposed that the zebrin II-immunonegative P3- stripe corresponds to the lateral vermal B zone of the mammalian cerebellum and that the border between the avian homologs of the mammalian vermis and hemispheres is located immediately lateral to P3-. Thus, there seem to be transverse zones in chicken that are plausible homologs of those identified in mammals, together with an LZ that is characteristic of birds. J. Comp. Neurol. 518:2221-2239, 2010. (C) 2010 Wiley-Liss, Inc.
  • Takuma Kitanishi, Jun Sakai, Shinichi Kojima, Yoshito Saitoh, Kaoru Inokuchi, Masahiro Fukaya, Masahiko Watanabe, Norio Matsuki, Maki K Yamada
    Genes to cells : devoted to molecular & cellular mechanisms 15 (7) 737 - 47 1356-9597 2010/06 [Refereed][Not invited]
     
    Actin reorganization in dendritic spines is hypothesized to underlie neuronal plasticity. Actin-related proteins, therefore, might serve as useful markers of plastic changes in dendritic spines. Here, we utilized memory deficits induced by fimbria-fornix transection (FFT) in rats as a dementia model to screen candidate memory-associated molecules by using a two-dimensional gel method. Comparison of protein profiles between the transected and control sides of hippocampi after unilateral FFT revealed a reduction in the F-actin capping protein (CapZ) signal on the FFT side. Subsequent immunostaining of brain sections and cultured hippocampal neurons revealed that CapZ localized in dendritic spines and the signal intensity in each spine varied widely. The CapZ content decreased after suppression of neuronal firing by tetrodotoxin treatment in cultured neurons, indicating rapid and activity-dependent regulation of CapZ accumulation in spines. To test input specificity of CapZ accumulation in vivo, we delivered high-frequency stimuli to the medial perforant path unilaterally in awake rats. This path selectively inputs to the middle molecular layer of the dentate gyrus, where CapZ immunoreactivity increased. We conclude that activity-dependent, synapse-specific regulation of CapZ redistribution might be important in both maintenance and remodeling of synaptic connections in neurons receiving specific spatial and temporal patterns of inputs.
  • Takatoshi Iijima, Eriko Miura, Masahiko Watanabe, Michisuke Yuzaki
    EUROPEAN JOURNAL OF NEUROSCIENCE 31 (9) 1606 - 1615 0953-816X 2010/05 [Refereed][Not invited]
     
    Many members of the C1q family, including complement C1q and adiponectin, and the structurally related tumor necrosis factor family are secreted and play crucial roles in intercellular signaling. Among them, the Cbln (precerebellin) and C1q-like (C1ql) subfamilies are highly and predominantly expressed in the central nervous system. Although the Cbln subfamily serve as essential trans-neuronal regulators of synaptic integrity in the cerebellum, the functions of the C1ql subfamily (C1ql1-C1ql4) remain unexplored. Here, we investigated the gene expression of the C1ql subfamily in the adult and developing mouse brain by reverse transcriptase-polymerase chain reaction and high-resolution in-situ hybridization. In the adult brain, C1ql1-C1ql3 mRNAs were mainly expressed in neurons but weak expression was seen in glia-like structures in the adult brain. The C1ql1 mRNA was predominantly expressed in the inferior olive, whereas the C1ql2 and C1ql3 mRNAs were strongly coexpressed in the dentate gyrus. Although the C1ql1 and C1ql3 mRNAs were detectable as early as embryonic day 13, the C1ql2 mRNA was observed at later embryonic stages. The C1ql1 mRNA was also expressed transiently in the external granular layer of the cerebellum. Biochemical characterization in heterologous cells revealed that all of the C1ql subfamily proteins were secreted and they formed both homomeric and heteromeric complexes. They also formed hexameric and higher-order complexes via their N-terminal cysteine residues. These results suggest that, like Cbln, the C1ql subfamily has distinct spatial and temporal expression patterns and may play diverse roles by forming homomeric and heteromeric complexes in the central nervous system.
  • Takatoshi Iijima, Eriko Miura, Masahiko Watanabe, Michisuke Yuzaki
    EUROPEAN JOURNAL OF NEUROSCIENCE 31 (9) 1606 - 1615 0953-816X 2010/05 [Refereed][Not invited]
     
    Many members of the C1q family, including complement C1q and adiponectin, and the structurally related tumor necrosis factor family are secreted and play crucial roles in intercellular signaling. Among them, the Cbln (precerebellin) and C1q-like (C1ql) subfamilies are highly and predominantly expressed in the central nervous system. Although the Cbln subfamily serve as essential trans-neuronal regulators of synaptic integrity in the cerebellum, the functions of the C1ql subfamily (C1ql1-C1ql4) remain unexplored. Here, we investigated the gene expression of the C1ql subfamily in the adult and developing mouse brain by reverse transcriptase-polymerase chain reaction and high-resolution in-situ hybridization. In the adult brain, C1ql1-C1ql3 mRNAs were mainly expressed in neurons but weak expression was seen in glia-like structures in the adult brain. The C1ql1 mRNA was predominantly expressed in the inferior olive, whereas the C1ql2 and C1ql3 mRNAs were strongly coexpressed in the dentate gyrus. Although the C1ql1 and C1ql3 mRNAs were detectable as early as embryonic day 13, the C1ql2 mRNA was observed at later embryonic stages. The C1ql1 mRNA was also expressed transiently in the external granular layer of the cerebellum. Biochemical characterization in heterologous cells revealed that all of the C1ql subfamily proteins were secreted and they formed both homomeric and heteromeric complexes. They also formed hexameric and higher-order complexes via their N-terminal cysteine residues. These results suggest that, like Cbln, the C1ql subfamily has distinct spatial and temporal expression patterns and may play diverse roles by forming homomeric and heteromeric complexes in the central nervous system.
  • Shinichi Sakuramoto, Shiro Kikuchi, Nobue Futawatari, Hiromitsu Moriya, Natsuya Katada, Keishi Yamashita, Masahiko Watanabe
    SURGERY 147 (5) 742 - 747 0039-6060 2010/05 [Refereed][Not invited]
     
    Background. During esophagojejunostomy using a circular stapler after laparoscopy-assisted gastrectomy, placement of the anvil head via the transabdominal approach proved difficult. The authors report on a method modified for laparoscopy-assisted, esophagojejunostomy performed by placing the pretilted anvil head via the transoral approach. Methods. Between November 2007 and December 2008, esophagojejunostomy was performed using the transoral, pretilted anvil head in 27 patients after laparoscopy-assisted gastrectomy. The anesthesiologist introduced the anvil while observing its passage through the pharynx. During the anastomosis, we kept the jejunum fixed in position with a silicone band Lig-A-Loops, thereby preventing the intestine from slipping off the shaft, of the stapler. Results. Esophagojejunal anastomosis Using the transoral anvil head was achieved successfully in 26 patients; for 1 patient, passage of the anvil head was difficult owing to esophageal stenosis. No other complications, such as hypopharyngeal perforation and/or esophageal mucosal injury, occurred during passage. No postoperative complications occurred, except for 1 patient who developed anastomotic stenosis, in whom mild relief was achieved using a bougie. Conclusion. Esophagojejunostomy using the transoral pretilted anvil head is a simple and safe technique. (Surgery 2010;147:742-7)
  • Carmelo Quarta, Luigi Bellocchio, Giacomo Mancini, Roberta Mazza, Cristina Cervino, Luzie J. Braulke, Csaba Fekete, Rocco Latorre, Cristina Nanni, Marco Bucci, Laura E. Clemens, Gerhard Heldmaier, Masahiko Watanabe, Thierry Leste-Lassere, Marlene Maitre, Laura Tedesco, Flaminia Fanelli, Stefan Reuss, Susanne Klaus, Raj Kamal Srivastava, Krisztina Monory, Alessandra Valerio, Annamaria Grandis, Roberto De Giorgio, Renato Pasquali, Enzo Nisoli, Daniela Cota, Beat Lutz, Giovanni Marsicano, Uberto Pagotto
    CELL METABOLISM 11 (4) 273 - 285 1550-4131 2010/04 [Refereed][Not invited]
     
    The endocannabinoid system (ECS) plays a critical role in obesity development. The pharmacological blockade of cannabinoid receptor type 1 (CB1) has been shown to reduce body weight and to alleviate obesity-related metabolic disorders. An unsolved question is at which anatomical level CB1 modulates energy balance and the mechanisms involved in its action. Here, we demonstrate that CB1 receptors expressed in forebrain and sympathetic neurons play a key role in the pathophysiological development of diet-induced obesity. Conditional mutant mice lacking CB1 expression in neurons known to control energy balance, but not in nonneuronal peripheral organs, displayed a lean phenotype and resistance to diet-induced obesity. This phenotype results from an increase in lipid oxidation and thermogenesis as a consequence of an enhanced sympathetic tone and a decrease in energy absorption. In conclusion, CB1 signaling in the forebrain and sympathetic neurons is a key determinant of the ECS control of energy balance.
  • Keiko Matsuda, Eriko Miura, Taisuke Miyazaki, Wataru Kakegawa, Kyoichi Emi, Sakae Narumi, Yugo Fukazawa, Aya Ito-Ishida, Tetsuro Kondo, Ryuichi Shigemoto, Masahiko Watanabe, Michisuke Yuzaki
    SCIENCE 328 (5976) 363 - 368 0036-8075 2010/04 [Refereed][Not invited]
     
    Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor delta 2 (GluD2), expressed by Purkinje cells, are essential for synapse integrity between these neurons in adult mice. Nevertheless, no endogenous binding partners for these molecules have been identified. We found that Cbln1 binds directly to the N-terminal domain of GluD2. GluD2 expression by postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via GluD2. These results indicate that the Cbln1-GluD2 complex is a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components.
  • Carmelo Quarta, Luigi Bellocchio, Giacomo Mancini, Roberta Mazza, Cristina Cervino, Luzie J. Braulke, Csaba Fekete, Rocco Latorre, Cristina Nanni, Marco Bucci, Laura E. Clemens, Gerhard Heldmaier, Masahiko Watanabe, Thierry Leste-Lassere, Marlene Maitre, Laura Tedesco, Flaminia Fanelli, Stefan Reuss, Susanne Klaus, Raj Kamal Srivastava, Krisztina Monory, Alessandra Valerio, Annamaria Grandis, Roberto De Giorgio, Renato Pasquali, Enzo Nisoli, Daniela Cota, Beat Lutz, Giovanni Marsicano, Uberto Pagotto
    CELL METABOLISM 11 (4) 273 - 285 1550-4131 2010/04 [Refereed][Not invited]
     
    The endocannabinoid system (ECS) plays a critical role in obesity development. The pharmacological blockade of cannabinoid receptor type 1 (CB1) has been shown to reduce body weight and to alleviate obesity-related metabolic disorders. An unsolved question is at which anatomical level CB1 modulates energy balance and the mechanisms involved in its action. Here, we demonstrate that CB1 receptors expressed in forebrain and sympathetic neurons play a key role in the pathophysiological development of diet-induced obesity. Conditional mutant mice lacking CB1 expression in neurons known to control energy balance, but not in nonneuronal peripheral organs, displayed a lean phenotype and resistance to diet-induced obesity. This phenotype results from an increase in lipid oxidation and thermogenesis as a consequence of an enhanced sympathetic tone and a decrease in energy absorption. In conclusion, CB1 signaling in the forebrain and sympathetic neurons is a key determinant of the ECS control of energy balance.
  • Yohei Okubo, Hiroshi Sekiya, Shigeyuki Namiki, Hirokazu Sakamoto, Sho Iinuma, Miwako Yamasaki, Masahiko Watanabe, Kenzo Hirose, Masamitsu Iino
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 (14) 6526 - 6531 0027-8424 2010/04 [Refereed][Not invited]
     
    Glutamate is the major neurotransmitter in the brain, mediating point-to-point transmission across the synaptic cleft in excitatory synapses. Using a glutamate imaging method with fluorescent indicators, we show that synaptic activity generates extrasynaptic glutamate dynamics in the vicinity of active synapses. These glutamate dynamics had magnitudes and durations sufficient to activate extrasynaptic glutamate receptors in brain slices. We also observed crosstalk between synapses-i.e., summation of glutamate released from neighboring synapses. Furthermore, we successfully observed that sensory input from the extremities induced extrasynaptic glutamate dynamics within the appropriate sensory area of the cerebral cortex in vivo. Thus, the present study clarifies the spatiotemporal features of extrasynaptic glutamate dynamics, and opens up an avenue to directly visualizing synaptic activity in live animals.
  • Keiko Matsuda, Eriko Miura, Taisuke Miyazaki, Wataru Kakegawa, Kyoichi Emi, Sakae Narumi, Yugo Fukazawa, Aya Ito-Ishida, Tetsuro Kondo, Ryuichi Shigemoto, Masahiko Watanabe, Michisuke Yuzaki
    SCIENCE 328 (5976) 363 - 368 0036-8075 2010/04 [Refereed][Not invited]
     
    Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor delta 2 (GluD2), expressed by Purkinje cells, are essential for synapse integrity between these neurons in adult mice. Nevertheless, no endogenous binding partners for these molecules have been identified. We found that Cbln1 binds directly to the N-terminal domain of GluD2. GluD2 expression by postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via GluD2. These results indicate that the Cbln1-GluD2 complex is a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components.
  • Yuichi Nakahara, Shigeo Imanishi, Kanako Mitsumasu, Yasushi Kanamori, Ken-ichi Iwata, Masahiko Watanabe, Takahiro Kikawada, Takashi Okuda
    CRYOBIOLOGY 60 (2) 138 - 146 0011-2240 2010/04 [Refereed][Not invited]
     
    Dry-preservation of nucleated cells from multicellular animals represents a significant challenge in life science As anhydrobionts can tolerate a desiccated state, their cells and organs are expected to show high desiccation tolerance in vitro. In the present study, we established cell lines derived from embryonic tissues of an anhydrobiotic chironomid, Polypedilum vanderplanki, designated as Pv11 and Pv210 Salinity stress induced the expression of a set of anhydrobiosis-related genes in both Pv11 and Pv210 cells, suggesting that at least a part of cells can autonomously control the physiological changes for the entry into anhydrobiosis. When desiccated with medium supplemented with 300 mM trehalose or sucrose and stored for 4 weeks in dry air (approximately 5% relative humidity), a small percentage of the cells was found to be viable upon rehydration, although surviving cells seemed not to be able to multiply We also attempted dry-preservation of organs isolated from P vanderplanki larvae, and found that a proportion of cells in some organs, including fat body, testis, nerve and dorsal vessel, tolerated in vitro desiccation (C) 2009 Elsevier Inc All rights reserved
  • Miwako Yamasaki, Minoru Matsui, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 30 (12) 4408 - 4418 0270-6474 2010/03 [Refereed][Not invited]
     
    Acetylcholine (ACh) plays important roles for higher brain functions, including arousal, attention, and cognition. These effects are mediated largely by muscarinic acetylcholine receptors (mAChRs). However, it remains inconclusive whether the mode of ACh-mAChR signaling is synaptic, so-called "wired," transmission mediated by ACh released into the synaptic cleft, or nonsynaptic, so-called " volume," transmission by ambient ACh. To address this issue, we examined cellular and subcellular distribution of M-1, the most predominant mAChR subtype in the cerebral cortex and hippocampus, and pursued its anatomical relationship with cholinergic varicosities in these regions of adult mice. M-1 was highly expressed in glutamatergic pyramidal neurons, whereas it was low or undetectable in various GABAergic interneuron subtypes. M-1 was preferentially distributed on the extrasynaptic membrane of pyramidal cell dendrites and spines. Cholinergic varicosities often made direct contact to pyramidal cell dendrites and synapses. At such contact sites, however, synapse-like specialization was infrequent, and no particular accumulation was found at around contact sites for both M-1 and presynpatic active zone protein Bassoon. These features contrasted with those of the glutamatergic system, in which AMPA receptor GluA2 and metabotropic receptor mGluR5 were recruited to the synaptic or perisynaptic membrane, respectively, and Bassoon was highly accumulated in the presynaptic terminals. These results suggest that M-1 is so positioned to sense ambient ACh released from cholinergic varicosities at variable distances, and to enhance the synaptic efficacy and excitability of pyramidal cells. These molecular-anatomical arrangements will provide the evidence for volume transmission, at least in M-1-mediated cortical cholinergic signaling.
  • Ikuo Matsuda, Masahiro Fukaya, Harumi Nakao, Kazuki Nakao, Hideyuki Matsumoto, Kensaku Mori, Masahiko Watanabe, Atsu Aiba
    NEUROSCIENCE RESEARCH 66 (3) 321 - 329 0168-0102 2010/03 [Refereed][Not invited]
     
    Semaphorin 3F (Sema3F) is a secreted type of the semaphorin family of axon guidance molecules Sema3F and its receptor Neuropilin-2 (Npn-2) mRNAs were distributed in a mutually exclusive manner throughout mouse brain development In order to examine physiological roles of Sema3F, we generated Sema3F knockout mice (KO) by gene targeting in embryonic stem (ES) cells We found that the loss of Sema3F expression did not significantly affect the mRNA expression of Npn-2 or the other putative Npn-2 ligands, namely. Sema3B, Sema3C, or Sema3G. The barrel structure of the somatosensory cortex and the cerebellar neuroanatomy were not significantly altered in Sema3F KO Finally, optical imaging of intrinsic signals of the dorsal olfactory bulb showed no significant differences in odor map between wildtype mice and Sema3F KO These data suggest that Sema3F plays a relatively restricted. if any, role in its receptor expression and postnatal development of these brain Structures (C) 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society All rights reserved
  • Hiroshi Katoh, Kanako Hosono, Yoshiya Ito, Tatsunori Suzuki, Yasufumi Ogawa, Hidefumi Kubo, Hiroki Kamata, Toshiaki Mishima, Hideaki Tamaki, Hiroyuki Sakagami, Yukihiko Sugimoto, Shuh Narumiya, Masahiko Watanabe, Masataka Majima
    AMERICAN JOURNAL OF PATHOLOGY 176 (3) 1469 - 1483 0002-9440 2010/03 [Refereed][Not invited]
     
    Bone marrow (BM)-derived hematopoietic cells, which are major components of tumor stroma, determine the tumor microenvironment and regulate tumor phenotypes. Cyclooxygenase (COX)-2 and endogenous prostaglandins are important determinants for tumor growth and tumor-associated angiogenesis; however, their contributions to stromal formation and angiogenesis remain unclear. in this study, we observed that Lewis lung carcinoma cells implanted in wild-type mice formed a tumor mass with extensive stromal formation that was markedly suppressed by COX-2 inhibition, which reduced the recruitment of BM cells. Notably, COX-2 inhibition attenuated CXCL12/CXCR4 expression as well as expression of several other chemokines. Indeed, in a Matrigel model, prostaglandin (PG) E(2) enhanced stromal formation and CXCL12/CXCR4 expression. in addition, a COX-2 inhibitor suppressed stromal formation and reduced expression of CXCL12/CXCR4 and a fibroblast marker (S100A4) in a micropore chamber model. Moreover, stromal formation after tumor implantation was suppressed in EP3(-/-) mice and EP4(-/-) mice, in which stromal expression of CXCL12/CXCR4 and S100A4 was reduced. The EP3 or EN knockout suppressed S100A4(+) fibroblasts, CXCL12(+), and/or CXCR4(+) stromal cells as well. Immunofluorescent analyses revealed that CXCL12(+)CXCR4(+)S100A4(+) fibroblasts mainly comprised stromal cells and most of these were recruited from the BM. Additionally, either EP3- or EP4-specific agonists stimulated CXCL12 expression by fibroblasts in vitro. The present results address the novel activities of COX-2/PGE(2)-EP3/EP4 signaling that modulate tumor biology and show that CXCL12/CXCR4 axis may play a crucial role in tumor stromal formation and angiogenesis under die control of prostaglandins. (Am J Pathol 2010, 176:1469-148.3,- DOI: 10.2353/ajpath.2010.090607)
  • Miwako Yamasaki, Minoru Matsui, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 30 (12) 4408 - 4418 0270-6474 2010/03 [Refereed][Not invited]
     
    Acetylcholine (ACh) plays important roles for higher brain functions, including arousal, attention, and cognition. These effects are mediated largely by muscarinic acetylcholine receptors (mAChRs). However, it remains inconclusive whether the mode of ACh-mAChR signaling is synaptic, so-called "wired," transmission mediated by ACh released into the synaptic cleft, or nonsynaptic, so-called " volume," transmission by ambient ACh. To address this issue, we examined cellular and subcellular distribution of M-1, the most predominant mAChR subtype in the cerebral cortex and hippocampus, and pursued its anatomical relationship with cholinergic varicosities in these regions of adult mice. M-1 was highly expressed in glutamatergic pyramidal neurons, whereas it was low or undetectable in various GABAergic interneuron subtypes. M-1 was preferentially distributed on the extrasynaptic membrane of pyramidal cell dendrites and spines. Cholinergic varicosities often made direct contact to pyramidal cell dendrites and synapses. At such contact sites, however, synapse-like specialization was infrequent, and no particular accumulation was found at around contact sites for both M-1 and presynpatic active zone protein Bassoon. These features contrasted with those of the glutamatergic system, in which AMPA receptor GluA2 and metabotropic receptor mGluR5 were recruited to the synaptic or perisynaptic membrane, respectively, and Bassoon was highly accumulated in the presynaptic terminals. These results suggest that M-1 is so positioned to sense ambient ACh released from cholinergic varicosities at variable distances, and to enhance the synaptic efficacy and excitability of pyramidal cells. These molecular-anatomical arrangements will provide the evidence for volume transmission, at least in M-1-mediated cortical cholinergic signaling.
  • Asami Tanimura, Maya Yamazaki, Yuki Hashimotodani, Motokazu Uchigashima, Shinya Kawata, Manabu Abe, Yoshihiro Kita, Kouichi Hashimoto, Takao Shimizu, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    NEURON 65 (3) 320 - 327 0896-6273 2010/02 [Refereed][Not invited]
     
    Endocannabinoids are released from postsynaptic neurons and cause retrograde suppression of synaptic transmission. Anandamide and 2-arachidonoylglycerol (2-AG) are regarded as two major endocannabinoids. To determine to what extent 2-AG contributes to retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase alpha (DGL alpha) and beta (DGL beta). We found that endocannabinoid-mediated retrograde synaptic suppression was totally absent in the cerebellum, hippocampus, and striatum of DGL alpha knockout mice, whereas the retrograde suppression was intact in DGL beta knockout brains. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGL alpha knockout brains, whereas the 2-AG content was normal in DGL beta knockout brains. Morphology of the brain and expression of molecules required for 2-AG production other than DGLs were normal in the two knockout mice. We conclude that 2-AG produced by DGL alpha, but not by DGL beta, mediates retrograde suppression at central synapses.
  • Norifumi Shioda, Yui Yamamoto, Masahiko Watanabe, Bert Binas, Yuji Owada, Kohji Fukunaga
    JOURNAL OF NEUROSCIENCE 30 (8) 3146 - 3155 0270-6474 2010/02 [Refereed][Not invited]
     
    Fatty acid binding proteins (FABPs) are essential for energy production and long-chain polyunsaturated fatty acid-related signaling in the brain and other tissues. Of various FABPs, heart-type fatty acid binding protein (H-FABP, FABP3) is highly expressed in neurons of mature brain and plays a role in arachidonic acid incorporation into brain and heart cells. However, the precise function of H-FABP in brain remains unclear. We previously demonstrated that H-FABP is associated with the dopamine D-2 receptor long isoform (D2LR) in vitro. Here, we confirm that H-FABP binds to dopamine D-2 receptor (D2R) in brain extracts and colocalizes immunohistochemically with D2R in the dorsal striatum. We show that H-FABP is highly expressed in acetylcholinergic interneurons and terminals of glutamatergic neurons in the dorsal striatum of mouse brain but absent in dopamine neuron terminals and spines in the same region. H-FABP knock-out (KO) mice showed lower responsiveness to methamphetamine-induced sensitization and enhanced haloperidol-induced catalepsy compared with wild-type mice, indicative of D2R dysfunction. Consistent with the latter, aberrant increased acetylcholine (ACh) release and depolarization-induced glutamate (Glu) release were observed in the dorsal striatum of H-FABP KO mice. Furthermore, phosphorylation of CaMKII (Ca2+/calmodulin-dependent protein kinase II) and ERK (extracellular signal-regulated kinase) was significantly increased in the dorsal striatum. We confirmed elevated ERK phosphorylation following quinpirole-mediated D2R stimulation in H-FABP-overexpressing SHSY-5Y human neuroblastoma cells. Together, H-FABP is highly expressed in ACh interneurons and glutamatergic terminals, thereby regulating dopamine D2R function in the striatum.
  • Zsuzsa S. Kocsis, Csilla S. Molnar, Masahiko Watanabe, Guy Daneels, Dieder Moechars, Zsolt Liposits, Erik Hrabovszky
    NEUROCHEMISTRY INTERNATIONAL 56 (3) 479 - 486 0197-0186 2010/02 [Refereed][Not invited]
     
    Recent immunohistochemical studies of the rat adenohypophysis identified type-2 vesicular glutamate transporter (VGLUT-2), a marker for glutamatergic neuronal phenotype, in high percentages of adenohypophysial gonadotrophs and thyrotrophs. The presence and molecular identity of amino acid neurotransmitters in the remaining hormone producing cell types are unknown. In the present study we addressed the putative synthesis of another glutamatergic marker, VGLUT1 by adenohypophysial cells. Immunohistochemical studies revealed VGLUT1 immunoreactivity in a small subset of polygonal medium-sized cells in the anterior lobe. Western blot analysis revealed a single major 60 kDa protein band in the adenohypophysis. Furthermore, the expression of VGLUT1 mRNA was confirmed by reverse transcription-polymerase chain reaction followed by sequence analysis of the amplicon. In contrast with rats which only showed VCLUT1 signal in the anterior lobe of the pituitary, mice contained high levels of VGLUT1 immunoreactivity in the intermediate, in addition to the anterior lobe. No signal was present in VGLUT1-knockout mice, providing evidence for specificity. In rats, results of colocalization studies with dual-immunofluorescent labeling provided evidence for VGLUT1 immunoreactivity in 45.9% of corticotrophs and 7.7% of luteinizing hormone beta-immunopositive gonadotrophs. Cells of the other peptide hormone phenotypes were devoid of VGLUT1 signal. A few cells in the adenohypophysis expressed both VGLUT1 and VGLUT2 immunoreactivities. The presence of the glutamate markers VGLUT1 and VGLUT2 in distinct populations of peptide hormone-secreting hypophysial cells highly indicates the involvement of endogenous glutamate release in autocrine/paracrine regulatory mechanisms. The biological function of adenohypophysial glutamate will require clarification. (C) 2009 Elsevier Ltd. All rights reserved.
  • Yasukazu Hozumi, Masahiko Watanabe, Kaoru Goto
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 58 (2) 119 - 129 0022-1554 2010/02 [Refereed][Not invited]
     
    The pituitary gland dynamically changes its hormone output under various pathophysiological conditions. One of the pathways implicated in the regulatory mechanism of this gland is a dopaminergic system that operates the phosphoinositide (PI) cycle to transmit downstream signal through second messengers. We have previously shown that diacylglycerol kinase beta (DGK beta) is coexpressed with dopamine D1 and D2 receptors in medium spiny neurons of the striatum, suggesting a plausible implication of DGK beta in dopaminergic transmission. However, it remains elusive whether DGK beta is involved in the dopaminergic system in the pituitary gland. The aim of this study is to investigate the expression and localization of DGK in the pituitary gland, together with the molecular components involved in the PI signaling cascade, including dopamine receptors, phospholipase C (PLC), and a major downstream molecule, protein kinase C (PKC). Here we show that DGK beta and the dopamine D2 receptor are coexpressed in the intermediate lobe and localize to the plasma membrane side by side. In addition, we reveal that PLC beta 4 and PKC alpha. are the subtypes expressed in the intermediate lobe among those families. These findings will substantiate and further extend our understanding of the molecular-anatomical pathway of PI signaling and the functional roles of DGK in the pituitary intermediate lobe. (J Histochem Cytochem 58:119-129, 2010)
  • Keiko Fujikawa, Takeshi Iwata, Kaoru Inoue, Masakazu Akahori, Hanako Kadotani, Masahiro Fukaya, Masahiko Watanabe, Qing Chang, Edward M. Barnett, Wojciech Swat
    PLOS ONE 5 (2) 1932-6203 2010/02 [Refereed][Not invited]
     
    Background: Glaucoma is a leading cause of blindness worldwide. Nonetheless, the mechanism of its pathogenesis has not been well-elucidated, particularly at the molecular level, because of insufficient availability of experimental genetic animal models. Methodology/Principal Findings: Here we demonstrate that deficiency of Vav2 and Vav3, guanine nucleotides exchange factors for Rho guanosine triphosphatases, leads to an ocular phenotype similar to human glaucoma. Vav2/Vav3-deficient mice, and to a lesser degree Vav2-deficient mice, show early onset of iridocorneal angle changes and elevated intraocular pressure, with subsequent selective loss of retinal ganglion cells and optic nerve head cupping, which are the hallmarks of glaucoma. The expression of Vav2 and Vav3 tissues was demonstrated in the iridocorneal angle and retina in both mouse and human eyes. In addition, a genome-wide association study screening glaucoma susceptibility loci using single nucleotide polymorphisms analysis identified VAV2 and VAV3 as candidates for associated genes in Japanese open-angle glaucoma patients. Conclusions/Significance: Vav2/Vav3-deficient mice should serve not only as a useful murine model of spontaneous glaucoma, but may also provide a valuable tool in understanding of the pathogenesis of glaucoma in humans, particularly the determinants of altered aqueous outflow and subsequent elevated intraocular pressure.
  • Keiko Fujikawa, Takeshi Iwata, Kaoru Inoue, Masakazu Akahori, Hanako Kadotani, Masahiro Fukaya, Masahiko Watanabe, Qing Chang, Edward M. Barnett, Wojciech Swat
    PLOS ONE 5 (2) e9050  1932-6203 2010/02 [Refereed][Not invited]
     
    Background: Glaucoma is a leading cause of blindness worldwide. Nonetheless, the mechanism of its pathogenesis has not been well-elucidated, particularly at the molecular level, because of insufficient availability of experimental genetic animal models. Methodology/Principal Findings: Here we demonstrate that deficiency of Vav2 and Vav3, guanine nucleotides exchange factors for Rho guanosine triphosphatases, leads to an ocular phenotype similar to human glaucoma. Vav2/Vav3-deficient mice, and to a lesser degree Vav2-deficient mice, show early onset of iridocorneal angle changes and elevated intraocular pressure, with subsequent selective loss of retinal ganglion cells and optic nerve head cupping, which are the hallmarks of glaucoma. The expression of Vav2 and Vav3 tissues was demonstrated in the iridocorneal angle and retina in both mouse and human eyes. In addition, a genome-wide association study screening glaucoma susceptibility loci using single nucleotide polymorphisms analysis identified VAV2 and VAV3 as candidates for associated genes in Japanese open-angle glaucoma patients. Conclusions/Significance: Vav2/Vav3-deficient mice should serve not only as a useful murine model of spontaneous glaucoma, but may also provide a valuable tool in understanding of the pathogenesis of glaucoma in humans, particularly the determinants of altered aqueous outflow and subsequent elevated intraocular pressure.
  • Hidefumi Kubo, Kanako Hosono, Tatsunori Suzuki, Yasufumi Ogawa, Hiroshi Kato, Hiroki Kamata, Yoshiya Ito, Hideki Amano, Tetsuki Kato, Hiroyuki Sakagami, Izumi Hayashi, Yukihiko Sugimoto, Shuh Narumiya, Masahiko Watanabe, Masataka Majima
    BIOMEDICINE & PHARMACOTHERAPY 64 (2) 101 - 106 0753-3322 2010/02 [Refereed][Not invited]
     
    Prostaglandin E(2) (PGE(2)) and prostaglandin E (EP) receptor signaling pathways have been implicated in the promotion Of tumor growth and angiogenesis. However, little is known about their roles in lymphangiogenesis during tumor development. The present study evaluates whether endogenous PGE2 exhibits a critical role in tumor-associated lymphangiogenesis. Treatment of male C57BL/6 mice with a cyclooxygenase-2 inhibitor, celecoxib, for seven days resulted in a 52.4% reduction in tumor size induced by subcutaneous injection of murine Lewis lung cells. Celecoxib treatment down-regulated the expression of vascular endothelial growth factor receptor (VEGFR)-3 in stromal tissues by 73.9%, and attenuated expression of podoplanin, a marker for lymphatic endothelial cells. To examine the role of host PGE receptor signaling, we tested four kinds of EP receptor knockout mice. At Day 7 after tumor cell implantation, EP3 receptor knockout mice, but not EP receptor knockout mice lacking EP1, EP2, or EP4. exhibited a 53.3% reduction in tumor weight, which was associated with a 74.5% reduction in VEGFR-3 mRNA expression in tumor stromal tissues. At Day 14, VEGFR-3 expression in EP3-/- mice remained significantly lower than that of their wild-type (WT) counterparts. The expression of VEGF-C in the tumor stromal tissues in EP3-/- mice were also reduced by 22.1% (Day 7) and 44.1% (Day 14), respectively. In addition, the level Of immunoreactive podoplanin in the tumor tissues from EP3-/- mice was less than that of WT. These results suggest that host EP3 receptor signaling regulates tumor-associated lymphangiogenesis by up-regulating expression of VEGF-C and its receptor, VEGFR-3, in tumor stromal tissues. Host EP3 blockade together with COX-2 inhibition may be a novel therapeutic strategy to suppress tumor-associated lymphangiogenesis. (C) 2009 Elsevier Masson SAS. All rights reserved.
  • Taisuke Miyazaki, Miwako Yamasaki, Tomonori Takeuchi, Kenji Sakimura, Masayoshi Mishina, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 68 E86 - E86 0168-0102 2010 [Refereed][Not invited]
  • Maya Yamazaki, Masahiro Fukaya, Kouichi Hashimoto, Miwako Yamasaki, Makoto Itakura, Masami Takahashi, Masanobu Kano, Masahiko Watanabe, Kenji Sakimura
    NEUROSCIENCE RESEARCH 68 E223 - E224 0168-0102 2010 [Refereed][Not invited]
  • Yohei Okubo, Hiroshi Sekiya, Shigeyuki Namiki, Hirokazu Sakamoto, Sho Iinuma, Miwako Yamasaki, Masahiko Watanabe, Kenzo Hirose, Masamitsu Iino
    JOURNAL OF PHARMACOLOGICAL SCIENCES 112 110P - 110P 1347-8613 2010 [Refereed][Not invited]
  • Miwako Yamasaki, Masahiro Fukaya, Manabu Abe, Kenji Sakimura, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 68 E56 - E56 0168-0102 2010 [Refereed][Not invited]
  • Nobue Futawatari, Shiro Kikuchi, Hiromitsu Moriya, Natsuya Katada, Shinichi Sakuramoto, Masahiko Watanabe
    SURGERY TODAY 40 (1) 64 - 67 0941-1291 2010/01 [Refereed][Not invited]
     
    We herein report our experience in performing a laparoscopy-assisted distal gastrectomy involving standard lymph node dissection for a patient with early gastric cancer and situs inversus. A superficial elevated lesion was found on the posterior wall of the antrum. The preoperative diagnosis was cStage IA (cT1, cN0, cH0, cP0, cM0). A laparoscopy-assisted distal gastrectomy with standard lymph node dissection (D1 + beta) for early gastric cancer was successfully performed by shifting the monitor to the right and left, and by applying sufficient care and time. The course of blood vessels was not abnormal except for the right/left inversion. Billroth I reconstruction was performed through a delta-shaped anastomosis. The postoperative course was favorable and the patient was discharged on postoperative day 12. Based on a histopathological examination, a 5.0 x 5.0-cm, mucosal, poorly differentiated adenocarcinoma (pT1, pN0, sH0, sP0, sM0, Stage IA) was diagnosed.
  • Takeshi Izumi, Yu Omura, Yukino Futami, Taku Yamaguchi, Takayuki Yoshida, Masahiko Watanabe, Mitsuhiro Yoshioka
    JOURNAL OF PHARMACOLOGICAL SCIENCES 112 98P - 98P 1347-8613 2010 [Refereed][Not invited]
  • Weiqiang Kong, Tomohiro Shiraishi, Yuki Mizushima, Noriyuki Katahira, Akira Fukuda, Masahiko Watanabe
    12TH INTERNATIONAL CONFERENCE ON ADVANCED COMMUNICATION TECHNOLOGY: ICT FOR GREEN GROWTH AND SUSTAINABLE DEVELOPMENT, VOLS 1 AND 2 1003 - 1008 1738-9445 2010 [Refereed][Not invited]
     
    State Transition Matrix (STM) is a flexible table-like modeling language that has been frequently used for specifying behavior of distributed systems. In this paper, we first present a formalization of the static and dynamic aspects of a STM design (i.e., design written in STM). Consequentially, based on this formalization, we investigate how a STM design can be formally analyzed using SAL, precisely SAL infinite bounded model checker, through a language translation. Specifically, the formal analysis is conducted focusing on four kinds of safety properties related to: (1) Invalid Cells, (2) Static Constraints, (3) Dynamic Constraints, and (4) Deadlock, respectively, since the fulfillment of these properties is commonly desired by industrial practitioners for a STM design. A simple Internet Connection Control system is used as our demonstration example.
  • Kouichi Hashimoto, Mika Tsujita, Kazuo Kitamura, Taisuke Miyazaki, Maya Yamazaki, Hee-Sup Shin, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    NEUROSCIENCE RESEARCH 68 E37 - E38 0168-0102 2010 [Refereed][Not invited]
  • Jonghan Shin, Gangadharan Gireesh, Seong-Wook Kim, Duk-Soo Kim, Sukyung Lee, Yeon-Soo Kim, Masahiko Watanabe, Hee-Sup Shin
    JOURNAL OF NEUROSCIENCE 29 (49) 15375 - 15385 0270-6474 2009/12 [Refereed][Not invited]
     
    Anxiety is among the most prevalent and costly diseases of the CNS, but its underlying mechanisms are not fully understood. Although attenuated theta rhythms have been observed in human subjects with increased anxiety, no study has been done on the possible physiological link between these two manifestations. We found that the mutant mouse for phospholipase C beta 4 (PLC-beta 4(-/-)) showed attenuated theta rhythm and increased anxiety, presenting the first animal model for the human condition. PLC-beta 4 is abundantly expressed in the medial septum, a region implicated in anxiety behavior. RNA interference-mediated PLC-beta 4 knockdown in the medial septum produced a phenotype similar to that of PLC-beta 4(-/-) mice. Furthermore, increasing cholinergic signaling by administering an acetylcholinesterase inhibitor cured the anomalies in both cholinergic theta rhythm and anxiety behavior observed in PLC-beta 4(-/-) mice. These findings suggest that (1) PLC-beta 4 in the medial septum is involved in controlling cholinergic theta oscillation and (2) cholinergic theta rhythm plays a critical role in suppressing anxiety. We propose that defining the cholinergic theta rhythm profile may provide guidance in subtyping anxiety disorders in humans for more effective diagnosis and treatments.
  • Ji-Young Kim, Hassan Marzban, Seung-Hyuk Chung, Masahiko Watanabe, Leonard M. Eisenman, Richard Hawkes
    JOURNAL OF COMPARATIVE NEUROLOGY 517 (2) 193 - 209 0021-9967 2009/11 [Refereed][Not invited]
     
    Transverse boundaries divide the mammalian cerebellar cortex into transverse zones, and within each zone the cortex is further subdivided into a symmetrical array of parasagittal stripes. This topography is highly conserved across the Mammalia. Bats have a remarkable cerebellum with presumed adaptations to flight and to echolocation, but nothing is known of its compartmentation. We have therefore used two Purkinje cell compartmentation antigens, zebrin II/aldolase C and phospholipase C beta 4, to reveal the topography of the cerebellum in microchiropteran bats. Three species of bat were studied, Lasiurus cinereus, Lasionycteris noctivagans, and Eptesicus fuscus. A reproducible pattern of zones and stripes was revealed that is similar across the three species. The architecture of the bat cerebellum conforms to the ground plan of other mammals. However, two exceptions to the highly conserved mammalian architectural plan were revealed. First, many Purkinje cells in lobule I express zebrin II. A zebrin II-immunopositive lobule I has not been seen previously in mammals but is characteristic of the avian cerebellum. Second, lobules VI-VII comprise the large central zone. Within the central zone two subdomains are evident, a small anterior subdomain (lobule VI) in which Purkinje cells are predominantly zebrin II-immunopositive/PLC beta 4-immunonegative, as in other mammals, and a posterior subdomain (lobule VII), in which alternating zebrin II/phospholipase C beta 4 stripes are prominent. J. Comp. Neurol. 517:193-209, 2009. (C) 2009 Wiley-Liss, Inc.
  • Noriko Ando, Yumi Iwamitsu, Masaru Kuranami, Shigemi Okazaki, Mei Wada, Kenji Yamamoto, Keiko Todoroki, Masahiko Watanabe, Hitoshi Miyaoka
    SUPPORTIVE CARE IN CANCER 17 (11) 1361 - 1370 0941-4355 2009/11 [Refereed][Not invited]
     
    The objective of this study was to determine how psychological characteristics, subjective symptoms, a family history of breast cancer, and age impact psychological distress in outpatients at the first hospital visit prior to breast cancer diagnosis. Participants were prediagnosed women with complaints of breast symptoms who either came to our hospital directly, or with a referral from another clinic. Patients were asked to complete questionnaires to determine the following: trait anxiety (State-Trait Anxiety Inventory), negative emotional suppression (Courtauld Emotional Control Scale), life stress events (Life Experiences Survey), and psychological distress (Profile of Mood States). We examined subjective symptoms (lumps, pain, abnormal nipple discharge, or deformed nipple) and family history of breast cancer based on answers to the interview sheet filled out by patients on their first visit. We analyzed a total of 154 patients who completed the questionnaires out of 237 eligible patients. A significant model was obtained by multiple regression analysis (adjusted R (2) = 0.574, p < 0.01) in which the standard partial regression coefficients for trait anxiety, suppression of anxiety, negative life change events, positive life change events, and subjective symptoms were statistically significant (beta = 0.623, 0.133, 0.155, 0.108, and 0.124, respectively; p < 0.05). Psychological distress prior to diagnosis was higher in patients who had high trait anxiety, suppression of anxiety, many life stress events, and subjective symptoms. In particular, trait anxiety had a large impact on psychological distress, underscoring the need for and importance of adequate psychological care.
  • Toshiharu Yasaka, David I. Hughes, Erika Polgar, Gergely G. Nagy, Masahiko Watanabe, John S. Riddell, Andrew J. Todd
    JOURNAL OF NEUROSCIENCE 29 (42) 13401 - 13409 0270-6474 2009/10 [Refereed][Not invited]
     
    Pure NMDA receptor (NMDAr)-mediated EPSCs, thought to correspond to "silent" glutamatergic synapses that lack AMPA receptors (AMPArs), have been observed in superficial spinal dorsal horn of neonatal but not adult rats. Recent anatomical studies suggest that AMPArs are present at virtually all glutamatergic synapses in this region in adults. We used antigen retrieval to examine colocalization of AMPArs and PSD-95 (a marker for glutamatergic synapses) in laminae I-II of neonatal and adult rats. We found a high degree of colocalization in all cases, which suggests that AMPArs are present in the great majority of glutamatergic synapses even in neonatal animals. We therefore reexamined evidence for silent synapses by performing blind whole-cell recordings from superficial dorsal horn neurons in slices from neonatal or adult rats, with focal stimulation to activate glutamatergic synapses. On some occasions in both neonatal (10 of 109, 9%) and adult (9 of 77, 12%) slices, NMDAr-mediated EPSCs were observed when the holding potential was raised to +50 mV at a stimulus strength that had failed to evoke AMPAr-mediated EPSCs. However, in all cases tested, AMPAr-mediated EPSCs were then observed when the cell was returned to-70 mV; this and other properties of the EPSCs suggest that they do not represent genuine silent synapses. When compared with previous findings, our results indicate that the appearance of silent synapses depends on experimental protocol. This suggests that pure NMDAr-mediated EPSCs seen in previous studies do not correspond to AMPAr-lacking synapses but result from another mechanism, for example, loss of labile AMPArs from recently formed synapses.
  • Etsuko Tarusawa, Ko Matsui, Timotheus Budisantoso, Elek Molnar, Masahiko Watanabe, Minoru Matsui, Yugo Fukazawa, Ryuichi Shigemoto
    JOURNAL OF NEUROSCIENCE 29 (41) 12896 - 12908 0270-6474 2009/10 [Refereed][Not invited]
     
    To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.
  • Osamu Katsumata, Naoki Ohara, Hideaki Tamaki, Tomoko Niimura, Hideaki Naganuma, Masahiko Watanabe, Hiroyuki Sakagami
    EUROPEAN JOURNAL OF NEUROSCIENCE 30 (8) 1509 - 1516 0953-816X 2009/10 [Refereed][Not invited]
     
    IQ-ArfGEF/BRAG1 is a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), which are implicated in membrane trafficking and actin cytoskeleton dynamics. In this study, we examined the immunohistochemical localization of IQ-ArfGEF/BRAG1 in the adult mouse retina using light and electron microscopy. IQ-ArfGEF/BRAG1 was distributed in a punctate manner and colocalized well with RIBEYE in both the outer and inner plexiform layers. Immunoelectron microscopic analysis showed that IQ-ArfGEF/BRAG1 was localized at the synaptic ribbons of photoreceptors. When heterologously expressed in HeLa cells, IQ-ArfGEF/BRAG1 was recruited to RIBEYE-containing clusters and formed an immunoprecipitable complex with RIBEYE. Furthermore, immunoprecipitation analysis showed that anti-IQ-ArfGEF/BRAG1 antibody efficiently pulled down RIBEYE from retinal lysates. These findings indicate that IQ-ArfGEF/BRAG1 is a novel component of retinal synaptic ribbons and forms a protein complex with RIBEYE.
  • Laia Canela, Victor Fernandez-Duenas, Catarina Albergaria, Masahiko Watanabe, Carme Lluis, Josefa Mallol, Enric I. Canela, Rafael Franco, Rafael Lujan, Francisco Ciruela
    JOURNAL OF NEUROCHEMISTRY 111 (2) 555 - 567 0022-3042 2009/10 [Refereed][Not invited]
     
    Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.
  • Hozumi Y, Watanabe M, Goto K
    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 0022-1554 2009/10/01 [Refereed][Not invited]
  • Etsuko Tarusawa, Ko Matsui, Timotheus Budisantoso, Elek Molnar, Masahiko Watanabe, Minoru Matsui, Yugo Fukazawa, Ryuichi Shigemoto
    JOURNAL OF NEUROSCIENCE 29 (41) 12896 - 12908 0270-6474 2009/10 [Refereed][Not invited]
     
    To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.
  • Naoyuki Yamasaki, Masahiko Watanabe, Kimihiro Masuyama, Yasuo Miyake, Hitoshi Kubo, Akihiko Fujii, Masanori Ozaki
    JAPANESE JOURNAL OF APPLIED PHYSICS 48 (10) 0021-4922 2009/10 [Refereed][Not invited]
     
    The optical and electrical properties of polymer composite films based on poly(2-methoxy-5-dodecyloxy-p-phenylenevinylene) (MDDOPPV) and poly(9,9-dioctylfluorene-co-benzothiadiazole) (F8BT) have been studied. We investigated the time-resolved photoluminescence of the composite films and clarified the rapid intermolecular energy transfer from F8BT to MDDOPPV. As a result of the utilization of the polymer composite thin film as the emission layer of a light-emitting diode, a 23-fold increase in luminance was observed in comparison to that with a non composite MDDOPPV emission layer. (C) 2009 The Japan Society of Applied Physics
  • Akira Iizuka, Kiyohiko Takayama, Takashi Torashima, Miwako Yamasaki, Chiho Koyama, Kazuhiro Mitsumura, Masahiko Watanabe, Hirozaku Hirai
    NEUROBIOLOGY OF DISEASE 35 (3) 457 - 465 0969-9961 2009/09 [Refereed][Not invited]
     
    Hotfoot5J mice are spontaneously occurring ataxic mice that lack delta 2 glutamate receptor (GluR82) protein in cerebellar Purkinje cells. Here we aimed to rescue the ataxic phenotype of hotfoot5J mice by lentiviral vector-mediated expression of recombinant GIuR82 in Purkinje cells. Lentiviral vectors expressing GluR delta 2 were injected into the cerebellar cortex of hotfoot5J mice 6 or 7 days after birth, and the effects were studied on postnatal day 30. The motor behavior of hotfoot5J mice treated with vectors expressing GluR82 was markedly rescued, whereas the ataxia of hotfoot5J mice treated with vectors expressing GFP was comparable to that of non-injected hotfoot5J littermates. Furthermore, the impaired release probability of glutamate from parallel fiber terminals and the failure of developmental elimination of surplus climbing fibers from Purkinje cells in hotfoot5J mice were completely rescued by GIuR82 expression. These results indicate the therapeutic potential of viral vector-based gene therapy for hereditary cerebellar ataxia and other neuronal disorders. (C) 2009 Elsevier Inc. All rights reserved.
  • K. Hashimoto, T. Yoshida, K. Sakimura, M. Mishina, M. Watanabe, M. Kano
    NEUROSCIENCE 162 (3) 601 - 611 0306-4522 2009/09 [Refereed][Not invited]
     
    The climbing fiber (CF) to Purkinje cell (PC) synapse in the cerebellum provides an ideal model for the study of developmental rearrangements of neural circuits. At birth, each PC is innervated by multiple CFs. These surplus CFs are eliminated during postnatal development, and mono innervation is attained by postnatal day 20 (P20) in mice. Earlier studies on spontaneous mutant mice and animals with "hypogranular" cerebella indicate that regression of surplus CFs requires normal generation of granule cells and their axons, parallel fibers (PFs), and normal formation of PF-PC synapses. Our understanding of how PF-PC synapse formation affects development of CF-PC synapse has been greatly advanced by analyses of mutant mice deficient in glutamate receptor 82 subunit (GluR delta 2), an orphan receptor expressed selectively in PCs. Deletion of GluR delta 2 results in impairment of PF-PC synapse formation, which leads to defects in development of CF-PC synapses. In this article, we review how impaired PF-PC synapse formation affects wiring of CFs to PCs based mostly on our data on GluR delta 2 knockout mice. We propose a new scheme that CF-PC synapses are shaped by the three consecutive events, namely functional differentiation of multiple CFs into one strong and a few weak inputs from P3 to P7, "early phase" of CF synapse elimination from P7 to around P11, and "late phase" of CF synapse elimination from around P12. Normal PF-PC synapse formation is required for the "late phase" of CF synapse elimination. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Seung-Hyuk Chung, Hassan Marzban, Masahiko Watanabe, Richard Hawkes
    CEREBELLUM 8 (3) 267 - 276 1473-4222 2009/09 [Refereed][Not invited]
     
    Unipolar brush cells (UBCs) are glutamatergic cerebellar interneurons of the granular layer. Previous studies have shown that there are two distinct subsets of UBCs present in the mice cerebellar cortex: calcium-binding protein calretinin (CR) positive and metabotropic glutamate receptor (mGluR)1 alpha positive. In this study, we identify phospholipase C (PLC) beta 4 as an antigenic marker of a novel subset of UBCs. Double immunolabeling reveals that none of the CR+ subset expresses PLC beta 4. In contrast, most members of the mGluR1 alpha subset also express PLC beta 4. In addition, 65% of the PLC beta 4+ subset does not express mGluR1 alpha. Thus, there are three distinct UBC subsets in the mouse cerebellum: CR+/PLC beta 4-/mGluR1 alpha-, PLC beta 4+/mGluR1 alpha-/CR-, and mGluR1 alpha+/PLC beta 4+/CR-. Each has a different topographical distribution, both between lobules and mediolaterally within the vermis. The development of PLC beta 4 expression in UBCs is exclusively postnatal-first seen only at P12 and mature at about 3 weeks. A distinct subset of PLC beta 4+ UBCs is also present in primary cerebellar cultures.
  • Kaori Akashi, Toshikazu Kakizaki, Haruyuki Kamiya, Masahiro Fukaya, Miwako Yamasaki, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura
    JOURNAL OF NEUROSCIENCE 29 (35) 10869 - 10882 0270-6474 2009/09 [Refereed][Not invited]
     
    GluN2B (GluR epsilon 2/NR2B) subunit is involved in synapse development, synaptic plasticity, and cognitive function. However, its roles in synaptic expression and function of NMDA receptors (NMDARs) in the brain remain mostly unknown because of the neonatal lethality of global knock-out mice. To address this, we generated conditional knock-out mice, in which GluN2B was ablated exclusively in hippocampal CA3 pyramidal cells. By immunohistochemistry, GluN2B disappeared and GluN1 (GluR zeta 1/NR1) was moderately reduced, whereas GluN2A (GluR epsilon 1/NR2A) and postsynaptic density-95 (PSD-95) were unaltered in the mutant CA3. This was consistent with protein contents in the CA3 crude fraction: 9.6% of control level for GluN2B, 47.7% for GluN1, 90.6% for GluN2A, and 98.0% for PSD-95. Despite the remaining NMDARs, NMDAR-mediated currents and long-term potentiation were virtually lost at various CA3 synapses. Then, we compared synaptic NMDARs by postembedding immunogold electron microscopy and immunoblot using the PSD fraction. In the mutant CA3, GluN1 was severely reduced in both immunogold (20.6-23.6%) and immunoblot (24.6%), whereas GluN2A and PSD-95 were unchanged in immunogold but markedly reduced in the PSD fraction (51.4 and 36.5%, respectively), indicating increased detergent solubility of PSD molecules. No such increased solubility was observed for GluN2B in the CA3 of GluN2A-knock-out mice. Furthermore, significant decreases were found in the ratio of filamentous to globular actin (49.5%) and in the density of dendritic spines (76.2%). These findings suggest that GluN2B is critically involved in NMDAR channel function, organization of postsynaptic macromolecular complexes, formation or maintenance of dendritic spines, and regulation of the actin cytoskeleton.
  • Daisuke Ichihara, Eijiro Okada, Kazuhiro Chiba, Yoshiaki Toyama, Hirokazu Fujiwara, Suketaka Momoshima, Yuji Nishiwaki, Takeshi Hashimoto, Jun Ogawa, Masahiko Watanabe, Takeshi Takahata, Morio Matsumoto
    JOURNAL OF ORTHOPAEDIC SCIENCE 14 (5) 602 - 610 0949-2658 2009/09 [Refereed][Not invited]
     
    Background. We conducted a prospective long-term follow-up study to assess associations between magnetic resonance imaging (MRI) findings and changes in clinical symptoms, as well as factors relating to the prognosis of symptoms. Methods. A total of 133 patients with acute whiplash injury between 1993 and 1996 participated in this follow-up study. They underwent neurological examinations by spine surgeons and second MRI scans of the cervical spine were obtained. They also filled out a questionnaire regarding cervical symptoms and the accident details. The items evaluated by MRI were (1) a decrease in the signal intensity of the intervertebral disc; (2) anterior compression of the dura and the spinal cord; (3) posterior disc protrusion; (4) disc space narrowing; and (5) foraminal stenosis. Relations between the presence/absence of degenerative changes on MRI, accident details, and patients' symptoms were assessed by calculating the adjusted odds ratio (OR). Results. Progression of some degenerative changes was recognized on MRI in 98.5% of the 133 whiplash injury patients, and clinical symptoms diminished in more than a half of the 133 patients. There were no statistically significant associations between MRI findings and changes in clinical symptoms. The prognosis for neck pain tended to be poor after accidents with double collisions (rear-end collision followed by front-end collision) [adjusted OR 5.83, 95% confidence interval (CI) 1.15-29.71] and accidents with serious car damage (2.87, 1.03-7.99). The prognosis for stiff shoulders tended to be poor in women (2.83, 1.23-6.51); and the prognosis for numbness in the upper extremities tended to be poor after accidents with serious car damage (3.39, 1.14-10.06). Conclusions. This study demonstrated that progression of degenerative changes of the cervical spine on MRI was not associated with clinical symptoms during the 10-year period after whiplash injury.
  • Takashi Torashima, Akira Iizuka, Hajime Horiuchi, Kazuhiro Mitsumura, Miwako Yamasaki, Chiho Koyama, Kiyohiko Takayama, Masae Iino, Masahiko Watanabe, Hirozaku Hirai
    EUROPEAN JOURNAL OF NEUROSCIENCE 30 (3) 355 - 365 0953-816X 2009/08 [Refereed][Not invited]
     
    The delta 2 glutamate receptor (GluR delta 2) is expressed predominantly in cerebellar Purkinje cells. GluR delta 2 knock-out mice show impaired synaptogenesis and loss of long-term depression (LTD) at parallel fiber/Purkinje cell synapses, and persistent multiple climbing fiber (CF) innervation of Purkinje cells, resulting in severe ataxia. To identify domains critical for GluR delta 2 function, we produced various GluR delta 2 deletion constructs. Using lentiviral vectors, those constructs were expressed in Purkinje cells of GluR delta 2-deficient mice at postnatal day (P) 6 or 7, and rescue of abnormal phenotypes was examined beyond P30. Most constructs failed to rescue the defects of GluR delta 2-deficient mice, mainly because they were not efficiently transferred to the postsynaptic sites. However, a construct carrying only the extracellular N-terminal domain (NTD) and the intracellular C-terminal domain (CTD) linked with the fourth transmembrane domain of GluR delta 2 (NTD-TM4-CTD) caused incomplete, but significant rescue of ataxia, consistent with relatively better transport of the construct to the synapses. Notably, the expression of NTD-TM4-CTD in GluR delta 2-deficient Purkinje cells restored abrogated LTD, and aberrant CF territory in the molecular layer. Although the expression of NTD-TM4-CTD failed to rescue persistent multiple CF innervation of GluR delta 2-deficient Purkinje cells, a similar construct in which only TM4 was replaced with a transmembrane domain of CD4 successfully rescued the multiple CF innervation, probably due to more efficient transport of the protein to postsynaptic sites. These results suggest that NTD and CTD are critical domains of GluR delta 2, which functions substantially without conventional ligand binding and ion channel structures.
  • Alejandro J. Pernia-Andrade, Ako Kato, Robert Witschi, Rita Nyilas, Istvan Katona, Tamas F. Freund, Masahiko Watanabe, Joerg Filitz, Wolfgang Koppert, Juergen Schuettler, Guangchen Ji, Volker Neugebauer, Giovanni Marsicano, Beat Lutz, Horacio Vanegas, Hanns Ulrich Zeilhofer
    SCIENCE 325 (5941) 760 - 764 0036-8075 2009/08 [Refereed][Not invited]
     
    Diminished synaptic inhibition in the spinal dorsal horn is a major contributor to chronic pain. Pathways that reduce synaptic inhibition in inflammatory and neuropathic pain states have been identified, but central hyperalgesia and diminished dorsal horn synaptic inhibition also occur in the absence of inflammation or neuropathy, solely triggered by intense nociceptive (C-fiber) input to the spinal dorsal horn. We found that endocannabinoids, produced upon strong nociceptive stimulation, activated type 1 cannabinoid (CB1) receptors on inhibitory dorsal horn neurons to reduce the synaptic release of gamma-aminobutyric acid and glycine and thus rendered nociceptive neurons excitable by nonpainful stimuli. Our results suggest that spinal endocannabinoids and CB1 receptors on inhibitory dorsal horn interneurons act as mediators of heterosynaptic pain sensitization and play an unexpected role in dorsal horn pain-controlling circuits.
  • Hirokazu Matsuzaki, Takeshi Izumi, Machiko Matsumoto, Hiroko Togashi, Taku Yamaguchi, Takayuki Yoshida, Masahiko Watanabe, Mitsuhiro Yoshioka
    EUROPEAN JOURNAL OF PHARMACOLOGY 615 (1-3) 76 - 82 0014-2999 2009/08 [Refereed][Not invited]
     
    Traumatic events in early life are associated with an increased risk of psychiatric diseases in adulthood. 5-hydroxytryptamine (5-HT)(1A) receptors are known to play a pivotal role in the 5-HTergic mechanisms associated with the etiology of stress-related disorders. The goal of the present study was to investigate whether early postnatal stress influences 5-HT1A receptor function in the medial prefrontal cortex in adult rats. Rats were subjected to aversive foot shock (FS) during the third week of the postnatal period (3wFS group). During the postadolescent period (10-14 weeks postnatal), immunohistochemical experiments were carried out to investigate c-Fos expression following the administration of R-(+)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a 5-HT1A receptor agonist. In the 3wFS group, the 8-OH-DPAT-induced c-Fos expression in the medial prefrontal cortex was significantly attenuated compared to that in the non-FS control group. A dual immunofluorescence study revealed that a small proportion of c-Fos positive cells co-express parvalbumin, and a relatively large proportion of c-Fos positive cells co-express glutaminase, suggesting that most c-Fos positive cells are glutamatergic neurons. We found that local perfusion of 8-OH-DPAT via a dialysis probe decreased extracellular 5-HT levels in the medial prefrontal cortex of the non-FS group, but not in the 3wFS group. However, the levels of 8-OH-DPAT-induced 5-HT syndrome were not significantly different between the non-FS and 3wFS groups. Therefore, aversive stress in the third week of the postnatal period attenuates 5-HT1A receptor function in the medial prefrontal cortex in adulthood and produces feedback inhibition of the raphe nuclei via postsynaptic 5-HT1A receptors. (C) 2009 Elsevier B.V. All rights reserved.
  • Yasukazu Hozumi, Masahiko Watanabe, Koichi Otani, Kaoru Goto
    BMC NEUROSCIENCE 10 1471-2202 2009/08 [Refereed][Not invited]
     
    Background: Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol to phosphatidic acid and comprises multiple isozymes of distinct properties. Of DGKs, mRNA signal for DGK beta is strongly detected in the striatum, and one of the transcripts derived from the human DGK beta locus is annotated in GenBank as being differentially expressed in bipolar disorder patients. Recently, we have reported that DGK beta is expressed in medium spiny neurons of the striatum and is highly concentrated at the perisynapse of dendritic spines. However, it remains elusive how DGK beta is implicated in pathophysiological role in neurons at the cellular level. Results: In the present study, we investigated the expression and subcellular localization of DGK beta in the hippocampus, together with its functional implication using transfected hippocampal neurons. DGK beta is expressed not only in projection neurons but also in interneurons and is concentrated at perisynaptic sites of asymmetrical synapses. Overexpression of wild-type DGK beta promotes dendrite outgrowth at 7 d in vitro (DIV) and spine maturation at 14 DIV in transfected hippocampal neurons, although its kinase-dead mutant has no effect. Conclusion: In the hippocampus, DGK beta is expressed in both projection neurons and interneurons and is accumulated at the perisynapse of dendritic spines in asymmetrical synapses. Transfection experiments suggest that DGK beta may be involved in the molecular machineries of dendrite outgrowth and spinogenesis through its kinase activity.
  • Kenjiro Hisano, Masahiko Watanabe, Yuji Morimoto
    JOURNAL OF ANESTHESIA 23 (3) 363 - 369 0913-8668 2009/08 [Refereed][Not invited]
     
    Edaravone, a free radical scavenger, has shown neuroprotection in both animals and humans. To evaluate the mechanism of this protection, we examined the effect of edaravone on neurons themselves against glutamate neurotoxicity. Neurons were collected from 18-day fetal rat brains and a culture of almost pure neurons was obtained after 14-day culture. The neurons were exposed to 50 mu M glutamate for 10 min, followed by normal culture for 24 h. Edaravone was added to the medium during the glutamate insult (prophylactic effect) or after the insult (treatment effect). First, the cell survival rate was measured by staining with trypan blue. Second, the cells were stained with 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, di-(acetoxymethyl ester) (C-DCDHF-DA) and the relative amount of reactive oxygen species (ROS) was measured by flow cytometry. Third, the cells were stained with Hoechst 33342 and propidium iodide and the numbers of apoptotic and necrotic cells were counted. A dose-dependent prophylactic effect was observed and the cell survival rate in 500 mu M edaravone was significantly higher than that without it. However, there was no treatment effect beyond 2 h after the insult. The amount of ROS under 500 mu M edaravone at 4 h after the glutamate insult was significantly lower than the control amount. Necrosis, but not apoptosis, was significantly inhibited by edaravone. Edaravone mainly showed a prophylactic effect on neurons against glutamate neurotoxicity, possibly through the inhibition of necrosis via the suppression of ROS production. However, for a protective effect, a higher, supraclinical concentration was required, compared to the concentrations producing a protective effect in glial and endothelial cells in previous studies.
  • Michael E. Hildebrand, Philippe Isope, Taisuke Miyazaki, Toshitaka Nakaya, Esperanza Garcia, Anne Feltz, Toni Schneider, Juergen Hescheler, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe, Stephane Dieudonne, Terrance P. Snutch
    JOURNAL OF NEUROSCIENCE 29 (31) 9668 - 9682 0270-6474 2009/08 [Refereed][Not invited]
     
    T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Ca(v)3.1 T-type currents via a G-protein-and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Ca(v)3.1 gene knock-out animals show that Ca(v)3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Ca(v)3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s.
  • Laura Fernandez-Alacid, Carolina Aguado, Francisco Ciruela, Ricardo Martin, Jose Colon, Maria Jose Cabanero, Martin Gassmann, Masahiko Watanabe, Ryuichi Shigemoto, Kevin Wickman, Bernhard Bettler, Jose Sanchez-Prieto, Rafael Lujan
    JOURNAL OF NEUROCHEMISTRY 110 (4) 1363 - 1376 0022-3042 2009/08 [Refereed][Not invited]
     
    Activation of G protein-gated inwardly-rectifying K+ (GIRK or Kir3) channels by metabotropic gamma-aminobutyric acid (B) (GABA(B)) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABA(B) receptors in cerebellar Purkinje cells at post- and pre-synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co-immunoprecipitation analysis demonstrated that GIRK subunits are co-assembled with GABA(B) receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, post-synaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The post-synaptic association of GIRK subunits with GABA(B) receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At pre-synaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABA(B) receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABA(B) receptors. The association of GIRK channels and GABA(B) receptors with excitatory synapses at both post- and pre-synaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum.
  • Hirokazu Matsuzaki, Takeshi Izumi, Machiko Matsumoto, Hiroko Togashi, Taku Yamaguchi, Takayuki Yoshida, Masahiko Watanabe, Mitsuhiro Yoshioka
    EUROPEAN JOURNAL OF PHARMACOLOGY 615 (1-3) 76 - 82 0014-2999 2009/08 [Refereed][Not invited]
     
    Traumatic events in early life are associated with an increased risk of psychiatric diseases in adulthood. 5-hydroxytryptamine (5-HT)(1A) receptors are known to play a pivotal role in the 5-HTergic mechanisms associated with the etiology of stress-related disorders. The goal of the present study was to investigate whether early postnatal stress influences 5-HT1A receptor function in the medial prefrontal cortex in adult rats. Rats were subjected to aversive foot shock (FS) during the third week of the postnatal period (3wFS group). During the postadolescent period (10-14 weeks postnatal), immunohistochemical experiments were carried out to investigate c-Fos expression following the administration of R-(+)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a 5-HT1A receptor agonist. In the 3wFS group, the 8-OH-DPAT-induced c-Fos expression in the medial prefrontal cortex was significantly attenuated compared to that in the non-FS control group. A dual immunofluorescence study revealed that a small proportion of c-Fos positive cells co-express parvalbumin, and a relatively large proportion of c-Fos positive cells co-express glutaminase, suggesting that most c-Fos positive cells are glutamatergic neurons. We found that local perfusion of 8-OH-DPAT via a dialysis probe decreased extracellular 5-HT levels in the medial prefrontal cortex of the non-FS group, but not in the 3wFS group. However, the levels of 8-OH-DPAT-induced 5-HT syndrome were not significantly different between the non-FS and 3wFS groups. Therefore, aversive stress in the third week of the postnatal period attenuates 5-HT1A receptor function in the medial prefrontal cortex in adulthood and produces feedback inhibition of the raphe nuclei via postsynaptic 5-HT1A receptors. (C) 2009 Elsevier B.V. All rights reserved.
  • Kenjiro Hisano, Masahiko Watanabe, Yuji Morimoto
    JOURNAL OF ANESTHESIA 23 (3) 363 - 369 0913-8668 2009/08 [Refereed][Not invited]
     
    Edaravone, a free radical scavenger, has shown neuroprotection in both animals and humans. To evaluate the mechanism of this protection, we examined the effect of edaravone on neurons themselves against glutamate neurotoxicity. Neurons were collected from 18-day fetal rat brains and a culture of almost pure neurons was obtained after 14-day culture. The neurons were exposed to 50 mu M glutamate for 10 min, followed by normal culture for 24 h. Edaravone was added to the medium during the glutamate insult (prophylactic effect) or after the insult (treatment effect). First, the cell survival rate was measured by staining with trypan blue. Second, the cells were stained with 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, di-(acetoxymethyl ester) (C-DCDHF-DA) and the relative amount of reactive oxygen species (ROS) was measured by flow cytometry. Third, the cells were stained with Hoechst 33342 and propidium iodide and the numbers of apoptotic and necrotic cells were counted. A dose-dependent prophylactic effect was observed and the cell survival rate in 500 mu M edaravone was significantly higher than that without it. However, there was no treatment effect beyond 2 h after the insult. The amount of ROS under 500 mu M edaravone at 4 h after the glutamate insult was significantly lower than the control amount. Necrosis, but not apoptosis, was significantly inhibited by edaravone. Edaravone mainly showed a prophylactic effect on neurons against glutamate neurotoxicity, possibly through the inhibition of necrosis via the suppression of ROS production. However, for a protective effect, a higher, supraclinical concentration was required, compared to the concentrations producing a protective effect in glial and endothelial cells in previous studies.
  • Kouichi Hashimoto, Ryoichi Ichikawa, Kazuo Kitamura, Masahiko Watanabe, Masanobu Kano
    NEURON 63 (1) 106 - 118 0896-6273 2009/07 [Refereed][Not invited]
     
    Functional neural circuits are formed by eliminating early-formed redundant synapses and strengthening necessary connections during development. In newborn mouse cerebellum, each Purkinje cell (PC) is innervated by multiple climbing fibers (CFs) with similar strengths. Subsequently, a single CF is selectively strengthened by postnatal day 7 (P7). We find that this competition among multiple CFs occurs on the soma before CFs form synapses along dendrites. Notably, inmost PCs, the single CF that has been functionally strengthened (the "winner" CF) undergoes translocation to dendrites while keeping its synapses on the soma. Synapses of the weaker CFs (the "loser" CFs) remain around the soma and form "pericellular nests" with synapses of the winner CFs. Then most perisomatic synapses are eliminated nonselectively by P15. Thus, our results suggest that the selective translocation of the winner CF to dendrites in each PC determines the single CF that survives subsequent synapse elimination and persistently innervates the PC.
  • Shingo Furuse, Tomoyuki Kawamata, Jun Yamamot, Yukitoshi Niiyama, Keiichi Omote, Masahiko Watanabe, Akiyoshi Namiki
    ANESTHESIOLOGY 111 (1) 173 - 186 0003-3022 2009/07 [Refereed][Not invited]
     
    Background: Bone cancer pain has a strong impact on the quality of life of patients, but it is difficult to treat. Therefore, development of a novel strategy for the treatment of bone cancer pain is needed for improvement of patient quality of life. This study examined whether selective spinal cannabinoid receptor 1 (CB1) activation alleviates bone cancer pain and also examined the spinal expression of CBI. Methods: A bone cancer pain model was made by implantation of sarcoma cells into the intramedullary space of the mouse femur. In behavioral experiments, the authors examined the effects of activation of spinal CB1 and inhibition of metabolism of endocannabinoid on bone cancer-related pain behaviors. Immunohistochemical experiments examined the distribution and localization of CB1 in the superficial dorsal horn of the spinal cord using specific antibodies. Results: Spinal CBI activation by exogenous administration of a CB1 agonist arachidonyl-2-chloroethylamide reduced bone cancer-related pain behaviors, including behaviors related to spontaneous pain and movement-evoked pain. in immunohistochemical experiments, although mu-opioid receptor 1 expression was reduced in the superficial dorsal horn ipsilateral to the site of implantation of sarcoma cells, CB1 expression was preserved. In addition, CBI was mainly expressed in the axon terminals, but not in the dendritic process in the superficial dorsal horn. Conclusion: Spinal CBI activation reduced bone cancer-related pain behavior. Presynaptic inhibition may contribute to the analgesic effects of spinal CBI activation. These findings may lead to novel strategies for the treatment of bone cancer pain.
  • Shingo Furuse, Tomoyuki Kawamata, Jun Yamamot, Yukitoshi Niiyama, Keiichi Omote, Masahiko Watanabe, Akiyoshi Namiki
    ANESTHESIOLOGY 111 (1) 173 - 186 0003-3022 2009/07 [Refereed][Not invited]
     
    Background: Bone cancer pain has a strong impact on the quality of life of patients, but it is difficult to treat. Therefore, development of a novel strategy for the treatment of bone cancer pain is needed for improvement of patient quality of life. This study examined whether selective spinal cannabinoid receptor 1 (CB1) activation alleviates bone cancer pain and also examined the spinal expression of CBI. Methods: A bone cancer pain model was made by implantation of sarcoma cells into the intramedullary space of the mouse femur. In behavioral experiments, the authors examined the effects of activation of spinal CB1 and inhibition of metabolism of endocannabinoid on bone cancer-related pain behaviors. Immunohistochemical experiments examined the distribution and localization of CB1 in the superficial dorsal horn of the spinal cord using specific antibodies. Results: Spinal CBI activation by exogenous administration of a CB1 agonist arachidonyl-2-chloroethylamide reduced bone cancer-related pain behaviors, including behaviors related to spontaneous pain and movement-evoked pain. in immunohistochemical experiments, although mu-opioid receptor 1 expression was reduced in the superficial dorsal horn ipsilateral to the site of implantation of sarcoma cells, CB1 expression was preserved. In addition, CBI was mainly expressed in the axon terminals, but not in the dendritic process in the superficial dorsal horn. Conclusion: Spinal CBI activation reduced bone cancer-related pain behavior. Presynaptic inhibition may contribute to the analgesic effects of spinal CBI activation. These findings may lead to novel strategies for the treatment of bone cancer pain.
  • Yoko Yamagata, Shizuka Kobayashi, Tatsuya Umeda, Akihiro Inoue, Hiroyuki Sakagami, Masahiro Fukaya, Masahiko Watanabe, Nobuhiko Hatanaka, Masako Totsuka, Takeshi Yagi, Kunihiko Obata, Keiji Imoto, Yuchio Yanagawa, Toshiya Manabe, Shigeo Okabe
    JOURNAL OF NEUROSCIENCE 29 (23) 7607 - 7618 0270-6474 2009/06 [Refereed][Not invited]
     
    Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII alpha) is an essential mediator of activity-dependent synaptic plasticity that possesses multiple protein functions. So far, the autophosphorylation site-mutant mice targeted at T286 and at T305/306 have demonstrated the importance of the autonomous activity and Ca2+/calmodulin-binding capacity of CaMKII alpha, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKII alpha, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKII alpha knock-in mouse that completely lacks its kinase activity by introducing K42R mutation and examined the effects on hippocampal synaptic plasticity and behavioral learning. In homozygous CaMKII alpha (K42R) mice, kinase activity was reduced to the same level as in CaMKII alpha-null mice, whereas CaMKII protein expression was well preserved. Tetanic stimulation failed to induce not only LTP but also sustained dendritic spine enlargement, a structural basis for LTP, at the Schaffer collateral-CA1 synapse, whereas activity-dependent postsynaptic translocation of CaMKII alpha was preserved. In addition, CaMKII alpha (K42R) mice showed a severe impairment in inhibitory avoidance learning, a form of memory that is dependent on the hippocampus. These results demonstrate that kinase activity of CaMKII alpha is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.
  • Nuria Cabello, Jorge Gandia, Daniela C. G. Bertarelli, Masahiko Watanabe, Carme Lluis, Rafael Franco, Sergi Ferre, Rafael Lujan, Francisco Ciruela
    JOURNAL OF NEUROCHEMISTRY 109 (5) 1497 - 1507 0022-3042 2009/06 [Refereed][Not invited]
     
    G protein-coupled receptors are known to form homo- and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu(5) receptors (mGlu(5)R) and dopamine D-2 receptors (D2R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu(5)R, D2R and adenosine A(2A) receptor (A(2A)R). Interestingly, by using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu(5)R, D2R and A(2A)R in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein-coupled receptor oligomers in general and the mGlu(5)R/D2R/A(2A)R oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders.
  • Yoko Yamagata, Shizuka Kobayashi, Tatsuya Umeda, Akihiro Inoue, Hiroyuki Sakagami, Masahiro Fukaya, Masahiko Watanabe, Nobuhiko Hatanaka, Masako Totsuka, Takeshi Yagi, Kunihiko Obata, Keiji Imoto, Yuchio Yanagawa, Toshiya Manabe, Shigeo Okabe
    JOURNAL OF NEUROSCIENCE 29 (23) 7607 - 7618 0270-6474 2009/06 [Refereed][Not invited]
     
    Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII alpha) is an essential mediator of activity-dependent synaptic plasticity that possesses multiple protein functions. So far, the autophosphorylation site-mutant mice targeted at T286 and at T305/306 have demonstrated the importance of the autonomous activity and Ca2+/calmodulin-binding capacity of CaMKII alpha, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKII alpha, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKII alpha knock-in mouse that completely lacks its kinase activity by introducing K42R mutation and examined the effects on hippocampal synaptic plasticity and behavioral learning. In homozygous CaMKII alpha (K42R) mice, kinase activity was reduced to the same level as in CaMKII alpha-null mice, whereas CaMKII protein expression was well preserved. Tetanic stimulation failed to induce not only LTP but also sustained dendritic spine enlargement, a structural basis for LTP, at the Schaffer collateral-CA1 synapse, whereas activity-dependent postsynaptic translocation of CaMKII alpha was preserved. In addition, CaMKII alpha (K42R) mice showed a severe impairment in inhibitory avoidance learning, a form of memory that is dependent on the hippocampus. These results demonstrate that kinase activity of CaMKII alpha is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.
  • Rita Nyilas, Laura C. Gregg, Ken Mackie, Masahiko Watanabe, Andreas Zimmer, Andrea G. Hohmann, Istvan Katona
    EUROPEAN JOURNAL OF NEUROSCIENCE 29 (10) 1964 - 1978 0953-816X 2009/05 [Refereed][Not invited]
     
    Cannabinoid administration suppresses pain by acting at spinal, supraspinal and peripheral levels. Intrinsic analgesic pathways also exploit endocannabinoids; however, the underlying neurobiological substrates of endocannabinoid-mediated analgesia have remained largely unknown. Compelling evidence shows that, upon exposure to a painful environmental stressor, an endocannabinoid molecule called 2-arachidonoylglycerol (2-AG) is mobilized in the lumbar spinal cord in temporal correlation with stress-induced antinociception. We therefore characterized the precise molecular architecture of 2-AG signaling and its involvement in nociception in the rodent spinal cord. Nonradioactive in situ hybridization revealed that dorsal horn neurons widely expressed the mRNA of diacylglycerol lipase-alpha (DGL-alpha), the synthesizing enzyme of 2-AG. Peroxidase-based immunocytochemistry demonstrated high levels of DGL-alpha protein and CB(1) cannabinoid receptor, a receptor for 2-AG, in the superficial dorsal horn, at the first site of modulation of the ascending pain pathway. High-resolution electron microscopy uncovered postsynaptic localization of DGL-alpha at nociceptive synapses formed by primary afferents, and revealed presynaptic positioning of CB(1) on excitatory axon terminals. Furthermore, DGL-alpha in postsynaptic elements receiving nociceptive input was colocalized with metabotropic glutamate receptor 5 (mGluR(5)), whose activation induces 2-AG biosynthesis. Finally, intrathecal activation of mGluR(5) at the lumbar level evoked endocannabinoid-mediated stress-induced analgesia through the DGL-2-AG-CB(1) pathway. Taken together, these findings suggest a key role for 2-AG-mediated retrograde suppression of nociceptive transmission at the spinal level. The striking positioning of the molecular players of 2-AG synthesis and action at nociceptive excitatory synapses suggests that pharmacological manipulation of spinal 2-AG levels may be an efficacious way to regulate pain sensation.
  • Wataru Kakegawa, Taisuke Miyazaki, Kazuhisa Kohda, Keiko Matsuda, Kyoichi Emi, Junko Motohashi, Masahiko Watanabe, Michisuke Yuzaki
    JOURNAL OF NEUROSCIENCE 29 (18) 5738 - 5748 0270-6474 2009/05 [Refereed][Not invited]
     
    The delta 2 glutamate receptor (GluR delta 2; GluD2), which is predominantly expressed on postsynaptic sites at parallel fiber (PF)-Purkinje cell synapses in the cerebellum, plays two crucial roles in the cerebellum: the formation of PF synapses and the regulation of long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Although the induction of LTD and motor learning absolutely require signaling via the cytoplasmic C-terminal domain of GluD2, the mechanisms by which GluD2 regulates PF synaptogenesis have remained unclear. Here, we examined the role of the extracellular N-terminal domain (NTD) of GluD2 on PF synaptogenesis by injecting Sindbis virus carrying wild-type (GluD2(wt)) or mutant GluD2 into the subarachnoid supracerebellar space of GluD2-null mice. Remarkably, the expression of GluD2(wt), but not of a mutant GluD2 lacking the NTD (GluD2(Delta NTD)), rapidly induced PF synapse formation and rescued gross motor dyscoordination in adult GluD2-null mice just 1 d after injection. In addition, although the kainate receptor GluR6 (GluK2) did not induce PF synaptogenesis, a chimeric GluK2 that contained the NTD of GluD2 (GluD2(NTD)-GluK2) did. Similarly, GluD2(wt) and GluD2(NTD)-GluK2, but not GluD2(Delta NTD), induced synaptogenesis in heterologous cells in vitro. In contrast, LTD was restored in GluD2-null Purkinje cells expressing a mutant GluD2 lacking the NTD. These results indicate that the NTD of GluD2 is necessary and sufficient for the function of GluD2 in the regulation of PF -Purkinje cell synaptogenesis. Furthermore, our results suggest that GluD2 differently regulates PF synaptogenesis and cerebellar LTD through the extracellular NTD and the cytoplasmic C-terminal end, respectively.
  • Wataru Kakegawa, Taisuke Miyazaki, Kazuhisa Kohda, Keiko Matsuda, Kyoichi Emi, Junko Motohashi, Masahiko Watanabe, Michisuke Yuzaki
    JOURNAL OF NEUROSCIENCE 29 (18) 5738 - 5748 0270-6474 2009/05 [Refereed][Not invited]
     
    The delta 2 glutamate receptor (GluR delta 2; GluD2), which is predominantly expressed on postsynaptic sites at parallel fiber (PF)-Purkinje cell synapses in the cerebellum, plays two crucial roles in the cerebellum: the formation of PF synapses and the regulation of long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Although the induction of LTD and motor learning absolutely require signaling via the cytoplasmic C-terminal domain of GluD2, the mechanisms by which GluD2 regulates PF synaptogenesis have remained unclear. Here, we examined the role of the extracellular N-terminal domain (NTD) of GluD2 on PF synaptogenesis by injecting Sindbis virus carrying wild-type (GluD2(wt)) or mutant GluD2 into the subarachnoid supracerebellar space of GluD2-null mice. Remarkably, the expression of GluD2(wt), but not of a mutant GluD2 lacking the NTD (GluD2(Delta NTD)), rapidly induced PF synapse formation and rescued gross motor dyscoordination in adult GluD2-null mice just 1 d after injection. In addition, although the kainate receptor GluR6 (GluK2) did not induce PF synaptogenesis, a chimeric GluK2 that contained the NTD of GluD2 (GluD2(NTD)-GluK2) did. Similarly, GluD2(wt) and GluD2(NTD)-GluK2, but not GluD2(Delta NTD), induced synaptogenesis in heterologous cells in vitro. In contrast, LTD was restored in GluD2-null Purkinje cells expressing a mutant GluD2 lacking the NTD. These results indicate that the NTD of GluD2 is necessary and sufficient for the function of GluD2 in the regulation of PF -Purkinje cell synaptogenesis. Furthermore, our results suggest that GluD2 differently regulates PF synaptogenesis and cerebellar LTD through the extracellular NTD and the cytoplasmic C-terminal end, respectively.
  • Keishi Yamashita, Akira Ooki, Masaki Mori, Myoung Sook Kim, David Sidransky, Masahiko Watanabe
    CANCER RESEARCH 69 0008-5472 2009/05 [Refereed][Not invited]
  • Jingping Zhang, Tai-Xiang Xu, Penelope J. Hallett, Masahiko Watanabe, Seth G. N. Grant, Ole Isacson, Wei-Dong Yao
    JOURNAL OF NEUROSCIENCE 29 (9) 2948 - 2960 0270-6474 2009/03 [Refereed][Not invited]
     
    Classical dopaminergic signaling paradigms and emerging studies on direct physical interactions between the D-1 dopamine (DA) receptor and the NMDA glutamate receptor predict a reciprocally facilitating, positive feedback loop. This loop, if not controlled, may cause concomitant overactivation of both D1 and NMDA receptors, triggering neurotoxicity. Endogenous protective mechanisms must exist. Here, we report that PSD-95, a prototypical structural and signaling scaffold in the postsynaptic density, inhibits D-1-NMDA receptor subunit 1 (NR1) NMDA receptor association and uncouples NMDA receptor-dependent enhancement of D-1 signaling. This uncoupling is achieved, at least in part, via a disinhibition mechanism by which PSD-95 abolishes NMDA receptor-dependent inhibition of D-1 internalization. Knockdown of PSD-95 immobilizes D-1 receptors on the cell surface and escalates NMDA receptor-dependent D-1 cAMP signaling in neurons. Thus, in addition to its role in receptor stabilization and synaptic plasticity, PSD-95 acts as a brake on the D-1-NMDA receptor complex and dampens the interaction between them.
  • Masami O. Kinoshita, Yoko Shinoda, Kazuhisa Sakai, Tsutomu Hashikawa, Masahiko Watanabe, Takeo Machida, Yoshio Hirabayashi, Shigeki Furuya
    NEUROSCIENCE LETTERS 453 (1) 21 - 26 0304-3940 2009/03 [Refereed][Not invited]
     
    In the adult rodent brain, constitutive neurogenesis occurs in two restricted regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus, where multipotent neural stem/progenitor cells generate new neurons. Using Western blotting and immunohistochemistry for established markers, we demonstrated that the expression of 3-phosphoglycerate dehydrogenase (Phgdh), an enzyme involved in de novo synthesis Of L-serine, was upregulated in the SVZ. The expression was selective to cells having morphological features and expressing markers of astrocyte-like primary neural stem cells (type B cells) and their progeny, actively proliferating progenitors (type C cells). By contrast, Phgdh protein expression was virtually absent in committed neuronal precursors (type A cells) derived from type C cells. High levels of Phgdh were also expressed by glial tube cells located in the rostral migratory stream (RMS). Interestingly, ensheathment of type A cells by these Phgdh-expressing cells was persistent in the SVZ and RMS, suggesting that L-serine mediates trophic support for type A cells via these glial cells. In vitro neurosphere assays confirmed that growth-factor-responsive, transient amplifying neural progenitors in the SVZ, but not differentiated neurons, expressed Phgdh. in the aged brain, a decline in Phgdh expression was evident in type B and C cells of the SVZ. These observations support the notion that availability of L-serine within neural stem/progenitor cells may be a critical factor for neurogenesis in developing and adult brain. (C) 2009 Elsevier Ireland Ltd. All rights reserved.
  • Yuriko Kawakami, Kazuyuki Yoshida, Jung Hoon Yang, Takeshi Suzuki, Norihiro Azuma, Kazuhisa Sakai, Tsutomu Hashikawa, Masahiko Watanabe, Kaori Yasuda, Satoru Kuhara, Yoshio Hirabayashi, Shigeki Furuya
    Neuroscience Research 63 (3) 184 - 193 0168-0102 2009/03 [Refereed][Not invited]
     
    Mutations in the d-3-phosphoglycerate dehydrogenase (PHGDH; EC 1.1.1.95) gene, which encodes an enzyme involved in de novo l-serine biosynthesis, are shown to cause human serine deficiency disorder. This disorder has been characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. Our previous work demonstrated that targeted disruption of mouse Phgdh leads to a marked decrease in serine and glycine, severe growth retardation of the central nervous system, and lethality after embryonic day 13.5. To clarify how a serine deficiency causes neurodevelopmental defects, we characterized changes in metabolites, gene expression and morphological alterations in the spinal cord of Phgdh knockout mice. BeadChip microarray analysis revealed significant dysregulation of genes involved in the cell cycle. Ingenuity Pathway Analysis also revealed a significant perturbation of regulatory networks that operate in the cell cycle progression. Moreover, morphological examinations of the knockout spinal cord demonstrated a marked deficit in dorsal horn neurons. Radial glia cells, native neural stem/progenitor cells, accumulated in the dorsal ventricular zone, but they did not proceed to a G0-like quiescent state. The present integrative study provides in vivo evidence that normal cell cycle progression and subsequent neurogenesis of radial glia cells are severely impaired by serine deficiency. © 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society.
  • 細胞内輸送と代謝 AP-4欠損神経細胞軸索におけるAMPA受容体のオートファゴソームへの集積
    松田 信爾, 三浦 会理子, 掛川 渉, 幸田 和久, 渡辺 雅彦, 柚崎 通介
    解剖学雑誌 (一社)日本解剖学会 84 (Suppl.) 67 - 67 0022-7722 2009/03
  • Andrew J. Todd, Erika Polgar, Christine Watt, Mark E. S. Bailey, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 29 (4) 718 - 726 0953-816X 2009/02 [Refereed][Not invited]
     
    alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPArs), which mediate fast excitatory glutamatergic transmission, are tetramers made from four subunits (GluR1-4 or GluRA-D). Although synaptic AMPArs are not normally detected by immunocytochemistry in perfusion-fixed tissue, they can be revealed by using antigen retrieval with pepsin. All AMPAr-positive synapses in spinal cord are thought to contain GluR2, while the other subunits have specific laminar distributions. GluR4 can be alternatively spliced such that it has a long or short cytoplasmic tail. We have reported that < 10% of AMPAr-containing synapses in lamina II have the long form of GluR4, and that these are often arranged in dorsoventrally orientated clusters. In this study, we test the hypothesis that GluR4-containing receptors are associated with dorsal dendrites of projection neurons in laminae III and IV that express the neurokinin 1 receptor (NK1r). Immunostaining for NK1r was carried out before antigen retrieval, and sections were then reacted to reveal GluR2 and either GluR4 (long form), GluR3 or GluR1. All NK1r-positive lamina III/IV neurons had numerous GluR2-immunoreactive puncta in their dendritic plasma membranes, and virtually all (97%) of the puncta tested were labelled (usually strongly) with the GluR4 antibody. Sizes of puncta varied, but many were elongated and they were significantly larger than nearby puncta that were not associated with the NK1r cells. None of the GluR2 puncta on these cells was positive for GluR1, while 85% were GluR3-immunoreactive. These results show that synaptic AMPArs on the dendrites of the lamina III/IV NK1r projection neurons contain GluR2, GluR3 and GluR4, but not GluR1 subunits.
  • Eriko Miura, Keiko Matsuda, James I. Morgan, Michisuke Yuzaki, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 29 (4) 693 - 706 0953-816X 2009/02 [Refereed][Not invited]
     
    Cbln1 (a.k.a. precerebellin) is secreted from cerebellar granule cells as homohexamer or in heteromeric complexes with Cbln3. Cbln1 plays crucial roles in regulating morphological integrity of parallel fiber (PF)-Purkinje cell (PC) synapses and synaptic plasticity. Cbln1-knockout mice display severe cerebellar phenotypes that are essentially indistinguishable from those in glutamate receptor GluR delta 2-null mice, and include severe reduction in the number of PF-PC synapses and loss of long-term depression of synaptic transmission. To understand better the relationship between Cbln1, Cbln3 and GluR delta 2, we performed light and electron microscopic immunohistochemical analyses using highly specific antibodies and antigen-exposing methods, i.e. pepsin pretreatment for light microscopy and postembedding immunogold for electron microscopy. In conventional immunohistochemistry, Cbln1 was preferentially associated with non-terminal portions of PF axons in the molecular layer but rarely overlapped with Cbln3. In contrast, antigen-exposing methods not only greatly intensified Cbln1 immunoreactivity in the molecular layer, but also revealed its high accumulation in the synaptic cleft of PF-PC synapses. No such synaptic accumulation was evident at other PC synapses. Furthermore, Cbln1 now came to overlap almost completely with Cbln3 and GluR delta 2 at PF-PC synapses. Therefore, the convergence of all three molecules provides the anatomical basis for a common signaling pathway regulating circuit development and synaptic plasticity in the cerebellum.
  • Keiko Matsuda, Tetsuro Kondo, Takatoshi Iijima, Shinji Matsuda, Masahiko Watanabe, Michisuke Yuzaki
    EUROPEAN JOURNAL OF NEUROSCIENCE 29 (4) 707 - 717 0953-816X 2009/02 [Refereed][Not invited]
     
    Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)-Purkinje cell synapses, but is also capable of inducing new PF synapses in adult cerebellum. Although Cbln1 is reportedly released from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear, partly because Cbln1 undergoes proteolysis to yield various fragments that are differentially detected by different antibodies. To circumvent this problem, we characterized the Cbln1-binding site using recombinant Cbln1. An immunohistochemical analysis revealed that recombinant Cbln1 preferentially bound to PF-Purkinje cell synapses in primary cultures and acute slice preparations in a saturable and replaceable manner. Specific binding was observed for intact Cbln1 that had formed a hexamer, but not for the N-terminal or C-terminal fragments of Cbln1 fused to other proteins. Similarly, mutant Cbln1 that had formed a trimer did not bind to the Purkinje cells. Immunoreactivity for the recombinant Cbln1 was observed in weaver cerebellum (which lacks granule cells) but was absent in pcd cerebellum (which lacks Purkinje cells), suggesting that the binding site was located on the postsynaptic sites of PF-Purkinje cell synapses. Finally, a subcellular fractionation analysis revealed that recombinant Cbln1 bound to the synaptosomal and postsynaptic density fractions. These results indicate that Cbln1, released from granule cells as hexamers, specifically binds to a putative receptor located at the postsynaptic sites of PF-Purkinje cell synapses, where it induces synaptogenesis.
  • Susana Mato, Elena Alberdi, Catherine Ledent, Masahiko Watanabe, Carlos Matute
    GLIA 57 (3) 295 - 306 0894-1491 2009/02 [Refereed][Not invited]
     
    Regulation of Ca2+ homeostasis plays a critical role in oligodendrocyte function and survival. Cannabinoid CB1 and CB2 receptors have been shown to regulate Ca2+ levels and/or K+ currents in a variety of cell types. In this study we investigated the effect of cannabinoid compounds on the Ca2+ influx elicited in cultured oligodendrocytes by transient membrane depolarization with an elevated extracellular K+ concentration (50 mM). The CB1 receptor agonist arachidonoyl-chloro-ethanolamide (ACEA) elicited a concentration -dependent inhibition of depolarization-evoked Ca2+ transients in oligodendroglial somata with a maximal effect (94 +/- 3)% and an EC50 of 1.3 +/- 0.03 mu M. This activity was mimicked by the CB1/CB2 agonist CP55,940, as well as by the endocannabinoids N-arachidonoyl-ethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), whereas the CB2 receptor selective agonist JWH133 was ineffective. The CB1 receptor antagonist AM251 (1 mu M) also reduced the Ca2+ response evoked by high extracellular K+ and did not prevent the inhibition elicited by ACEA (3 mu M). Nevertheless, the ability of ACEA and AEA to reduce depolarization-evoked Ca2+ transients was significantly reduced in oligodendrocytes from CB1 receptor knockout mice, as well as by pretreatment with pertussis toxin. Bath application of the inwardly rectifying K+ channels (Kir channels) blockers BaCl2 (300 mu M) and CsCl2 (1 mM) reduced the size of voltage-induced Ca2+ influx and partially prevented the inhibitory effect of ACEA. Our results indicate that cannabinoids inhibit depolarization-evoked Ca2+ transients in oligodendrocytes via CB1 receptor -dependent mechanisms that involve the activation of PTX-sensitive G(i/o) proteins and the blockade of Kir channels. (C) 2008 Wiley-Liss, Inc.
  • Eriko Miura, Keiko Matsuda, James I. Morgan, Michisuke Yuzaki, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 29 (4) 693 - 706 0953-816X 2009/02 [Refereed][Not invited]
     
    Cbln1 (a.k.a. precerebellin) is secreted from cerebellar granule cells as homohexamer or in heteromeric complexes with Cbln3. Cbln1 plays crucial roles in regulating morphological integrity of parallel fiber (PF)-Purkinje cell (PC) synapses and synaptic plasticity. Cbln1-knockout mice display severe cerebellar phenotypes that are essentially indistinguishable from those in glutamate receptor GluR delta 2-null mice, and include severe reduction in the number of PF-PC synapses and loss of long-term depression of synaptic transmission. To understand better the relationship between Cbln1, Cbln3 and GluR delta 2, we performed light and electron microscopic immunohistochemical analyses using highly specific antibodies and antigen-exposing methods, i.e. pepsin pretreatment for light microscopy and postembedding immunogold for electron microscopy. In conventional immunohistochemistry, Cbln1 was preferentially associated with non-terminal portions of PF axons in the molecular layer but rarely overlapped with Cbln3. In contrast, antigen-exposing methods not only greatly intensified Cbln1 immunoreactivity in the molecular layer, but also revealed its high accumulation in the synaptic cleft of PF-PC synapses. No such synaptic accumulation was evident at other PC synapses. Furthermore, Cbln1 now came to overlap almost completely with Cbln3 and GluR delta 2 at PF-PC synapses. Therefore, the convergence of all three molecules provides the anatomical basis for a common signaling pathway regulating circuit development and synaptic plasticity in the cerebellum.
  • Masashi Sanda, Akifumi Kamata, Osamu Katsumata, Kohji Fukunag, Masahiko Watanabe, Hisatake Kondo, Hiroyuki Sakagami
    BRAIN RESEARCH 1251 7 - 15 0006-8993 2009/01 [Refereed][Not invited]
     
    IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor for Arf1 and Arf6, is localized at the postsynaptic density (PSD) and interacts with PSD-95. In this study, we identified a novel interaction of IQ-ArfGEF/BRAG1 with insulin receptor tyrosine kinase substrate of 53 kDa (IRSp53), also known as brain-specific angiogenesis inhibitor 1-associated protein 2. The interaction was mediated by the binding of the C-terminal proline-rich sequence of IQArfGEF/BRAG1 to the SH3 domain of IRSp53. IQ-ArfGEF/BRAG1 and IRSp53 were colocalized at the PSD of excitatory synapses of certain neuronal populations. Our present findings suggest that IQ-ArfGEF/BRAG1 may play roles downstream of NMDA receptors through the interaction with multivalent PSD proteins such as IRSp53 and PSD-95. (c) 2008 Elsevier B.V. All rights reserved.
  • Cesar Quiroz, Rafael Lujan, Motokazu Uchigashima, Ana Patricia Simoes, Talia N. Lerner, Janusz Borycz, Anil Kachroo, Paula M. Canas, Marco Orru, Michael A. Schwarzschild, Diane L. Rosin, Anatol C. Kreitzer, Rodrigo A. Cunha, Masahiko Watanabe, Sergi Ferre
    THESCIENTIFICWORLDJOURNAL 9 1321 - 1344 1537-744X 2009 [Refereed][Not invited]
     
    Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D-1 and D-2 receptors, respectively. Adenosine A(2A) receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D-2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A(2A) receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A(2A) receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A(2A) receptors could provide a new target for the treatment of neuropsychiatric disorders.
  • Masanobu Kano, Takako Ohno-Shosaku, Yuki Hashimotodani, Motokazu Uchigashima, Masahiko Watanabe
    PHYSIOLOGICAL REVIEWS 89 (1) 309 - 380 0031-9333 2009/01 [Refereed][Not invited]
     
    Kano M, Ohno-Shosaku T, Hashimotodani Y, Uchigashima M, Watanabe M. Endocannabinoid-Mediated Control of Synaptic Transmission. Physiol Rev 89: 309-380, 2009; doi: 10.1152/physrev.00019.2008. - The discovery of cannabinoid receptors and subsequent identification of their endogenous ligands (endocannabinoids) in early 1990s have greatly accelerated research on cannabinoid actions in the brain. Then, the discovery in 2001 that endocannabinoids mediate retrograde synaptic signaling has opened up a new era for cannabinoid research and also established a new concept how diffusible messengers modulate synaptic efficacy and neural activity. The last 7 years have witnessed remarkable advances in our understanding of the endocannabinoid system. It is now well accepted that endocannabinoids are released from postsynaptic neurons, activate presynaptic cannabinoid CB 1 receptors, and cause transient and long-lasting reduction of neurotransmitter release. In this review, we aim to integrate our current understanding of functions of the endocannabinoid system, especially focusing on the control of synaptic transmission in the brain. We summarize recent electrophysiological studies carried out on synapses of various brain regions and discuss how synaptic transmission is regulated by endocannabinoid signaling. Then we refer to recent anatomical studies on subcellular distribution of the molecules involved in endocannabinoid signaling and discuss how these signaling molecules are arranged around synapses. In addition, we make a brief overview of studies on cannabinoid receptors and their intracellular signaling, biochemical studies on endocannabinoid metabolism, and behavioral studies on the roles of the endocannabinoid system in various aspects of neural functions.
  • Andrew N. Iwaniuk, Hassan Marzban, Janelle M. P. Pakan, Masahiko Watanabe, Richard Hawkes, Douglas R. W. Wylie
    JOURNAL OF CHEMICAL NEUROANATOMY 37 (1) 55 - 63 0891-0618 2009/01 [Refereed][Not invited]
     
    The parasagittal organization of the mammalian cerebellar cortex into zones has been well characterized by immunohistochemical, hodological and physiological studies in recent years. The pattern of these parasagittal bands across the cerebellum is highly conserved across mammals, but whhether a similar conservation of immunohistochemically defined parasagittal bands occurs within birds has remained uncertain. Here, we examine the compartmentation of the cerebellar cortex of a group of birds with unique cerebellar morphology-hummingbirds (Trochilidae). Immunohistochemical techniques were used to characterize the expression of zebrin II (aldolase C) and phospholipase C beta 4 (PLC beta 4) in the cerebellar cortex of two hummingbird species. A series of zebrin II immunopositive/immunonegative parasagittal stripes was apparent across most folia representing three major transverse zones: an anterior zone with a central stripe flanked by three lateral stripes on either side; a central zone of high/low immunopositive stripes; and a posterior zone with a central stripe flanked by four to six lateral stripes on either side. In addition, both folia I and X were uniformly immunopositive. The pattern of PLC beta 4 immunoreactivity was largely complementary-PLC beta 4 positive stripes were zebrin II negative and vice versa. The similarity of zebrin II expression between the hummingbirds and the pigeon indicates that the neurochemical compartmentation of the cerebellar cortex in birds is highly conserved, but species differences in the number and width of stripes do occur. (C) 2008 Elsevier B.V. All rights reserved.
  • Daniel Garcia-Ovejero, Angel Arevalo-Martin, Stefania Petrosino, Fabian Docagne, Carlos Hagen, Tiziana Bisogno, Masahiko Watanabe, Carmen Guaza, Vincenzo Di Marzo, Eduardo Molina-Holgado
    NEUROBIOLOGY OF DISEASE 33 (1) 57 - 71 0969-9961 2009/01 [Refereed][Not invited]
     
    Endocannabinoids are lipid mediators with protective effects in many diseases of the nervous system. We have studied the modulation of the endocannabinoid system after a spinal cord contusion in rats. In early stages, lesion induced increases of anandamide and palmitoylethanolamide (PEA) levels, an upregulation of the synthesizing enzyme NAPE-phospholipase D and a downregulation of the degradative enzyme FAAH. In delayed stages, lesion induced increases in 2-arachidonoylglycerol and a strong upregulation of the synthesizing enzyme DAGL-alpha. that is expressed by neurons, astrocytes and immune infiltrates. The degradative enzyme MAGL was also moderately increased but only 7 days after the lesion. We have studied the cellular targets for the newly formed endocannabinoids using RT-PCR and immunohistochemistry against CB(1) and CB(2) receptors. We observed that CB, was constitutively expressed by neurons and oligodendrocytes and induced in reactive astrocytes. CB(2) receptor was strongly upregulated after lesion, and mostly expressed by immune infiltrates and astrocytes. The endocannabinoid system may represent an interesting target for new therapeutical approaches to spinal cord injury. (c) 2008 Elsevier Inc. All rights reserved.
  • Fumiaki Fukushima, Kazuhito Nakao, Toru Shinoe, Masahiro Fukaya, Shin-ichi Muramatsu, Kenji Sakimura, Hirotaka Kataoka, Hisashi Mori, Masahiko Watanabe, Toshiya Manabe, Masayoshi Mishina
    PLOS ONE 4 (1) 1932-6203 2009/01 [Refereed][Not invited]
     
    Synchronized discharges in the hippocampal CA3 recurrent network are supposed to underlie network oscillations, memory formation and seizure generation. In the hippocampal CA3 network, NMDA receptors are abundant at the recurrent synapses but scarce at the mossy fiber synapses. We generated mutant mice in which NMDA receptors were abolished in hippocampal CA3 pyramidal neurons by postnatal day 14. The histological and cytological organizations of the hippocampal CA3 region were indistinguishable between control and mutant mice. We found that mutant mice lacking NMDA receptors selectively in CA3 pyramidal neurons became more susceptible to kainate-induced seizures. Consistently, mutant mice showed characteristic large EEG spikes associated with multiple unit activities (MUA), suggesting enhanced synchronous firing of CA3 neurons. The electrophysiological balance between fast excitatory and inhibitory synaptic transmission was comparable between control and mutant pyramidal neurons in the hippocampal CA3 region, while the NMDA receptor-slow AHP coupling was diminished in the mutant neurons. In the adult brain, inducible ablation of NMDA receptors in the hippocampal CA3 region by the viral expression vector for Cre recombinase also induced similar large EEG spikes. Furthermore, pharmacological blockade of CA3 NMDA receptors enhanced the susceptibility to kainate-induced seizures. These results raise an intriguing possibility that hippocampal CA3 NMDA receptors may suppress the excitability of the recurrent network as a whole in vivo by restricting synchronous firing of CA3 neurons.
  • Levente Deli, Gabor Wittmann, Imre Kallo, Ronald M. Lechan, Masahiko Watanabe, Zsolt Liposits, Csaba Fekete
    ENDOCRINOLOGY 150 (1) 98 - 103 0013-7227 2009/01 [Refereed][Not invited]
     
    Hypophysiotropic TRH-synthesizing neurons of the hypothalamic paraventricular nucleus (PVN) have a critical role in the regulation of the energy homeostasis through control of the hypothalamic-pituitary-thyroid axis. Recently, endocannabinoids have been shown to exert inhibitory effects on TRH neurons via the type 1 cannabinoid receptor (CB1). To understand the anatomical basis for this regulatory mechanism, we determined whether CB1 is contained in axons innervating hypophysiotropic TRH neurons using a recently developed antiserum against the C-terminal portion of mouse CB1. CB1-immunoreactive axons densely innervated the parvicellular subdivisions of the PVN where the hypophysiotropic TRH neurons are located. By double-labeling immunocytochemistry, CB1-immunoreactive varicosities were observed in juxtaposition to the vast majority of TRH neurons in the PVN. At the ultrastructural level, CB1-immunoreactivity was observed in the preterminal portion of axons establishing both symmetric and asymmetric synaptic specializations with the perikarya and dendrites of TRH neurons in the PVN. These data demonstrate that CB1 is abundantly present in axons that are in synaptic association with hypophysiotropic TRH neurons, indicating an important role for endocannabinoids in the regulation of the hypothalamic-pituitarythyroid axis. The presence of both symmetric and asymmetric type CB1 synapses on TRH neurons in the PVN suggests that endocannabinoids may influence both excitatory and inhibitory inputs of these neurons. (Endocrinology 150: 98-103, 2009)
  • Yoko Yamagata, Shizuka Kobayashi, Tatsuya Umeda, Akihiro Inoue, Hiroyuki Sakagami, Masahiro Fukaya, Masahiko Watanabe, Nobuhiko Hatanaka, Masako Totsuka, Takeshi Yagi, Kunihiko Obata, Keiji Imoto, Yuchio Yanagawa, Toshiya Manabe, Shigeo Okabe
    NEUROSCIENCE RESEARCH 65 S144 - S144 0168-0102 2009 [Refereed][Not invited]
  • Eriko Miura, Takatoshi Iijima, Tetsuro Kondo, Masahiko Watanabe, Michisuke Yuzaki
    NEUROSCIENCE RESEARCH 65 S83 - S83 0168-0102 2009 [Refereed][Not invited]
  • Taisuke Miyazaki, Miwako Yamasaki, Kouichi Tanaka, Masahiko Watanabe
    JOURNAL OF PHYSIOLOGICAL SCIENCES 59 198 - 198 1880-6546 2009 [Refereed][Not invited]
  • Yohei Okubo, Hiroshi Sekiya, Shigeyuki Namiki, Hirokazu Sakamoto, Sho Iinuma, Miwako Yamasaki, Masahiko Watanabe, Kenzo Hirose, Masamitsu Iino
    NEUROSCIENCE RESEARCH 65 S142 - S143 0168-0102 2009 [Refereed][Not invited]
  • Kaori Akashi, Toshikazu Kakizaki, Haruyuki Kamiya, Masahiro Fukaya, Miwako Yamasaki, Manabu Abe, Masahiko Watanabe, Kenji Sakimura
    NEUROSCIENCE RESEARCH 65 S139 - S140 0168-0102 2009 [Refereed][Not invited]
  • Masahiko Watanabe, Masahiro Fukaya, Miwako Yamasaki
    NEUROSCIENCE RESEARCH 65 S140 - S141 0168-0102 2009 [Refereed][Not invited]
  • Miwako Yamasaki, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 65 S47 - S47 0168-0102 2009 [Refereed][Not invited]
  • Miwako Yamasaki, Masahiko Watanabe
    JOURNAL OF PHYSIOLOGICAL SCIENCES 59 142 - 142 1880-6546 2009 [Refereed][Not invited]
  • Hiroaki Shiraishi, Tetuo Mikami, Junko Aida, Ken-Ichi Nakamura, Naotaka Izumiyama-Shimomura, Tomio Arai, Masahiko Watanabe, Isao Okayasu, Kaiyo Takubo
    SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY 44 (5) 538 - 544 0036-5521 2009 [Refereed][Not invited]
     
    Objective. Telomere shortening is thought to be associated with genetic instability. The purpose of this study was to measure telomere length in a series of Barrett's adenocarcinomas (BAs), focusing on the telomere/centromere fluorescent intensity ratio (TCR) with tissue quantitative fluorescent in situ hybridization (Q-FISH). Material and methods. A total of 11 cases of BA were evaluated for upper esophagus (UE), lower esophagus (LE), Barrett's mucosa (BM), BA, and gastric cardiac mucosa (GC). Q-FISH was performed using two kinds of peptide nucleic acid probe, specific for telomeres and centromeres. The sections were analyzed with a CCD camera and original software (Tissue Telo) for measuring TCR. In addition, Laser Capture Microdissection and GeneScan were implemented for evaluation of genetic instability. Results. The TCR values in BM and, to a lesser extent, BA were significantly lower than those in the other tissues, particularly in heterozygosity (LOH)-positive cases. However, no significant difference was evident between microsatellite instability (MSI)-positive and -negative groups. Conclusions. In our study of BA series, telomere length appeared to change with the degree of histological atypia, with decreases linked to LOH.
  • Keiichi Tamai, Masafumi Toyoshima, Nobuyuki Tanaka, Noriko Yamamoto, Yuji Owada, Hiroshi Kiyonari, Kazuko Murata, Yoshiyuki Ueno, Masao Ono, Tooru Shimosegawa, Nobuo Yaegashi, Masahiko Watanabe, Kazuo Sugamura
    AMERICAN JOURNAL OF PATHOLOGY 173 (6) 1806 - 1817 0002-9440 2008/12 [Refereed][Not invited]
     
    The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the brs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked brs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs(flox/flox);SynI-cre mice. Notably, the hrs(flox/flox);SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs(flox/flox);SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates. (Am J Pathol 2008, 173:1806-1817; DOI: 10.2353/ajpath.2008.080684)
  • Yoshiaki Shinohara, Hajime Hirase, Masahiko Watanabe, Makoto Itakura, Masami Takahashi, Ryuichi Shigemoto
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (49) 19498 - 19503 0027-8424 2008/12 [Refereed][Not invited]
     
    Left-right asymmetry of the brain has been studied mostly through psychological examination and functional imaging in primates, leaving its molecular and synaptic aspects largely unaddressed. Here, we show that hippocampal CA1 pyramidal cell synapses differ in size, shape, and glutamate receptor expression depending on the laterality of presynaptic origin. CA1 synapses receiving neuronal input from the right CA3 pyramidal cells are larger and have more perforated PSD and a GluR1 expression level twice as high as those receiving input from the left CA3. The synaptic density of GluR1 increases as the size of a synapse increases, whereas that of NR2B decreases because of the relatively constant NR2B expression in CA1 regardless of synapse size. Densities of other major glutamate receptor subunits show no correlation with synapse size, thus resulting in higher net expression in synapses having right input. Our study demonstrates universal left-right asymmetry of hippocampal synapses with a fundamental relationship between synaptic area and the expression of glutamate receptor subunits.
  • Tie-Jun Sten Shi, Su-Xing Leslie Liu, Henrik Hammarberg, Masahiko Watanabe, Zhi-Qing David Xu, Tomas Hokfelt
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (50) 20004 - 20008 0027-8424 2008/12 [Refereed][Not invited]
     
    Treatment of neuropathic pain is a major clinical problem. This study shows expression of phospholipase beta 3 (PLC beta 3) in mouse and human DRG neurons, mainly in small ones and mostly with a nonpeptidergic phenotype. After spared nerve injury, the pain threshold was strongly reduced, and systemic treatment of such animals with the unselective PLC inhibitor U73122 caused a rapid and long-lasting (48-h) increase in pain threshold. Thus, inhibition of PLC may provide a way to treat neuropathic pain.
  • Erika Polgar, Khulood M. Al-Khater, Safa Shehab, Masahiko Watanabe, Andrew J. Todd
    JOURNAL OF NEUROSCIENCE 28 (49) 13150 - 13160 0270-6474 2008/12 [Refereed][Not invited]
     
    Although most projection neurons in lamina I express the neurokinin 1 receptor (NK1r), we have identified a population of large multipolar projection cells that lack the NK1r, are characterized by the high density of gephyrin puncta that coat their cell bodies and dendrites, and express the transcription factor Fos in response to noxious chemical stimulation. Here we show that these cells have a very high density of glutamatergic input from axons with strong immunoreactivity for vesicular glutamate transporter 2 that are likely to originate from excitatory interneurons. However, they receive few contacts from peptidergic primary afferents or transganglionically labeled A delta nociceptors. Unlike most glutamatergic synapses in superficial laminas, those on the gephyrin-coated cells contain the GluR4 subunit of the AMPA receptor. A noxious heat stimulus caused Fos expression in 38% of the gephyrin-coated cells but in 85% of multipolar NK1r-immunoreactive cells. These findings are consistent with the suggestion that there is a correlation between function and morphology for lamina I neurons but indicate that there are at least two populations of multipolar neurons that differ in receptor expression, excitatory inputs, and responses to noxious stimulation. Although there are only similar to 10 gephyrin-coated cells on each side per segment in the lumbar enlargement, they constitute similar to 18% of the lamina I component of the spinothalamic tract at this level, which suggests that they play an important role in transmission of nociceptive information to the cerebral cortex. Our results also provide the first evidence that postsynaptic GluR4-containing AMPA receptors are involved in spinal nociceptive transmission.
  • Carolina Ladera, Maria del Carmen Godino, Maria Jose Cabanero, Magdalena Torres, Masahiko Watanabe, Rafael Lujan, Jose Sanchez-Prieto
    JOURNAL OF NEUROCHEMISTRY 107 (6) 1506 - 1517 0022-3042 2008/12 [Refereed][Not invited]
     
    Neuronal G protein-gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post-synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABA(B) receptors. In this study, we show for the first time that GABA(B) receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABA(B) receptors reduces glutamate release and the Ca(2+) influx mediated by N-type Ca(2+) channels in a mode insensitive to the GIRK channel blocker tertiapin-Q and consistent with direct inhibition of this voltage-gated Ca(2+) channel. However, by means of weak stimulation protocols, we reveal that GABA(B) receptors also reduce glutamate release mediated by P/Q-type Ca(2+) channels, and that these responses are reversed by the GIRK channel blocker tertiapin-Q. Consistent with the functional interaction between GABA(B) receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre-synaptic boutons of asymmetric synapses co-express GABA(B) receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post-synaptic level, also occurs at glutamatergic nerve terminals.
  • Yasukazu Hozumi, Masahiro Fukaya, Naoko Adachi, Naoaki Saito, Koichi Otani, Hisatake Kondo, Masahiko Watanabe, Kaoru Goto
    EUROPEAN JOURNAL OF NEUROSCIENCE 28 (12) 2409 - 2422 0953-816X 2008/12 [Refereed][Not invited]
     
    Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers, i.e. diacylglycerol (DAG) and inositol 1,4,5-trisphosphate. The former activates protein kinase C and the latter mobilizes Ca(2+) from intracellular store. DAG kinase (DGK) then phosphorylates DAG to produce another second messenger (phosphatidic acid). Of 10 mammalian DGK isozymes, DGK beta is expressed in dopaminergic projection fields with the highest level in the striatum and its particular splice variant is differentially expressed in patients with bipolar disorder. To gain molecular anatomical evidence for its signaling role, we investigated the cellular expression and subcellular localization of DGK beta in the striatum of rat brain. DGK beta was expressed in medium spiny neurons constituting the striatonigral and striatopallidal pathways, whereas striatal interneurons were below the detection threshold. DGK beta was distributed in somatodendritic elements of medium spiny neurons and localized in association with the smooth endoplasmic reticulum and plasma membrane or in the narrow cytoplasmic space between them. In particular, DGK beta exhibited dense accumulation at perisynaptic sites on dendritic spines forming asymmetrical synapses. The characteristic anatomical localization was consistent with exclusive enrichment of DGK beta in the microsomal and postsynaptic density fractions. Intriguingly, DGK beta was very similar in immunohistochemical and immunochemical distribution to Gq-coupled receptors, such as metabotropic glutamate receptors 1 and 5, and also to other downstream molecules involving DAG metabolism, such as phospholipase C beta and DAG lipase. These findings suggest that abundant DGK beta is provided to perisynaptic sites of medium spiny neurons so that it can effectively produce phosphatidic acid upon activation of Gq-coupled receptors and modulate the cellular state of striatal output neurons.
  • Masanori Tachikawa, Jun Fujinawa, Masato Takahashi, Yasuyuki Kasai, Masahiro Fukaya, Kazuhisa Sakai, Maya Yamazaki, Masatoshi Tomi, Masahiko Watanabe, Kenji Sakimura, Tetsuya Terasaki, Ken-ichi Hosoya
    JOURNAL OF NEUROCHEMISTRY 107 (3) 768 - 778 0022-3042 2008/11 [Refereed][Not invited]
     
    Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a K(m) value of 269 mu M/412 mu M which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that of D-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB.
  • Ruben Nogueiras, Christelle Veyrat-Durebex, Paula M. Suchanek, Marcella Klein, Johannes Tschoep, Charles Caldwell, Stephen C. Woods, Gabor Wittmann, Masahiko Watanabe, Zsolt Liposits, Csaba Fekete, Ofer Reizes, Francoise Rohner-Jeanrenaud, Matthias H. Tschoep
    DIABETES 57 (11) 2977 - 2991 0012-1797 2008/11 [Refereed][Not invited]
     
    OBJECTIVE-Blockade of the CB1 receptor is one of the promising strategies for the treatment of obesity. Although antagonists suppress food intake and reduce body weight, the role of central versus peripheral CB1 activation on weight loss and related metabolic parameters remains to be elucidated. We therefore specifically assessed and compared the respective potential relevance of central nervous system (CNS) versus peripheral CB1 receptors in the regulation of energy homeostasis and lipid and glucose metabolism in diet-induced obese (DIO) rats. RESEARCH DESIGN AND METHODS-Both lean and DIO rats were used for our experiments. The expression of key enzymes involved in lipid metabolism was measured by real-time PCR, and euglycemic-hyperinsulinemic clamps were used for insulin sensitivity and glucose metabolism studies. RESULTS-Specific CNS-CB1 blockade decreased body weight and food intake but, independent of those effects, had no beneficial influence on peripheral lipid and glucose metabolism. Peripheral treatment with CB1 antagonist (Rimonabant) also reduced food intake and body weight but, in addition, independently triggered lipid mobilization pathways in white adipose tissue and cellular glucose uptake. Insulin sensitivity and skeletal muscle glucose uptake were enhanced, while hepatic glucose production was decreased during peripheral infusion of the CB1 antagonist. However, these effects depended on the antagonist-elicited reduction of food intake. CONCLUSIONS-Several relevant metabolic processes appear to independently benefit from peripheral blockade of CB I, while CNS-CB1 blockade alone predominantly affects food intake and body weight. Diabetes 57:2977-2991, 2008
  • Masahiro Fukaya, Motokazu Uchigashima, Sachi Nomura, Yuta Hasegawa, Hisaya Kikuchi, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 28 (9) 1744 - 1759 0953-816X 2008/11 [Refereed][Not invited]
     
    Upon activation of receptors coupled to the Gq subclass of G proteins, phospholipase C (PLC)beta hydrolyses membrane phospholipid to yield a pair of second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Of four PLC beta isoforms, PLC beta 1 is transcribed predominantly in the telencephalon and its gene inactivation in mice impairs metabotropic glutamate receptor- and muscarinic acetylcholine receptor-dependent hippocampal oscillations, endocannabinoid production in the hippocampus and barrel formation in the somatosensory cortex. Here we examined cellular and subcellular distributions of PLC beta 1 in adult mouse brains. In the telencephalon, high levels of PLC beta 1 were observed in principal neurons, including pyramidal cells in the cortex and hippocampus, granule cells and mossy cells in the dentate gyrus, and medium spiny neurons in the caudate-putamen, whereas most interneurons had low levels of or were negative for PLC beta 1 and, instead, expressed PLC beta 4. By immunofluorescence, tiny clusters of PLC beta 1 were distributed in somatodendritic compartments of principal neurons and positioned close to those of metabotropic glutamate receptor 5, muscarinic acetylcholine receptor M1 and diacylglycerol lipase-alpha, respectively. Immunoelectron microscopy revealed that PLC beta 1 was often associated with the smooth endoplasmic reticulum, cell membrane or postsynaptic density. In particular, it was highly accumulated at the perisynapse of dendritic spines forming asymmetrical synapses. In the cerebellum, PLC beta 1 was generally low but was enriched in axons and dendrites of basket cells. These results suggest that PLC beta 1 is the key effector in telencephalic principal neurons and cerebellar interneurons. Furthermore, the well-orchestrated molecular arrangement appears to be the anatomical basis for the specificity, efficiency and convergence of the neuronal phosphoinositide signaling system.
  • Masahiro Fukaya, Motokazu Uchigashima, Sachi Nomura, Yuta Hasegawa, Hisaya Kikuchi, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 28 (9) 1744 - 1759 0953-816X 2008/11 [Refereed][Not invited]
     
    Upon activation of receptors coupled to the Gq subclass of G proteins, phospholipase C (PLC)beta hydrolyses membrane phospholipid to yield a pair of second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Of four PLC beta isoforms, PLC beta 1 is transcribed predominantly in the telencephalon and its gene inactivation in mice impairs metabotropic glutamate receptor- and muscarinic acetylcholine receptor-dependent hippocampal oscillations, endocannabinoid production in the hippocampus and barrel formation in the somatosensory cortex. Here we examined cellular and subcellular distributions of PLC beta 1 in adult mouse brains. In the telencephalon, high levels of PLC beta 1 were observed in principal neurons, including pyramidal cells in the cortex and hippocampus, granule cells and mossy cells in the dentate gyrus, and medium spiny neurons in the caudate-putamen, whereas most interneurons had low levels of or were negative for PLC beta 1 and, instead, expressed PLC beta 4. By immunofluorescence, tiny clusters of PLC beta 1 were distributed in somatodendritic compartments of principal neurons and positioned close to those of metabotropic glutamate receptor 5, muscarinic acetylcholine receptor M1 and diacylglycerol lipase-alpha, respectively. Immunoelectron microscopy revealed that PLC beta 1 was often associated with the smooth endoplasmic reticulum, cell membrane or postsynaptic density. In particular, it was highly accumulated at the perisynapse of dendritic spines forming asymmetrical synapses. In the cerebellum, PLC beta 1 was generally low but was enriched in axons and dendrites of basket cells. These results suggest that PLC beta 1 is the key effector in telencephalic principal neurons and cerebellar interneurons. Furthermore, the well-orchestrated molecular arrangement appears to be the anatomical basis for the specificity, efficiency and convergence of the neuronal phosphoinositide signaling system.
  • Peizhong Mao, Yuan-Xiang Tao, Masahiro Fukaya, Feng Tao, Dechun Li, Masahiko Watanabe, Roger A. Johns
    IUBMB LIFE 60 (10) 684 - 692 1521-6543 2008/10 [Refereed][Not invited]
     
    Membrane-associated guanylate kinases (MAGUKs) act as scaffolds to coordinate signaling events through their multiple domains at the plasma membrane. The MAGUK SH3 domain is noncanonical and its function remains unclear. To identify potential binding partners of MAGUK SH3, the synapse-associated protein 102 (SAP102) SH3 domain was used as bait in a yeast two-hybrid screen of a mouse embryonic cDNA library. A mouse homologue of the Drosophila discs large tumor suppressor (Dig, also known as SAP97) bound preferentially to SAP102 SH3. The 4347bp cDNA sequence encoded an 893 amino acid protein with 94% identity to mouse SAP97. A deleted region (33-aa) strongly suggests this is a novel splice variant, which we call Embryonic-dlg/SAP97 (E-dlg). The interaction of SAP102 and F-dig was confirmed in mammalian cells. E-dig can also hind to potassium channel Kvl.4 in a pull-down assay. E-dlg wits highly expressed in embryonic and some adult mouse tissues, such as brain, kidney, and ovary. Furthermore. in situ hybridization showed that E-dlg was mostly expressed in olfactory bulb and cerebellum. (C) 2008 IUBMB.
  • Hiroshi Hasegawa, Tomoyuki Nakano, Yasukazu Hozumi, Michiaki Takagi, Toshihiko Ogino, Masashi Okada, Ken Iseki, Hisatake Kondo, Masahiko Watanabe, Alberto M. Martelli, Kaoru Goto
    JOURNAL OF CELLULAR BIOCHEMISTRY 105 (3) 756 - 765 0730-2312 2008/10 [Refereed][Not invited]
     
    Diacylglycerol kinase (DGK) converts diacylglycerol (DG) to phosphatidic acid, both of which act as second messengers to mediate a variety of cellular mechanisms. Therefore, DGK contributes to the regulation of these messengers in cellular signal transduction. Of DGK isozymes cloned, DGK zeta is characterized by a nuclear localization signal that overlaps with a sequence similar to the myristoylated alanine-rich C-kinase substrate. Previous studies showed that nuclear DG is differentially regulated from plasma membrane DG and that the nuclear DG levels fluctuate in correlation with cell cycle progression, suggesting the importance of nuclear DG in cell cycle control. In this connection, DGK zeta has been shown to localize to the nucleus in fully differentiated cells, such as neurons and lung cells, although it remains elusive how DGK behaves during the cell cycle in proliferating cells. Here we demonstrate that DGK zeta localizes to the nucleus during interphase including G1, S, and G2 phases and is associated with chromatin although it dissociates from condensed chromatin during mitotic phase in NIH3T3 cells. Furthermore, this localization pattern is also observed in proliferating spermatogonia in the testis. These results suggest a reversible association of DGK zeta with histone or its related proteins in cell cycle, plausibly dependent on their post-translational modifications. J. Cell. Biochem. 105: 756-765, 2008. (c) 2008 Wiley-Liss, Inc.
  • Annarita Patrizi, Bibiana Scelfo, Laura Viltono, Federica Briatore, Masahiro Fukaya, Masahiko Watanabe, Piergiorgio Strata, Frederique Varoqueaux, Nils Brose, Jean-Marc Fritschy, Marco Sassoe-Pognetto
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (35) 13151 - 13156 0027-8424 2008/09 [Refereed][Not invited]
     
    GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of GABA(A)alpha 1 knockout (KO) mice express transiently the alpha 3 subunit, leading to the assembly of functional GABA(A) receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the alpha 3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. Notably, the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking GABAA receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that GABAA receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation.
  • Nobue Futawatari, Shiro Kikuchi, Shinichi Sakuramoto, Mitsuhiro Kida, Masahiko Watanabe
    ANTICANCER RESEARCH 28 (5B) 2907 - 2912 0250-7005 2008/09 [Refereed][Not invited]
     
    Background: The objective of the present study was to measure the volume of early gastric cancer using 3-dimensional endoscopic ultrasonography (3D-EUS) and to ascertain the clinical significance of this new diagnostic method. Patients and Methods: The study comprised 100 patients with early gastric (cancer Who underwent preoperative 3D-EUS followed by surgical resection. The relationships of lymph node metastasis to 10 clinicopathological factors (gender, age, tumor location, ulcer. depth, macroscopic type, tumor histology, lymphatic invasion and venous invasion) and preoperative tumor volume (log tumor volume) as measured by 3D-EUS were determined. Results: Median tumor volume was 414.75 mm(3) (range, 7.8-2,683.8 mm(3)) and median log tumor volume was 2.617 (range, 1.444-3.429). Univariate analyses showed that three factors (lymphatic invasion, p=0.0001; venous invasion, p=0.0216, log tumor volume, p=0.0121) correlated significatitly with lymph node metastasis. Multivariate analysis showed only two factors (lymphatic invasion, p=0.011, regression coefficient=0.104,- log tumor volume, p=0.020. regression coefficient=30.414) representing independent predictors for lymph node metastasis. Conclusion: Tumor volume a., measured by 3D-EUS represents an independent risk factor for lymph node metastasis in early gastric cancer. This new method can be used as a reliable diagnostic tool for earl-v gastric cancer.
  • Kei Watase, Curtis F. Barrett, Taisuke Miyazaki, Taro Ishiguro, Kinya Ishikawa, Yuanxin Hu, Toshinori Unno, Yaling Sun, Sayumi Kasai, Masahiko Watainabe, Christopher M. Gomez, Hidehiro Mizusawa, Richard W. Tsien, Huda Y. Zoghbi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (33) 11987 - 11992 0027-8424 2008/08 [Refereed][Not invited]
     
    Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (Ca(v)) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of Ca(v)2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein. We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine (Sca6(84Q)) developed progressive motor impairment and aggregation of mutant Ca(v)2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca2(+) channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca6(84Q) neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant Ca(v)2.1 channels.
  • Hidemi Shimizu, Masahiro Fukaya, Miwako Yamasaki, Masahiko Watanabe, Toshiya Manabe, Haruyuki Kamiya
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (33) 11998 - 12003 0027-8424 2008/08 [Refereed][Not invited]
     
    Presynaptic Ca2+ stores have been suggested to regulate Ca2+ dynamics within the nerve terminals at certain types of the synapse. However, little is known about their mode of activation, molecular identity, and detailed subcellular localization. Here, we show that the ryanodine-sensitive stores exist in axons and amplify presynaptic Ca2+ accumulation at the hippocampal mossy fiber synapses, which display robust presynaptic forms of plasticity. Caffeine, a potent drug inducing Ca2+ release from ryanodine-sensitive stores, causes elevation of presynaptic Ca2+ levels and enhancement of transmitter release from the mossy fiber terminals. The blockers of ryanodine receptors, TMB-8 or ryanodine, reduce presynaptic Ca2+ transients elicited by repetitive stimuli of mossy fibers but do not affect those evoked by single shocks, suggesting that ryanodine receptors amplify presynaptic Ca2+ dynamics in an activity dependent manner. Furthermore, we generated the specific antibody against the type 2 ryanodine receptor (RyR2; originally referred to as the cardiac type) and examined the cellular and subcellular localization using immunohistochemistry. RyR2 is highly expressed in the stratum lucidum of the CA3 region and mostly colocalizes with axonal marker NF160 but not with terminal marker VGLUT1. Immunoelectron microscopy revealed that RyR2 is distributed around smooth ER within the mossy fibers but is almost excluded from their terminal portions. These results suggest that axonal localization of RyR2 at sites distant from the active zones enables use dependent Ca2+ release from intracellular stores within the mossy fibers and thereby facilitates robust presynaptic forms of plasticity at the mossy fiber-CA3 synapse.
  • Hidetoshi Kassai, Toshio Terashima, Masahiro Fukaya, Kazuki Nakao, Mizuho Sakahara, Masahiko Watanabe, Atsu Aiba
    EUROPEAN JOURNAL OF NEUROSCIENCE 28 (2) 257 - 267 0953-816X 2008/07 [Refereed][Not invited]
     
    Rac1 is a member of Rho family GTPases and regulates multiple cellular functions through actin cytoskeleton reorganization. During cerebral corticogenesis, Rac1 has been assumed to be involved in neuronal migration, neurite formation, polarization and axonal guidance. Here we show the specific role of Rac1, regulating midline crossing of commissural axons during cortical development by using cortex-restricted Rac1-knockout mice. In the knockout mice, Rac1 was eliminated from the beginning of corticogenesis exclusively in the dorsal telencephalon where progenitors of cortical projection neurons are located. Cortical lamination was distorted only mildly in the knockout mice, being preserved with six layers of neurons. However, cortex-restricted Rac1 deletion exhibited striking agenesis of commissural axons including the corpus callosum and anterior commissure without affecting other corticofugal axons including corticospinal and corticothalamic projections. Of note, the commissural axons of the knockout mice were potent in extending their process, but failed to cross the midline. Therefore, these findings indicate that Rac1 specifically controls the midline crossing of the commissural fibers, but not axonal formation of corticospinal or corticothalamic fibers during cortical development.
  • Seiichiro Yamamoto, Kenichi Yoshimura, Fumio Konishi, Masahiko Watanabe
    JAPANESE JOURNAL OF CLINICAL ONCOLOGY 38 (7) 497 - 500 0368-2811 2008/07 [Refereed][Not invited]
     
    Recently reported randomized controlled trials demonstrated that laparoscopic surgery (LS) was comparable or superior to open surgery with regard to the long-term outcome for colon and rectosigmoidal carcinoma; however, controversy persists with regard to the appropriateness of LS for patients with rectal carcinoma. To examine the technical and oncological feasibility of LS for rectal carcinoma, a phase II trial was started in patients with a preoperative diagnosis of Stage 0/I rectal carcinoma, under the direction of the Japan Society of Laparoscopic Colorectal Surgery. Surgeons in 39 specialized institutions will recruit 350 patients. The primary end-point in the first stage is the anastomotic leakage rate by double-stapling technique and that in the second stage is overall survival. Secondary end-points are relapse-free survival, short-term clinical outcome, adverse events, the rate of histologically curative operation, the proportion of completion of LS and the conversion rate.
  • Takayoshi Arai, Ryuta Takashita, Yoko Endo, Masahiko Watanabe, Akira Yanagisawa
    JOURNAL OF ORGANIC CHEMISTRY 73 (13) 4903 - 4906 0022-3263 2008/07 [Refereed][Not invited]
     
    A catalytic asymmetric Henry reaction has been developed with use of a sulfonyldiamine-CuCl complex as a catalyst. A series of new binaphthyl-containing sulfonyldiamine ligands (2a-h) were readily synthesized in two steps starting from commercially available chiral 1,2-diamines. The (R,R)-diamine-(R)-binaphthyl ligand (2d)-CuCl complex smoothly catalyzed the enantioselective Henry reaction with the assistance of pyridine to give the corresponding adduct with high enantiomeric excess (up to 93%). Moreover, the 2d-CuCl-pyridine system promotes the diastereoselective Henry reaction in syn-selective manner to give the adduct in up to 99% yield with 92:8 syn/anti selectivity. The enantiomeric excess of the syn-adduct was 84% ee.
  • Tayo Katano, Hidemasa Furue, Emiko Okuda-Ashitaka, Mitsuo Tagaya, Masahiko Watanabe, Megumu Yoshimura, Seiji Ito
    EUROPEAN JOURNAL OF NEUROSCIENCE 27 (12) 3161 - 3170 0953-816X 2008/06 [Refereed][Not invited]
     
    Central sensitization, similar to long-term potentiation in the hippocampus, refers to the increased synaptic efficacy established in somatosensory neurons in the dorsal horn of the spinal cord following tissue injury or nerve damage. In the course of inflammation, many proteins including glutamate receptors are assumed to be dynamically reorganized in the postsynaptic density (PSD) and involved in persistent pain. Mechanical hyperalgesia induced by intraplantar injection of complete Freund's adjuvant (CFA) was inhibited at 4 h, but not at 24 h, by indomethacin, an inhibitor of prostanoid synthesis. To elucidate the nature of the molecule(s) involved in the late phase of inflammatory pain, we analysed the PSD fraction prepared from the lumbar spinal cord of rats before and 24 h after CFA injection by conducting two-dimensional differential gel electrophoresis. N-ethylmaleimide-sensitive fusion protein (NSF) was identified as a downregulated protein in the PSD by MALDI-TOF MS and immunoblotting. Concomitant with the decrease in NSF, GluR2 and GluR3 were decreased and GluR1 was conversely increased in the PSD fraction 24 h after CFA injection. In vivo patch-clamp recordings of rats 24 h after CFA injection showed that excitatory postsynaptic currents of dorsal horn neurons evoked by pinch stimuli to inflamed skin were inwardly rectified and inhibited by 60% by philanthotoxin-433, a selective inhibitor of the Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. These results suggest that peripheral inflammation gives rise to central sensitization in the spinal cord through subunit composition switch of AMPA receptors in the late phase.
  • Aya Ito-Ishida, Eriko Miura, Kyoichi Emi, Keiko Matsuda, Takatoshi Iijima, Tetsuro Kondo, Kazuhisa Kohda, Masahiko Watanabe, Michisuke Yuzaki
    JOURNAL OF NEUROSCIENCE 28 (23) 5920 - 5930 0270-6474 2008/06 [Refereed][Not invited]
     
    Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers ( PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders.
  • Lucius Cueni, Marco Canepari, Rafael Lujan, Yann Emmenegger, Masahiko Watanabe, Chris T. Bond, Paul Franken, John P. Adelman, Anita Luthi
    NATURE NEUROSCIENCE 11 (6) 683 - 692 1097-6256 2008/06 [Refereed][Not invited]
     
    T-type Ca(2+) channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca(2+)-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca(2+) concentration increases were dominated by Ca(2+) influx through T channels and shaped rhythmic bursting via competition between Ca(2+)-dependent small-conductance (SK)-type K1 channels and Ca(2+) uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca(2+) sequestration by sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2(-/-) (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca(2+) signaling triad to regulate oscillatory dynamics related to sleep.
  • Daiki D. Horikawa, Takekazu Kunieda, Wataru Abe, Masahiko Watanabe, Yuichi Nakahara, Fumiko Yukuhiro, Tetsuya Sakashita, Nobuyuki Hamada, Seiichi Wada, Tomoo Funayama, Chihiro Katagiri, Yasuhiko Kobayashi, Seigo Higashi, Takashi Okuda
    ASTROBIOLOGY 8 (3) 549 - 556 1531-1074 2008/06 [Refereed][Not invited]
     
    Studies on the ability of multicellular organisms to tolerate specific environmental extremes are relatively rare compared to those of unicellular microorganisms in extreme environments. Tardigrades are extremotolerant animals that can enter an ametabolic dry state called anhydrobiosis and have high tolerance to a variety of extreme environmental conditions, particularly while in anhydrobiosis. Although tardigrades have been expected to be a potential model animal for astrobiological studies due to their excellent anhydrobiotic and extremotolerant abilities, few studies of tolerance with cultured tardigrades have been reported, possibly due to the absence of a model species that can be easily maintained under rearing conditions. We report the successful rearing of the herbivorous tardigrade, Ramazzottius varieornatus, by supplying the green alga Chlorella vulgaris as food. The life span was 35 +/- 16.4 d, deposited eggs required 5.7 +/- 1.1 d to hatch, and animals began to deposit eggs 9 d after hatching. The reared individuals of this species had an anhydrobiotic capacity throughout their life cycle in egg, juvenile, and adult stages. Furthermore, the reared adults in an anhydrobiotic state were tolerant of temperatures of 90 degrees C and - 196 degrees C, and exposure to 99.8% acetonitrile or irradiation with 4000 Gy (4)He ions. Based on their life history traits and tolerance to extreme stresses, R. varieornatus may be a suitable model for astrobiological studies of multicellular organisms.
  • Tomonori Takeuchi, Gen Ohtsuki, Takashi Yoshida, Masahiro Fukaya, Tasuku Wainai, Manami Yamashita, Yoshito Yamazaki, Hisashi Mori, Kenji Sakimura, Susumu Kawamoto, Masahiko Watanabe, Tomoo Hirano, Masayoshi Mishina
    PLOS ONE 3 (5) 1932-6203 2008/05 [Refereed][Not invited]
     
    In the cerebellum, Delphilin is expressed selectively in Purkinje cells (PCs) and is localized exclusively at parallel fiber (PF) synapses, where it interacts with glutamate receptor (GluR) delta 2 that is essential for long-term depression (LTD), motor learning and cerebellar wiring. Delphilin ablation exerted little effect on the synaptic localization of GluR delta 2. There were no detectable abnormalities in cerebellar histology, PC cytology and PC synapse formation in contrast to GluR delta 2 mutant mice. However, LTD induction was facilitated at PF-PC synapses in Delphilin mutant mice. Intracellular Ca(2+) required for the induction of LTD appeared to be reduced in the mutant mice, while Ca(2+) influx through voltage-gated Ca(2+) channels and metabotropic GluR1-mediated slow synaptic response were similar between wild-type and mutant mice. We further showed that the gain-increase adaptation of the optokinetic response (OKR) was enhanced in the mutant mice. These findings are compatible with the idea that LTD induction at PF-PC synapses is a crucial rate-limiting step in OKR gain-increase adaptation, a simple form of motor learning. As exemplified in this study, enhancing synaptic plasticity at a specific synaptic site of a neural network is a useful approach to understanding the roles of multiple plasticity mechanisms at various cerebellar synapses in motor control and learning.
  • Chihiro Takasaki, Rieko Okada, Akira Mitani, Masahiro Fukaya, Miwako Yamasaki, Yuri Fujihara, Tetsuo Shirakawa, Kohichi Tanaka, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 28 (19) 4995 - 5006 0270-6474 2008/05 [Refereed][Not invited]
     
    Glutamate transporters are involved in neural differentiation, neuronal survival, and synaptic transmission. In the present study, we examined glutamate transporter 1 (GLT1) expression in the neonatal somatosensory cortex of C57BL/6 mice, and pursued its role in somatosensory development by comparing barrel development between GLT1 knock-out and control mice. During the first few neonatal days, a critical period for barrels, GLT1 expression is strikingly upregulated in cortical astrocytes, whereas it was downregulated in neuronal elements to below the detection threshold. GLT1 knock-out neonates developed normally in terms of body growth, cortical histoarchitecture, barrel formation, and critical period termination. However, when row C whiskers were lesioned during the critical period, reduction of lesioned row C barrels and reciprocal expansion of intact row B/D barrels were both milder in GLT1 knock-out mice than in control littermates. Accordingly, the map plasticity index, calculated as (B + D)/2C, was significantly lowered in GLT1 knock-out mice. We also found that extracellular glutamate levels in the neonatal somatosensory cortex were significantly elevated in GLT1 knockout mice. Diminished lesion-induced plasticity was further found in mutant mice lacking glutamate-aspartate transporter (GLAST), an astrocyte-specific glutamate transporter throughout development. Therefore, glutamate transporters regulate critical period plasticity by enhancing expansion of active barrels and shrinkage of inactive barrels. Because cortical contents of glutamate receptors and GLAST were unaltered in GLT1 knock-out mice, this action appears to be mediated, at least partly, by keeping the ambient glutamate level low. Considering an essential role of glutamate receptors in the formation of whisker-related thalamocortical synapse patterning, glutamate transporters thus facilitate their activity-dependent remodeling.
  • Hideki Miwa, Masahiro Fukaya, Ayako M. Watabe, Masahiko Watanabe, Toshiya Manabe
    JOURNAL OF PHYSIOLOGY-LONDON 586 (10) 2539 - 2550 0022-3751 2008/05 [Refereed][Not invited]
     
    The NMDA-type glutamate receptor is a heteromeric complex composed of the NR1 and at least one of the NR2 subunits. Switching from the NR2B to the NR2A subunit is thought to underlie functional alteration of the NMDA receptor during synaptic maturation, and it is generally believed that it results in preferential localization of NR2A subunits on the synaptic site and that of NR2B subunits on the extracellular site in the mature brain. It has also been proposed that activation of the NR2A and NR2B subunits results in long-term potentiation (LTP) and long-term depression (LTD), respectively. Furthermore, recent reports suggest that synaptic and extrasynaptic receptors may have distinct roles in synaptic plasticity as well as in gene expression associated with neuronal death. Here, we have investigated whether NR2B subunit-containing receptors are present and functional at mature synapses in the lateral nucleus of the amygdala (LA) and the CA1 region of the hippocampus, comparing their properties between the two brain regions. We have found, in contrast to the above hypotheses, that the NR2B subunit significantly contributes to synaptic transmission as well as LTP induction. Furthermore, its contribution is greater in the LA than in the CA1 region, and biophysical properties of NMDA receptors and the NR2B/NR2A ratio are different between the two brain regions. These results indicate that NR2B subunit-containing NMDA receptors accumulate on the synaptic site and are responsible for the unique properties of synaptic function and plasticity in the amygdala.
  • Chihiro Takasaki, Rieko Okada, Akira Mitani, Masahiro Fukaya, Miwako Yamasaki, Yuri Fujihara, Tetsuo Shirakawa, Kohichi Tanaka, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 28 (19) 4995 - 5006 0270-6474 2008/05 [Refereed][Not invited]
     
    Glutamate transporters are involved in neural differentiation, neuronal survival, and synaptic transmission. In the present study, we examined glutamate transporter 1 (GLT1) expression in the neonatal somatosensory cortex of C57BL/6 mice, and pursued its role in somatosensory development by comparing barrel development between GLT1 knock-out and control mice. During the first few neonatal days, a critical period for barrels, GLT1 expression is strikingly upregulated in cortical astrocytes, whereas it was downregulated in neuronal elements to below the detection threshold. GLT1 knock-out neonates developed normally in terms of body growth, cortical histoarchitecture, barrel formation, and critical period termination. However, when row C whiskers were lesioned during the critical period, reduction of lesioned row C barrels and reciprocal expansion of intact row B/D barrels were both milder in GLT1 knock-out mice than in control littermates. Accordingly, the map plasticity index, calculated as (B + D)/2C, was significantly lowered in GLT1 knock-out mice. We also found that extracellular glutamate levels in the neonatal somatosensory cortex were significantly elevated in GLT1 knockout mice. Diminished lesion-induced plasticity was further found in mutant mice lacking glutamate-aspartate transporter (GLAST), an astrocyte-specific glutamate transporter throughout development. Therefore, glutamate transporters regulate critical period plasticity by enhancing expansion of active barrels and shrinkage of inactive barrels. Because cortical contents of glutamate receptors and GLAST were unaltered in GLT1 knock-out mice, this action appears to be mediated, at least partly, by keeping the ambient glutamate level low. Considering an essential role of glutamate receptors in the formation of whisker-related thalamocortical synapse patterning, glutamate transporters thus facilitate their activity-dependent remodeling.
  • Yukiko Tomioka, Taisuke Miyazaki, Satoshi Taharaguchi, Saori Yoshino, Masami Morimatsu, Toshimitsu Uede, Etsuro Ono, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 27 (8) 2115 - 2132 0953-816X 2008/04 [Refereed][Not invited]
     
    Pseudorabies virus is an alphaherpesvirus causing fatal neurological diseases in animals. Pseudorabies virus carries a gene encoding immediate-early (IE) protein IE180, which controls the transcription of other viral and host cell genes. Previously, we reported that transgenic expression of IE180 in mice causes severe ataxia and cerebellar deformity. Here we identified profound abnormalities in adult IE180 transgenic mice, including malpositioning of Purkinje cells (PCs), granule cells (GCs) and Bergmann glia (BG), impaired dendritogenesis and synaptogenesis in PCs, disoriented BG fibers, absence of molecular layer interneurons, and increased apoptosis of neurons and glia. In accordance with the cellular defects, we found the expression of IE180 in PCs, GCs and astrocytes during cerebellar development. We next examined transgenic mice expressing truncated IE180 mutants: dlN132 lacking the acidic transcriptional active domain, dlC629 lacking the nuclear localization signal and dlC1081 having all known domains but lacking the carboxyl-terminal sequence. Despite similar expression levels of the transgenes, ataxia and cerebellar defects were only manifested in the dlC1081 transgenic mice but their phenotypes were milder compared with the IE180 transgenic mice. In the dlC1081 transgenic mice, cerebellar neurons and glia were normally positioned but cerebellar size was severely reduced due to GC deficits. Interestingly, dlC1081 was mainly expressed in the GCs with low expression in a few BG. Taken together, the present findings clarified a causal relationship between cerebellar pathology and cellular expression of IE180, and further afforded an experimental insight into different symptomatic severity as a consequence of different cellular defects caused by such cytotoxic viral agents.
  • Carolina Aguado, Jose Colon, Francisco Ciruela, Falk Schlaudraff, Maria Jose Cabanero, Cydne Perry, Masahiko Watanabe, Birgit Liss, Kevin Wickman, Rafael Lujan
    JOURNAL OF NEUROCHEMISTRY 105 (2) 497 - 511 0022-3042 2008/04 [Refereed][Not invited]
     
    G protein-gated inwardly rectifying potassium (GIRK/Kir3) channels regulate cellular excitability and neurotransmission. In this study, we used biochemical and morphological techniques to analyze the cellular and subcellular distributions of GIRK channel subunits, as well as their interactions, in the mouse cerebellum. We found that GIRK1, GIRK2, and GIRK3 subunits co-precipitated with one another in the cerebellum and that GIRK subunit ablation was correlated with reduced expression levels of residual subunits. Using quantitative RT-PCR and immunohistochemical approaches, we found that GIRK subunits exhibit overlapping but distinct expression patterns in various cerebellar neuron subtypes. GIRK1 and GIRK2 exhibited the most widespread and robust labeling in the cerebellum, with labeling particularly prominent in granule cells. A high degree of molecular diversity in the cerebellar GIRK channel repertoire is suggested by labeling seen in less abundant neuron populations, including Purkinje neurons (GIRK1/GIRK2/GIRK3), basket cells (GIRK1/GIRK3), Golgi cells (GIRK2/GIRK4), stellate cells (GIRK3), and unipolar brush cells (GIRK2/GIRK3). Double-labeling immunofluorescence and electron microscopies showed that GIRK subunits were mainly found at post-synaptic sites. Altogether, our data support the existence of rich GIRK molecular and cellular diversity, and provide a necessary framework for functional studies aimed at delineating the contribution of GIRK channels to synaptic inhibition in the cerebellum.
  • Yukiko Tomioka, Taisuke Miyazaki, Satoshi Taharaguchi, Saori Yoshino, Masami Morimatsu, Toshimitsu Uede, Etsuro Ono, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 27 (8) 2115 - 2132 0953-816X 2008/04 [Refereed][Not invited]
     
    Pseudorabies virus is an alphaherpesvirus causing fatal neurological diseases in animals. Pseudorabies virus carries a gene encoding immediate-early (IE) protein IE180, which controls the transcription of other viral and host cell genes. Previously, we reported that transgenic expression of IE180 in mice causes severe ataxia and cerebellar deformity. Here we identified profound abnormalities in adult IE180 transgenic mice, including malpositioning of Purkinje cells (PCs), granule cells (GCs) and Bergmann glia (BG), impaired dendritogenesis and synaptogenesis in PCs, disoriented BG fibers, absence of molecular layer interneurons, and increased apoptosis of neurons and glia. In accordance with the cellular defects, we found the expression of IE180 in PCs, GCs and astrocytes during cerebellar development. We next examined transgenic mice expressing truncated IE180 mutants: dlN132 lacking the acidic transcriptional active domain, dlC629 lacking the nuclear localization signal and dlC1081 having all known domains but lacking the carboxyl-terminal sequence. Despite similar expression levels of the transgenes, ataxia and cerebellar defects were only manifested in the dlC1081 transgenic mice but their phenotypes were milder compared with the IE180 transgenic mice. In the dlC1081 transgenic mice, cerebellar neurons and glia were normally positioned but cerebellar size was severely reduced due to GC deficits. Interestingly, dlC1081 was mainly expressed in the GCs with low expression in a few BG. Taken together, the present findings clarified a causal relationship between cerebellar pathology and cellular expression of IE180, and further afforded an experimental insight into different symptomatic severity as a consequence of different cellular defects caused by such cytotoxic viral agents.
  • Masahiko Watanabe
    Tohoku Journal of Experimental Medicine 214 (3) 175 - 190 0040-8727 2008/03/07 [Refereed][Not invited]
     
    Cerebellar Purkinje cells (PCs) play a principal role in motor coordination and motor learning. To fulfill these functions, PCs receive and integrate two types of excitatory inputs, climbing fiber (CF) and parallel fiber (PF). CFs are projection axons from the inferior olive, and convey error signals to PCs. On the other hand, PFs are T-shaped axons of cerebellar granule cells, and convey sensory and motor information carried through the pontocerebellar and spinocerebellar mossy fiber pathways. The most remarkable feature of PC circuits is the highly territorial innervation by these two excitatory afferents. A single climbing CF powerfully and exclusively innervates proximal PC dendrites, whereas hundreds of thousands of PFs innervate distal PC dendrites. Recent studies using gene-manipulated mice have been elucidating that the PC circuitry is formed and maintained by molecular mechanisms that fuel homosynaptic competition among CFs and heterosynaptic competition between CFs and PFs. GluRδ2 (a PC-specific glutamate receptor) and precerebellin or Cbln1 (a granule cell-derived secretory protein) cooperatively work for selective strengthening of PF-PC synapses, and prevent excessive distal extension of CFs that eventually causes multiple innervation at distal. dendrites. In contrast, P/Q-type Ca2+ channels, which mediate Ca2+ influx upon CF activity, selectively strengthen the innervation by a single main CF, and expel PFs and other CFs from proximal dendrites that it innervates. Therefore, we now understand that owing to these mechanisms, territorial innervation by CFs and PFs is properly structured and mono-innervation by CFs is established. Several key issues for future study are also discussed. © 2008 Tohoku University Medical Press.
  • Misato Yasumura, Takeshi Uemura, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe, Masayoshi Mishina
    NEUROSCIENCE LETTERS 433 (2) 146 - 151 0304-3940 2008/03 [Refereed][Not invited]
     
    Glutamate receptor (GluR) delta 2 selectively expressed in cerebellar Purkinje cells (PCs) plays key roles in cerebellar long-term depression (LTD), motor learning and formation of parallel fiber (PF)-PC synapses. We have recently shown that the PDZ [postsynaptic density (PSD)-95/Discs large/zona occludens-1]-binding domain at the C-terminal, the T site, is essential for LTD induction and the regulation of climbing fiber (CF) territory, but is dispensable for synaptic localization of GluR delta 2, PF-PC synapse formation and CF elimination process. To investigate the functional roles of the S segment, the second PDZ-binding domain in the middle of the C-terminal cytoplasmic region, we generated GluR delta 2 Delta S mice carrying mutant GluR delta 2 lacking this segment. The amount of GluR delta 2 Delta S in mutant mice was reduced compared with that of GluR delta 2 in wild-type mice. However, the extent of decrease was much larger in the PSD fractions than in cerebellar homogenates, suggesting the requirement of the S segment for efficient synaptic localization. Furthermore, mismatched PF synapses and free spines emerged and CF-innervation territory on PC dendrites expanded in GluR delta 2 Delta S mice. On the other hand, the performance in the rotarod test was comparable between wild-type and GluR delta 2 Delta S mice. These results suggest that the S segment and T site, the two PDZ-binding domains in the C-terminal cytoplasmic region, are differentially involved in diverse GluR delta 2 functions. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
  • Shinji Matsuda, Eriko Miura, Keiko Matsuda, Wataru Kakegawa, Kazuhisa Kohda, Masahiko Watanabe, Michisuke Yuzaki
    NEURON 57 (5) 730 - 745 0896-6273 2008/03 [Refereed][Not invited]
     
    AP-4 is a member of the adaptor protein complexes, which control vesicular trafficking of membrane proteins. Although AP-4 has been suggested to contribute to basolateral sorting in epithelial cells, its function in neurons is unknown. Here, we show that disruption of the gene encoding the 0 subunit of AP-4 resulted in increased accumulation of axonal autophagosomes, which contained AMPA receptors and transmembrane AMPA receptor regulatory proteins (TARPs), in axons of hippocampal neurons and cerebellar Purkinje cells both in vitro and in vivo. AP-4 indirectly associated with the AMPA receptor via TARPs, and the specific disruption of the interaction between AP-4 and TARPs caused the mislocalization of endogenous AMPA receptors in axons of wild-type neurons. These results indicate that AP-4 may regulate proper somatodendritic-specific distribution of its cargo proteins, including AMPA receptor-TARP complexes and the autophagic pathway in neurons.
  • Aniko Ludanyi, Lorand Eross, Sandor Czirjak, Janos Vajda, Peter Halasz, Masahiko Watanabe, Miklos Palkovits, Zsofia Magloczky, Tamas F. Freund, Istvan Katona
    JOURNAL OF NEUROSCIENCE 28 (12) 2976 - 2990 0270-6474 2008/03 [Refereed][Not invited]
     
    Endocannabinoid signaling is a key regulator of synaptic neurotransmission throughout the brain. Compelling evidence shows that its perturbation leads to development of epileptic seizures, thus indicating that endocannabinoids play an intrinsic protective role in suppressing pathologic neuronal excitability. To elucidate whether long-term reorganization of endocannabinoid signaling occurs in epileptic patients, we performed comparative expression profiling along with quantitative electron microscopic analysis in control (postmortem samples from subjects with no signs of neurological disorders) and epileptic (surgically removed from patients with intractable temporal lobe epilepsy) hippocampal tissue. Quantitative PCR measurements revealed that CB1 cannabinoid receptor mRNA was downregulated to one-third of its control value in epileptic hippocampus. Likewise, the cannabinoid receptor-interacting protein-1a mRNA was decreased, whereas 1b isoform levels were unaltered. Expression of diacylglycerol lipase-alpha, an enzyme responsible for 2-arachidonoylglycerol synthesis, was also reduced by similar to 60%, whereas its related beta isoform levels were unchanged. Expression level of N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and fatty acid amide hydrolase, metabolic enzymes of anandamide, and 2-arachidonoylglycerol's degrading enzyme monoacylglycerol lipase did not change. The density of CB1 immunolabeling was also decreased in epileptic hippocampus, predominantly in the dentate gyrus, where quantitative electron microscopic analysis did not reveal changes in the ratio of CB1-positive GABAergic boutons, but uncovered robust reduction in the fraction of CB1-positive glutamatergic axon terminals. These findings show that a neuroprotective machinery involving endocannabinoids is impaired in epileptic human hippocampus and imply that downregulation of CB1 receptors and related molecular components of the endocannabinoid system may facilitate the deleterious effects of increased network excitability.
  • Masahiko Watanabe
    TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE 214 (3) 175 - 190 0040-8727 2008/03 [Refereed][Not invited]
     
    Cerebellar Purkinje cells (PCs) play a principal role in motor coordination and motor learning. To fulfill these functions, PCs receive and integrate two types of excitatory inputs, climbing fiber (CF) and parallel fiber (PF). CFs are projection axons from the inferior olive, and convey error signals to PCs. On the other hand, PFs are T-shaped axons of cerebellar granule cells, and convey sensory and motor information carried through the pontocerebellar and spinocerebellar mossy fiber pathways. The most remarkable feature of PC circuits is the highly territorial innervation by these two excitatory afferents. A single climbing CF powerfully and exclusively innervates proximal PC dendrites, whereas hundreds of thousands of PFs innervate distal PC dendrites. Recent studies using gene-manipulated mice have been elucidating that the PC circuitry is formed and maintained by molecular mechanisms that fuel homosynaptic competition among CFs and heterosynaptic competition between CFs and PFs. GluR delta 2 (a PC-specific glutamate receptor) and precerebellin or Cbln1 (a granule cell-derived secretory protein) cooperatively work for selective strengthening of PF-PC synapses, and prevent excessive distal extension of CFs that eventually causes multiple innervation at distal dendrites. In contrast, P/Q-type Ca2+ channels, Aid which mediate Ca2+ influx upon CF activity, selectively strengthen the innervation by a single main CF, and expel PFs and other CFs from proximal dendrites that it innervates. Therefore, we now understand that owing to these mechanisms, territorial innervation by CFs and PFs is properly structured and mono-innervation by CFs is established. Several key issues for future study are also discussed.
  • Koichi Itabashi, Kazunori Furuta, Tsuyoshi Takahashi, Yoshiya Ito, Hiroyuki Katagiri, Koshi Sato, Akira Kakita, Masahiko Watanabe
    HEPATO-GASTROENTEROLOGY 55 (82-83) 568 - 573 0172-6390 2008/03 [Refereed][Not invited]
     
    Background/Aims: The present study examined the effect of urinary trypsin inhibitor (UTI) on liver injury in hypotensive brain-dead rats. Methods: Brain death was induced by inflating a balloon catheter placed in the epidural space. UTI (100,000 units/kg/hour) was intravenously administered from 30 min until 6 hours after the induction of brain death. Systemic hemodynamics and hepatic tissue flow (HTF) were measured, and blood samples and hepatic tissue specimens for morphological examinations were obtained during the experiments. Results: The induction of brain death caused a 30% decrease in both mean arterial pressure and HTF, and an increase in the serum transaminase level in comparison with sham-operated rats. Brain death also increased the serum concentration of cytokine-induced neutrophil chemoattractant (CINC) (4.4-fold), as well as the number of CINC-positive cells (4.4-fold) and sequestered neutrophils in the sinusoids (3.1-fold). Post-treatment of brain-dead rats with UTI restored the HTF and reduced serum transaminase level. UTI decreased plasma CINC level and the number of neutrophils and CINC-positive cells in the sinusoids. Conclusions: The results suggest that treatment with UTI after the establishment of brain death improved the viability of the liver in hypotensive brain-dead rats by inhibiting CINC production.
  • Fumihiro Watanabe, Taisuke Miyazaki, Tomonori Takeuchi, Masahiro Fukaya, Takanori Nomura, Shigeru Noguchi, Hisashi Mori, Kenji Sakimura, Masahiko Watanabe, Masayoshi Mishina
    EUROPEAN JOURNAL OF NEUROSCIENCE 27 (4) 836 - 854 0953-816X 2008/02 [Refereed][Not invited]
     
    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is widely expressed in the brain, and plays key roles in various cellular processes in response to both extracellular and intracellular stimuli. Here, we explored the role of FAK in cerebellar development. In the mouse cerebellum, FAK was found to be distributed as tiny cytoplasmic aggregates in various neuronal and glial elements, including Purkinje cells (PCs), Bergmann glia (BG), parallel fiber (PF)-terminals and climbing fiber (CF)-terminals. The neuron/glia-specific ablation of FAK impaired cerebellar foliation, such as variable decreases in foliation sizes and the lack of intercrural and precentral fissures. Some of the BG cells became situated ectopically in the molecular layer. Furthermore, the FAK ablation altered the innervation territories of CFs and PFs on PCs. CF innervation regressed to the basal portion of proximal dendrites and somata, whereas ectopic spines protruded from proximal dendrites and PFs expanded their territory by innervating the ectopic spines. Furthermore, the persistence of surplus CFs innervating PC somata caused multiple innervation. When FAK was selectively ablated in PCs, diminished dendritic innervation and persistent somatic innervation by CFs were observed, whereas cerebellar foliation and cell positioning of BG were normally retained. These results suggest that FAK in various neuronal and glial elements is required for the formation of normal histoarchitecture and cytoarchitecture in the cerebellum, and for the construction of proper innervation territory and synaptic wiring in PCs.
  • Hiroyuki Sakagami, Masashi Sanda, Masahiro Fukaya, Taisuke Miyazaki, Jun Sukegawa, Teruyuki Yanagisawa, Tatsuo Suzuki, Kohji Fukunaga, Masahiko Watanabe, Hisatake Kondo
    NEUROSCIENCE RESEARCH 60 (2) 199 - 212 0168-0102 2008/02 [Refereed][Not invited]
     
    ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates dendritic differentiation possibly through the organization of actin cytoskeleton and membrane traffic. Here, we characterized IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor (GEF) for Arf6, in the mouse brain. In vivo Arf pull down assay demonstrated that IQ-ArfGEF/BRAG1 activated Arf6 more potently than Arf1. IQ-ArfGEF/BRAG1 mRNA was abundantly expressed in the brain with higher levels in forebrain structures and cerebellar granule cells. In hippocampal neurons, IQ-ArfGEF/BRAG1 mRNA was localized not only at neuronal cell bodies but also at dendritic processes, indicating its dendritic transport and localization. Immunoprecipitation and in vitro binding experiments revealed that IQ-ArfGEF/BRAG1 formed a protein complex with N-methyl-D-aspartate (NMDA)-type glutamate receptors through the interaction with a postsynaptic density (PSD) scaffold protein, PSD-95. Immunohistochemical analysis demonstrated that IQ-ArfGEF/BRAG1 was localized preferentially at the postsynaptic density of asymmetrical synapses on dendritic spines, but was lacking at GABAA receptor-carrying inhibitory synapses. Taken together, IQ-ArfGEF/BRAG1 forms a postsynaptic protein complex containing PSD-95 and NMDA receptors at excitatory synapses, where it may function as a GEF for Arf6. (C) 2007 Published by Elsevier Ireland Ltd and the Japan Neuroscience Society.
  • Mike T. Lin, Rafael Lujan, Masahiko Watanabe, John P. Adelman, James Maylie
    NATURE NEUROSCIENCE 11 (2) 170 - 177 1097-6256 2008/02 [Refereed][Not invited]
     
    Long-term potentiation (LTP) of synaptic strength at Schaffer collateral synapses has largely been attributed to changes in the number and biophysical properties of AMPA receptors (AMPARs). Small-conductance Ca2(+)-activated K+ channels (SK2 channels) are functionally coupled with NMDA receptors (NMDARs) in CA1 spines such that their activity modulates the shape of excitatory postsynaptic potentials (EPSPs) and increases the threshold for induction of LTP. Here we show that LTP induction in mouse hippocampus abolishes SK2 channel activity in the potentiated synapses. This effect is due to SK2 channel internalization from the postsynaptic density (PSD) into the spine. Blocking PKA or cell dialysis with a peptide representing the C-terminal domain of SK2 that contains three known PKA phosphorylation sites blocks the internalization of SK2 channels after LTP induction. Thus the increase in AMPARs and the decrease in SK2 channels combine to produce the increased EPSP underlying LTP.
  • Wataru Kakegawa, Taisuke Miyazaki, Kyoichi Emi, Keiko Matsuda, Kazuhisa Kohda, Junko Motohashi, Masayoshi Mishina, Shigenori Kawahara, Masahiko Watanabe, Michisuke Yuzaki
    JOURNAL OF NEUROSCIENCE 28 (6) 1460 - 1468 0270-6474 2008/02 [Refereed][Not invited]
     
    The delta 2 glutamate receptor (GluR delta 2) is predominantly expressed in Purkinje cells and plays crucial roles in cerebellar functions: GluR delta 2(-/-) mice display ataxia and impaired motor learning. In addition, long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is abrogated, and synapse formation with PFs and climbing fibers (CFs) is severely disturbed in GluR delta 2(-/-) Purkinje cells. Recently, we demonstrated that abrogated LTD was restored in GluR delta 2(-/-) Purkinje cells by the virus-mediated expression of the wild-type GluR delta 2 transgene (Tgwt) but not by that of mutant GluR delta 2 lacking the C-terminal seven residues to which several PDZ proteins bind (Tg(Delta CT7)). These results indicated that the C terminus of GluR delta 2 conveys the signal(s) necessary for LTD. In contrast, other phenotypes of GluR delta 2(-/-) cerebellum, especially morphological abnormalities at PF and CF synapses, could not be rescued by virus-mediated transient expression. Thus, whether these phenotypes are mediated by the same signaling pathway remains unclear. To address these issues and to further delineate the function of GluR delta 2 in vivo, we generated transgenic mice that expressed Tg(Delta CT7) on a GluR delta 2(-/-) background. Interestingly, although Tg(Delta CT7) restored abnormal PF and CF synapse formation almost completely, it could not rescue abrogated LTD in GluR Delta 2(-/-) Purkinje cells. Furthermore, although the gross motor discoordination of GluR Delta 2(-/-) mice was restored, the cerebellar motor learning underlying delayed eyeblink conditioning remained impaired. These results indicate that LTD induction and motor learning are regulated by signaling via the C-terminal end of GluR delta 2, whereas other functions may be differentially regulated by other regions of GluR delta 2.
  • Takahiro Kikawada, Ayako Saito, Yasushi Kanamori, Mika Fujita, Katarzyna Snigorska, Masahiko Watanabe, Takashi Okuda
    BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 1778 (2) 514 - 520 0005-2736 2008/02 [Refereed][Not invited]
     
    Aquaporin, AQP, is a channel protein that allows water to permeate across cell membranes. Larvae of the sleeping chironomid, Polypedilum vanderplanki, can withstand complete dehydration by entering anhydrobiosis, a state of suspended animation; however, the mechanism by which water flows out of the larval body during dehydration is still unclear. We isolated two cDNAs (PvAqp1 and PvAqp2) encoding water-selective aquaporins from the chironomid. When expressed in Xenopus oocytes, PvAQP1 and PvAQP2 facilitated permeation of water but not glycerol. Northern blots and in situ hybridization showed that expression of PvAqp1 was dehydration-inducible and ubiquitous whereas that of PvAqp2 was dehydration-repressive and fat body-specific. These data suggest distinct roles for these aquaporins in P vanderplanki, i.e., PvAqp2 controls water homeostasis of fat body during normal conditions and PvAqp1 is involved in the removal of water during induction of anhydrobiosis. (c) 2007 Elsevier B.V. All rights reserved.
  • Erika Polgar, Masahiko Watanabe, Bettina Hartmann, Seth G. N. Grant, Andrew J. Todd
    MOLECULAR PAIN 4 1744-8069 2008/01 [Refereed][Not invited]
     
    Background: Glutamate receptors of the AMPA type ( AMPArs) mediate fast excitatory transmission in the dorsal horn and are thought to underlie perception of both acute and chronic pain. They are tetrameric structures made up from 4 subunits (GluR1- 4), and subunit composition determines properties of the receptor. Antigen retrieval with pepsin can be used to reveal the receptors with immunocytochemistry, and in this study we have investigated the subunit composition at synapses within laminae I-III of the dorsal horn. In addition, we have compared staining of AMPArs with that for PSD-95, a major constituent of glutamatergic synapses. We also examined tissue from knock-out mice to confirm the validity of the immunostaining. Results: As we have shown previously, virtually all AMPAr-immunoreactive puncta were immunostained for GluR2. In laminae I-II, similar to 65% were GluR1- positive and similar to 60% were GluR3-positive, while in lamina III the corresponding values were 34% ( GluR1) and 80% ( GluR3). Puncta stained with antibody against the C-terminus of GluR4 ( which only detects the long form of this subunit) made up 23% of the AMPAr-containing puncta in lamina I, similar to 8% of those in lamina II and 46% of those in lamina III. Some overlap between GluR1 and GluR3 was seen in each region, but in lamina I GluR1 and GluR4 were present in largely non-overlapping populations. The GluR4 puncta often appeared to outline dendrites of individual neurons in the superficial laminae. Virtually all of the AMPAr-positive puncta were immunostained for PSD-95, and 98% of PSD-95 puncta contained AMPAr-immunoreactivity. Staining for GluR1, GluR2 and GluR3 was absent in sections from mice in which these subunits had been knocked out, while the punctate staining for PSD-95 was absent in mice with a mutation that prevents accumulation of PSD-95 at synapses. Conclusion: Our results suggest that virtually all glutamatergic synapses in laminae I-III of adult rat spinal cord contain AMPArs. They show that synapses in laminae I-II contain GluR2 together with GluR1 and/or GluR3, while the long form of GluR4 is restricted to specific neuronal populations, which may include some lamina I projection cells. They also provide further evidence that immunostaining for AMPAr subunits following antigen retrieval is a reliable method for detecting these receptors at glutamatergic synapses.
  • Rita Nyilas, Barna Dudok, Gabriella M. Urban, Ken Mackie, Masahiko Watanabe, Benjamin F. Cravatt, Tamas F. Freund, Istvan Katona
    JOURNAL OF NEUROSCIENCE 28 (5) 1058 - 1063 0270-6474 2008/01 [Refereed][Not invited]
     
    Endocannabinoids are regarded as retrograde signaling molecules at various types of synapses throughout the CNS. The lipid derivatives anandamide and 2-arachidonoylglycerol (2-AG) are generally thought to be the key molecular players in this process. Previous anatomical and electrophysiological studies provided compelling evidence that the biosynthetic enzyme of 2-AG is indeed localized in the postsynaptic plasma membrane, whereas its target, the CB1 cannabinoid receptor, and the enzyme responsible for its inactivation are both found presynaptically. This molecular architecture of 2-AG signaling is a conserved feature of most synapses and supports the retrograde signaling role of 2-AG. Conversely, the molecular and neuroanatomical organization of synaptic anandamide signaling remains largely unknown. In contrast to its predicted role in retrograde signaling, here we show that N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD), a biosynthetic enzyme of anandamide and its related bioactive congeners, the N-acylethanolamines (NAEs), is concentrated presynaptically in several types of hippocampal excitatory axon terminals. Furthermore, high-resolution quantitative immunogold labeling demonstrates that this calcium-sensitive enzyme is localized predominantly on the intracellular membrane cisternae of axonal calcium stores. Finally, the highest density of NAPE-PLD is found in mossy terminals of granule cells, which do not express CB1 receptors. Together, these findings suggest that anandamide and related NAEs are also present at glutamatergic synapses, but the sites of their synthesis and action are remarkably different from 2-AG, indicating distinct physiological roles for given endocannabinoids in the regulation of synaptic neurotransmission and plasticity.
  • Ferenc Matyas, Gabriella M. Urban, Masahiko Watanabe, Ken Mackie, Andreas Zimmer, Tamas F. Freund, Istvan Katona
    NEUROPHARMACOLOGY 54 (1) 95 - 107 0028-3908 2008/01 [Refereed][Not invited]
     
    Intact endogenous cannabinoid signaling is involved in several aspects of drug addiction. Most importantly, endocannabinoids exert pronounced influence on primary rewarding effects of abused drugs, including exogenous cannabis itself, through the regulation of drug-induced increase in bursting activity of dopaminergic: neurons in the ventral tegmental area (VTA). Previous electrophysiological studies have proposed that these dopaminergic neurons may release endocannabinoids in an activity-dependent manner to regulate their various synaptic inputs; however, the underlying molecular and anatomical substrates have so far been elusive. To facilitate understanding of the neurobiological mechanisms involving endocannabinoid signaling in drug addiction, we carried out detailed analysis of the molecular architecture of the endocannabinoid system in the VTA. In situ hybridization for sn-1-diacylglycerol lipase-alpha (DGL-alpha), the biosynthetic enzyme of the most abundant endocannabinoid, 2-arachidonoylglycerol (2-AG), revealed that DGL-a was expressed at moderate to high levels by most neurons of the VTA. Immunostaining for DGL-a resulted in a widespread punctate pattern at the light microscopic level, whereas high-resolution electron microscopic analysis demonstrated that this pattern is due to accumulation of the enzyme adjacent to postsynaptic specializations of several distinct morphological types of glutamatergic and GABAergic synapses. These axon terminal types carried presynaptic CB1 cannabinoid receptors on the opposite side of DGL-alpha-containing synapses and double immunostaining confirmed that DGL-alpha is present on the plasma membrane of both tyrosine hydroxylase (TH)-positive (dopaminergic) and TH-negative dendrites. These findings indicate that retrograde synaptic signaling mediated by 2-AG via CB1 may influence the drug-reward circuitry at multiple types of synapses in the VTA. (c) 2007 Elsevier Ltd. All rights reserved.
  • Aya Ishida, Keiko Matsuda, Taisuke Miyazaki, Eriko Miura, Takatoshi Iijima, Tetsuro Kondo, Masahiko Watanabe, Michisuke Yuzaki
    NEUROSCIENCE RESEARCH 61 S78 - S78 0168-0102 2008 [Refereed][Not invited]
  • Jun Nishiyama, Eriko Miura, Noboru Mizushima, Masahiko Watanabe, Michisuke Yuzaki
    NEUROSCIENCE RESEARCH 61 S166 - S166 0168-0102 2008 [Refereed][Not invited]
  • Misato Yasumura, Takeshi Uemura, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe, Masayoshi Mishina
    NEUROSCIENCE RESEARCH 61 S221 - S221 0168-0102 2008 [Refereed][Not invited]
  • Harumi Nakao, Kouichi Hashimoto, Miwako Yamasaki, Kazuki Nakao, Masahiko Watanabe, Masanobu Kano, Atsu Ailba
    NEUROSCIENCE RESEARCH 61 S147 - S147 0168-0102 2008 [Refereed][Not invited]
  • Takeshi Uemura, Sho Kakizavva, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe, Masamitsu Iino, Masayoshi Mishina
    JOURNAL OF PHARMACOLOGICAL SCIENCES 106 80P - 80P 1347-8613 2008 [Refereed][Not invited]
  • Takeshi Uemura, Sho Kakizawa, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe, Masamitsu Lino, Masayoshi Mishina
    NEUROSCIENCE RESEARCH 61 S219 - S219 0168-0102 2008 [Refereed][Not invited]
  • Misato Yasumura, Takeshi Uemura, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe, Masayoshi Mishina
    JOURNAL OF PHARMACOLOGICAL SCIENCES 106 133P - 133P 1347-8613 2008 [Refereed][Not invited]
  • Ruka Echigo, Kazuki Nakao, Masahiro Fukaya, Masahiko Watanabe, Atsu Aiba
    Kobe Journal of Medical Sciences 54 (6) E272 - E278 0023-2513 2008 [Refereed][Not invited]
     
    We generated a versatile mouse line, L7-tTA knock-in mouse, in which tetracycline-responsive transcriptional activator (tTA) gene was introduced into exon 2 of L7 locus. Since L7 is specifically expressed in cerebellar Purkinje cells, we expected Purkinje cell-restricted expression of tTA gene in the knock-in mice. In situ hybridization analysis exhibited that tTA mRNAs in those mice were expressed only in Purkinje cells. Introduction of transgene consisting of tetracycline-responsive element that is a binding site for tTA and ideal cDNAs into L7-tTA knock-in mouse would result in specific expression of cDNA encoding proteins in Purkinje cells and its expression could be controlled by doxycycline administration. L7-tTA knock-in mice would provide us with opportunity to elucidate the role of specific genes in cerebellar Purkinje cells.
  • Hiroto Yoshida, Takeshi Kishida, Masahiko Watanabe, Joji Ohshita
    CHEMICAL COMMUNICATIONS (45) 5963 - 5965 1359-7345 2008 [Refereed][Not invited]
     
    Acyl and fluorenyl moieties are facilely installed into neighbouring positions of aromatic skeletons via C-C bond cleavage reaction using arynes.
  • Shinji Matsuda, Eriko Miura, Wataru Kakegawa, Keiko Matsuda, Kazuhisa Kohda, Masahiko Watanabe, Michisuke Yuzaki
    NEUROSCIENCE RESEARCH 61 S21 - S21 0168-0102 2008 [Refereed][Not invited]
  • Takeshi Harada, Yoshie Hirai, Miwako Yamasaki, Kouichi Hashimoto, Harumi Nakao, Toshihide Tabata, Masahiko Watanabe, Masanobu Kano, Atsu Aiba
    NEUROSCIENCE RESEARCH 61 S57 - S57 0168-0102 2008 [Refereed][Not invited]
  • Gwenael Labouebe, Marta Lomazzi, Hans G. Cruz, Cyril Creton, Rafael Lujan, Meng Li, Yuchio Yanagawa, Kunihiko Obata, Masahiko Watanabe, Kevin Wickman, Stephanie B. Boyer, Paul A. Slesinger, Christian Luescher
    NATURE NEUROSCIENCE 10 (12) 1559 - 1568 1097-6256 2007/12 [Refereed][Not invited]
     
    Agonists of GABA(B) receptors exert a bi-directional effect on the activity of dopamine (DA) neurons of the ventral tegmental area, which can be explained by the fact that coupling between GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK) channels is significantly weaker in DA neurons than in GABA neurons. Thus, low concentrations of agonists preferentially inhibit GABA neurons and thereby disinhibit DA neurons. This disinhibition might confer reinforcing properties on addictive GABA(B) receptor agonists such as gamma-hydroxybutyrate (GHB) and its derivatives. Here we show that, in DA neurons of mice, the low coupling efficiency reflects the selective expression of heteromeric GIRK2/3 channels and is dynamically modulated by a member of the regulator of G protein signaling (RGS) protein family. Moreover, repetitive exposure to GHB increases the GABA(B) receptor-GIRK channel coupling efficiency through downregulation of RGS2. Finally, oral self-administration of GHB at a concentration that is normally rewarding becomes aversive after chronic exposure. On the basis of these results, we propose a mechanism that might underlie tolerance to GHB.
  • Yuji Kamikubo, Toshihide Tabata, Sho Kakizawa, Daisuke Kawakami, Masahiko Watanabe, Akihiko Ogura, Masamitsu Iino, Masanobu Kano
    JOURNAL OF PHYSIOLOGY-LONDON 585 (2) 549 - 563 0022-3751 2007/12 [Refereed][Not invited]
     
    Long-term depression (LTD) of excitatory transmission at cerebellar parallel fibre-Purkinje cell synapses is a form of synaptic plasticity crucial for cerebellar motor learning. Around the postsynaptic membrane of these synapses, B-type gamma-aminobutyric acid receptor (GABA(B)R), a G(i/o) protein-coupled receptor for the inhibitory transmitter GABA is concentrated and closely associated with type-1 metabotropic glutamate receptors (mGluR1) whose signalling is a key factor for inducing LTD. We found that in cultured Purkinje cells, GABA(B)R activation enhanced LTD of a glutamate-evoked current (LTDglu), increasing the magnitude of depression. It has been reported that parallel fibre-Purkinje cell synapses receive a micromolar level of GABA spilt over from the synaptic terminals of the neighbouring GABAergic interneurons. This level of GABA was able to enhance LTDglu. Our pharmacological analyses revealed that the beta gamma subunits but not the alpha subunit of G(i/o) protein mediated GABA(B)R-mediated LTDglu enhancement. G(i/o) protein activation was sufficient to enhance LTDglu. In this respect, LTDglu enhancement is clearly distinguished from the previously reported GABA(B)R-mediated augmentation of an mGluR1-coupled slow excitatory postsynaptic potential. Baclofen application for only the induction period of LTDglu was sufficient to enhance LTDglu, suggesting that GABA(B)R signalling may modulate mechanisms underlying LTDglu induction. Baclofen augmented mGluR1-coupled Ca2+ release from the intracellular stores in a G(i/o) protein-dependent manner. Therefore, GABA(B)R-mediated LTDglu enhancement is likely to result from augmentation of mGluR1 signalling. Furthermore, pharmacological inhibition of GABA(B)R reduced the magnitude of LTD at parallel fibre-Purkinje cell synapses in cerebellar slices. These findings demonstrate a novel mechanism that would facilitate cerebellar motor learning.
  • Jun Nishiyama, Erika Miura, Noboru Mizushima, Masahiko Watanabe, Michisuke Yuzaki
    AUTOPHAGY 3 (6) 591 - 596 1554-8627 2007/11 [Refereed][Not invited]
     
    Autophagy (macroautophagy) is an evolutionally conserved process by which cytoplasmic proteins and organelles are surrounded by unique double membranes and are subsequently degraded upon fusion with lysosomes. Many autophagy-related genes (Atg) have been identified in yeast; a ubiquitin-like Atg 12-Atg5 system is also essential for the elongation of the isolation membrane in mammalian cells. Nevertheless, the regulation of autophagy in neurons remains largely unknown. In this study, we crossed conditional knockout mice Atg5(flox/flox) with pcp2-Cre transgenic mice, which express Cre recombinase through a Purkinje cell-specific promoter, pcp2. In Atg5(flox/flox); pcp2-Cre mice, the Atg5 gene was excised as early as postnatal day 6; Purkinje cells started to degenerate after approximately 8 weeks, and the animals showed an ataxic gait from around 10 months. Initially, however, the Purkinje cells showed axonal swelling around its terminals from as early as 4 weeks after birth. An electron microscopic analysis revealed the accumulation of autophagosome-like double-membrane structures in the swollen regions, together with numerous membranous organelles, such as tubular or sheet-like smooth endoplasmic reticulum and vesicles. These results suggest that Atg5 plays important roles in the maintenance of axon morphology and membrane structures, and its loss of function leads to the swelling of axons, followed by progressive neurodegeneration in mammalian neurons.
  • Hiroshi Nishiyama, Masahiro Fukaya, Masahiko Watanabe, David J. Linden
    NEURON 56 (3) 472 - 487 0896-6273 2007/11 [Refereed][Not invited]
     
    We performed two-photon in vivo imaging of cerebellar climbing fibers (CFs; the terminal arbor of olivocerebellar axons) in adult mice. CF ascending branches innervate Purkinje cells while CIF transverse branches show a near complete failure to form conventional synapses. Time-lapse imaging over hours or days revealed that ascending branches were very stable. However, transverse branches were highly dynamic, exhibiting rapid elongation and retraction and varicosity turnover. Thus, different branches of the same axon, with different innervation patterns, display branch type-specific motility in the adult cerebellum. Furthermore, dynamic changes in transverse branch length were almost completely suppressed by pharmacological stimulation of olivary firing.
  • Atsushi Ikeda, Taisuke Miyazaki, Sho Kakizawa, Yasushi Okuno, Soken Tsuchiya, Akira Myomoto, Shin-Ya Saito, Tetsuji Yamamoto, Tetsuo Yamazaki, Masamitsu Iino, Gozoh Tsujimoto, Masahiko Watanabe, Hiroshi Takeshima
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 363 (3) 835 - 839 0006-291X 2007/11 [Refereed][Not invited]
     
    Junctional membrane complexes (JMCs) generated by junctophilins are required for Ca (2+) -Mediated communication between cell-surface and intracellular channels in excitable cells. Knockout mice lacking neural junctophilins (JP-DKO) show severe motor defects and irregular cerebellar plasticity due to abolished channel crosstalk in Purkinje cells (PCs). To precisely understand aberrations in JP-DKO mice, we further analyzed the mutant PCs. During the induction of cerebellar plasticity via electrical stimuli, JP-DKO PCs showed insufficient depolarizing responses. Immunochemistry detected mild impairment in synaptic maturation and hyperphosphorylation of protein kinase C gamma in JP-DKO PCs. Moreover, gene expression was slightly altered in the JP-DKO cerebellum. Therefore, the mutant PCs bear marginal but widespread abnormalities, all of which likely cause cerebellar motor defects in JP-DKO mice. (C) 2007 Elsevier Inc. All rights reserved.
  • Takeshi Uemura, Sho Kakizawa, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe, Masamitsu Iino, Masayoshi Mishina
    JOURNAL OF NEUROSCIENCE 27 (44) 12096 - 12108 0270-6474 2007/10 [Refereed][Not invited]
     
    Glutamate receptor ( GluR) delta 2 selectively expressed in cerebellar Purkinje cells ( PCs) plays key roles in long-term depression ( LTD) induction at parallel fiber ( PF)-PC synapses, motor learning, the matching and connection of PF-PC synapses in developing and adult cerebella, the elimination of multiple climbing fibers ( CFs) during development, and the regulation of CF territory on PCs. However, it remains unsolved how GluR delta 2 regulates cerebellar synaptic plasticity, PF-PC synapse formation, and CF wiring. One possible signaling mechanism through GluR delta 2 is signaling by protein-protein interactions. The C-terminal region of GluR delta 2 contains at least three domains for protein-protein interactions. The PDZ ( postsynaptic density-95/Discs large/zona occludens 1)-binding domain at the C terminal, named as the T site, interacts with several postsynaptic density proteins. Here, we generated GluR delta 2 Delta T mice carrying mutant GluR delta 2 lacking the T site. There were no significant differences in the amount of receptor proteins at synapses, histological features, and the fine structures of PF-PC synapses between wild-type and GluR delta 2 Delta T mice. However, LTD induction at PF-PC synapses and improvement in the accelerating rotarod test were impaired in GluR delta 2 Delta T mice. Furthermore, CF territory expanded distally and ectopic innervation of CFs occurred at distal dendrites in GluR delta 2 Delta T mice, but the elimination of surplus CF innervation at proximal dendrites appeared to proceed normally. These results suggest that the C-terminal T site of GluR delta 2 is essential for LTD induction and the regulation of CF territory but is dispensable for PF-PC synapse formation and the elimination of surplus CFs at proximal dendrites during development.
  • Kohtarou Konno, Machiko Matsumoto, Hiroko Togashi, Taku Yamaguchi, Takeshi Izumi, Masahiko Watanabe, Toshihiko Iwanaga, Mitsuhiro Yoshioka
    BRAIN RESEARCH 1172 60 - 66 0006-8993 2007/10 [Refereed][Not invited]
     
    Recent studies have focused on the serotonergic mechanism mediated via serotonin (5-HT) receptors underlying regulation of emotional stress during the developmental period. The present study was undertaken to elucidate whether early postnatal stress affects rat brain development and influences the serotonergic function in the midbrain median raphe nuclei (MRN) and dorsal raphe nuclei (DRN) in the adult, focusing on the response to unconditioned fear stress. Rats received aversive foot shock (FS) stimuli at the third week of the postnatal period (3wFS), but not those at the second week (2wFS), had increased percentage of time spent on open arms, estimated by the elevated plus maze test, at the postadolescent period (10-12 weeks old). The anxiolytic behavior observed in 3wFS was similar to that in rats having electrolytic lesion of the MRN, but not the DRN. In addition, the number of MRN 5-HT-immunoreactive cells in 3wFS remarkably was reduced compared to the non-FS control and 2wFS groups. These data suggest that aversive stress at the third week is attributable to the serotonergic function in the MRN underlying regulation of unconditioned fear stress. In other words, the "critical period" appears to be the time of neural circuit development of the MRN serotonergic system, which may be implicated in lifelong susceptibility to emotional stress. Published by Elsevier B.V.
  • Yuki Hashimotodani, Takako Ohno-Shosaku, Masahiko Watanabe, Masanobu Kano
    JOURNAL OF PHYSIOLOGY-LONDON 584 (2) 373 - 380 0022-3751 2007/10 [Refereed][Not invited]
     
    Endocannabinoids are released from postsynaptic neurons, activate presynaptic cannabinoid receptors and cause various forms of short-term and long-term synaptic plasticity throughout the brain. Using hippocampal and cerebellar neurons, we have revealed that endocannabinoid release can be induced through two different pathways. One is independent of phospholipase C,3 (PLC,3) and driven by Ca2+ elevation alone (Ca2+-driven endocannabinoid release, CaER), and the other is PLC beta-dependent and driven by activation of G(q/11)-coupled receptors (receptor-driven endocannabinoid release, RER). CaER is induced by activation of either voltage-gated Ca2+ channels or NMDA receptors. RER is functional even at resting Ca2+ levels (basal RER), but markedly enhanced by a small Ca2+ elevation (Ca2+-assisted RER). In Ca2+-assisted RER, PLC beta serves as a coincidence detector of receptor activation and Ca2+ elevation. We have also demonstrated that Ca2+-assisted RER is essential for the endocannabinoid release triggered by synaptic activity. Our anatomical data show that a set of receptors and enzymes required for RER are well organized so that the excitatory input can trigger RER effectively. Certain forms of spike-timing-dependent plasticity (STDP) are reported to depend on endocannabinoid signalling. The NMDA receptor and PLC,3 might play key roles in the endocannabinoid-dependent forms of STDP as coincidence detectors with different timing dependences.
  • Kohtarou Konno, Machiko Matsumoto, Hiroko Togashi, Taku Yamaguchi, Takeshi Izumi, Masahiko Watanabe, Toshihiko Iwanaga, Mitsuhiro Yoshioka
    BRAIN RESEARCH 1172 60 - 66 0006-8993 2007/10 [Refereed][Not invited]
     
    Recent studies have focused on the serotonergic mechanism mediated via serotonin (5-HT) receptors underlying regulation of emotional stress during the developmental period. The present study was undertaken to elucidate whether early postnatal stress affects rat brain development and influences the serotonergic function in the midbrain median raphe nuclei (MRN) and dorsal raphe nuclei (DRN) in the adult, focusing on the response to unconditioned fear stress. Rats received aversive foot shock (FS) stimuli at the third week of the postnatal period (3wFS), but not those at the second week (2wFS), had increased percentage of time spent on open arms, estimated by the elevated plus maze test, at the postadolescent period (10-12 weeks old). The anxiolytic behavior observed in 3wFS was similar to that in rats having electrolytic lesion of the MRN, but not the DRN. In addition, the number of MRN 5-HT-immunoreactive cells in 3wFS remarkably was reduced compared to the non-FS control and 2wFS groups. These data suggest that aversive stress at the third week is attributable to the serotonergic function in the MRN underlying regulation of unconditioned fear stress. In other words, the "critical period" appears to be the time of neural circuit development of the MRN serotonergic system, which may be implicated in lifelong susceptibility to emotional stress. Published by Elsevier B.V.
  • Stephan Niemann, Hiroaki Kanki, Yasuyuki Fukui, Keizo Takao, Masahiro Fukaya, Meri N. Hynynen, Michael J. Churchill, Jeremy M. Shefner, Roderick T. Bronson, Robert H. Brown, Masahiko Watanabe, Tsuyoshi Miyakawa, Shigeyoshi Itohara, Yasunori Hayashi
    EUROPEAN JOURNAL OF NEUROSCIENCE 26 (6) 1407 - 1420 0953-816X 2007/09 [Refereed][Not invited]
     
    NR3B is a modulatory subunit of the NMDA receptor, abundantly expressed in both cranial and spinal somatic motoneurons and at lower levels in other regions of the brain as well. Recently, we found the human NR3B gene (GRIN3B) to be highly genetically heterogeneous, and that similar to 10% of the normal European-American population lacks NR3B due to homozygous occurrence of a null allele in the gene. Therefore, it is especially important to understand the phenotypic consequences of the genetic loss of NR3B in both humans and animal models. We here provide results of behavioral analysis of mice genetically lacking NR3B, which is an ideal animal model due to homogeneity in genetic and environmental background. The NR3B(-/-) mice are viable and fertile. Consistent with the expression of NR3B in somatic motoneurons, the NR3B(-/-) mice showed a moderate but significant impairment in motor learning or coordination, and decreased activity in their home cages. Remarkably, the NR3B(-/-) mice showed a highly increased social interaction with their familiar cage mates in their home cage but moderately increased anxiety-like behaviour and decreased social interaction in a novel environment, consistent with the inhibitory role of NR3B on the functions of NMDA receptors. This work is the first reporting of the functional significance of NR3B in vivo and may give insight into the contribution of genetic variability of NR3B in the phenotypic heterogeneity among human population.
  • Judit K. Makara, Istvan Katona, Gabor Nyiri, Beata Nemeth, Catherine Ledent, Masahiko Watanabe, Jan de Vente, Tamas F. Freund, Norbert Hajos
    JOURNAL OF NEUROSCIENCE 27 (38) 10211 - 10222 0270-6474 2007/09 [Refereed][Not invited]
     
    Several types of neurons are able to regulate their synaptic inputs via releasing retrograde signal molecules, such as endocannabinoids or nitric oxide (NO). Here we show that, during activation of cholinergic receptors, retrograde signaling by NO controls CB1 cannabinoid receptor (CB1R)-dependent depolarization-induced suppression of inhibition (DSI). Spontaneously occurring IPSCs were recorded in CA(1) pyramidal neurons in the presence of carbachol, and DSI was induced by a 1-s-long depolarization step. We found that, in addition to the inhibition of CB(1)Rs, blocking the NO signaling pathway at various points also disrupted DSI. Inhibitors of NO synthase (NOS) or NO-sensitive guanylyl cyclase (NO-sGC) diminished DSI, whereas a cGMP analog or an NO donor inhibited IPSCs and partially occluded DSI in a CB1R-dependent manner. Furthermore, an NO scavenger applied extracellularly or postsynaptically also decreased DSI, whereas L-arginine, the precursor for NO, prolonged it. DSI of electrically evoked IPSCs was also blocked by an inhibitor of NOS in the presence, but not in the absence, of carbachol. In line with our electrophysiological data, double immunohistochemical staining revealed an NO-donor-induced cGMP accumulation in CB1R-positive axon terminals. Using electron microscopy, we demonstrated the postsynaptic localization of neuronal NOS at symmetrical synapses formed by CB1R-positive axon terminals on pyramidal cell bodies, whereas NO-sGC was found in the presynaptic terminals. These electrophysiological and anatomical results in the hippocampus suggest that NO is involved in depolarization-induced CB1R-mediated suppression of IPSCs as a retrograde signal molecule and that operation of this cascade is conditional on cholinergic receptor activation.
  • Yoshimasa Kosaka, Hiroshi Inoue, Takahiro Ohmachi, Takeshi Yokoe, Toshifumi Matsumoto, Koshi Mimori, Fumiaki Tanaka, Masahiko Watanabe, Masaki Mori
    ANNALS OF SURGICAL ONCOLOGY 14 (9) 2543 - 2549 1068-9265 2007/09 [Refereed][Not invited]
     
    Background: Tripartite motif-containing 29 (TRIM29) belongs to the TRIM protein family, which has unique structural characteristics, including multiple zinc finger motifs and a leucine zipper motif. TRIM29, also known as ataxia telangiectasia group D complementing gene, possesses radiosensitivity suppressor functions. Although TRIM29 has been reported to be underexpressed in prostate and breast cancer, its expression in gastrointestinal cancer has not been studied. Methods: By use of real-time reverse transcriptase-polymerase chain reaction, we analyzed TRIM29 mRNA expression status with respect to various clinicopathological parameters in 124 patients with gastric cancer. An immunohistochemical study was also conducted. Results: The expression of TRIM29 was far higher in gastric cancer tumor tissue. Increased TRIM29 mRNA expression was markedly associated with such parameters as histological grade, large tumor size, extent of tumor invasion, and lymph node metastasis. In the TRIM29 high-expression group, it was an independent predictor for lymph node metastasis. Furthermore, patients with high TRIM29 mRNA expression showed a far poorer survival rate than those with low TRIM29 mRNA expression. Conclusions: TRIM29 expression may serve as a good marker of lymph node metastasis in gastric cancer.
  • Gabor Wittmann, Levente Deli, Imre Kallo, Erik Hrabovszky, Masahiko Watanabe, Zsolt Liposits, Csaba Fekete
    JOURNAL OF COMPARATIVE NEUROLOGY 503 (2) 270 - 279 0021-9967 2007/07 [Refereed][Not invited]
     
    Type 1 cannabinoid receptor (CB1) is the principal receptor for endocannabinoids in the brain; it mainly occurs in preterminal/terminal axons and mediates retrograde neuronal signaling mechanisms. A large body of physiological and electrophysiological evidence indicates the critical role of CB1 in the regulation of hypothalamic functions. Conversely, the distribution of CB1-containing axons in the hypothalamus is essentially unknown. Therefore, we have analyzed the distribution and the ultrastructural characteristics of the CBI-immunoreactive (IR) axons in the mouse hypothalamus by using an antiserum. against the C-terminal 31 amino acids of the mouse CB1. We found that CB1-IR axons innervated densely the majority of hypothalamic nuclei, except for the suprachiasmatic and lateral mammillary nuclei, in which only scattered CB1-IR fibers occurred. CB1-IR innervation of the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the external zone of the median eminence corroborated the important role of CB1 in the regulation of energy homeostasis and neuroendocrine functions. Ultrastructural studies to characterize the phenotype of CB1-IR fibers established that most CB1 immunoreactivity appeared in the preterminal and terminal portions of axons. The CB1-IR boutons formed axospinous, axo-dendritic, and axosomatic synapses. Analysis of labeled synapses in the paraventricular and arcuate nuclei detected approximately equal numbers of symmetric and asymmetric specializations. In conclusion, the study revealed the dense and differential CB1-IR innervation of most hypothalamic nuclei and the median eminence of the mouse brain. At the ultrastructural level, CB1-IR axons established communication with hypothalamic neurons via symmetric and asymmetric synapses indicating the occurrence of retrograde signaling by endocannabinoids in hypothalamic neuronal networks.
  • Motokazu Uchigashima, Masahiro Fukaya, Masahiko Watanabe, Haruyuki Kamiya
    JOURNAL OF NEUROSCIENCE 27 (30) 8088 - 8100 0270-6474 2007/07 [Refereed][Not invited]
     
    Hippocampal mossy fibers of young rodents have been reported to corelease inhibitory neurotransmitter GABA in addition to excitatory transmitter glutamate. In this study, we aimed at re-evaluating this corelease hypothesis of both inhibitory and excitatory transmitters in the hippocampus. Electrophysiological examination revealed that, in juvenile mice and rats of the two to 3 weeks old, stimulation at the granule cell layer of the dentate gyrus elicited monosynaptic GABAergic IPSCs in CA3 neurons in the presence of ionotropic glutamate receptor (iGluR) blockers, only when rather strong stimuli were given. The group II mGluR agonist (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclo-propyl)glycine (DCG-IV), which selectively suppresses transmission at the mossy fiber-CA3 synapse, abolished almost all postsynaptic responses elicited by the weak stimuli, whereas those by strong stimuli were inhibited only slightly. In addition, the minimal stimulation elicited GABAergic IPSCs in neonatal mice of the first postnatal week, whereas these responses are not sensitive to DCG-IV. Immunohistochemical examination revealed that mossy fiber terminals expressed GABA and the GABA-synthesizing enzyme GAD67, although the expression levels were much weaker than those in the inhibitory interneurons. Notably, the expression levels of the vesicular GABA transporter were much lower than those of GABA and GAD67, and almost below detection threshold. These results suggest that mossy fiber synapses are purely glutamatergic and apparent monosynaptic IPSCs so far reported are evoked by costimulation of inhibitory interneurons, at least in young mice and rats. Hippocampal mossy fiber terminals synthesize and store GABA, but have limited ability in vesicular release for GABA in the developing rodents.
  • Kazuhiro Nakamura, Hirokazu Hirai, Takashi Torashima, Taisuke Miyazaki, Hiromichi Tsurui, Yan Xiu, Mareki Ohtsuji, Qing Shun Lin, Kazuyuki Tsukamoto, Hiroyuki Nishimura, Masao Ono, Masahiko Watanabe, Sachiko Hirose
    MOLECULAR AND CELLULAR BIOLOGY 27 (14) 5128 - 5134 0270-7306 2007/07 [Refereed][Not invited]
     
    The immune and nervous systems display considerable overlap in their molecular repertoire. Molecules originally shown to be critical for immune responses also serve neuronal functions that include normal brain development, neuronal differentiation, synaptic plasticity, and behavior. We show here that Fc gamma RIIB, a low-affinity immunoglobulin G Fc receptor, and CD3 are involved in cerebellar functions. Although membranous CD3 and Fc gamma RIIB are crucial regulators on different cells in the immune system, both CD3 epsilon and Fc gamma RIIB; are expressed on Purkinje cells in the cerebellum. Both CD3 epsilon-deficient mice and Fc gamma RIIB-deficient mice showed an impaired development of Purkinje neurons. In the adult, rotarod performance of these mutant mice was impaired at high speed. In the two knockout mice, enhanced paired-pulse facilitation of parallel fiber-Purkinje cell synapses was shared. These results indicate that diverse immune molecules play critical roles in the functional establishment in the cerebellum.
  • Motokazu Uchigashima, Masahiro Fukaya, Masahiko Watanabe, Haruyuki Kamiya
    JOURNAL OF NEUROSCIENCE 27 (30) 8088 - 8100 0270-6474 2007/07 [Refereed][Not invited]
     
    Hippocampal mossy fibers of young rodents have been reported to corelease inhibitory neurotransmitter GABA in addition to excitatory transmitter glutamate. In this study, we aimed at re-evaluating this corelease hypothesis of both inhibitory and excitatory transmitters in the hippocampus. Electrophysiological examination revealed that, in juvenile mice and rats of the two to 3 weeks old, stimulation at the granule cell layer of the dentate gyrus elicited monosynaptic GABAergic IPSCs in CA3 neurons in the presence of ionotropic glutamate receptor (iGluR) blockers, only when rather strong stimuli were given. The group II mGluR agonist (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclo-propyl)glycine (DCG-IV), which selectively suppresses transmission at the mossy fiber-CA3 synapse, abolished almost all postsynaptic responses elicited by the weak stimuli, whereas those by strong stimuli were inhibited only slightly. In addition, the minimal stimulation elicited GABAergic IPSCs in neonatal mice of the first postnatal week, whereas these responses are not sensitive to DCG-IV. Immunohistochemical examination revealed that mossy fiber terminals expressed GABA and the GABA-synthesizing enzyme GAD67, although the expression levels were much weaker than those in the inhibitory interneurons. Notably, the expression levels of the vesicular GABA transporter were much lower than those of GABA and GAD67, and almost below detection threshold. These results suggest that mossy fiber synapses are purely glutamatergic and apparent monosynaptic IPSCs so far reported are evoked by costimulation of inhibitory interneurons, at least in young mice and rats. Hippocampal mossy fiber terminals synthesize and store GABA, but have limited ability in vesicular release for GABA in the developing rodents.
  • Yoshiko Takagishi, Kouichi Hashimoto, Tetsuro Kayahara, Masahiko Watanabe, Hiroyuki Otsuka, Akira Mizoguchi, Masanobu Kano, Yoshiharu Murata
    DEVELOPMENTAL NEUROBIOLOGY 67 (7) 909 - 923 1932-8451 2007/06 [Refereed][Not invited]
     
    Myosin Va is an actin-based molecular motor that is involved in organelle transport and membrane trafficking. Here, we explored the role of myosin Va in the formation of synaptic circuitry by examining climbing fiber (CF) innervation of Purkinje cells (PCs) in the cerebella of dilate-neurological (d-n) mice and dilute-opisthotonus (dop) rats that have mutations in dilute-encoded myosin Va. Anterograde labeling of CFs with biotinylated dextran amine (BDA) revealed that they arborized poorly and that their tips extended only half way through the thickness of the molecular layer (NIL) in adult d-n mice. Using immunohistochemistry specific for vesicular glutamate transporter 2 (VGluT2) to visualize CF synaptic terminals, we found that during development and in adulthood, these terminals did not ascend as far along the proximal shaft dendrites of PCs in d-n mice and dop rats as they did in normal animals. An irregular distribution of BDA-labeled bulbous varicosities and VGluT2 spots along CF branches were also noted in these animals. Finally, VGluT2-positive CF terminals were occasionally localized on the PC somata of adult d-n cerebella. These phenotypes are consistent with our electrophysiological findings that CF-mediated excitatory postsynaptic currents (EPSCs) were significantly smaller in amplitude and faster in decay in adult d-n mice, and that the regression of multiple CFs was slightly delayed in developing d-n mice. Taken together, our results suggest that myosin Va is essential for terminal CF extension and for the establishment of CF synapses within the proper dendritic territories Of PCs. (c) 2007 Wiley Periodicals, Inc
  • Hassan Marzban, Seunghyuk Chung, Masahiko Watanabe, Richard Hawkes
    JOURNAL OF COMPARATIVE NEUROLOGY 502 (5) 857 - 871 0021-9967 2007/06 [Refereed][Not invited]
     
    Mediolateral boundaries divide the mouse cerebellar cortex into four transverse zones, and within each zone the cortex is further subdivided into a symmetrical array of parasagittal stripes. Various expression markers reveal this complexity, and detailed maps have been constructed based on the differential expression of zebrin II/aldolase C in a Purkinje cell subset. Recently, phospholipase (PL) C beta 4 expression in adult mice was shown to be restricted to, and coextensive with, the zebrin II-immunonegative Purkinje cell subset. The Purkinje cell expression of PLC 04 during embryogenesis and postnatal development begins just before birth in a subset of Purkinje cells that are clustered to form a reproducible array of parasagittal stripes. Double label and serial section immunocytochemistry revealed that the early PLC beta 4-immunoreactive clusters in the neonate are complementary to those previously identified by neurogranin expression. The PLC beta 4 expression pattern can be traced continuously from embryo to adult, revealing the continuity of the topographical map from perinatal to adult cerebella. The only exception, as has been seen for other antigenic markers, is that transient PLC beta 4 expression (which subsequently disappears) is seen in some Purkinje cell stripes during the second postnatal week. Furthermore, the data confirm that some adult Purkinje cell stripes are composite in origin, being derived from two or more distinct embryonic clusters. Thus, the zone and stripe topography of the cerebellum is conserved from embryo to adult, confirming that the early- and late-antigenic markers share a common cerebellar topography.
  • P. Brumovsky, M. Watanabe, T. Hokfelt
    NEUROSCIENCE 147 (2) 469 - 490 0306-4522 2007/06 [Refereed][Not invited]
     
    The expression of two vesicular glutamate transporters (VGLUTs), VGLUT(1) and VGLUT(2), was studied with immunohistochemistry in lumbar dorsal root ganglia (DRGs), the lumbar spinal cord and the skin of the adult mouse. About 12% and 65% of the total number of DRG neuron profiles (NPs) expressed VGLUT(1) and VGLUT(2), respectively. VGLUT(1)-immunoreactive (IR) NPs were usually medium- to large-sized, in contrast to a majority of small- or medium-sized VGLUT(2)-IR NPs. Most VGLUT(1)-IR NPs did not coexpress calcitonin gene-related peptide (CGRP) or bound isolectin B4 (IB4). In contrast, similar to 31% and similar to 42% of the VGLUT(2)-IR DRG NPs were also CGRP-IR or bound IB4, respectively. Conversely, virtually all CGRP-IR and IB4-binding NPs coexpressed VGLUT(2). Moderate colocalization between VGLUT(1) and VGLUT(2) was also observed. Sciatic nerve transection induced a decrease in the overall number of VGLUT(1)- and VGLUT(2)-IR NPs (both ipsi- and contralaterally) and, in addition, a parallel, unilateral increase of VGLUT(2)-like immunoreactivity (LI) in a subpopulation of mostly small NPs. In the spinal cord, strong VGLUT(1)-LI was detected, particularly in deep dorsal horn layers and in the ventral horns. VGLUT(2)-LI was abundant throughout the gray spinal matter, 'radiating' into/from the white matter. A unilateral dorsal rhizotomy reduced VGLUT(1)-LI, while apparently leaving unaffected the VGLUT(2)-LI. Transport through axons for both VGLUTs was confirmed by their accumulation after compression of the sciatic nerve or dorsal roots. In the hind paw skin, abundant VGLUT(2)-IR nerve fibers were observed, sometimes associated with Merkel cells. Lower numbers of VGLUT(1)-IR fibers were also detected in the skin. Some VGLUT(1)-IR and VGLUT(2)-IR fibers were associated with hair follicles. Based on these data and those by Morris et al. [Morris JL, Konig P, Shimizu T, Jobling P, Gibbins IL (2005) Most peptide-containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate. J Comp Neurol 483:1-16], we speculate that virtually all DRG neurons in adult mouse express VGLUTs and use glutamate as transmitter. (c) 2007 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Masahiko Watanabe, Yuichi Nakahara, Tetsuya Sakashita, Kikawada A. B. Ad B. Takahiro, Aklhlko Fujita, Nobuyuki Hamada, Dalki D. Horikawa, Seiichi Wada, Yasuhiko Kobayashi, Takashi Okuda
    JOURNAL OF INSECT PHYSIOLOGY 53 (6) 573 - 579 0022-1910 2007/06 [Refereed][Not invited]
     
    High tolerance against various extreme environments exhibited by some anhydrobionts might be due to being almost completely desiccated, a state where little or no chemical reactions occur. We have shown that anhydrobiotic larvae of Polypedillan vanderplanki have higher tolerance against both high- and low-linear energy transfer (LET) radiation than hydrated larvae. It is of great interest to know how the desiccating larvae gain radiation tolerance. We therefore examined effects of high-LET radiation on four kinds of larvae: (1) normal hydrated (intact) larva, (2) intermediates between the anhydrobiotic and normal hydrated state, (3) almost completely dehydrated (anhydrobiotic) larvae, and (4) immediately rehydrated larvae that are assumed to have a similar molecular profile to anhydrobiotic larvae. The intermediates and immediately rehydrated larvae survived longer after high-LET radiation than intact larvae, indicating that radiation tolerance could be enhanced even in hydrated larvae. Physiological changes toward anhydrobiosis, e.g. accumulation of protectants or increasing damage repair capacity, correlate with improved radiation tolerance in hydrated larvae. In addition, almost complete desiccation further enhanced radiation tolerance, possibly in a different way from the hydrated larvae. (c) 2007 Elsevier Ltd. All rights reserved.
  • Motokazu Uchigashima, Madoka Narushima, Masahiro Fukaya, Istvan Katona, Masanobu Kano, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 27 (14) 3663 - 3676 0270-6474 2007/04 [Refereed][Not invited]
     
    Endogenous cannabinoids (endocannabinoids) mediate retrograde signals for short- and long-term suppression of transmitter release at synapses of striatal medium spiny ( MS) neurons. An endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is synthesized from diacylglycerol (DAG) after membrane depolarization and Gq-coupled receptor activation. To understand 2-AG-mediated retrograde signaling in the striatum, we determined precise subcellular distributions of the synthetic enzyme of 2-AG, DAG lipase-alpha(DAGL alpha), and its upstream metabotropic glutamate receptor 5 (mGluR5) and muscarinic acetylcholine receptor 1 (M-1). DAGL alpha, mGluR5, and M-1 were all richly distributed on the somatodendritic surface of MS neurons, but their subcellular distributions were different. Although mGluR5 and DAGL alpha levels were highest in spines and accumulated in the perisynaptic region, M-1 level was lowest in spines and was rather excluded from the mGluR5-rich perisynaptic region. These subcellular arrangements suggest that mGluR5 and M-1 might differentially affect endocannabinoid-mediated, depolarization-induced suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE) in MS neurons. Indeed, mGluR5 activation enhanced both DSI and DSE, whereas M-1 activation enhanced DSI only. Importantly, DSI, DSE, and receptor-driven endocannabinoid-mediated suppression were all abolished by the DAG lipase inhibitor tetrahydrolipstatin, indicating 2-AG as the major endocannabinoid mediating retrograde suppression at excitatory and inhibitory synapses of MS neurons. Accordingly, CB1 cannabinoid receptor, the main target of 2-AG, was present at high levels on GABAergic axon terminals of MS neurons and parvalbumin-positive interneurons and at low levels on excitatory corticostriatal afferents. Thus, endocannabinoid signaling molecules are arranged to modulate the excitability of the MS neuron effectively depending on cortical activity and cholinergic tone as measured by mGluR5 and M-1 receptors, respectively.
  • Chihiro Takasaki, Eriko Miura, Masahiko Watanabe
    BIOMEDICAL RESEARCH-TOKYO 28 (2) 61 - 69 0388-6107 2007/04 [Refereed][Not invited]
     
    3-Phosphoglycerate dehydrogenase (Phgdh) is the initial step enzyme in the phosphorylated pathway of L-serine biosynthesis. We have previously revealed in the brain that Phgdh is preferentially expressed in glial cells, but not in neurons, and that glia-borne L-serine exerts strong neurotrophic actions to neuronal survive, differentiation, and development. To investigate whether such an L-serine-meditated intercellular relationship is constructed in peripheral organs and tissues, we examined the kidney, which is one of the organs with the highest expression of Phgdh mRNA in the body. We found that Phgdh was distributed highly in the renal papilla and inner layer of the outer zone and moderately in the cortex, whereas it was almost negative in the outer layer of the outer zone. This heterogeneous distribution was due to selective expression in distinct tubular segments, i.e., the Bowman's capsule, proximal tubule, and thin limbs of the Henle's loop. Interestingly, neutral amino acid transporter ASCT1, which preferentially transports alanine, serine, cysteine, and threonine, was selectively expressed in Phgdh-negative tubular segments, i.e., the distal tubule and collecting duct. Therefore, either Phgdh or ASCT1 is provided to each segment of renal tubules, suggesting that metabolic interplay mediated by L-serine biosynthesis and supply may exist in the kidney too.
  • Sho Kakizawa, Yasushi Kishimoto, Kouichi Hashimoto, Taisuke Miyazaki, Kazuharu Furutani, Hidemi Shimizu, Masahiro Fukaya, Miyuki Nishi, Hiroyuki Sakagami, Atsushi Ikeda, Hisatake Kondo, Masanobu Kano, Masahiko Watanabe, Masamitsu Iino, Hiroshi Takeshima
    EMBO JOURNAL 26 (7) 1924 - 1933 0261-4189 2007/04 [Refereed][Not invited]
     
    Functional crosstalk between cell-surface and intracellular ion channels plays important roles in excitable cells and is structurally supported by junctophilins (JPs) in muscle cells. Here, we report a novel form of channel crosstalk in cerebellar Purkinje cells (PCs). The generation of slow afterhyperpolarization (sAHP) following complex spikes in PCs required ryanodine receptor (RyR)-mediated Ca2+ induced Ca2+ release and the subsequent opening of small-conductance Ca2+ activated K+ (SK) channels in somatodendritic regions. Despite the normal expression levels of these channels, sAHP was abolished in PCs from mutant mice lacking neural JP subtypes (JP-DKO), and this defect was restored by exogenously expressing JPs or enhancing SK channel activation. The stimulation paradigm for inducing long-term depression (LTD) at parallel fiber-PC synapses adversely established long-term potentiation in the JP-DKO cerebellum, primarily due to the sAHP deficiency. Furthermore, JP-DKO mice exhibited impairments of motor coordination and learning, although normal cerebellar histology was retained. Therefore, JPs support the Ca2+-mediated communication between voltage-gated Ca2+ channels, RyRs and SK channels, which modulates the excitability of PCs and is fundamental to cerebellar LTD and motor functions.
  • Motokazu Uchigashima, Madoka Narushima, Masahiro Fukaya, Istvan Katona, Masanobu Kano, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 27 (14) 3663 - 3676 0270-6474 2007/04 [Refereed][Not invited]
     
    Endogenous cannabinoids (endocannabinoids) mediate retrograde signals for short- and long-term suppression of transmitter release at synapses of striatal medium spiny ( MS) neurons. An endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is synthesized from diacylglycerol (DAG) after membrane depolarization and Gq-coupled receptor activation. To understand 2-AG-mediated retrograde signaling in the striatum, we determined precise subcellular distributions of the synthetic enzyme of 2-AG, DAG lipase-alpha(DAGL alpha), and its upstream metabotropic glutamate receptor 5 (mGluR5) and muscarinic acetylcholine receptor 1 (M-1). DAGL alpha, mGluR5, and M-1 were all richly distributed on the somatodendritic surface of MS neurons, but their subcellular distributions were different. Although mGluR5 and DAGL alpha levels were highest in spines and accumulated in the perisynaptic region, M-1 level was lowest in spines and was rather excluded from the mGluR5-rich perisynaptic region. These subcellular arrangements suggest that mGluR5 and M-1 might differentially affect endocannabinoid-mediated, depolarization-induced suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE) in MS neurons. Indeed, mGluR5 activation enhanced both DSI and DSE, whereas M-1 activation enhanced DSI only. Importantly, DSI, DSE, and receptor-driven endocannabinoid-mediated suppression were all abolished by the DAG lipase inhibitor tetrahydrolipstatin, indicating 2-AG as the major endocannabinoid mediating retrograde suppression at excitatory and inhibitory synapses of MS neurons. Accordingly, CB1 cannabinoid receptor, the main target of 2-AG, was present at high levels on GABAergic axon terminals of MS neurons and parvalbumin-positive interneurons and at low levels on excitatory corticostriatal afferents. Thus, endocannabinoid signaling molecules are arranged to modulate the excitability of the MS neuron effectively depending on cortical activity and cholinergic tone as measured by mGluR5 and M-1 receptors, respectively.
  • Chihiro Takasaki, Eriko Miura, Masahiko Watanabe
    BIOMEDICAL RESEARCH-TOKYO 28 (2) 61 - 69 0388-6107 2007/04 [Refereed][Not invited]
     
    3-Phosphoglycerate dehydrogenase (Phgdh) is the initial step enzyme in the phosphorylated pathway of L-serine biosynthesis. We have previously revealed in the brain that Phgdh is preferentially expressed in glial cells, but not in neurons, and that glia-borne L-serine exerts strong neurotrophic actions to neuronal survive, differentiation, and development. To investigate whether such an L-serine-meditated intercellular relationship is constructed in peripheral organs and tissues, we examined the kidney, which is one of the organs with the highest expression of Phgdh mRNA in the body. We found that Phgdh was distributed highly in the renal papilla and inner layer of the outer zone and moderately in the cortex, whereas it was almost negative in the outer layer of the outer zone. This heterogeneous distribution was due to selective expression in distinct tubular segments, i.e., the Bowman's capsule, proximal tubule, and thin limbs of the Henle's loop. Interestingly, neutral amino acid transporter ASCT1, which preferentially transports alanine, serine, cysteine, and threonine, was selectively expressed in Phgdh-negative tubular segments, i.e., the distal tubule and collecting duct. Therefore, either Phgdh or ASCT1 is provided to each segment of renal tubules, suggesting that metabolic interplay mediated by L-serine biosynthesis and supply may exist in the kidney too.
  • Wataru Kakegawa, Taisuke Miyazaki, Hirokazu Hirai, Junko Motohashi, Masayoshi Mishina, Masahiko Watanabe, Michisuke Yuzaki
    JOURNAL OF PHYSIOLOGY-LONDON 579 (3) 729 - 735 0022-3751 2007/03 [Refereed][Not invited]
     
    The delta 2 glutamate receptor (GluR delta 2) plays a crucial role in cerebellar functions; mice with a disrupted GluR delta 2 gene (GluR delta 2(-/-)) display impaired synapse formation and abrogated long-term depression (LTD). However, the mechanisms by which GluR delta 2 functions have remained unclear. Because a GluR delta 2 mutation in lurcher mice causes channel activities characterized by Ca2+ permeability, GluR delta 2 was previously suggested to serve as a Ca2+-permeable channel in Purkinje cells. To test this hypothesis, we introduced a GluR delta 2 transgene, which had a mutation (Gln618Arg) in the putative channel pore, into GluR delta 2(-/-) mice. Interestingly, the mutant transgene rescued the major functional and morphological abnormalities of GluR delta 2(-/-) Purkinje cells, such as enhanced paired-pulse facilitation, impaired LTD at parallel fibre synapses, and sustained innervation by multiple climbing fibres. These results indicate that the conserved glutamine residue in the channel pore, which is crucial for all Ca2+-permeable glutamate receptors, is not essential for the function of GluR delta 2.
  • Kentaro Takagaki, Hiroshi Shima, Nobuhiro Tanuma, Miyuki Nomura, Takeshi Satoh, Masahiko Watanabe, Kunimi Kikuchi
    MOLECULAR AND CELLULAR BIOCHEMISTRY 296 (1-2) 177 - 184 0300-8177 2007/02 [Refereed][Not invited]
     
    Dual-specificity phosphatases (DSPs), which dephosphorylate proteins at Ser/Thr as well as Tyr residues, are thought to be involved in critical signaling events such as control of MAP kinases (MAPKs). We have isolated the cDNA for a novel DSP and termed it low molecular mass DSP-4 (LDP-4). LDP-4 is composed of 211 amino acids with a predicted molecular mass of 23.9-kDa. Northern blot analysis using various mouse tissues showed that the LDP-4 transcript was expressed exclusively in brain. In situ hybridization showed that brain expression of LDP-4 was ubiquitous except for the hippocampus. When expressed in COS-7 cells, FLAG-tagged LDP-4 protein was present within the nucleus and Golgi apparatus. LDP-4 expression did not reduce phosphorylation levels of MAPKs, but rather evoked activation of JNK and p38.
  • Erika Polgar, Annie D. Campbell, Lynsey M. MacIntyre, Masahiko Watanabe, Andrew J. Todd
    MOLECULAR PAIN 3 1744-8069 2007/02 [Refereed][Not invited]
     
    Background: There is a population of large neurons with cell bodies in laminae III and IV of the spinal dorsal horn which express the neurokinin 1 receptor (NK1r) and have dendrites that enter the superficial laminae. Although it has been shown that these are all projection neurons and that they are innervated by substance P-containing (nociceptive) primary afferents, we know little about their responses to noxious stimuli. In this study we have looked for phosphorylation of extracellular signal-regulated kinases (ERKs) in these neurons in response to different types of noxious stimulus applied to one hindlimb of anaesthetised rats. The stimuli were mechanical (repeated pinching), thermal (immersion in water at 52 degrees C) or chemical (injection of 2% formaldehyde). Results: Five minutes after each type of stimulus we observed numerous cells with phosphorylated ERK (pERK) in laminae I and IIo, together with scattered positive cells in deeper laminae. We found that virtually all of the lamina III/IV NK1r-immunoreactive neurons contained pERK after each of these stimuli and that in the great majority of cases there was internalisation of the NK1r on the dorsal dendrites of these cells. In addition, we also saw neurons in lamina III that were pERK-positive but lacked the NK1r, and these were particularly evident in animals that had had the pinch stimulus. Conclusion: Our results demonstrate that lamina III/IV NK1r-immunoreactive neurons show receptor internalisation and ERK phosphorylation after mechanical, thermal or chemical noxious stimuli.
  • Sachi Nomura, Masahiro Fukaya, Takao Tsujioka, Dianqing Wu, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 25 (3) 659 - 672 0953-816X 2007/02 [Refereed][Not invited]
     
    Phospholipase C beta 3 (PLC beta 3) and PLC beta 4 are the two major isoforms in cerebellar Purkinje cells (PCs), displaying reciprocal expression across the cerebellum. Here, we examined subcellular distribution of PLC beta 3 in the mouse cerebellum by producing specific antibody. PLC beta 3 was detected as a particulate pattern of immunostaining in various PC elements. Like PLC beta 4, PLC beta 3 was richly distributed in somatodendritic compartments, where it was colocalized with molecules constituting the metabotropic glutamate receptor (mGluR1) signalling pathway, i.e. mGluR1 alpha, G alpha q/G alpha 11 subunits of Gq protein, inositol 1,4,5-trisphosphate receptor IP3R1, Homer1, protein kinase C PKC gamma, and diacylglycerol lipase DAGL alpha. Unlike PLC beta 4, PLC beta 3 was also distributed at low to moderate levels in PC axons, which were intense for IP3R1 and PKC gamma, low for G alpha q/G alpha 11, and negative for mGluR1 alpha, Homer1, and DAGL alpha. By immunoelectron microscopy, PLC beta 3 was preferentially localized around the smooth endoplasmic reticulum in spines, dendrites, and axons of PCs, and also accumulated at the perisynapse of parallel fibre-PC synapses. Consistent with the ultrastructural localization, PLC beta 3 was biochemically enriched in the microsomal and postsynaptic density fractions. These results suggest that PLC beta 3 plays a major role in mediating mGluR1-dependent synaptic transmission, plasticity, and integration in PLC beta 3-dominant PCs, through eliciting Ca2+ release, protein phosphorylation, and endocannabinoid production at local somatodendritic compartments. Because PLC beta 3 can be activated by G beta gamma subunits liberated from Gi/o and Gs proteins as well, axonal PLC beta 3 seems to modulate the conduction of action potentials through mediating local Ca2+ release and protein phosphorylation upon activation of a variety of G protein-coupled receptors other than mGluR1.
  • Miwako Masugi-Tokita, Etsuko Tarusawa, Masahiko Watanabe, Elek Molnar, Kazushi Fujimoto, Ryuichi Shigemoto
    JOURNAL OF NEUROSCIENCE 27 (8) 2135 - 2144 0270-6474 2007/02 [Refereed][Not invited]
     
    The number of AMPA receptor ( AMPAR) is the major determinant of synaptic strength at glutamatergic synapses, but little is known about the absolute number and density of AMPARs in individual synapses. Using SDS-digested freeze-fracture replica labeling, which has high detection efficiency comparable with electrophysiological noise analysis for functional AMPAR, we analyzed three kinds of excitatory synapses in the molecular layer of the adult rat cerebellum. In parallel fiber ( PF) - Purkinje cell ( PC) synapses, we found large variability in the number ( 38.1 +/- 34.4 particles per synapse, mean +/- SD; range, 2 - 178 particles per synapse) and density ( 437 +/- 277 particles/mu m(2); range, 48 - 1210 particles/ mu m(2)) of immunogold- labeled AMPARs. Two- dimensional view and high sensitivity of this method revealed irregular- shaped small AMPAR clusters within synapses. Climbing fiber ( CF) - PC synapses had higher number of AMPAR labeling ( 68.6 +/- 34.5 particles per synapse) than PF - PC and PF - interneuron synapses ( 36.8 +/- 14.4 particles per synapse). Furthermore, AMPAR density at CF - PC and PF - interneuron synapses was approximately five times higher and more uniform than that at PF - PC synapses. These results suggest input- and target- dependent regulation of AMPAR- mediated synaptic strength.
  • Takatoshi Iijima, Eriko Miura, Keiko Matsuda, Yuichi Kamekawa, Masahiko Watanabe, Michisuke Yuzaki
    EUROPEAN JOURNAL OF NEUROSCIENCE 25 (4) 1049 - 1057 0953-816X 2007/02 [Refereed][Not invited]
     
    Cbln1, a member of the C1q and tumor necrosis factor superfamily, plays crucial roles as a cerebellar granule cell-derived transneuronal regulator of synapse integrity and plasticity in Purkinje cells. Although other Cbln family members, Cbln2-Cbln4, have distinct spatial and temporal patterns of expression throughout the CNS, their biochemical and biological properties have remained largely uncharacterized. Here, we demonstrated that in mammalian heterologous cells, Cbln2 and Cbln4 were secreted as N-linked glycoproteins, like Cbln1. In contrast, despite the presence of a functional signal sequence, Cbln3 was not secreted when expressed alone but was retained in the endoplasmic reticulum (ER) or cis-Golgi because of its N-terminal domain. All members of the Cbln family formed not only homomeric but also heteromeric complexes with each other in vitro. Accordingly, when Cbln1 and Cbln3 were co-expressed in heterologous cells, a proportion of the Cbln1 proteins was retained in the ER or cis-Golgi; conversely, some Cbln3 proteins were secreted together with Cbln1. Similarly, in wild-type granule cells expressing Cbln1 and Cbln3, Cbln3 proteins were partially secreted and reached postsynaptic sites on Purkinje cell dendrites, while Cbln3 was almost completely degraded in cbln1-null granule cells. These results indicate that like Cbln1, Cbln2 and Cbln4 may also serve as transneuronal regulators of synaptic functions in various brain regions. Furthermore, heteromer formation between Cbln1 and Cbln3 in cerebellar granule cells may modulate each other's trafficking and signaling pathways; similarly, heteromerization of other Cbln family proteins may also have biological significance in other neurons.
  • Kentaro Takagaki, Hiroshi Shima, Nobuhiro Tanuma, Miyuki Nomura, Takeshi Satoh, Masahiko Watanabe, Kunimi Kikuchi
    MOLECULAR AND CELLULAR BIOCHEMISTRY 296 (1-2) 177 - 184 0300-8177 2007/02 [Refereed][Not invited]
     
    Dual-specificity phosphatases (DSPs), which dephosphorylate proteins at Ser/Thr as well as Tyr residues, are thought to be involved in critical signaling events such as control of MAP kinases (MAPKs). We have isolated the cDNA for a novel DSP and termed it low molecular mass DSP-4 (LDP-4). LDP-4 is composed of 211 amino acids with a predicted molecular mass of 23.9-kDa. Northern blot analysis using various mouse tissues showed that the LDP-4 transcript was expressed exclusively in brain. In situ hybridization showed that brain expression of LDP-4 was ubiquitous except for the hippocampus. When expressed in COS-7 cells, FLAG-tagged LDP-4 protein was present within the nucleus and Golgi apparatus. LDP-4 expression did not reduce phosphorylation levels of MAPKs, but rather evoked activation of JNK and p38.
  • Sachi Nomura, Masahiro Fukaya, Takao Tsujioka, Dianqing Wu, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 25 (3) 659 - 672 0953-816X 2007/02 [Refereed][Not invited]
     
    Phospholipase C beta 3 (PLC beta 3) and PLC beta 4 are the two major isoforms in cerebellar Purkinje cells (PCs), displaying reciprocal expression across the cerebellum. Here, we examined subcellular distribution of PLC beta 3 in the mouse cerebellum by producing specific antibody. PLC beta 3 was detected as a particulate pattern of immunostaining in various PC elements. Like PLC beta 4, PLC beta 3 was richly distributed in somatodendritic compartments, where it was colocalized with molecules constituting the metabotropic glutamate receptor (mGluR1) signalling pathway, i.e. mGluR1 alpha, G alpha q/G alpha 11 subunits of Gq protein, inositol 1,4,5-trisphosphate receptor IP3R1, Homer1, protein kinase C PKC gamma, and diacylglycerol lipase DAGL alpha. Unlike PLC beta 4, PLC beta 3 was also distributed at low to moderate levels in PC axons, which were intense for IP3R1 and PKC gamma, low for G alpha q/G alpha 11, and negative for mGluR1 alpha, Homer1, and DAGL alpha. By immunoelectron microscopy, PLC beta 3 was preferentially localized around the smooth endoplasmic reticulum in spines, dendrites, and axons of PCs, and also accumulated at the perisynapse of parallel fibre-PC synapses. Consistent with the ultrastructural localization, PLC beta 3 was biochemically enriched in the microsomal and postsynaptic density fractions. These results suggest that PLC beta 3 plays a major role in mediating mGluR1-dependent synaptic transmission, plasticity, and integration in PLC beta 3-dominant PCs, through eliciting Ca2+ release, protein phosphorylation, and endocannabinoid production at local somatodendritic compartments. Because PLC beta 3 can be activated by G beta gamma subunits liberated from Gi/o and Gs proteins as well, axonal PLC beta 3 seems to modulate the conduction of action potentials through mediating local Ca2+ release and protein phosphorylation upon activation of a variety of G protein-coupled receptors other than mGluR1.
  • Madoka Narushima, Motokazu Uchigashima, Masahiro Fukaya, Minoru Matsui, Toshiya Manabe, Kouichi Hashimoto, Masahiko Watanabe, Masanobu Kano
    JOURNAL OF NEUROSCIENCE 27 (3) 496 - 506 0270-6474 2007/01 [Refereed][Not invited]
     
    Tonically active cholinergic interneurons in the striatum modulate activities of striatal outputs from medium spiny (MS) neurons and significantly influence overall functions of the basal ganglia. Cellular mechanisms of this modulation are not fully understood. Here we show that ambient acetylcholine (ACh) derived from tonically active cholinergic interneurons constitutively upregulates depolarizationinduced release of endocannabinoids from MS neurons. The released endocannabinoids cause transient suppression of inhibitory synaptic inputs to MS neurons through acting retrogradely onto presynaptic CB1 cannabinoid receptors. The effects were mediated by postsynaptic M-1 subtype of muscarinic ACh receptors, because the action of a muscarinic agonist to release endocannabinoids and the enhancement of depolarization-induced endocannabinoid release by ambient ACh were both deficient in M-1 knock-out mice and were blocked by postsynaptic infusion of guanosine-5'-O-(2-thiodiphosphate). Suppression of spontaneous firings of cholinergic interneurons by inhibiting I-h current reduced the depolarization-induced release of endocannabinoids. Conversely, elevation of ambient ACh concentration by inhibiting choline esterase significantly enhanced the endocannabinoid release. Paired recording from a cholinergic interneuron and an MS neuron revealed that the activity of single cholinergic neuron could influence endocannabinoid-mediated signaling in neighboring MS neurons. These results clearly indicate that striatal endocannabinoid-mediated modulation is under the control of cholinergic interneuron activity. By immunofluorescent and immunoelectron microscopic examinations, we demonstrated that M-1 receptor was densely distributed in perikarya and dendrites of dopamine D-1 or D-2 receptor-positive MS neurons. Thus, we have disclosed a novel mechanism by which the muscarinic system regulates striatal output and may contribute to motor control.
  • Avital Leibovici, Candace Rossignol, Jennifer A. Montrowl, Jeffery D. Erickson, Helene Varoqui, Masahiko Watanabe, Farrukh A. Chaudhry, May Kristin L. Bredahl, Kevin J. Anderson, Michael D. Weiss
    DEVELOPMENTAL NEUROSCIENCE 29 (3) 268 - 274 0378-5866 2007 [Refereed][Not invited]
     
    The neutral amino acid transporters SNAT1-3 and ASCT1 play critical roles in the recycling of glutamine, and subsequently glutamate, via the glutamine-glutamate cycle. Hypoxiaischemia was induced in rat pups using the Rice-Vannucci model. Brains were harvested at 1 h, 24 h and 7 days after ischemia. The expression of NAATs was evaluated using immunoblotting, real-time PCR, and immunohistochemistry. Results were compared with age-matched controls and shams. SNAT1 mRNA decreased at 1 h after injury in both hemispheres when compared with the control animals and correlated with a decrease in protein expression at 24 h in the hippocampus and cortex. SNAT1 protein expression increased globally at 7 days after injury and specifically in the hippocampus. Finally, SNAT2 and 3 demonstrated subtle changes in various brain regions after injury. These data suggest that neutral amino acid transporters remain largely intact after hypoxia-ischemia. Copyright (c) 2006 S. Karger AG, Basel
  • Miwa Hideki, Fukaya Masahiro, Watabe Ayako M, Watanabe Masahiko, Manabe Toshiya
    Proceedings of Annual Meeting of the Physiological Society of Japan 一般社団法人 日本生理学会 2007 019 - 019 2007 
    The amygdala is a crucial brain structure for the acquisition and expression of fear memory.The N-methyl-D-aspartate (NMDA)-type glutamate receptor channel, composed of the NR1 and NR2 subunits, plays a key role in synaptic plasticity in the CNS. NR2 subunits (NR2A-NR2D) are differentially expressed, depending on developmental stages and brain regions, but their functional roles are still unclear. In this study, we investigated the properties of synaptic NMDA receptors in the lateral nucleus of the amygdala (LA), comparing them with those of synaptic NMDA receptors in the CA1. We show that the electrophysiological properties of NMDA receptors and NR2A/NR2B ratio in the LA are distict from those of NMDA receptors in the CA1 and that the NR2B subunit contributes to synaptic transmission and long-term potentiation (LTP) induction more significantly in the LA than in the CA1. Our data suggest that these properties of NMDA receptors and the NR2B subunit in the LA are responsible for amygdala synaptic function and plasticity. [J Physiol Sci. 2007;57 Suppl:S19]
  • Takatoshi Iijima, Eriko Miura, Keiko Matsuda, Yuichi Kamekawa, Masahiko Watanabe, Michisuke Yuzaki
    NEUROSCIENCE RESEARCH 58 S72 - S72 0168-0102 2007 [Refereed][Not invited]
  • Miwako Yamasaki, Yutaka Iizuka, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 58 S77 - S77 0168-0102 2007 [Refereed][Not invited]
  • Motokazu Uchigashima, Madoka Narushima, Masahiro Fukaya, Istven Katona, Masanobu Kano, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 58 S75 - S75 0168-0102 2007 [Refereed][Not invited]
  • Masahiko Watanabe, Akira Fukuda, Michihiro Matsumoto, Hirokazu Yatsu, Ichiro Hosotani, Shigeyuki Kido
    USIC 2007: PROCEEDINGS OF THE SEVENTH INTERNATIONAL CONFERENCE ON QUALITY SOFTWARE 137 - + 2007 [Refereed][Not invited]
     
    In this paper, we deal with the kind of distributed embedded software that is constructed from tasks that communicate with each other by sending and receiving multicast messages. These tasks must share resources. So, it is important to schedule a timetable describing when the tasks occupy the resources so that there is no resource competition and the tasks occupy the resources within pre-specified periods. We call this scheduling process resource scheduling. Because tasks must satisfy many constraints and there are message delays in the communication of the tasks, resource scheduling is a difficult task. So, resource scheduling design methods are necessary. In this paper, we propose a resource scheduling design method that allows multicast messages whose receivers can be more than three tasks and an efficient verification method for the method.
  • Taisuke Miyazaki, Kouichi Tanaka, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 58 S132 - S132 0168-0102 2007 [Refereed][Not invited]
  • Daiki D. Horikawa, Tetsuya Sakashita, Chihiro Katagiri, Masahiko Watanabe, Takahiro Kikawada, Yuichi Nakahara, Nobuyuki Hamada, Seiichi Wada, Tomoo Funayama, Seigo Higashi, Yasuhiko Kobayashi, Takashi Okuda, Mikinori Kuwabara
    INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 82 (12) 843 - 848 0955-3002 2006/12 [Refereed][Not invited]
     
    Purpose: Tardigrades are known to survive high doses of ionizing radiation. However, there have been no reports about radiation effects in tardigrades under culture conditions. In this study, we investigated tolerance of the tardigrade, Milnesium tardigradum, against gamma-rays and heavy ions by determining short-term or long-term survival, and reproductive ability after irradiation. Materials and methods: Hydrated and anhydrobiotic animals were exposed to gamma-rays (1000-7000 Gy) or heavy ions (1000-8000 Gy) to evaluate short-term survival at 2, 24 and 48 h post-irradiation. Long-term survival and reproduction were observed up to 31 days after irradiation with gamma-rays (1000-4000 Gy). Results: At 48 h after irradiation, median lethal doses were 5000 Gy (gamma-rays) and 6200 Gy (heavy ions) in hydrated animals, and 4400 Gy (gamma-rays) and 5200 Gy (heavy ions) in anhydrobiotic ones. Gamma-irradiation shortened average life span in a dose-dependent manner both in hydrated and anhydrobiotic groups. No irradiated animals laid eggs with one exception in which a hydrated animal irradiated with 2000 Gy of gamma-rays laid 3 eggs, and those eggs failed to hatch, whereas eggs produced by non-irradiated animals hatched successfully. Conclusion: M. tardigradum survives high doses of ionizing radiation in both hydrated and anhydrobiotic states, but irradiation with > 1000 Gy makes them sterile.
  • Masahiko Watanabe, Tetsuya Sakashita, Akihiko Fujita, Takahiro Kikawada, Yuichi Nakahara, Nobuyuki Hamada, Daiki D. Horikawa, Seiichi Wada, Tomoo Funayama, Yasuhiko Kobayashi, Takashi Okuda
    INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 82 (12) 835 - 842 0955-3002 2006/12 [Refereed][Not invited]
     
    Purpose: Anhydrobiotic larvae of Polypedilum vanderplanki are known to show an extremely high tolerance against a range of stresses. We have recently reported that this insect withstands exposure to high doses of gamma-rays (linear energy transfer [LET] 0.2 keV/mu m). However, its tolerance against high LET radiation remains unknown. The aim of this study is to characterize the tolerance to high-LET radiations of P. vanderplanki. Materials and methods: Larval survival and subsequent metamorphoses were compared between anhydrobiotic (dry) and non-anhydrobiotic (wet) samples after exposure to 1-7000 Gy of three types of heavy ions delivered from the azimuthally varying field (AVF) cyclotron with LET values ranging from 16.2-321 keV/mu m. The tolerance against 4 He ions was also compared among three chironomid species. Results: At all LET values measured, dry larvae consistently showed greater radiation tolerance than hydrated larvae, perhaps due to the presence of high concentrations of the disaccharide trehalose in anhydrobiotic animals, and the radiation-induced damage became evident at lower doses as development progressed. Relative biological effectiveness (RBE) values based on the median inhibitory doses reached a maximum at 116 keV/mu m (C-12), and the maximum RBE clearly increased as development progressed. Lower D-0 ( dose to reduce survival from relative value 1.00-0.37 on the exponential part of the survival curve), and higher D-q (quasi-threshold dose) were found in individuals exposed to He-4 ions, compared to gamma-rays, and in P. vanderplanki larvae compared to non-anhydrobiotic chironomids. Conclusion: Anhydrobiosis potentiates radiation tolerance in terms of larval survival, pupation and adult emergence of P. vanderplanki exposed to high-LET radiations as well as to low-LET radiation. P. vanderplanki larvae might have more efficient DNA damage repair after radiation than other chironomid species.
  • Yuta Nemoto, Tetuo Mikami, Kiyomi Hana, Shirou Kikuchi, Nobuyuki Kobayashi, Masahiko Watanabe, Isao Okayasu
    PATHOLOGY INTERNATIONAL 56 (12) 724 - 731 1320-5463 2006/12 [Refereed][Not invited]
     
    The aim of the present study was to determine whether expression of molecules associated with cell cycle regulation and apoptosis might reflect tumor grade and patients' prognosis of gastrointestinal stromal tumor (GIST). Forty-nine cases of gastric GIST were divided into three grades; low, intermediate, and high risk. Ki-67, cyclin A, cyclin D1, cyclin E, p16(Ink4), p21(Waf1), p27(Kip1), cyclin-dependent kinase (cdk)2, cdk4 and single-strand DNA (ssDNA) were immunohistochemically stained and assessed. Ki-67, ssDNA, cyclin A and cdk2 had higher labeling indices (LI) in high-risk than in low-risk cases. Cyclin E expression was greater in the intermediate- than in the low-risk grade. On Kaplan-Meier analysis, tumor size, necrosis, cellularity, Ki-67, ssDNA, and cyclin A LI were significantly correlated with disease-free survival. Necrosis, cellularity, and Ki-67 LI were significant as prognostic factors on univariate, and Ki-67 LI on multivariate Cox hazard tests. Within the high-risk grade, high cellularity and low p27(Kip1) subgroups had the worst prognosis. The histological grade is related to cell turnover, assessed in terms of Ki-67, ssDNA, cyclin A, cyclin E, and cdk2 levels. Ki-67, ssDNA, and cyclin A are useful for prediction of prognosis, with cellularity and p27(Kip1) expression as further prognostic factors in high-risk cases.
  • Shiro Kikuchi, Nobue Futawatari, Shinichi Sakuramoto, Masahiko Watanabe, Katsumi Kubota, Mayumi Sasaki, Koichiro Murata, Mitsuhiro Kida
    ANTICANCER RESEARCH 26 (6C) 4641 - 4645 0250-7005 2006/11 [Refereed][Not invited]
     
    Background: The possibilities and limitations of computer tomography (CT) examination for detecting primary gastric cancer tumors have been poorly understood. The aim of the present study was to evaluate the pre-operative assessment of gastric cancer tumors using 32-multidetector row-CT Patients and Methods: A prospective study of the pre-operative assessment of gastric cancer tumors using 32-multidetector row-CT was carried out. Seventy-four patients with adenocarcinoma of the stomach (T1 tumors, 38; T2 and T3 tumors, 36) underwent abdominal CTs to assess the feasibility of using MD-CT to assess primary lesions. Results: In 35 (47%) out of the 74 patients, the primary lesions could be detected on 2-D images obtained by CT In these patients, virtual endoscopic images of these tumors could be created. Twenty-seven advanced cancer tumors (75%) were assessed based on 2-D CT images and 27 larger tumors (> 40 mm) (69%) were assessed based on 2-D CT images. Significant differences were found with respect to depth of tumor (p < 0.0001) and tumor size (p < 0.0001) between tumors that could or could not be assessed on MD-CT Conclusion: The findings of the present study show that CT assessment of tumors is feasible in patients with advanced gastric cancer. Future studies are required to fully explore the ability of MD-CT to assess tumor volume in advanced gastric cancer cases and to determine the optimum application of this approach.
  • Hayato Sasaki, Yasukazu Hozumi, Hiroshi Hasegawa, Tsukasa Ito, Michiaki Takagi, Toshihiko Ogino, Masahiko Watanabe, Kaoru Goto
    Cell and tissue research 326 (1) 35 - 42 0302-766X 2006/10 [Refereed][Not invited]
     
    The dorsal root ganglion (DRG) and dorsal horn of the spinal cord are areas through which primary afferent information passes enroute to the brain. Previous studies have reported that, during normal neuronal activity, the regional distribution of a second messenger, diacylglycerol (DG), which is derived from phosphoinositide turnover, is diverse in these areas. However, the way that DG is regulated in these organs remains unknown. The present study was performed to investigate mRNA expression and protein localization of DG kinase (DGK) isozymes, which play a central role in DG metabolism. Gene expression for DGK isozymes was detected with variable regional distributions and intensities in the spinal cord. Among the isozymes, most intense signals were found for DGKzeta and DGKiota in the DRG. By immunohistochemical analysis, DGKzeta immunoreactivity was detected heterogeneously in the nucleus and cytoplasm of small DRG neurons with variable levels of distribution, whereas it was detected exclusively in the cytoplasm of large neurons. On the other hand, DGKiota immunoreactivity was distributed solely in the cytoplasm of most of the DRG neurons. Double-immunofluorescent imaging of these isozymes showed that they coexisted in a large population of DRG neurons at distinct subcellular sites, i.e., DGKzeta in the nucleus and DGKiota in the cytoplasm. Thus, DGK isozymes may have different functional roles at distinct subcellular sites. Furthermore, the heterogeneous subcellular localization of DGKzeta between the nucleus and cytoplasm implies the possible translocation of this isozyme in small DRG neurons under various conditions.
  • Madoka Narushima, Motokazu Uchigashima, Kouichi Hashimoto, Masahiko Watanabe, Masanobu Kano
    EUROPEAN JOURNAL OF NEUROSCIENCE 24 (8) 2246 - 2252 0953-816X 2006/10 [Refereed][Not invited]
     
    Endogenous cannabinoids (endocannabinoids) act as retrograde inhibitory messengers in various regions of the brain. We have recently reported that endocannabinoids mediate short-term retrograde suppression of excitatory synaptic transmission from the neocortex to medium spiny (MS) neurons, the major projection neurons from the striatum. However, it remains unclear whether endocannabinoids modulate inhibitory transmission in the striatum. Here we show that depolarization of MS neurons induces transient suppression of inhibition that is mediated by retrograde endocannabinoid signalling. By paired recording from a fast-spiking (FS) interneuron and an MS neuron, we demonstrated that FS-MS inhibitory synapses undergo endocannabinoid-mediated retrograde suppression. We verified that GABAergic inhibitory terminals immunopositive for parvalbumin (PV), a marker for FS interneurons, expressed CB1 receptors. These PV-CB1 double-positive terminals surrounded dopamine D1 receptor-positive and D2 receptor-positive MS neurons; these constitute direct and indirect pathways, respectively. These results suggest that endocannabinoid-mediated retrograde suppression of inhibition influences information flow along both direct and indirect pathways, depending on the activity of MS neurons.
  • Masahiro Fukaya, Mika Tsujita, Maya Yamazaki, Etsuko Kushiya, Manabu Abe, Kaori Akashi, Rie Natsume, Masanobu Kano, Haruyuki Kamiya, Masahiko Watanabe, Kenji Sakimura
    EUROPEAN JOURNAL OF NEUROSCIENCE 24 (8) 2177 - 2190 0953-816X 2006/10 [Refereed][Not invited]
     
    Transmembrane alpha-amino-3-hydroxyl-5-isoxazolepropionate (AMPA) receptor regulatory proteins (TARPs) play pivotal roles in AMPA receptor trafficking and gating. Here we examined cellular and subcellular distribution of TARP gamma-8 in the mouse brain. Immunoblot and immunofluorescence revealed the highest concentration of gamma-8 in the hippocampus. Immunogold electron microscopy demonstrated dense distribution of gamma-8 on the synaptic and extrasynaptic surface of hippocampal neurons with very low intracellular labeling. Of the neuronal surface, gamma-8 was distributed at the highest level on asymmetrical synapses of pyramidal cells and interneurons, whereas their symmetrical synapses selectively lacked immunogold labeling. Then, the role of gamma-8 in AMPA receptor expression was pursued in the hippocampus using mutant mice defective in the gamma-8 gene. In the mutant cornu ammonis (CA)1 region, synaptic and extrasynaptic AMPA receptors on dendrites and spines were severely reduced to 35-37% of control levels, whereas reduction was mild for extrasynaptic receptors on somata (74%) and no significant decrease was seen for intracellular receptors within spines. In the mutant CA3 region, synaptic AMPA receptors were reduced mildly at asymmetrical synapses in the stratum radiatum (67% of control level), and showed no significant decrease at mossy fiber-CA3 synapses. Therefore, gamma-8 is abundantly distributed on hippocampal excitatory synapses and extrasynaptic membranes, and plays an important role in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines, particularly, in the CA1 region. Variable degrees of reduction further suggest that other TARPs may also mediate this function at different potencies depending on hippocampal subregions, input sources and neuronal compartments.
  • Masahiro Fukaya, Mika Tsujita, Maya Yamazaki, Etsuko Kushiya, Manabu Abe, Kaori Akashi, Rie Natsume, Masanobu Kano, Haruyuki Kamiya, Masahiko Watanabe, Kenji Sakimura
    EUROPEAN JOURNAL OF NEUROSCIENCE 24 (8) 2177 - 2190 0953-816X 2006/10 [Refereed][Not invited]
     
    Transmembrane alpha-amino-3-hydroxyl-5-isoxazolepropionate (AMPA) receptor regulatory proteins (TARPs) play pivotal roles in AMPA receptor trafficking and gating. Here we examined cellular and subcellular distribution of TARP gamma-8 in the mouse brain. Immunoblot and immunofluorescence revealed the highest concentration of gamma-8 in the hippocampus. Immunogold electron microscopy demonstrated dense distribution of gamma-8 on the synaptic and extrasynaptic surface of hippocampal neurons with very low intracellular labeling. Of the neuronal surface, gamma-8 was distributed at the highest level on asymmetrical synapses of pyramidal cells and interneurons, whereas their symmetrical synapses selectively lacked immunogold labeling. Then, the role of gamma-8 in AMPA receptor expression was pursued in the hippocampus using mutant mice defective in the gamma-8 gene. In the mutant cornu ammonis (CA)1 region, synaptic and extrasynaptic AMPA receptors on dendrites and spines were severely reduced to 35-37% of control levels, whereas reduction was mild for extrasynaptic receptors on somata (74%) and no significant decrease was seen for intracellular receptors within spines. In the mutant CA3 region, synaptic AMPA receptors were reduced mildly at asymmetrical synapses in the stratum radiatum (67% of control level), and showed no significant decrease at mossy fiber-CA3 synapses. Therefore, gamma-8 is abundantly distributed on hippocampal excitatory synapses and extrasynaptic membranes, and plays an important role in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines, particularly, in the CA1 region. Variable degrees of reduction further suggest that other TARPs may also mediate this function at different potencies depending on hippocampal subregions, input sources and neuronal compartments.
  • Eriko Miura, Takatoshi Iijima, Michisuke Yuzaki, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 24 (3) 750 - 760 0953-816X 2006/08 [Refereed][Not invited]
     
    Cbln1 belongs to the C1q and tumour necrosis factor superfamily, and plays crucial roles as a cerebellar granule cell-derived transneuronal regulator for synapse integrity and plasticity in Purkinje cells. Although Cbln2-Cbln4 are also expressed in the brain and could form heteromeric complexes with Cbln1, their precise expressions remain unclear. Here, we investigated gene expression of the Cbln family in developing and adult C57BL mouse brains by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and high-resolution in situ hybridization (ISH) analyses. In the adult brain, spatial patterns of mRNA expression were highly differential depending on Cbln subtypes. Notably, particularly high levels of Cbln mRNAs were expressed in some nuclei and neurons, whereas their postsynaptic targets often lacked or were low for any Cbln mRNAs, as seen for cerebellar granule cells/Purkinje cells, entorhinal cortex/hippocampus, intralaminar group of thalamic nuclei/caudate-putamen, and dorsal nucleus of the lateral lemniscus/central nucleus of the inferior colliculus. In the developing brain, Cbln1, 2, and 4 mRNAs appeared as early as embryonic day 10-13, and exhibited transient up-regulation during the late embryonic and neonatal periods. For example, Cbln2 mRNA was expressed in the cortical plate of the developing neocortex, displaying a high rostromedial to low caudolateral gradient. In contrast, Cbln3 mRNA was selective to cerebellar granule cells throughout development, and its onset was as late as postnatal day 7-10. These results will provide a molecular-anatomical basis for future studies that characterize roles played by the Cbln family.
  • Toshiko R. Matsugami, Kentaro Tanemura, Michihiro Mieda, Reiko Nakatomi, Keiko Yamada, Takashi Kondo, Masaharu Ogawa, Kunihiko Obata, Masahiko Watanabe, Tsutomu Hashikawa, Kohichi Tanaka
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 (32) 12161 - 12166 0027-8424 2006/08 [Refereed][Not invited]
     
    Previous in vitro studies have shown that the neurotransmitter glutamate is important in brain development. Paradoxically, loss-of-function mouse models of glutamatergic signaling that are generated by genetic deletion of glutamate receptors or glutamate release show normal brain assembly. We examined the direct consequences on brain development of extracellular glutamate buildup due to the depletion of the glutamate transporters GLAST and GLT1. GLAST/GLT1 double knockout mice show multiple brain defects, including cortical, hippocampal, and olfactory bulb disorganization with perinatal mortality. Here, we report abnormal formation of the neocortex in GLAST/GLT1 mutants. Several essential aspects of neuronal development, such as stem cell proliferation, radial migration, neuronal differentiation, and survival of SP neurons, were impaired. These results provide direct in vivo evidence that GLAST and GLT1 are necessary for brain development through regulation of extracellular glutamate concentration and show that an important mechanism is likely to be maintenance of glutamate-mediated synaptic transmission.
  • Eriko Miura, Takatoshi Iijima, Michisuke Yuzaki, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 24 (3) 750 - 760 0953-816X 2006/08 [Refereed][Not invited]
     
    Cbln1 belongs to the C1q and tumour necrosis factor superfamily, and plays crucial roles as a cerebellar granule cell-derived transneuronal regulator for synapse integrity and plasticity in Purkinje cells. Although Cbln2-Cbln4 are also expressed in the brain and could form heteromeric complexes with Cbln1, their precise expressions remain unclear. Here, we investigated gene expression of the Cbln family in developing and adult C57BL mouse brains by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and high-resolution in situ hybridization (ISH) analyses. In the adult brain, spatial patterns of mRNA expression were highly differential depending on Cbln subtypes. Notably, particularly high levels of Cbln mRNAs were expressed in some nuclei and neurons, whereas their postsynaptic targets often lacked or were low for any Cbln mRNAs, as seen for cerebellar granule cells/Purkinje cells, entorhinal cortex/hippocampus, intralaminar group of thalamic nuclei/caudate-putamen, and dorsal nucleus of the lateral lemniscus/central nucleus of the inferior colliculus. In the developing brain, Cbln1, 2, and 4 mRNAs appeared as early as embryonic day 10-13, and exhibited transient up-regulation during the late embryonic and neonatal periods. For example, Cbln2 mRNA was expressed in the cortical plate of the developing neocortex, displaying a high rostromedial to low caudolateral gradient. In contrast, Cbln3 mRNA was selective to cerebellar granule cells throughout development, and its onset was as late as postnatal day 7-10. These results will provide a molecular-anatomical basis for future studies that characterize roles played by the Cbln family.
  • Masahiko Watanabe, Tetsuya Sakashita, Akihiko Fujita, Takahiro Kikawada, Daiki D. Horikawa, Yuichi Nakahara, Seiichi Wada, Tomoo Funayama, Nobuyuki Hamada, Yasuhiko Kobayashi, Takashi Okuda
    INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 82 (8) 587 - 592 0955-3002 2006/08 [Refereed][Not invited]
     
    Purpose: Anhydrobiotic organisms are known to have an extremely high tolerance against a range of stresses. However, the functional role of anhydrobiosis in radiation tolerance is poorly understood, especially in development following irradiation. The present study aims to evaluate effects of anhydrobiosis on radiation tolerance in an anhydrobiotic insect, Polypedilum vanderplanki. Materials and methods: Larval survival ( 48 h), anhydrobiotic ability, metamorphosis and reproduction after exposure to 1-9000 Gy of gamma-rays at the larval stage were compared between anhydrobiotic (dry) and normal (wet) phases. Results: Wet larvae were killed in a dose-dependent manner at doses higher than 2000 Gy, and all died within 8 h after 4000 Gy exposure. In contrast, dry larvae survived even 5000 Gy, and some of them still tolerated 7000 Gy and were alive at 48 h after rehydration. Moreover, greater radiotolerance of dry larva, compared to wet ones, was demonstrated in terms of metamorphoses. However, anhydrobiosis did not protect against radiation damage in terms of producing viable offspring. Conclusion: These results indicate that anhydrobiosis enhances radiotolerance, resulting in increases of successful metamorphoses.
  • K Yoshimura, M Shibata, M Koike, K Gotoh, M Fukaya, M Watanabe, Y Uchiyama
    AUTOPHAGY 2 (3) 200 - 208 1554-8627 2006/07 [Refereed][Not invited]
     
    Atg4B, a mammalian homologue of yeast Atg4, has been shown to play an important role in the processing of LC3, a mammalian homologue of yeast Atg8, but the tissue distribution of Atg4B remains unknown. To better understand the role of Atg4B in rat tissue cells, we prepared antibodies against Atg4B, and PC 12 cells in which the expression of Atg4B was knocked down by RNA interference. In the RNA interference-treated PC 12 cells, for which the expression of Atg4B was 10% of wild-type PC 12 cells, the expression of cytosolic LC3-1 was similar to that in wild-type cells. Knockdown cell lysates, however, suppressed the cleavage of recombinant proLC3 to LC3-1. Moreover, the expression of Atg4B protein and mRNA was ubiquitous in rat tissues; however, the expression levels were not identical, but were dependent on the tissue, with the expression high in brain and testicular tissue, and low in muscular and heart tissue. In brain tissue, the expression of Atg4B protein and mRNA was higher in neurons, especially in the cerebellum and olfactory bulb, as evidenced by immunohistochemistry and in situ hybridization. These lines of evidence suggest that Atg4B plays a major role in the processing of LC3 and is widely distributed in rat tissues. In particular, in brain tissues, autophagy may be deeply associated with the metabolism of neurons, especially in the cerebellum.
  • Shigeki Moriguchi, Miyuki Nishi, Shinji Komazaki, Hiroyuki Sakagami, Taisuke Miyazaki, Haruko Masumiya, Shin-ya Saito, Masahiko Watanabe, Hisatake Kondo, Hiromu Yawo, Kohji Fukunaga, Hiroshi Takeshima
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 (28) 10811 - 10816 0027-8424 2006/07 [Refereed][Not invited]
     
    Junctional membrane complexes (JMCs) composed of the plasma membrane and endoplasmic/sarcoplasmic reticulum seem to be a structural platform for channel crosstalk. Junctophilins (JPs) contribute to JMC formation by spanning the sarcoplasmic reticulum membrane and binding with the plasma membrane in muscle cells. In this article, we report that mutant JP double-knockout (JP-DKO) mice lacking neural JP subtypes exhibited an irregular hindlimb reflex and impaired memory. Electrophysiological experiments indicated that the activation of small-conductance Ca2+-activated K+ channels responsible for afterhyperpolarization in hippocampal neurons requires endoplasmic reticulum Ca2+ release through ryanodine receptors, triggered by NMDA receptor-mediated Ca2+ influx. We propose that in JP-DKO neurons lacking afterhyperpolarization, the functional communications between NMDA receptors, ryanodine receptors, and small-conductance Ca2+-activated K+ channels are disconnected because of JMC disassembly. Moreover, JP-DKO neurons showed an impaired long-term potentiation and hyperactivation of Ca2+/calmodulin-dependent protein kinase II. Therefore, JPs seem to have an essential role in neural excitability fundamental to plasticity and integrated functions.
  • Taisuke Miyazaki, Kouichi Hashimoto, Atsushi Uda, Hiroyuki Sakagami, Yoshitaka Nakamura, Shin-ya Saito, Miyuki Nishi, Hideaki Kume, Akira Tohgo, Izumi Kaneko, Hisatake Kondo, Kohji Fukunaga, Masanobu Kano, Masahiko Watanabe, Hiroshi Takeshima
    FEBS LETTERS 580 (17) 4057 - 4064 0014-5793 2006/07 [Refereed][Not invited]
     
    By DNA cloning, we have identified the BSRP (brain-specific receptor-like proteins) family of three members in mammalian genomes. BSRPs were predominantly expressed in the soma and dendrites of neurons and localized in the endoplasmic reticulum (ER). Expression levels of BSRPs seemed to fluctuate greatly during postnatal cerebellar maturation. Triple-knockout mice lacking BSRP members exhibited motor discoordination, and Purkinje cells (PCs) were often innervated by multiple climbing fibers with different neuronal origins in the mutant cerebellum. Moreover, the phosphorylation levels of protein kinase C alpha (PKC alpha) were significantly downregulated in the mutant cerebellum. Because cerebellar maturation and plasticity require metabotropic glutamate receptor signaling and resulting PKC activation, BSRPs are likely involved in ER functions supporting PKCa activation in PCs. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Taisuke Miyazaki, Kouichi Hashimoto, Atsushi Uda, Hiroyuki Sakagami, Yoshitaka Nakamura, Shin-ya Saito, Miyuki Nishi, Hideaki Kume, Akira Tohgo, Izumi Kaneko, Hisatake Kondo, Kohji Fukunaga, Masanobu Kano, Masahiko Watanabe, Hiroshi Takeshima
    FEBS LETTERS 580 (17) 4057 - 4064 0014-5793 2006/07 [Refereed][Not invited]
     
    By DNA cloning, we have identified the BSRP (brain-specific receptor-like proteins) family of three members in mammalian genomes. BSRPs were predominantly expressed in the soma and dendrites of neurons and localized in the endoplasmic reticulum (ER). Expression levels of BSRPs seemed to fluctuate greatly during postnatal cerebellar maturation. Triple-knockout mice lacking BSRP members exhibited motor discoordination, and Purkinje cells (PCs) were often innervated by multiple climbing fibers with different neuronal origins in the mutant cerebellum. Moreover, the phosphorylation levels of protein kinase C alpha (PKC alpha) were significantly downregulated in the mutant cerebellum. Because cerebellar maturation and plasticity require metabotropic glutamate receptor signaling and resulting PKC activation, BSRPs are likely involved in ER functions supporting PKCa activation in PCs. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Takanobu Nakazawa, Shoji Komai, Ayako M. Watabe, Yuji Kiyama, Masahiro Fukaya, Fumiko Arima-Yoshida, Reiko Horai, Katsuko Sudo, Kazumi Ebine, Mina Delawary, June Goto, Hisashi Umemori, Tohru Tezuka, Yoichiro Iwakura, Masahiko Watanabe, Tadashi Yamamoto, Toshiya Manabe
    EMBO JOURNAL 25 (12) 2867 - 2877 0261-4189 2006/06 [Refereed][Not invited]
     
    Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine-phosphorylated in the brain, with Tyr-1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr-1472 site to phenylalanine ( Y1472F) and show that Tyr-1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear-related learning and reduced amygdaloid long-term potentiation. NMDA receptor-mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr-1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.
  • E Miura, M Fukaya, T Sato, K Sugihara, M Asano, K Yoshioka, M Watanabe
    JOURNAL OF NEUROCHEMISTRY 97 (5) 1431 - 1446 0022-3042 2006/06 [Refereed][Not invited]
     
    The c-Jun N-terminal kinase (JNK) is one of the three major mitogen-activated protein kinases (MAPKs) playing key roles in various cellular processes in response to both extracellular and intracellular stimuli. JNK/SAPK-associated protein 1 (JSAP1 also referred to as JIP3) is a JNK-associated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression in mouse brains. JSAP1 mRNA was expressed in developing and adult brains, showing spatial patterns similar to JNK1-3 mRNAs. In embryos, JSAP1 immunolabeling was intense for progenitor cells in the ventricular zone throughout the brain and in the external granular layer of the cerebellum, and for neurons and glial cells differentiating in the mantle zone. In adults, JSAP1 was distributed in various neurons and Bergmann glia, with higher levels in striatal cholinergic interneurons, telencephalic parvalbumin-positive interneurons and cerebellar Purkinje cells. In these neurons, JSAP1 was observed as tiny particulate staining in spines, dendrites, perikarya and axons, where it was often associated with the smooth endoplasmic reticulum (sER) and cell membrane. Immunoblots revealed enriched distribution in the microsomal fraction and cytosolic fraction. Therefore, the characteristic cellular expression and subcellular distribution of JSAP1 might be beneficial for cells to efficiently link external stimuli to the JNK MAPK pathway and other intracellular machineries.
  • P Brumovsky, K Hygge-Blakeman, MJ Villar, M Watanabe, Z Wiesenfeld-Hallin, T Hokfelt
    JOURNAL OF CHEMICAL NEUROANATOMY 31 (4) 243 - 262 0891-0618 2006/06 [Refereed][Not invited]
     
    The distribution of galanin was studied in the lumbar 5 dorsal root ganglia (DRGs) and spinal cord, superior cervical ganglia (SCGs), and skin of transgenic mice overexpressing galanin under the doparnine beta-hydroxylase (DBH) promoter (GalOE-DBH mice) and in wild type (WT) mice. The DRGs and spinal cord were analysed before and after a unilateral, complete transection (axotomy) of the sciatic nerve and after dorsal rhizotomy. Both galanin protein and transcript were studied by, respectively, immunohistochemistry and in situ hybridization. Increased galanin expression was observed in several small, medium-sized and large DRG neuron profiles (NPs) in the naive transgenic mouse, frequently in neurons lacking calcitonin gene-related peptide (CGRP) and isolectin B4-binding. This lack of coexistence was particularly evident in the medium-sized/large NPs. In the dorsal horn of the spinal cord, no differences were detected between GalOE-DBH and WT mice, both displaying a strong galanin-positive neuropil in the superficial laminae of the dorsal horn, but the transgenic mice showed a more abundant galanin-positive innervation of the ventral horn. A 12-day dorsal rhizotomy, surprisingly, failed to alter the galanin staining patterns in the dorsal (and ventral) dorsal horn. Unilateral axotomy induced upregulation of galanin in DRG NPs of all sizes in both types of mouse. In the hindpaw skin, a profuse galanin-positive fiber plexus was observed in sweat glands and around blood vessels of the transgenic mice, being much more restricted in WT mice. Finally, GalOE mice exhibited a strong galanin-like immunoreactivity in most SCG NPs. The overexpression of the peptide in DRGs and SCGs was paralleled by increased mRNA levels. The present results show that overexpression of galanin under the control of the DBH promoter does not only occur, as expected in these mice, in noradrenline/adrenaline neurons but also in DRG neurons, particularly in large and medium-sized NPs. To what extent and how this overexpression pattern is related to the previously shown elevated pain threshold under normal and lesion conditions is discussed Grass, S., Crawley, J.N., Xu, X.J., Wiesenfeld-Hallin, Z., 2003a. Reduced spinal cord sensitization to C-fibre stimulation in mice over-expressing galanin. Eur. J. Neurosci. 17,1829-1832; Hygge-Blakeman, K., Brumovsky, P., Hao, JX, Xu, X.J., Hokfelt, T., Crawley, J.N., Wiesenfeld-Hallin, Z., 2004. Galanin over-expression decreases the development of neuropathic pain-like behaviour in mice after partial sciatic nerve injury. Brain Res. 1025, 152-1581. (c) 2006 Elsevier B.V. All rights reserved.
  • T Asashima, S Hori, S Ohtsuki, M Tachikawa, M Watanabe, C Mukai, S Kitagaki, N Miyakoshi, T Terasaki
    PHARMACEUTICAL RESEARCH 23 (6) 1235 - 1242 0724-8741 2006/06 [Refereed][Not invited]
     
    Purpose: The purpose of this study was to clarify the localization and function of the ATP-binding cassette transporter G2 (ABCG2; BCRP/MXR/ABCP) in retinal capillary endothelial cells, which form the inner blood-retinal barrier, as an efflux transport system. Methods: The expression was determined by reverse transcriptase polymerase chain reaction and Western blotting. The localization was identified by immunostaining. The transport function of ABCG2 was measured by flow cytometry. Results: Western blotting indicated that ABCG2 was expressed as a glycosylated disulfide-linked complex in the mouse retina and in peripheral tissues, including liver, kidney, and small intestine. Double immunolabeling of ABCG2 and glucose transporter 1 suggested that ABCG2 was localized on the luminal membrane of mouse retinal capillary endothelial cells. ABCG2 mRNA and protein were found to be expressed in a conditionally immortalized rat retinal capillary endothelial cell line, TR-iBRB, and rat retina. Treatment with Ko143, an ABCG2 inhibitor, restored the accumulation of pheophorbide a and protoporphyrin IX in TR-iBRB cells. Conclusion: ABCG2 is expressed on the luminal membrane of retinal capillary endothelial cells, where ABCG2 acts as the efflux transporter for photosensitive toxins such as pheophorbide a and protoporphyrin IX. ABCG2 could play an important role at the inner blood-retinal barrier in restricting the distribution of phototoxins and xenobiotics in retinal tissue.
  • T Akagi, K Ishida, T Hanasaka, S Hayashi, M Watanabe, T Hashikawa, K Tohyama
    JOURNAL OF NEUROSCIENCE METHODS 153 (2) 276 - 282 0165-0270 2006/06 [Refereed][Not invited]
     
    We examined each step of the protocol for ultracryotomy for central nervous system tissue in order to define and overcome some of the methodological difficulties. The following three steps emerged as critical for the method's success: (1) pretreatment of grids to render them hydrophilic immediately before use; (2) careful collection of ultrathin cryosections during ultracryotomy; (3) removal of the appropriate amount of excess poly(vinyl alcohol)-uranyl acetate (PVA-UA) prior to drying after staining with PVA-UA. By taking account of the three critical steps described above, we succeeded in obtaining ultrathin cryosections, including serial sections, with excellent preservation of ultrastructure, as well as semithin cryosections which are useful for evaluating the quality of the samples and for selecting areas of interest for ultrastructural analysis. Cytoplasmic organelles in neurons and glial cells, and the fine structure of synapses and myelinated fibers were well preserved. The localization of gold particles after immunostaining for astrocytic glutamate transporter (GLAST), metabotropic glutamate receptor 1 (mGluR1) and neurofilament protein was consistent with previous reports and ultrastructure was well-preserved in all cases. These findings should be helpful to researchers wishing to carry out ultrastructural and immunogold analyses of cryosections of nervous tissue. (c) 2005 Elsevier B.V. All rights reserved.
  • Eriko Miura, Masahiro Fukaya, Tokiharu Sato, Kazushi Sugihara, Masahide Asano, Katsuji Yoshioka, Masahiko Watanabe
    Journal of Neurochemistry 97 (5) 1431 - 1446 0022-3042 2006/06 [Refereed][Not invited]
     
    The c-Jun N-terminal kinase (JNK) is one of the three major mitogen-activated protein kinases (MAPKs) playing key roles in various cellular processes in response to both extracellular and intracellular stimuli. JNK/SAPK-associated protein 1 (JSAP1 also referred to as JIP3) is a JNK-associated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression in mouse brains. JSAP1 mRNA was expressed in developing and adult brains, showing spatial patterns similar to JNK1-3 mRNAs. In embryos, JSAP1 immunolabeling was intense for progenitor cells in the ventricular zone throughout the brain and in the external granular layer of the cerebellum, and for neurons and glial cells differentiating in the mantle zone. In adults, JSAP1 was distributed in various neurons and Bergmann glia, with higher levels in striatal cholinergic interneurons, telencephalic parvalbumin-positive interneurons and cerebellar Purkinje cells. In these neurons, JSAP1 was observed as tiny particulate staining in spines, dendrites, perikarya and axons, where it was often associated with the smooth endoplasmic reticulum (sER) and cell membrane. Immunoblots revealed enriched distribution in the microsomal fraction and cytosolic fraction. Therefore, the characteristic cellular expression and subcellular distribution of JSAP1 might be beneficial for cells to efficiently link external stimuli to the JNK MAPK pathway and other intracellular machineries. © 2006 International Society for Neurochemistry.
  • Eriko Miura, Masahiro Fukaya, Tokiharu Sato, Kazushi Sugihara, Masahide Asano, Katsuji Yoshioka, Masahiko Watanabe
    Journal of Neurochemistry 97 (5) 1431 - 1446 0022-3042 2006/06 [Refereed][Not invited]
     
    The c-Jun N-terminal kinase (JNK) is one of the three major mitogen-activated protein kinases (MAPKs) playing key roles in various cellular processes in response to both extracellular and intracellular stimuli. JNK/SAPK-associated protein 1 (JSAP1 also referred to as JIP3) is a JNK-associated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression in mouse brains. JSAP1 mRNA was expressed in developing and adult brains, showing spatial patterns similar to JNK1-3 mRNAs. In embryos, JSAP1 immunolabeling was intense for progenitor cells in the ventricular zone throughout the brain and in the external granular layer of the cerebellum, and for neurons and glial cells differentiating in the mantle zone. In adults, JSAP1 was distributed in various neurons and Bergmann glia, with higher levels in striatal cholinergic interneurons, telencephalic parvalbumin-positive interneurons and cerebellar Purkinje cells. In these neurons, JSAP1 was observed as tiny particulate staining in spines, dendrites, perikarya and axons, where it was often associated with the smooth endoplasmic reticulum (sER) and cell membrane. Immunoblots revealed enriched distribution in the microsomal fraction and cytosolic fraction. Therefore, the characteristic cellular expression and subcellular distribution of JSAP1 might be beneficial for cells to efficiently link external stimuli to the JNK MAPK pathway and other intracellular machineries. © 2006 International Society for Neurochemistry.
  • N Kitanaka, Y Owada, R Okuyama, H Sakagami, MR Nourani, S Aiba, H Furukawa, M Watanabe, M Ono, T Ohteki, H Kondo
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 345 (1) 459 - 466 0006-291X 2006/06 [Refereed][Not invited]
     
    Fatty acids and their metabolites have recently been shown to modulate various functions of dendritic cells (DCs) including their differentiation and cytokine production, although the mechanisms underlying their cellular functions are not fully understood. In view of our previous finding that epidermal-type fatty acid binding protein (E-FABP) was exclusively expressed in splenic DCs among FABP family, we examined the phenotype of E-FABP-null mutant mice in order to elucidate the functional significance of E-FABP expression in DCs. Although E-FABP-null mutant mice showed no apparent abnormalities in the population density and subset distribution of DCs as well as the microscopic morphology in the spleen, DCs isolated from E-FABP-null spleen showed enhanced production of IL-12p70, a key cytokine for innate immune responses, in response to appropriate stimuli as compared with wild-type. In real-time PCR, the expression level of IL-12p35 mRNA after LPS stimuli was much higher in mutant DCs when compared with wild-type, while no apparent change of IL-12p40 mRNA level was detected. Phosphorylated forms of p38 mitogen-activated protein kinase (p38MAPK) and I kappa Bot, molecules critical for IL-12 production, were detected at higher levels in E-FABP-null-mutant DCs after LPS stimuli when compared with wild-type counterparts. Collectively, it is suggested that E-FABP may be a novel negative regulator of IL-12 production in DCs, and this regulation may be exerted via its involvement in the p38MAPK-mediated transcription of IL-12p35. (c) 2006 Elsevier Inc. All rights reserved.
  • T Yoshida, M Fukaya, M Uchigashima, E Miura, H Kamiya, M Kano, M Watanabe
    JOURNAL OF NEUROSCIENCE 26 (18) 4740 - 4751 0270-6474 2006/05 [Refereed][Not invited]
     
    2-Arachidonoyl-glycerol ( 2-AG) is an endocannabinoid that is released from postsynaptic neurons, acts retrogradely on presynaptic cannabinoid receptor CB1, and induces short- and long-term suppression of transmitter release. To understand the mechanisms of the 2-AG-mediated retrograde modulation, we investigated subcellular localization of a major 2-AG biosynthetic enzyme, diacylglycerol lipase-alpha( DAGL alpha), by using immunofluorescence and immunoelectron microscopy in the mouse brain. In the cerebellum, DAGL alpha was predominantly expressed in Purkinje cells. DAGL alpha was detected on the dendritic surface and occasionally on the somatic surface, with a distal-to-proximal gradient from spiny branchlets toward somata. DAGL alpha was highly concentrated at the base of spine neck and also accumulated with much lower density on somatodendritic membrane around the spine neck. However, DAGL alpha was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGL alpha was also accumulated in spines. In contrast to the distribution in Purkinje cells, DAGL alpha was distributed in the spine head, neck, or both, whereas somatodendritic membrane was labeled very weakly. These results indicate that DAGL alpha is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two neurons. The preferential spine targeting should enable efficient 2-AG production on excitatory synaptic activity and its swift retrograde modulation onto nearby presynaptic terminals expressing CB1. Furthermore, different fine localization within and around spines suggests that the distance between postsynaptic 2-AG production site and presynaptic CB1 is differentially controlled depending on neuron types.
  • Sarna, JR, H Marzban, M Watanabe, R Hawkes
    JOURNAL OF COMPARATIVE NEUROLOGY 496 (3) 303 - 313 0021-9967 2006/05 [Refereed][Not invited]
     
    Transverse boundaries divide the cerebellar cortex into four transverse zones, and within each zone the cortex is further subdivided into a symmetrical array of parasagittal stripes. Several molecules believed to mediate long-term depression at the parallel fiber-Purkinje cell synapse are known to be expressed in stripes. We have therefore explored the distributions of phospholipase C beta 3 and phospholipase C beta 4, key components in the transduction of type 1 metabotropic glutamate receptor-mediated responses. The data reveal that both phospholipase C beta isotypes are expressed strongly in the mouse cerebellum in subsets of Purkinje cells. The two distributions are distinct and largely nonoverlapping. The pattern of phospholipase C beta 3 expression is unique, revealing stripes in three of the four transverse zones and a uniform distribution in the fourth. In contrast, phospholipase C beta 4 appears to be confined largely to the Purkinje cells that are pbospholipase C beta 3-negative. PLC beta 3 is restricted to the zebrin II-immunopositive Purkinje cell subset. Not all zebrin II-immunoreactive Purkinje cells express PLC beta 3: in lobules IX and X it is restricted to that zebrin II-immunopositive subset that also expresses the small heat shock protein HSP25. PLC beta 4 expression is restricted to, and coextensive with, the zebrin II-immunonegative Purkinje cell subset. These nonoverlapping expression patterns suggest that long-term depression may be manifested differently between cerebellar modules.
  • T Yoshida, M Fukaya, M Uchigashima, E Miura, H Kamiya, M Kano, M Watanabe
    JOURNAL OF NEUROSCIENCE 26 (18) 4740 - 4751 0270-6474 2006/05 [Refereed][Not invited]
     
    2-Arachidonoyl-glycerol ( 2-AG) is an endocannabinoid that is released from postsynaptic neurons, acts retrogradely on presynaptic cannabinoid receptor CB1, and induces short- and long-term suppression of transmitter release. To understand the mechanisms of the 2-AG-mediated retrograde modulation, we investigated subcellular localization of a major 2-AG biosynthetic enzyme, diacylglycerol lipase-alpha( DAGL alpha), by using immunofluorescence and immunoelectron microscopy in the mouse brain. In the cerebellum, DAGL alpha was predominantly expressed in Purkinje cells. DAGL alpha was detected on the dendritic surface and occasionally on the somatic surface, with a distal-to-proximal gradient from spiny branchlets toward somata. DAGL alpha was highly concentrated at the base of spine neck and also accumulated with much lower density on somatodendritic membrane around the spine neck. However, DAGL alpha was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGL alpha was also accumulated in spines. In contrast to the distribution in Purkinje cells, DAGL alpha was distributed in the spine head, neck, or both, whereas somatodendritic membrane was labeled very weakly. These results indicate that DAGL alpha is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two neurons. The preferential spine targeting should enable efficient 2-AG production on excitatory synaptic activity and its swift retrograde modulation onto nearby presynaptic terminals expressing CB1. Furthermore, different fine localization within and around spines suggests that the distance between postsynaptic 2-AG production site and presynaptic CB1 is differentially controlled depending on neuron types.
  • T Nakano, Y Hozumi, H Ali, S Saino-Saito, H Kamii, S Sato, T Kayama, M Watanabe, H Kondo, K Goto
    EUROPEAN JOURNAL OF NEUROSCIENCE 23 (6) 1427 - 1435 0953-816X 2006/03 [Refereed][Not invited]
     
    Diacylglycerol kinase (DGK) is an enzyme that phosphorylates a second messenger diacylglycerol (DG) and is involved in a variety of pathophysiological cellular responses. We have previously reported that DGK zeta may be involved in the selective vulnerability of hippocampal CA1 neurons in transient forebrain ischemia. In this study we aimed to further elucidate functional implications of DGK isozymes in the cerebral cortex suffering from infarction using a focal ischemic model. In the early phase of 90 min of middle cerebral artery occlusion, DGK zeta-immunoreactivity is reduced rapidly in the nucleus of cortical neurons in the ischemic core, while DGK iota and other neuronal proteins such as MAP-2 and NeuN remain intact. This suggests that rapid disappearance of DGK zeta in ischemic neurons is a quite early event precedent to neuronal degeneration in response to ischemia. Furthermore, in the late inflammatory phase of infarction DGK zeta-immunoreactivity is detected in non-neuronal cells including factor VIII-positive endothelial cells and ED-1-positive phagocytic cells. The present study suggests that DGK zeta may play roles in various processes of ischemic brain damage including neuronal death and non-neuronal inflammatory response.
  • Y Kawamura, M Fukaya, T Maejima, T Yoshida, E Miura, M Watanabe, T Ohno-Shosaku, M Kano
    JOURNAL OF NEUROSCIENCE 26 (11) 2991 - 3001 0270-6474 2006/03 [Refereed][Not invited]
     
    Endocannabinoids work as retrograde messengers and contribute to short-term and long-term modulation of synaptic transmission via presynaptic cannabinoid receptors. It is generally accepted that the CB1 cannabinoid receptor (CB1) mediates the effects of endocan-nabinoid in inhibitory synapses. For excitatory synapses, however, contributions of CB1, "CB3," and some other unidentified receptors have been suggested. In the present study we used electrophysiological and immunohistochemical techniques and examined the type(s)of cannabinoid receptor functioning at hippocampal and cerebellar excitatory synapses. Our electrophysiological data clearly demonstrate the predominant contribution of CB1. At hippocampal excitatory synapses on pyramidal neurons the cannabinoid-induced synaptic suppression was reversed by a CB1-specificant agonist, N-(piperidin-1-y1)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1Hpyrazole3-carboxamide (AM251), and was absent in CB1 knock-out mice. At climbing fiber (CF) and parallel fiber (PF) synapses on cerebellar Purkinje cells the cannabinoid-dependent suppression was absent in CB1 knock-out mice. The presence of CB1 at presynaptic terminals was confirmed by immunohistochemical experiments with specific antibodies against CB1. In immunoelectron microscopy the densities of CB1-positive signals in hippocampal excitatory terminals and cerebellar PF terminals were much lower than in inhibitory terminals but were clearly higher than the background. Along the long axis of PFs, the CB1 was localized at a much higher density on the perisynaptic membrane than on the extrasynaptic and synaptic regions. In contrast, CB1 density was low in CF terminals and was not significantly higher than the background. Despite the discrepancy between the electrophysiological and morphological data for CB1 expression on CFs, these results collectively indicate that CB1 is responsible for cannabinoid-dependent suppression of excitatory transmission in the hippocampus and cerebellum.
  • A Tohgo, M Eiraku, T Miyazaki, E Miura, S Kawaguchi, M Nishi, M Watanabe, T Hirano, M Kengaku, H Takeshima
    MOLECULAR AND CELLULAR NEUROSCIENCE 31 (2) 326 - 333 1044-7431 2006/02 [Refereed][Not invited]
     
    DNER is a transmembrane protein carrying extracellular EGF repeats and is strongly expressed in Purkinje cells (PCs) in the cerebellum. Current study indicated that DNER functions as a new Notch ligand and mediates the functional communication via cell-cell interaction. By producing and analyzing knockout mice lacking DNER, we demonstrate its essential roles in functional and morphological maturation of the cerebellum. The knockout mice exhibited motor discoordination in the fixed bar and rota-rod tests. The cerebellum from the knockout mice showed significant retardation in morphogenesis and persistent abnormality in fissure organization. Histochemical and electrophysiological analyses detected that PCs retained multiple innervations from climbing fibers (CFs) in the mutant cerebellum. Synaptic transmission from parallel fibers (PFs) or CFs to PCs was apparently normal, while glutamate clearance at the PF-PC synapses was significantly impaired in the mutant mice. Moreover, the protein level of GLAST, the glutamate transporter predominantly expressed in Bergmann glia (BG), was reduced in the mutant cerebellum. Our results indicate that DNER takes part in stimulation of BG maturation via intercellular communication and is essential for precise cerebellar development. (c) 2005 Elsevier Inc. All rights reserved.
  • Takayuki Yoshida, Masahiro Fukaya, Motokazu Uchigashima, Eriko Miura, Haruyuki Kamiya, Masanobu Kano, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 55 S77 - S77 0168-0102 2006 [Refereed][Not invited]
  • Masahiro Fukaya, Mika Tsujita, Maya Yamazaki, Etsuko Kushiya, Manabu Abe, Kaori Akashi, Masanobu Kano, Haruyuki Kamiya, Kenji Sakimura, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 55 S172 - S172 0168-0102 2006 [Refereed][Not invited]
  • Miwako Yamasaki, Kouichi Hashimoto, Taisuke Miyazaki, Masahiko Watanabe, Masanobu Kano
    NEUROSCIENCE RESEARCH 55 S174 - S174 0168-0102 2006 [Refereed][Not invited]
  • Tomonori Takeuchi, Gen Ohtsuki, Takashi Yoshida, Masahiro Fukaya, Tasuku Wainai, Hisashi Mori, Kenji Sakimura, Susumu Kawamoto, Masahiko Watanabe, Tomoo Hirano, Masayoshi Mishina
    NEUROSCIENCE RESEARCH 55 S98 - S98 0168-0102 2006 [Refereed][Not invited]
  • Eriko Miura, Masahiro Fukaya, Tokiharu Sato, Kazushi Sugihara, Masahide Asano, Katsuji Yoshioka, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 55 S179 - S179 0168-0102 2006 [Refereed][Not invited]
  • Takatoshi Iijima, Eriko Miura, Keiko Matsuda, Tetsuro Kondo, Masahiko Watanabe, Michisuke Yuzaki
    NEUROSCIENCE RESEARCH 55 S78 - S78 0168-0102 2006 [Refereed][Not invited]
  • Etsuko Tarusawa, Yugo Fukazawa, Elek Molnar, Masahiko Watanabe, Ryuichi Shigemoto
    NEUROSCIENCE RESEARCH 55 S172 - S172 0168-0102 2006 [Refereed][Not invited]
  • S Kakizawa, T Miyazaki, D Yanagihara, M Iino, M Watanabe, M Kano
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 (52) 19180 - 19185 0027-8424 2005/12 [Refereed][Not invited]
     
    Activity-dependent synaptic modification occurs in both developing and mature animals. For reliable information transfer and storage, however, once established, synapses must be maintained stably. We investigated how chronic blockade of neuronal activity or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors affects excitatory climbing fiber (CF) to Purkinje cell (PC) synapses in adult mouse cerebellum. Both treatments caused reduced glutamate concentration transient at the synaptic cleft, decreased frequency of quantal excitatory postsynaptic current, and diminished CF innervation of PC shaft dendrites but no change in CF's release probability. These results indicate that, in the mature cerebellum, AMPA receptor-mediated excitatory postsynaptic activity maintains CF's functional glutamate-release sites and its innervation of PC shaft dendrites.
  • T Iwanaga, M Goto, M Watanabe
    BIOMEDICAL RESEARCH-TOKYO 26 (6) 271 - 278 0388-6107 2005/12 [Refereed][Not invited]
     
    L-Glutamate transport by intestinal epithelial cells is an initial step of the entire glutamate metabolism pathway in the gut mucosa. The present study examined the cellular distribution of glutamate transporters in the digestive tract of adult mice using immunohistochemistry and in situ hybridization technique. Expression of EAAC1 mRNA was more intense in the ileum, where the epithelium in crypts and the basal half of intestinal villi showed high levels of transcripts, suggesting an essential role of EAAC1 in differentiating or premature epithelial cells. Electron-microscopically, EAAC1 immunoreactivity was predominantly localized in the striated border of enterocytes. Immunoreactivity for GLT-1 was found in the lateral membrane of epithelial cells at the bottom of gastric glands and at the intestinal crypts, and also in the lateral membrane of secretory cells at the duodenal gland. GLAST immunoreactivity was restricted to the fundic and pyloric glands, and was especially intense in the neck portion of both glands. However, in situ hybridization analysis failed to confirm the expression of GLT-1 and GLAST at the mRNA level, possibly due to limited sensitivity. The strong and specific luminal localization of EAAC1 in the intestinal epithelium suggests that EAAC1 is a predominant transporter of glutamate, at least in the lower part of the small intestine.
  • Masahiro Fukaya, Maya Yamazaki, Kenji Sakimura, Masahiko Watanabe
    Neuroscience Research 53 (4) 376 - 383 0168-0102 2005/12 [Refereed][Not invited]
     
    The γ subunit of voltage-dependent Ca2+ channels (VDCCs) is characterized by molecular diversity and regulation of AMPA-type glutamate receptors as well as VDCCs. In the present study, we examined expressions for the VDCCγ1-8 subunit mRNAs in developing and adult mouse brains by in situ hybridization. In adult brains, the γ2 and γ7 subunit mRNAs were widely expressed in various grey matter regions with the highest level in cerebellar Purkinje cells and granule cells. The γ3 and γ8 subunit mRNAs predominated in the telencephalon, with the latter being at striking levels in the hippocampus. The γ4 subunit mRNA was enriched in the olfactory bulb, striatum, thalamus and hypothalamus. The γ5 subunit mRNA was abundant in the olfactory bulb, hippocampal CA2, thalamus, inferior colliculus and Bergmann glia. Transcripts of these subunits were detected in embryonic brains: some showed well-preserved spatial patterns (γ2, γ5, γ7 and γ8), while others underwent developmental up- (γ3) or down-regulation (γ4). In contrast, the γ1 and γ6 subunit mRNAs were negative or very low throughout brain development. Therefore, the present study has revealed spatial diversity in gene expression for individual VDCCγ subunits, presumably reflecting functional diversity of this protein family and their differential involvement in neural function. © 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • M Fukaya, M Yamazaki, K Sakimura, M Watanabe
    NEUROSCIENCE RESEARCH 53 (4) 376 - 383 0168-0102 2005/12 [Refereed][Not invited]
     
    The gamma subunit of voltage-dependent Ca2+ channels (VDCCs) is characterized by molecular diversity and regulation of AMPA-type glutamate receptors as well as VDCCs. In the present study, we examined expressions for the VDCC gamma l-8 subunit mRNAs in developing and adult mouse brains by in situ hybridization. In adult brains, the gamma 2 and gamma 7 subunit mRNAs were widely expressed in various grey matter regions with the highest level in cerebellar Purkinje cells and granule cells. The gamma 3 and gamma 8 subunit mRNAs predominated in the telencephalon, with the latter being at striking levels in the hippocampus. The gamma 4 subunit mRNA was enriched in the olfactory bulb, striatum, thalamus and hypothalamus. The gamma 5 subunit mRNA was abundant in the olfactory bulb, hippocampal CA2, thalamus, inferior colliculus and Bergmann glia. Transcripts of these subunits were detected in embryonic brains: some showed well-preserved spatial patterns (gamma 2, gamma 5, gamma 7 and gamma 8), while others underwent developmental up- (gamma 3) or down-regulation (gamma 4). In contrast, the gamma 1 and gamma 6 subunit mRNAs were negative or very low throughout brain development. Therefore, the present study has revealed spatial diversity in gene expression for individual VDCC gamma subunits, presumably reflecting functional diversity of this protein family and their differential involvement in neural function. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • T Iwanaga, M Goto, M Watanabe
    BIOMEDICAL RESEARCH-TOKYO 26 (6) 271 - 278 0388-6107 2005/12 [Refereed][Not invited]
     
    L-Glutamate transport by intestinal epithelial cells is an initial step of the entire glutamate metabolism pathway in the gut mucosa. The present study examined the cellular distribution of glutamate transporters in the digestive tract of adult mice using immunohistochemistry and in situ hybridization technique. Expression of EAAC1 mRNA was more intense in the ileum, where the epithelium in crypts and the basal half of intestinal villi showed high levels of transcripts, suggesting an essential role of EAAC1 in differentiating or premature epithelial cells. Electron-microscopically, EAAC1 immunoreactivity was predominantly localized in the striated border of enterocytes. Immunoreactivity for GLT-1 was found in the lateral membrane of epithelial cells at the bottom of gastric glands and at the intestinal crypts, and also in the lateral membrane of secretory cells at the duodenal gland. GLAST immunoreactivity was restricted to the fundic and pyloric glands, and was especially intense in the neck portion of both glands. However, in situ hybridization analysis failed to confirm the expression of GLT-1 and GLAST at the mRNA level, possibly due to limited sensitivity. The strong and specific luminal localization of EAAC1 in the intestinal epithelium suggests that EAAC1 is a predominant transporter of glutamate, at least in the lower part of the small intestine.
  • H Sakagami, A Kamata, H Nishimura, J Kasahara, Y Owada, Y Takeuchi, M Watanabe, K Fukunaga, H Kondo
    EUROPEAN JOURNAL OF NEUROSCIENCE 22 (11) 2697 - 2707 0953-816X 2005/12 [Refereed][Not invited]
     
    Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) including CaMKI, II and IV, are thought to regulate a variety of neuronal functions. Unlike CaMKII, which is regulated by autophosphorylation, CaMKI as well as CaMKIV are activated by CaMKK. In this study, we examined the cellular and subcellular localization of CaMKI delta, a recently identified fourth isoform of CaMKI, in the mature brain. In situ hybridization analysis demonstrated wide expression of CaMKI delta mRNA in the adult mouse brain with prominent expression in the hippocampal pyramidal cells. FLAG-tagged CaMKI delta was localized at the cytoplasm and neurites without nuclear immunoreactivity in approximately 80% of the transfected primary hippocampal neurons. The stimulation with either KCl depolarization or glutamate triggered the nuclear localization of FLAG-tagged CaMKI delta by two-fold with a peak at 1 min. In contrast, the catalytically inactive mutants of CaMKI delta remained cytoplasmic without nuclear translocation during KCl depolarization, indicating the requirement of its activation for the nuclear translocation. Furthermore, we showed that immunoprecipitated CaMKI delta could phosphorylate cAMP response element binding protein (CREB)alpha in vitro and that the over-expression of CaMKI delta enhanced GAL4-CREB-luciferase activity in PC12 cells stimulated by KCl depolarization. Our present study provides the first evidence for the possible involvement of CaMKI delta in nuclear functions through its nuclear translocation in response to stimuli that trigger intracellular Ca2+ influx.
  • H Hirai, Z Pang, DH Bao, T Miyazaki, LY Li, E Miura, J Parris, YQ Rong, M Watanabe, M Yuzaki, JI Morgan
    NATURE NEUROSCIENCE 8 (11) 1534 - 1541 1097-6256 2005/11 [Refereed][Not invited]
     
    Cbln1 is a cerebellum-specific protein of previously unknown function that is structurally related to the C1q and tumor necrosis factor families of proteins. We show that Cbln1 is a glycoprotein secreted from cerebellar granule cells that is essential for three processes in cerebellar Purkinje cells: the matching and maintenance of pre- and postsynaptic elements at parallel fiber-Purkinje cell synapses, the establishment of the proper pattern of climbing fiber-Purkinje cell innervation, and induction of long-term depression at parallel fiber-Purkinje cell synapses. Notably, the phenotype of cbln1-null mice mimics loss-of-function mutations in the orphan glutamate receptor, GluR delta 2, a gene selectively expressed in Purkinje neurons. Therefore, Cbln1 secreted from presynaptic granule cells may be a component of a transneuronal signaling pathway that controls synaptic structure and plasticity.
  • Y Wu, R Kawakami, Y Shinohara, M Fukaya, K Sakimura, M Mishina, M Watanabe, Ito, I, R Shigemoto
    JOURNAL OF NEUROSCIENCE 25 (40) 9213 - 9226 0270-6474 2005/10 [Refereed][Not invited]
     
    Input-dependent left-right asymmetry of NMDA receptor epsilon 2 (NR2B) subunit allocation was discovered in hippocampal Schaffer collateral (Sch) and commissural fiber pyramidal cell synapses (Kawakami et al., 2003). To investigate whether this asymmetrical epsilon 2 allocation is also related to the types of the postsynaptic cells, we compared postembedding immunogold labeling for epsilon 2 in left and right Sch synapses on pyramidal cells and interneurons. To facilitate the detection of epsilon 2 density difference, we used epsilon 1 (NR2A) knock-out (K0) mice, which have a simplified NMDA receptor subunit composition. The labeling density for epsilon 2 but not zeta 1 (NR1) and subtype 2/3 glutamate receptor (GluR2/3) in Sch-CA1 pyramidal cell synapses was significantly different between the left and right hippocampus with opposite directions in strata oriens and radiatum; the left to right ratio of epsilon 2 labeling density was 1:1.50 in stratum oriens and 1.44:1 in stratum radiatum. Nosignificant difference, however, was detected in CA1 stratum radiatum between the left and right Sch-GluR4-positive (mostly parvalbumin-positive) and Sch-GluR4-negative interneuron synapses. Consistent with the anatomical asymmetry, the amplitude ratio of NMDA EPSCs to non-NMDA EPSCs in pyramidal cells was approximately two times larger in right than left stratum radiatum and vice versa in stratum oriens of epsilon 1 K0 mice. Moreover, the amplitude of long-term potentiation in the Sch-CA1 synapses of left stratum radiatum was significantly larger than that in the right corresponding synapses. These results indicate that the asymmetry of epsilon 2 distribution is target cell specific, resulting in the left-right difference in NMDA receptor content and plasticity in Sch-CA1 pyramidal cell synapses in epsilon 1 K0 mice.
  • M Tachikawa, M Watanabe, S Hori, M Fukaya, S Ohtsuki, T Asashima, T Terasaki
    JOURNAL OF NEUROCHEMISTRY 95 (1) 294 - 304 0022-3042 2005/10 [Refereed][Not invited]
     
    Using in situ hybridization for the mouse brain, we analyzed developmental changes in gene expression for the ATP-binding cassette (ABC) transporter subfamilies ABCA1-4 and 7, and ABCG1, 2, 4, 5 and 8. In the embryonic brains, ABCA1 and A7 were highly expressed in the ventricular (or germinal) zone, whereas ABCA2, A3 and G4 were enriched in the mantle (or differentiating) zone. At the postnatal stages, ABCA1 was detected in both the gray and white matter and in the choroid plexus. On the other hand, ABCA2, A3 and A7 were distributed in the gray matter. In addition, marked up-regulation of ABCA2 occurred in the white matter at 14 days-of-age when various myelin protein genes are known to be up-regulated. In marked contrast, ABCA4 was selective to the choroid plexus throughout development. ABCG1 was expressed in both the gray and white matters, whereas ABCG4 was confined to the gray matter. ABCG2 was diffusely and weakly detected throughout the brain at all stages examined. Immunohistochemistry of ABCG2 showed its preferential expression on the luminal membrane of brain capillaries. Expression signals for ABCG5 and G8 were barely detected at any stages. The distinct spatio-temporal expressions of individual ABCA and G transporters may reflect their distinct cellular expressions in the developing and adult brains, presumably, to regulate and maintain lipid homeostasis in the brain.
  • T Nakashima, M Tomi, M Tachikawa, M Watanabe, T Terasaki, K Hosoya
    GLIA 52 (1) 47 - 52 0894-1491 2005/10 [Refereed][Not invited]
     
    In high-energy metabolic tissues like the retina, creatine may play an important role in energy storage and in transmission of phosphate-bound energy substrates. To prove this, we investigated creatine synthesis in Muller glia. We also characterized the localization of the creatine synthetic enzyme, S-adenosyl-L-methionine:N-guanidinoacetate methyltransferase (GAMT) in the retina. Reverse transcription-polymerase chain reaction analysis revealed that L-arginine:glycine amidinotransferase and GAMT mRNAs were expressed in the retina and the Muller cell line, TR-MUL5. [C-14] Creatine was detected after incubation of isolated rat retina or TR-MUL5 cells with [C-14]glycine, L-arginine and L-methionine, suggesting creatine synthesis in Muller glia. Western blot analysis also revealed expression of GAMT protein in the rat retina and TR-MUL5 cells. Furthermore, confocal immunofluorescent microscopy of dual-labeled rat retinal sections demonstrated co-localization of GAMT with glutamine synthetase. Taken together, the results of the present study indicate creatine synthesis in Muller glia, implying an important role of creatine in energy metabolism in the retina. (c) 2005 Wiley-Liss, Inc.
  • T Klausberger, LF Marton, J O'Neill, JHJ Huck, Y Dalezios, P Fuentealba, WY Suen, E Papp, T Kaneko, M Watanabe, J Csicsvari, P Somogyi
    JOURNAL OF NEUROSCIENCE 25 (42) 9782 - 9793 0270-6474 2005/10 [Refereed][Not invited]
     
    In the hippocampal CA1 area, a relatively homogenous population of pyramidal cells is accompanied by a diversity of GABAergic interneurons. Previously, we found that parvalbumin-expressing basket, axo-axonic, bistratified, and oriens-lacunosum moleculare cells, innervating different domains of pyramidal cells, have distinct firing patterns during network oscillations in vivo. A second family of interneurons, expressing cholecystokinin but not parvalbumin, is known to target the same domains of pyramidal cells as do the parvalbumin cells. To test the temporal activity of these independent and parallel GABAergic inputs, we recorded the precise spike timing of identified cholecystokinin interneurons during hippocampal network oscillations in anesthetized rats and determined their molecular expression profiles and synaptic targets. The cells were cannabinoid receptor type 1 immunopositive. Contrary to the stereotyped firing of parvalbumin interneurons, cholecystokinin-expressing basket and dendrite-innervating cells discharge, on average, with 1.7 +/- 2.0 Hz during high-frequency ripple oscillations in an episode- dependent manner. During theta oscillations, cholecystokinin-expressing interneurons fire with 8.8 +/- 3.3 Hz at a characteristic time on the ascending phase of theta waves (155 +/- 81), when place cells start firing in freely moving animals. The firing patterns of some interneurons recorded in drug- free behaving rats were similar to cholecystokinin cells in anesthetized animals. Our results demonstrate that cholecystokinin- and parvalbumin- expressing interneurons make different contributions to network oscillations and play distinct roles in different brain states. We suggest that the specific spike timing of cholecystokinin interneurons and their sensitivity to endocannabinoids might contribute to differentiate subgroups of pyramidal cells forming neuronal assemblies, whereas parvalbumin interneurons contribute to synchronizing the entire network.
  • Y Takayasu, M Iino, W Kakegawa, H Maeno, K Watase, K Wada, D Yanagihara, T Miyazaki, O Komine, M Watanabe, K Tanaka, S Ozawa
    JOURNAL OF NEUROSCIENCE 25 (38) 8788 - 8793 0270-6474 2005/09 [Refereed][Not invited]
     
    Glutamate transporters are essential for terminating excitatory neurotransmission. Two distinct glutamate transporters, glutamate aspartate transporter ( GLAST) and excitatory amino acid transporter 4 ( EAAT4), are expressed most abundantly in the molecular layer of the cerebellar cortex. GLAST is expressed in Bergmann glial processes surrounding excitatory synapses on Purkinje cell dendritic spines, whereas EAAT4 is concentrated on the extrasynaptic regions of Purkinje cell spine membranes. To clarify the functional significance of the coexistence of these transporters, we analyzed the kinetics of EPSCs in Purkinje cells of mice lacking either GLAST or EAAT4. There was no difference in the amplitude or the kinetics of the rising and initial decay phase of EPSCs evoked by stimulations of climbing fibers and parallel fibers between wild-type and EAAT4-deficient mice. However, long-lasting tail currents of the EPSCs appeared age dependently in most of Purkinje cells in EAAT4-deficient mice. These tail currents were never seen in mice lacking GLAST. In the GLAST-deficient mice, however, the application of cyclothiazide that reduces desensitization of AMPA receptors increased the peak amplitude of the EPSC and prolonged its decay more markedly than in both wild-type and EAAT4-deficient mice. The results indicate that these transporters play differential roles in the removal of synaptically released glutamate. GLAST contributes mainly to uptake of glutamate that floods out of the synaptic cleft at early times after transmitter release. In contrast, the main role of EAAT4 is to remove low concentrations of glutamate that escape from the uptake by glial transporters at late times and thus prevents the transmitter from spilling over to neighboring synapses.
  • T Abe, S Matsumura, T Katano, T Mabuchi, K Takagi, L Xu, A Yamamoto, K Hattori, T Yagi, M Watanabe, T Nakazawa, T Yamamoto, M Mishina, Y Nakai, S Ito
    EUROPEAN JOURNAL OF NEUROSCIENCE 22 (6) 1445 - 1454 0953-816X 2005/09 [Refereed][Not invited]
     
    Despite abundant evidence implicating the importance of N-methyl-D-aspartate (NMDA) receptors in the spinal cord for pain transmission, the signal transduction coupled to NMDA receptor activation is largely unknown for the neuropathic pain state that lasts over periods of weeks. To address this, we prepared mice with neuropathic pain by transection of spinal nerve L5. Wild-type, NR2A-deficient, and NR2D-deficient mice developed neuropathic pain; in addition, phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 was observed in the superficial dorsal horn of the spinal cord 1 week after nerve injury. Neuropathic pain and NR2B phosphorylation at Tyr1472 were attenuated by the NR2B-selective antagonist CP-101,606 and disappeared in mice lacking Fyn kinase, a Src-family tyrosine kinase. Concomitant with the NR2B phosphorylation, an increase in neuronal nitric oxide synthase activity was visualized in the superficial dorsal horn of neuropathic pain mice by NADPH diaphorase histochemistry. Electron microscopy showed that the phosphorylated NR2B was localized at the postsynaptic density in the spinal cord of mice with neuropathic pain. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, and PGE receptor subtype EP1-selective antagonist reduced the NR2B phosphorylation in these mice. Conversely, EP1-selective agonist stimulated Fyn kinase-dependent nitric oxide formation in the spinal cord. The present study demonstrates that Tyr1472 phosphorylation of NR2B subunits by Fyn kinase may have dual roles in the retention of NMDA receptors in the postsynaptic density and in activation of nitric oxide synthase, and suggests that PGE(2) is involved in the maintenance of neuropathic pain via the EP1 subtype.
  • S Hatakeyama, M Watanabe, Y Fujii, KL Nakayama
    CANCER RESEARCH 65 (17) 7874 - 7879 0008-5472 2005/09 [Refereed][Not invited]
     
    Given that expression of c-Myc is up-regulated in many human malignancies, targeted inactivation of this oncoprotein is a potentially effective strategy for cancer treatment The ubiquitin-proteasome pathway of protein degradation is highly specific and can be engineered to achieve the elimination of undesirable proteins such as oncogene products. We have now generated a ftision protein (designated Max-U) that is composed both of May, which forms a heterodimer with c-Myc, and of CRW, which is a U box-type ubiquitin ligase (E3). Max-U physically interacted with c-Myc in transfected cells and promoted the ubiquitylation of c-Myc in vitro. It also reduced the stability of c-Myc in vivo, resulting in suppression of transcriptional activity dependent on c-Myc. Expression of Max-U reduced both the abundance of endogenous c-Myc in and the proliferation rate of a Burkitt lymphoma cell line. Furthermore, expression of Max-U but not that of a catalytically inactive mutant thereof markedly inhibited both the anchorage-independent growth in vitro of NIH 3T3 cells that overexpress c-Myc as well as tumor formation by these cells in nude mice. These findings indicate that the targeted destruction of c-Myc by an artificial E3 may represent an effective therapeutic strategy for certain human malignancies.
  • M Taniguchi, T Masuda, M Fukaya, H Kataoka, M Mishina, H Yaginuma, M Watanabe, T Shimizu
    GENES TO CELLS 10 (8) 785 - 792 1356-9597 2005/08 [Refereed][Not invited]
     
    The semaphorin gene family contains a large number of secreted type or transmembrane type proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. Here we report a novel member of murine class 3 semaphorin genes, semaphorin 3G (Sema3G), mapped on chromosome 14. In adulthood, Sema3G is mainly expressed in the lung and kidney, and a little in the brain. Interestingly, in the adult rodent brain Sema3G is expressed only in the granular layer of the cerebellum, as determined by Northern blot and in situ hybridization analyses. We also found that Sema3G binds Neuropilin-2, but not Neuropilin-1, and induces the repulsion of sympathetic axons, but not dorsal root ganglion axons, indicating that Sema3G utilizes Neuropilin-2 as a receptor to repel specific types of axons.
  • M Hoshino, S Nakamura, K Mori, T Kawauchi, M Terao, YV Nishimura, A Fukuda, T Fuse, N Matsuo, M Sone, M Watanabe, H Bito, T Terashima, CVE Wright, Y Kawaguchi, K Nakao, YI Nabeshima
    NEURON 47 (2) 201 - 213 0896-6273 2005/07 [Refereed][Not invited]
     
    The molecular machinery governing glutamatergic-GABAergic neuronal subtype specification is unclear. Here we describe a cerebellar mutant, cerebelless, which lacks the entire cerebellar cortex in adults. The primary defect of the mutant brains was a specific inhibition of GABAergic neuron production from the cerebellar ventricular zone (VZ), resulting in secondary and complete loss of external germinal layer, pontine, and olivary nuclei during development. We identified the responsible gene, Ptf1a, whose expression was lost in the cerebellar VZ but was maintained in the pancreas in cerebelless. Lineage tracing revealed that two types of neural precursors exist in the cerebellar VZ: Ptf1a-expressing and -nonexpressing precursors, which generate GABAergic and glutamatergic neurons, respectively. Introduction of Ptf1a into glutamatergic neuron precursors in the dorsal telencephalon generated GABAergic neurons with representative morphological and migratory features. Our results suggest that Ptf1a is involved in driving neural precursors to differentiate into GABAergic neurons in the cerebellum.
  • K Kanbara, K Okamoto, S Nomura, T Kaneko, R Shigemoto, H Azuma, Y Katsuoka, M Watanabe
    JOURNAL OF ANDROLOGY 26 (4) 485 - 493 0196-3635 2005/07 [Refereed][Not invited]
     
    The GABAergic system, a major inhibitory regulator in the central nervous system, may also play important roles in peripheral nonneuronal tissues and cells. Recent studies showed that GABA, receptor is expressed in testis and sperm. To understand the role of the GABAergic system in spermiogenesis, we examined cellular localization of GABA and GABA(B) receptor subunits in rat spermatics by immunocytochemistry. Immunoreactivity for GABA was detected around acrosomal granules of spermatids during the Golgi and cap phases. GABA(B(1)) immunoreactivity was observed in the acrosomal vesicle of spermatids in Golgi phase, and during cap phase, this reactivity expanded to the entire region of the acrosome covering the nuclear membrane. The level of reactivity decreased gradually with maturation of spermatics. In contrast, GABA(B(2)) immunoreactivity was not observed in spermatics during Golgi phase but was detected in the equatorial region during cap phase. Both GABA immunoreactivity and GABA(B(2)) immunoreactivity were transferred to the residual cytoplasm during the release of spermatozoa. Electron microscopic immunocytochemistry revealed that, during cap phase, GABA and GABA(B(1)) were distributed within the whole acrosomal vesicle but not in the acrosomal granule. GABA(B(2)) immunoreactivity was observed in the narrow space between the inner acrosomal and nuclear membrane and was limited to the equatorial region of the spermatid head. These results indicate that the GABAergic system might be involved in regulation of spermiogenesis.
  • M Kinoshita, M Fukaya, T Tojima, S Kojima, H Ando, M Watanabe, A Urano, E Ito
    JOURNAL OF COMPARATIVE NEUROLOGY 484 (2) 249 - 259 0021-9967 2005/04 [Refereed][Not invited]
     
    Retinotectal transmission has not yet been well characterized at the cellular level in the optic tectum. To address this issue, we used a teleost, the rainbow trout, and characterized periventricular neurons as postsynaptic cells expected to receive the retinotectal inputs to the optic tectum. The somata of periventricular neurons are localized in the upper zone of the stratum periventriculare (SPV), whereas the lower zone of the SPV comprises the cell body layer of radial glial cells. Ca2+ imaging identified functional ionotropic glutamate receptors in periventricular neurons. We also cloned cDNAs encoding the NR1 subunit of N-methyl-D-aspartic acid (NMDA) receptors and the GluR2 subunit of (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors, and detected their mRNAs in periventricular neurons by in situ hybridization. The presence of the receptor subunit proteins was also confirmed in the dendrites of periventricular neurons by immunoblotting and immunohistochemistry. On the other hand, radial glial cells in the lower zone of the SPV did not respond to glutamate applications, and mRNA and immunoreactivities of ionotropic glutamate receptors were not detected in glial cells. The present findings suggest that glutamatergic transmission at synapses between retinotectal afferents and periventricular neurons is mediated by the functional NMDA and AMPA receptors. (C) 2005 Wiley-Liss, Inc.
  • M Kinoshita, M Fukaya, T Tojima, S Kojima, H Ando, M Watanabe, A Urano, E Ito
    JOURNAL OF COMPARATIVE NEUROLOGY 484 (2) 249 - 259 0021-9967 2005/04 [Refereed][Not invited]
     
    Retinotectal transmission has not yet been well characterized at the cellular level in the optic tectum. To address this issue, we used a teleost, the rainbow trout, and characterized periventricular neurons as postsynaptic cells expected to receive the retinotectal inputs to the optic tectum. The somata of periventricular neurons are localized in the upper zone of the stratum periventriculare (SPV), whereas the lower zone of the SPV comprises the cell body layer of radial glial cells. Ca2+ imaging identified functional ionotropic glutamate receptors in periventricular neurons. We also cloned cDNAs encoding the NR1 subunit of N-methyl-D-aspartic acid (NMDA) receptors and the GluR2 subunit of (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors, and detected their mRNAs in periventricular neurons by in situ hybridization. The presence of the receptor subunit proteins was also confirmed in the dendrites of periventricular neurons by immunoblotting and immunohistochemistry. On the other hand, radial glial cells in the lower zone of the SPV did not respond to glutamate applications, and mRNA and immunoreactivities of ionotropic glutamate receptors were not detected in glial cells. The present findings suggest that glutamatergic transmission at synapses between retinotectal afferents and periventricular neurons is mediated by the functional NMDA and AMPA receptors. (C) 2005 Wiley-Liss, Inc.
  • M Fukaya, Y Hayashi, M Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 21 (5) 1432 - 1436 0953-816X 2005/03 [Refereed][Not invited]
     
    The NR3B NMDA receptor subunit is selective to somatic motoneurons in the adult nervous system. Here we report its developmental expression in the mouse brain and spinal cord by in situ hybridization. NR3B mRNA was detected in few neural regions during embryonic and neonatal periods. It first appeared in motoneurons at postnatal day (P)10-P14, and attained the maximal level at P21 and adult stage. This developmental profile was reciprocal with that of NR2 subunits, of which NR2A mRNA was most predominant in embryonic and neonatal motoneurons and downregulated by P14. Interestingly, mRNA of the NR1 subunit, which is required for functional NMDA receptors, displayed a 'V'-shaped change, decreasing with the early postnatal decline of NR2 mRNAs and increasing with the subsequent appearance of NR3B mRNA. Therefore, the major regulatory subunit of NMDA receptors is likely to switch from NR2 to NR3B in somatic motoneurons during the early postnatal period.
  • M Fukaya, Y Hayashi, M Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 21 (5) 1432 - 1436 0953-816X 2005/03 [Refereed][Not invited]
     
    The NR3B NMDA receptor subunit is selective to somatic motoneurons in the adult nervous system. Here we report its developmental expression in the mouse brain and spinal cord by in situ hybridization. NR3B mRNA was detected in few neural regions during embryonic and neonatal periods. It first appeared in motoneurons at postnatal day (P)10-P14, and attained the maximal level at P21 and adult stage. This developmental profile was reciprocal with that of NR2 subunits, of which NR2A mRNA was most predominant in embryonic and neonatal motoneurons and downregulated by P14. Interestingly, mRNA of the NR1 subunit, which is required for functional NMDA receptors, displayed a 'V'-shaped change, decreasing with the early postnatal decline of NR2 mRNAs and increasing with the subsequent appearance of NR3B mRNA. Therefore, the major regulatory subunit of NMDA receptors is likely to switch from NR2 to NR3B in somatic motoneurons during the early postnatal period.
  • T Takeuchi, T Miyazaki, M Watanabe, H Mori, K Sakimura, M Mishina
    JOURNAL OF NEUROSCIENCE 25 (8) 2146 - 2156 0270-6474 2005/02 [Refereed][Not invited]
     
    Precise topological matching of presynaptic and postsynaptic specializations is essential for efficient synaptic transmission. Furthermore, synaptic connections are subjected to rearrangements throughout life. Here we examined the role of glutamate receptor (GluR) delta2 in the adult brain by inducible and cerebellar Purkinje cell (PC)-specific gene targeting under the pure C57BL/6 genetic background. Concomitant with the decrease of postsynaptic GluRdelta2 proteins, presynaptic active zones shrank progressively and postsynaptic density (PSD) expanded, resulting in mismatching between presynaptic and postsynaptic specializations at parallel fiber-PC synapses. Furthermore, GluRdelta2 and PSD-93 proteins were concentrated at the contacted portion of mismatched synapses, whereas AMPA receptors were distributed in both the contacted and dissociated portions. When GluRdelta2 proteins were diminished, PC spines lost their synaptic contacts. We thus identified postsynaptic GluRdelta2 as a key regulator of the presynaptic active zone and PSD organization at parallel fiber-PC synapses in the adult brain.
  • H Hirai, T Miyazaki, W Kakegawa, S Matsuda, M Mushina, M Watanabe, M Yuzaki
    EMBO REPORTS 6 (1) 90 - 95 1469-221X 2005/01 [Refereed][Not invited]
     
    The delta2 glutamate receptor (GluRdelta2) has a crucial role in cerebellar functions; disruption of GluRdelta2 alleles in mice (delta2(-/-)) impairs synapse formation and long-term depression, which is thought to underlie motor learning in the cerebellum, and consequently leads to motor discoordination. However, it has been unclear whether GluRdelta2 is activated by glutamate analogues. Here we introduced a GluRdelta2 transgene, which had a mutation (Arg514Lys) in the putative ligand-binding motif conserved in all mammalian ionotropic glutamate receptors (iGluRs) and their ancestral bacterial periplasmic amino-acid-binding proteins, into delta2(-/-) mice. Surprisingly, a mutant GluRdelta2 transgene, as well as a wild-type GluRdelta2 transgene, rescued all abnormal phenotypes of delta2(-/-) mice. Therefore, these results indicate that the conserved arginine residue, which is crucial for the binding of iGluRs to glutamate analogues, is not essential for the restoration of GluRdelta2 functions in delta2(-/-) mice.
  • M Yamazaki, T Ohno-Shosaku, M Fukaya, M Kano, M Watanabe, K Sakimura
    NEUROSCIENCE RESEARCH 50 (4) 369 - 374 0168-0102 2004/12 [Refereed][Not invited]
     
    Stargazin (gamma-2) is disrupted in the ataxic and epileptic mutant mouse, stargazer (stg). The striking defect in the stg cerebellum is the lack of functional AMPA receptors on granule cells. Recently, it has been reported that gamma-2 and its related molecules are crucial for the surface expression, synaptic targeting and recycling of AMPA receptors, being termed collectively as the transmembrane AMPA receptor regulatory proteins (TARPs). However, it is still unclear whether TARPs directly modulate AMPA receptor activity. Here we report that coexpression of GluRalpha1 (GluR1) with gamma-2 using HEK293 cells and Xenopus oocytes markedly enhanced glutamate-induced currents. This effect was far beyond the increase of AMPA receptor surface expression and accompanied by increased glutamate affinity and subunit cooperativity. Other member of TARPs (gamma-3, gamma-4, and gamma-8) also enhanced the current response through the AMPA receptors. The enhancing effect by gamma-2 coexpression was further observed for homomeric GluRalpha2 (GluR2) channels, which, when expressed alone, are known to produce only a small or negligible current response. These results suggest that gamma-2 not only promotes AMPA receptor surface expression but also directly modulates AMPA receptor activity. (C) 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • M Nakamura, K Sato, M Fukaya, K Araishi, A Aiba, M Kano, M Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 20 (11) 2929 - 2944 0953-816X 2004/12 [Refereed][Not invited]
     
    Upon activation of cell surface receptors coupled to the Gq subclass of G proteins, phospholipase C (PLC) beta hydrolyses membrane phospholipid to yield a pair of second messengers, inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. PLCbeta4 has been characterized as the isoform enriched in cerebellar Purkinje cells (PCs) and the retina and involved in motor and visual functions. Here we examined cellular and subcellular distributions of PLCbeta4 in adult mouse brains. Immunohistochemistry showed that high levels of PLCbeta4 were detected in the somatodendritic domain of neuronal populations expressing the metabotropic glutamate receptor (mGluR) type 1alpha, including olfactory periglomerular cells, neurons in the bed nucleus anterior commissure, thalamus, substantia nigra, inferior olive, and unipolar brush cells and PCs in the cerebellum. Low to moderate levels were detected in many other mGluR1alpha-positive neurons and in a few mGluR1alpha-negative neurons. In PCs, immunogold electron microscopy localized PLCbeta4 to the perisynapse, at which mGluR1alpha is concentrated, and to the smooth endoplasmic reticulum in dendrites and spines, an intracellular Ca2+ store gated by IP3 receptors. In the cerebellum, immunoblot demonstrated its concentrated distribution in the post-synaptic density and microsomal fractions, where mGluR1alpha and type 1 IP3 receptor were also greatly enriched. Furthermore, PLCbeta4 formed coimmunoprecipitable complexes with mGluR1alpha, type 1 IP3 receptor and Homer 1. These results suggest that PLCbeta4 is preferentially localized in the perisynapse and smooth endoplasmic reticulum as a component of the physically linked phosphoinositide signaling complex. This close molecular relationship might provide PLCbeta4 with a high-fidelity effector function to mediate various neuronal responses under physiological and pathophysiological conditions.
  • GG Nagy, M Watanabe, M Fukaya, AJ Todd
    EUROPEAN JOURNAL OF NEUROSCIENCE 20 (12) 3301 - 3312 0953-816X 2004/12 [Refereed][Not invited]
     
    Glutamate is the main excitatory neurotransmitter in the spinal cord and acts on several types of receptor, including N-methyl-D-aspartate (NMDA) receptors, which play an important role in synaptic plasticity and chronic pain. Three families of NMDA receptor subunit have been identified: NR1, NR2 (A-D) and NR3 (A and B). NMDA receptors are heteromeric channels that contain NR1 with at least one NR2 subunit. There is extensive evidence that NMDA receptors are present in spinal cord but little is known about their synaptic distribution. We have used an antigen-unmasking method involving pepsin treatment to reveal NR1, NR2A and NR2B subunits and have compared their distribution with that of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor GluR2 subunit, which is thought to be present at most glutamatergic synapses throughout the spinal cord. After pepsin treatment, punctate labelling was seen with antibodies against each of these subunits. Although NR1 puncta were present throughout the grey matter, NR2A was concentrated in laminae III-IV and NR2B in laminae I-II. The majority of puncta labelled with each NMDA receptor antibody were GluR2-immunoreactive, which suggests that they were present at synapses, and this was confirmed with electron microscopy for the NR1 and NR2A antibodies. However, many GluR2-immunoreactive puncta did not show NMDA receptor immunoreactivity. In laminae I-II, most NR2B puncta were also NR1-immunoreactive and a similar arrangement was found for NR2A/NR1 in laminae III-IV. These results suggest that many, but not all, glutamatergic synapses in the spinal cord possess NMDA receptors and that subunit composition varies in different regions.
  • M Nakamura, K Sato, M Fukaya, K Araishi, A Aiba, M Kano, M Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 20 (11) 2929 - 2944 0953-816X 2004/12 [Refereed][Not invited]
     
    Upon activation of cell surface receptors coupled to the Gq subclass of G proteins, phospholipase C (PLC) beta hydrolyses membrane phospholipid to yield a pair of second messengers, inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. PLCbeta4 has been characterized as the isoform enriched in cerebellar Purkinje cells (PCs) and the retina and involved in motor and visual functions. Here we examined cellular and subcellular distributions of PLCbeta4 in adult mouse brains. Immunohistochemistry showed that high levels of PLCbeta4 were detected in the somatodendritic domain of neuronal populations expressing the metabotropic glutamate receptor (mGluR) type 1alpha, including olfactory periglomerular cells, neurons in the bed nucleus anterior commissure, thalamus, substantia nigra, inferior olive, and unipolar brush cells and PCs in the cerebellum. Low to moderate levels were detected in many other mGluR1alpha-positive neurons and in a few mGluR1alpha-negative neurons. In PCs, immunogold electron microscopy localized PLCbeta4 to the perisynapse, at which mGluR1alpha is concentrated, and to the smooth endoplasmic reticulum in dendrites and spines, an intracellular Ca2+ store gated by IP3 receptors. In the cerebellum, immunoblot demonstrated its concentrated distribution in the post-synaptic density and microsomal fractions, where mGluR1alpha and type 1 IP3 receptor were also greatly enriched. Furthermore, PLCbeta4 formed coimmunoprecipitable complexes with mGluR1alpha, type 1 IP3 receptor and Homer 1. These results suggest that PLCbeta4 is preferentially localized in the perisynapse and smooth endoplasmic reticulum as a component of the physically linked phosphoinositide signaling complex. This close molecular relationship might provide PLCbeta4 with a high-fidelity effector function to mediate various neuronal responses under physiological and pathophysiological conditions.
  • F Nakatsu, M Okada, F Mori, N Kumazawa, H Iwasa, G Zhu, Y Kasagi, H Kamiya, A Harada, K Nishimura, A Takeuchi, T Miyazaki, M Watanabe, S Yuasa, T Manabe, K Wakabayashi, S Kaneko, T Saito, H Ohno
    JOURNAL OF CELL BIOLOGY 167 (2) 293 - 302 0021-9525 2004/10 [Refereed][Not invited]
     
    A P-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B(-/-) mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B(-/-) mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.
  • HW Kitamura, H Hamanaka, M Watanabe, K Wada, C Yamazaki, SC Fujita, T Manabe, N Nukina
    NEUROSCIENCE LETTERS 369 (3) 173 - 178 0304-3940 2004/10 [Refereed][Not invited]
     
    Human apolipoprotein E (apoE) comprises three isoforms, apoE2, apoE3 and apoE4, and apoE4 has been reported as a risk factor of Alzheimer's disease (AD). One of the clinical symptoms of AD is disorder of memory that has been suggested to be related with synaptic plasticity such as long-term potentiation (UP). Here, we show the enhancement of hippocampal UP at younger age in knock-in mice lacking mouse apoE, but instead expressing human apoE4. The enhancement of UP in apoE4 knock-in mice is age-dependent, and it disappears in adult apoE4 knock-in mice. In apoE3 knock-in mice UP is unaltered, thus human apoE4, but not apoE3, specifically modulates synaptic plasticity at younger age. Since basal synaptic transmission and distribution of glutamate receptors, as well as presynaptic functions, are intact in apoE4 knock-in mice, postsynaptic functional modification of UP through lipid homeostasis is suggested. ApoE4 knock-in mice would be a useful animal model of human apoE4 carriers, and our finding that UP is enhanced in younger apoE4 knock-in mice is in accord with the previous report showing higher intelligence in young human apoE4 carriers. (C) 2004 Elsevier Ireland Ltd. All rights reserved.
  • S Hori, S Ohtsuki, M Tachikawa, N Kimura, T Kondo, M Watanabe, E Nakashima, T Terasaki
    JOURNAL OF NEUROCHEMISTRY 90 (3) 526 - 536 0022-3042 2004/08 [Refereed][Not invited]
     
    The purpose of the present study was to clarify the expression, transport properties and regulation of ATP-binding cassette G2 (ABCG2) transporter at the rat blood-brain barrier (BBB). The rat homologue of ABCG2 (rABCG2) was cloned from rat brain capillary fraction. In rABCG2-transfected HEK293 cells, rABCG2 was detected as a glycoprotein complex bridged by disulfide bonds, possibly a homodimer. The protein transported mitoxantrone and BODIPY-prazosin. In rat brain capillary fraction, rABCG2 protein was also detected as a glycosylated and disulfide-linked complex. Immunohistochemical analysis revealed that rABCG2 was localized mainly on the luminal side of rat brain capillaries, suggesting that rABCG2 is involved in brain-to-blood efflux transport. For the regulation study, conditionally immortalized rat brain capillary endothelial (TR-BBB13), astrocyte (TR-AST4) and pericyte (TR-PCT1) cell lines were used as an in vitro BBB model. Following treatment of TR-BBB13 cells with conditioned medium of TR-AST4 cells, the Ko143 (an ABCG2-specific inhibitor)-sensitive transport activity and rABCG2 mRNA level were significantly increased, whereas conditioned medium of TR-PCT1 cells had no effect. These results suggest that rat brain capillaries express functional rABCG2 protein and that the transport activity of the protein is up-regulated by astrocyte-derived soluble factor(s) concomitantly with the induction of rABCG2 mRNA.
  • M Abe, M Fukaya, T Yagi, M Mishina, M Watanabe, K Sakimura
    JOURNAL OF NEUROSCIENCE 24 (33) 7292 - 7304 0270-6474 2004/08 [Refereed][Not invited]
     
    In NMDA receptors, GluRepsilon/NR2 subunits strictly require the GluRzeta1/NR1 subunit to exit from endoplasmic reticulum ( ER) to the cell surface in vitro and to the postsynapse in vivo, whereas Cterminus-dependent self-surface delivery has been demonstrated for the GluRzeta1 subunit in vitro. To test whether this leads to C terminus-dependent self-postsynaptic expression in neurons in vivo, we investigated the GluRzeta1 subunit in cerebellar granule cells lacking two major GluRepsilon subunits, GluRepsilon1/NR2Aand GluRepsilon3/NR2C. In the mutant cerebellum, synaptic labeling for the GluRzeta1 subunit containing the C2 (GluRzeta1-C2) or C2' (GluRzeta1-C2') cassette was reduced at mossy fiber-granule cell synapses to the extrasynaptic level. The loss was not accompanied by decreased transcription and translation levels, increased extrasynaptic labeling, or ER accumulation. Quantitative immunoblot revealed substantial reductions in the mutant cerebellum of GluRzeta1-C2 and GluRzeta1-C2'. The most severe deficit was observed in the postsynaptic density (PSD) fraction: mutant levels relative to the wild-type level were 12.3 +/- 3.3% for GluRzeta1-C2 and 17.0 +/- 4.6% for GluRzeta1-C2'. The GluRzeta1 subunit carrying the C1 cassette (GluRzeta1-C1) was, although low in cerebellar content, also reduced to 12.7 +/- 3.5% in the mutant PSD fraction. Considering a trace amount of other GluRepsilon subunits in the mutant cerebellum, the severe reductions thus represent that the GluRzeta1 subunit, by itself, is virtually unable to accumulate at postsynaptic sites, regardless of C-terminal forms. By protein turnover analysis, the degradation of the GluRzeta1 subunit was accelerated in the mutant cerebellum, being particularly rapid for that carrying the C2 cassette. Therefore, accompanying expression of GluRepsilon subunits is essential for postsynaptic localization and protein stability of the GluRzeta1 subunit.
  • セマフォリン3Fノックアウトマウスの作成と解析(Generation and analysis of Semaphorin 3F knockout mice)
    松田 育雄, 中尾 晴美, 深谷 昌弘, 渡辺 雅彦, 中尾 和貴, 饗場 篤
    神経化学 43 (2-3) 519 - 519 0037-3796 2004/08
  • NMDA受容体NR2Bサブユニットのリン酸化残基(Tyr-1472)改変マウスの解析(NMDAR2B tyrosine-phosphorylation modulates synaptic plasticity and memory formation in amygdala)
    中澤 敬信, 駒井 章治, 城山 優治, 渡部 文子, 深谷 昌弘, 手塚 徹, 渡辺 雅彦, 真鍋 俊也, 山本 雅
    神経化学 43 (2-3) 377 - 377 0037-3796 2004/08
  • 成熟マウス小脳におけるシナプス後膜AMPA受容体の活動に依存したシナプス前終末の機能維持(Postsynaptic AMPA receptor activity maintains presynaptic function in the adult cerebellum)
    柿澤 昌, 宮崎 太輔, 飯野 正光, 渡辺 雅彦, 狩野 方伸
    神経化学 43 (2-3) 470 - 470 0037-3796 2004/08
  • M Tachikawa, M Fukaya, T Terasaki, S Ohtsuki, M Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 20 (1) 144 - 160 0953-816X 2004/07 [Refereed][Not invited]
     
    The creatine/phosphocreatine shuttle system, as catalysed reversibly by creatine kinases, is thought to be essential for the storing and buffering of high phosphate-bound energy in tissues with high energy demand. In the present study, we aimed to clarify the cellular system of creatine biosynthesis and its energy metabolism in the mouse brain by immunohistochemistry for creatine biosynthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT), ubiquitous mitochondrial creatine kinase (uCK-Mi) and brain-type cytoplasmic creatine kinase (CK-B). GAMT was expressed highly in oligodendrocytes and olfactory ensheathing glia and moderately in astrocytes, whereas GAMT was very low in neurons and microglia. By contrast, uCK-Mi was expressed selectively in neurons and localized in their mitochondria in dendrites, cell bodies, axons and terminals. The distinct and almost complementary distribution of GAMT and uCK-Mi suggests that the creatine in neuronal mitochondria is derived not only from the circulation, but also from local glial cells associated with these neuronal elements. By contrast, CK-B was selective to astrocytes among glial populations, and was exclusive to inhibitory neurons among neuronal populations. Interestingly, these cells with high CK-B immunoreactivity are known to be highly resistant to acute energy loss, such as hypoxia and hypoglycemia. Considering that phosphocreatine generates ATP much faster than the processes of glycolysis and oxidative phosphorylation, the highly regulated cellular expressions of creatine biosynthetic and metabolic enzymes suggest that the creatine/phosphocreatine shuttle system plays a role in brain energy homeostasis through a novel neuron-glial relationship.
  • S Oshima, K Yamada, T Shirakawa, M Watanabe
    NEUROSCIENCE LETTERS 365 (2) 97 - 101 0304-3940 2004/07 [Refereed][Not invited]
     
    The high-affinity choline transporter CHT1 works for choline uptake in the presynaptic terminals of cholinergic neurons. We examined its expression in the hypoglossal nucleus after unilateral hypoglossal nerve transection in mice by fluorescent in situ hybridization. One week after axotomy, CHT1 mRNA expression was lost in all hypoglossal motoneurons in the lesioned side. Two weeks after axotomy, CHT1 mRNA started to be re-expressed in a few motoneurons that recovered connections to tongue muscles as revealed by retrograde labeling with Fast Blue. After 4 weeks, most of axotomized hypoglossal motoneurons were reconnected and re-expressed CHT1 mRNA as strongly as control neurons, and the regenerating cholinergic axons established mature neuromuscular junctions. These results suggest that the establishment of motor innervation is critical for CHT1 mRNA expression in hypoglossal neurons after axotomy. (C) 2004 Elsevier Ireland Ltd. All rights reserved.
  • K Oguro, T Miyawaki, H Yokota, K Kato, T Kamiya, Y Katayama, M Fukaya, M Watanabe, K Shimazaki
    NEUROSCIENCE LETTERS 364 (2) 101 - 105 0304-3940 2004/07 [Refereed][Not invited]
     
    Developing animals are known to be resistant to cerebral ischemia. To investigate the mechanisms by which developing animals exhibit ischemic resistance, we examined the changes in intracellular calcium ([Ca2+](i)) after oxygen-glucose deprivation (OGD) using hippocampal slices from gerbils. We found that increases of [Ca2+](i) in hippocampal CA1 neurons is significantly less after OGD in developing gerbils than in adults. Western blot analysis of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors (AMPARs) showed that GluR2 expression, but not that of the other AMPARs is significantly higher in developing gerbils than in adults. Expression of the anti-apoptotic proteins such as HSP70, Bcl-X-L, and plasma membrane Ca2+-ATPase type1 (PMCA1) are not higher in the developing gerbils than in adults. These results suggest that the higher expression of GluR2 is important for the smaller increases in [Ca2+](i) and enhanced resistance to ischemia-induced neuronal damage in developing animals. (C) 2004 Elsevier Ireland Ltd. All rights reserved.
  • S Oshima, K Yamada, T Shirakawa, M Watanabe
    NEUROSCIENCE LETTERS 365 (2) 97 - 101 0304-3940 2004/07 [Refereed][Not invited]
     
    The high-affinity choline transporter CHT1 works for choline uptake in the presynaptic terminals of cholinergic neurons. We examined its expression in the hypoglossal nucleus after unilateral hypoglossal nerve transection in mice by fluorescent in situ hybridization. One week after axotomy, CHT1 mRNA expression was lost in all hypoglossal motoneurons in the lesioned side. Two weeks after axotomy, CHT1 mRNA started to be re-expressed in a few motoneurons that recovered connections to tongue muscles as revealed by retrograde labeling with Fast Blue. After 4 weeks, most of axotomized hypoglossal motoneurons were reconnected and re-expressed CHT1 mRNA as strongly as control neurons, and the regenerating cholinergic axons established mature neuromuscular junctions. These results suggest that the establishment of motor innervation is critical for CHT1 mRNA expression in hypoglossal neurons after axotomy. (C) 2004 Elsevier Ireland Ltd. All rights reserved.
  • T Nakashima, M Tomi, K Katayama, M Tachikawa, M Watanabe, T Terasaki, K Hosoya
    JOURNAL OF NEUROCHEMISTRY 89 (6) 1454 - 1461 0022-3042 2004/06 [Refereed][Not invited]
     
    The purpose of this study was to elucidate the mechanisms of blood-to-retina creatine transport across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The creatine transport across the BRB in vivo and creatine uptake in an in vitro model of the inner BRB (TR-iBRB2 cells) were examined using [C-14]creatine. Identification and localization of the creatine transporter (CRT) were carried out by RT-PCR, western blot, and immunoperoxidase electron microscopic analyses. An in vivo intravenous administration study suggested that [C-14]creatine is transported from the blood to the retina against the creatine concentration gradient that exists between the retina and blood. [C-14]Creatine uptake by TR-iBRB2 cells was saturable, Na+- and Cl--dependent and inhibited by CRT inhibitors, suggesting that CRT is involved in creatine transport at the inner BRB. RT-PCR and western blot analyses demonstrated that CRT is expressed in rat retina and TR-iBRB2 cells. Moreover, using an immunoperoxidase electron microscopic analysis, CRT immunoreactivity was found at both the luminal and abluminal membranes of the rat retinal capillary endothelial cells. In conclusion, CRT is expressed at the inner BRB and plays a role in blood-to-retina creatine transport across the inner BRB.
  • GG Nagy, M Al-Ayyan, D Andrew, M Fukaya, M Watanabe, AJ Todd
    JOURNAL OF NEUROSCIENCE 24 (25) 5766 - 5777 0270-6474 2004/06 [Refereed][Not invited]
     
    Glutamate, the principal excitatory neurotransmitter in the spinal cord, acts primarily through AMPA receptors. Although all four AMPA subunits are expressed by spinal neurons, we know little about their distribution at glutamatergic synapses. We used an antigen-unmasking technique to reveal the synaptic distribution of glutamate receptor (GluR) 1-4 subunits with confocal microscopy. After pepsin treatment, punctate staining was seen with antibodies against each subunit: GluR2-immunoreactive puncta were distributed throughout the gray matter, whereas GluR1-immunoreactive puncta were restricted to the dorsal horn and were most numerous in laminas I-II. Punctate staining for GluR3 and GluR4 was found in all laminas but was weak in superficial dorsal horn. Colocalization studies showed that GluR2 was present at virtually all (98%) puncta that were GluR1, GluR3, or GluR4 immunoreactive and that most (>90%) immunoreactive puncta in laminas IV, V, and IX showed GluR2, GluR3, and GluR4 immunoreactivity. Evidence that these puncta represented synaptic receptors was obtained with electron microscopy and by examining the association of GluR2- and GluR1-immunoreactive puncta with glutamatergic boutons ( identified with vesicular glutamate transporters or markers for unmyelinated afferents). The great majority (96%) of these boutons were associated with GluR2- immunoreactive puncta. Our findings suggest that GluR2 is almost universally present at AMPA-containing synapses, whereas GluR1 is preferentially associated with primary afferent terminals. We also found a substantial, rapid increase in staining for synaptic GluR1 subunits phosphorylated on the S845 residue in the ipsilateral dorsal horn after peripheral noxious stimulation. This finding demonstrates plastic changes, presumably contributing to central sensitization, at the synaptic level.
  • H Kawano, T Nakatani, T Mori, S Ueno, M Fukaya, A Abe, M Kobayashi, F Toda, M Watanabe, Matsuoka, I
    MOLECULAR BRAIN RESEARCH 125 (1-2) 60 - 75 0169-328X 2004/06 [Refereed][Not invited]
     
    Processes of neuronal differentiation involve activation of a set of neuronal specific genes and cessation of cell proliferation in postmitotic neurons. Previous studies revealed that bone morphogenetic protein (BMP) and retinoic acid (RA) play important roles in the differentiation of peripheral sympathetic neurons such as the synergistic induction of responsiveness to specific neurotrophic factors. In the present study, while trying to clarify the mechanism of the BMP/RA-actions, we identified a novel neural-specific protein, BMP/RA-inducible neural-specific protein-1 (BRINP1) which shows no similarity to other known proteins. Subsequently, two homologous proteins, BRINP2 and BRINP3, making up the BRINP family, are identified. Individual BRINP genes have distinct regulatory mechanisms of expression within the nervous system. In rodent brain, BRINP1 is expressed from earlier developmental stage, i.e. E9.5, and widely expressed in various neuronal layers and nuclei of the adult animal, while BRINP2 and BRINP3 were detectable from E11.5 and expressed in rather limited regions in a complementary manner. During the course of perinatal development of sympathetic neurons, BRINP1 is induced from earlier embryonic stage and further increased toward adult stage, while BRINP3 expressed from earlier stage is replaced by BRINP2 expression which increases postnatally in accordance with the action of BMP2 and RA. Furthermore, when expressed in nonneuronal cells, all three BRINP family proteins suppressed the cell cycle progression. Possible physiological functions of BRINP family members in the development of the nervous system are discussed. (C) 2004 Elsevier B.V All rights reserved.
  • Y Fukudome, T Ohno-Shosaku, M Matsui, Y Omori, M Fukaya, H Tsubokawa, MM Taketo, M Watanabe, T Manabe, M Kano
    EUROPEAN JOURNAL OF NEUROSCIENCE 19 (10) 2682 - 2692 0953-816X 2004/05 [Refereed][Not invited]
     
    The cholinergic system in the CNS plays important roles in higher brain functions, primarily through muscarinic acetylcholine receptors. At cellular levels, muscarinic activation produces various effects including modulation of synaptic transmission. Here we report that muscarinic activation suppresses hippocampal inhibitory transmission through two distinct mechanisms, namely a cannabinoid-dependent and cannabinoid-independent mechanism. We made paired whole-cell recordings from cultured hippocampal neurons of rats and mice, and monitored inhibitory postsynaptic currents (IPSCs). When cannabinoid receptor type 1 (CB1) was blocked, oxotremorine M (oxo-M), a muscarinic agonist, suppressed IPSCs in a subset of neuron pairs. This suppression was associated with an increase in paired-pulse ratio, blocked by the M-2-prefering antagonist gallamine, and was totally absent in neuron pairs from M-2-knockout mice. When CB1 receptors were not blocked, oxo-M suppressed IPSCs in a gallamine-resistant manner in cannabinoid-sensitive pairs. This suppression was associated with an increase in paired-pulse ratio, blocked by the CB1 antagonist AM281, and was completely eliminated in neuron pairs from M-1/M-3-compound-knockout mice. Our immunohistochemical examination showed that M-2 and CB1 receptors were present at inhibitory presynaptic terminals of mostly different origins. These results indicate that two distinct mechanisms mediate the muscarinic suppression. In a subset of synapses, activation of M-2 receptors at presynaptic terminals suppresses GABA release directly. In contrast, in a different subset of synapses, activation of M-1/M-3 receptors causes endocannabinoid production and subsequent suppression of GABA release by activating presynaptic CB1 receptors. Thus, the muscarinic system can influence hippocampal functions by controlling different subsets of inhibitory synapses through the two distinct mechanisms.
  • Kotaro Hama, Junken Aoki, Masahiro Fukaya, Yasuhiro Kishi, Teruyuki Sakai, Rika Suzuki, Hideo Ohta, Takao Yamori, Masahiko Watanabe, Jerold Chun, Hiroyuki Arai
    The Journal of biological chemistry 17 279 (17) 17634 - 9 0021-9258 2004/04/23 [Refereed][Not invited]
     
    Autotaxin (ATX) is a tumor cell motility-stimulating factor originally isolated from melanoma cell supernatant that has been implicated in regulation of invasive and metastatic properties of cancer cells. Recently, we showed that ATX is identical to lysophospholipase D, which converts lysophosphatidylcholine to a potent bioactive phospholipid mediator, lysophosphatidic acid (LPA), raising the possibility that autocrine or paracrine production of LPA by ATX contributes to tumor cell motility. Here we demonstrate that LPA and ATX mediate cell motility-stimulating activity through the LPA receptor, LPA(1). In fibroblasts isolated from lpa(1)(-/-) mice, but not from wild-type or lpa(2)(-/-), cell motility stimulated with LPA and ATX was completely absent. In the lpa(1)(-/-) cells, LPA-stimulated lamellipodia formation was markedly diminished with a concomitant decrease in Rac1 activation. LPA stimulated the motility of multiple human cancer cell lines expressing LPA(1), and the motility was attenuated by an LPA(1)-selective antagonist, Ki16425. The present study suggests that ATX and LPA(1) represent potential targets for cancer therapy.
  • J Somogyi, A Baude, Y Omori, H Shimizu, S El Mestikawy, M Fukaya, R Shigemoto, M Watanabe, P Somogyi
    EUROPEAN JOURNAL OF NEUROSCIENCE 19 (3) 552 - 569 0953-816X 2004/02 [Refereed][Not invited]
     
    Vesicular glutamate transporter type 3 (VGLUT3) containing neuronal elements were characterized using antibodies to VGLUT3 and molecular cell markers. All VGLUT3-positive somata were immunoreactive for CCK, and very rarely, also for calbindin; none was positive for parvalbumin, calretinin, VIP or somatostatin. In the CA1 area, 26.8 +/- 0.7% of CCK-positive interneuron somata were VGLUT3-positive, a nonoverlapping 22.8 +/- 1.9% were calbindin-positive, 10.7 +/- 2.5% VIP-positive and the rest were only CCK-positive. The patterns of coexpression were similar in the CA3 area, the dentate gyrus and the isocortex. Immunoreactivity for VGLUT3 was undetectable in pyramidal and dentate granule cells. Boutons colabelled for VGLUT3, CCK and GAD were most abundant in the cellular layers of the hippocampus and in layers II-III of the isocortex. Large VGLUT3-labelled boutons at the border of strata radiatum and lacunosum-moleculare in the CA1 area were negative for GAD, but were labelled for vesicular monoamine transporter type 2, plasmalemmal serotonin transporter or serotonin. No colocalization was found in terminals between VGLUT3 and parvalbumin, vesicular acetylcholine transporter and group III (mGluR7a,b; mGluR8a,b) metabotropic glutamate receptors. In stratum radiatum and the isocortex, VGLUT3-positive but GAD-negative boutons heavily innervated the soma and proximal dendrites of some VGLUT3- or calbindin-positive interneurons. The results suggest that boutons coexpressing VGLUT3, CCK and GAD originate from CCK-positive basket cells, which are VIP-immunonegative. Other VGLUT3-positive boutons immunopositive for serotonergic markers but negative for GAD probably originate from the median raphe nucleus and innervate select interneurons. The presumed amino acid substrate of VGLUT3 may act on presynaptic kainate or group II metabotropic glutamate receptors.
  • T Miyazaki, K Hashimoto, HS Shin, M Kano, M Watanabe
    JOURNAL OF NEUROSCIENCE 24 (7) 1734 - 1743 0270-6474 2004/02 [Refereed][Not invited]
     
    Synapse formation depends critically on the competition among inputs of multiple sources to individual neurons. Cerebellar Purkinje cells have highly organized synaptic wiring from two distinct sources of excitatory afferents. Single climbing fibers innervate proximal dendrites of Purkinje cells, whereas numerous parallel fibers converge on their distal dendrites. Here, we demonstrate that the P/Q-type Ca2+ channel alpha1A, a major Ca2+ channel subtype in Purkinje cells, is crucial for this organized synapse formation. In the alpha1A knock-out mouse, many ectopic spines were protruded from proximal dendrites and somata of Purkinje cells. Innervation territory of parallel fibers was expanded proximally to innervate the ectopic spines, whereas that of climbing fibers was regressed to the basal portion of proximal dendrites and somata. Furthermore, multiple climbing fibers consisting of a strong climbing fiber and one or a few weaker climbing fibers, persisted in the majority of Purkinje cells and were cowired to the same somata, proximal dendrites, or both. Therefore, the lack of alpha1A results in the persistence of parallel fibers and surplus climbing fibers, which should normally be expelled from the compartment innervated by the main climbing fiber. These results suggest that a P/Q-type Ca2+ channel alpha1A fuels heterosynaptic competition between climbing fibers and parallel fibers and also fuels homosynaptic competition among multiple climbing fibers. This molecular function facilitates the distal extension of climbing fiber innervation along the dendritic tree of the Purkinje cell and also establishes climbing fiber monoinnervation of individual Purkinje cells.
  • T Miyazaki, K Hashimoto, HS Shin, M Kano, M Watanabe
    JOURNAL OF NEUROSCIENCE 24 (7) 1734 - 1743 0270-6474 2004/02 [Refereed][Not invited]
     
    Synapse formation depends critically on the competition among inputs of multiple sources to individual neurons. Cerebellar Purkinje cells have highly organized synaptic wiring from two distinct sources of excitatory afferents. Single climbing fibers innervate proximal dendrites of Purkinje cells, whereas numerous parallel fibers converge on their distal dendrites. Here, we demonstrate that the P/Q-type Ca2+ channel alpha1A, a major Ca2+ channel subtype in Purkinje cells, is crucial for this organized synapse formation. In the alpha1A knock-out mouse, many ectopic spines were protruded from proximal dendrites and somata of Purkinje cells. Innervation territory of parallel fibers was expanded proximally to innervate the ectopic spines, whereas that of climbing fibers was regressed to the basal portion of proximal dendrites and somata. Furthermore, multiple climbing fibers consisting of a strong climbing fiber and one or a few weaker climbing fibers, persisted in the majority of Purkinje cells and were cowired to the same somata, proximal dendrites, or both. Therefore, the lack of alpha1A results in the persistence of parallel fibers and surplus climbing fibers, which should normally be expelled from the compartment innervated by the main climbing fiber. These results suggest that a P/Q-type Ca2+ channel alpha1A fuels heterosynaptic competition between climbing fibers and parallel fibers and also fuels homosynaptic competition among multiple climbing fibers. This molecular function facilitates the distal extension of climbing fiber innervation along the dendritic tree of the Purkinje cell and also establishes climbing fiber monoinnervation of individual Purkinje cells.
  • Kazuyuki Yoshida, Shigeki Furuya, Soh Osuka, Junya Mitoma, Yoko Shinoda, Masahiko Watanabe, Norihiro Azuma, Hideyuki Tanaka, Tsutomu Hashikawa, Shigeyoshi Itohara, Yoshio Hirabayashi
    Journal of Biological Chemistry 279 (5) 3573 - 3577 0021-9258 2004/01/30 [Refereed][Not invited]
     
    D-3-Phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95) is the first committed enzyme Of L-serine biosynthesis in the phosphorylated pathway. To determine the physiological importance of Phgdh-dependent L-serine biosynthesis in vivo, we generated Phgdh-deficient mice using targeted gene disruption in embryonic stem cells. The absence of Phgdh led to a drastic reduction of L-serine metabolites such as phosphatidyl-L-serine and sphingolipids. Phgdh null embryos have small bodies with abnormalities in selected tissues and died after days post-coitum 13.5. Striking abnormalities were evident in the central nervous system in which the Phgdh null mutation culminated in hypoplasia of the telencephalon, diencephalon, and mesencephalon; in particular, the olfactory bulbs, ganglionic eminence, and cerebellum appeared as indistinct structures. These observations demonstrate that the Phgdh-dependent phosphorylated pathway is essential for normal embryonic development, especially for brain morphogenesis.
  • N Kinoshita, K Kimura, N Matsumoto, M Watanabe, M Fukaya, C Ide
    GENES TO CELLS 9 (1) 1 - 14 1356-9597 2004/01 [Refereed][Not invited]
     
    Sept2 is a member of the septin family of GTPases. Septins form filaments in a GTP-form dependent manner, and are involved in cytokinesis from yeast to mammals; however, some mammalian septins, including Sept2, are expressed in the brain, a tissue in which almost all the cells are postmitotic. Recently, some functions of mammalian septin other than cytokinesis such as vesicle transport have been reported. However, mammalian septin's physiological functions are still unclear. The present study revealed that Sept2 co-localizes with the astrocyte glutamate transporter GLAST in the Bergmann glial processes facing axons and synapses. Biochemical analyses demonstrated that Sept2 bound directly to the carboxy-terminal region of GLAST in a GDP-form dependent manner. Expression of constitutive GDP-form Sept2 mutant reduced the glutamate uptake activity of GLAST via internalization of GLAST from cell surface. Thus Sept2 may regulate GLAST-mediated glutamate uptake by astrocytes, which is important for appropriate transmitter signalling in the cerebellum.
  • K Hosoya, T Nakashima, M Tachikawa, T Terasaki, M Watanabe, M Tomi
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 124 85 - 88 0031-6903 2004 [Refereed][Not invited]
     
    Creatine may play an essential role in ATP homeostasis in the retina. The purpose of this study was to elucidate the mechanisms of supplying of creatine from blood across the blood-retinal barrier (BRB) and biosynthesis of creatine in the retina. In vivo intravenous administration analysis revealed that creatine is transported across the BRB and accumulated in the retina. Creatine uptake study using TR-iBRB2 cells, which are an in vitro model of the inner BRB, suggested that creatine transporter (CRT). is involved in creatine transport at the inner BRB. Western blot and immunoperoxidase electron microscopic analyses demonstrated that CRT is expressed in the retina and TR-iBRB2 cells and localized in rat retinal endothelial cells. HPLC study revealed that creatine is synthesized in the retina and TR-MUL5 cells, which are a rat Muller cell line. RT-PCR and Western blot analyses demonstrated that L-arginine:glycine amidinotransferase (AGAT) and S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT), which are enzymes for creatine biosynthesis, are expressed in the retina and TR-MUL5 cells. Confocal immunofluorescent microscopy analysis demonstrated that GAMT is predominantly expressed in retinal Muller cells. These findings are consistent with the involvement of CRT-mediated creatine transport at the inner BRB and biosynthesis of creatine in Muller cells.
  • F Ferraguti, P Cobden, M Pollard, D Cope, R Shigemoto, M Watanabe, P Somogyi
    HIPPOCAMPUS 14 (2) 193 - 215 1050-9631 2004 [Refereed][Not invited]
     
    In the hippocampal CA1 region, metabotropic glutamate subtype 1 (mGluR1) receptors have been implicated in a variety of phyiological responses to glutamate, which include modulation of synaptic transmission and plasticity, as well as neuronal excitability and synchronization. The mGluR1alpha isoform is characteristically expressed only by nonprincipal cells, and it is particularly enriched in somatostatin (SS)-containing interneurons in stratum oriens-alveus. Anatomical and physiological data have indicated the presence of mGluR1alpha in several distinct classes of interneurons with their somata located also in strata pyramidale, radiatum, and lacunosum moleculare. Each different interneuron subtype, as defined by functionally relevant criteria, including input/ output characteristics and expression of selective molecular markers, subserves distinct functions in local hippocampal circuits. We have investigated which of the different CA1 interneuron classes express mGluR1alpha by immunofluorescent labeling, combining antibodies to mGluR1alpha, calcium-binding proteins, and neuropeptides, and by intracellular labeling in vitro. Several types of interneuron that are immunopositive for mGluR1alpha each targeted different domains of pyramidal cells and included (1) O-LM interneurons, found to coexpress both SS and parvalbumin (PV); (2) interneurons with target selectivity for other interneurons, expressing vasoactive intestinal polypeptide (VIP) and/or the calcium-binding protein calretinin; (3) pro-cholecystokinin-immunopositive interneurons probably non-basket and dendrite-targeting; and (4) an as-yet unidentified SS-immunoreactive but PV-immunonegative interneuron class, possibly corresponding to oriens-bistratified cells. Estimation of the relative proportion of mGluR1alpha-positive interneurons showed 43%, 46%, and 30% co-labeling with SS, VIP, or PV, respectively. The identification of the specific subclasses of CA1 interneurons expressing mGluR1alpha provides the network basis for assessing the contribution of this receptor to the excitability of the hippocampus. (C) 2004 Wiley-Liss, Inc.
  • Noboru Yamashita, Kazuhisa Sakai, Shigeki Furuya, Masahiko Watanabe
    Archives of Histology and Cytology 66 (5) 429 - 436 0914-9465 2003/12 [Refereed][Not invited]
     
    Non-essential amino acid L-serine functions as a highly potent, glia-derived neurotrophic factor, because it is a precursor for syntheses of proteins, other amino acids, membrane lipids, and nucleotides, and also because its biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH) is preferentially expressed in particular glial cells within the brain. Here we pursued 3PGDH expression in peripheral nerves and its change after crush injury. In the pathway of rat sciatic nerves, 3PGDH was selectively expressed in non-neuronal elements: Schwann sheaths and endoneurial fibroblasts in sciatic nerves, satellite cells in dorsal root ganglia, and astrocytes and oligodendrocytes in the spinal ventral horn. In contrast, 3PGDH was immunonegative in axons, somata of spinal motoneurons and ganglion cells, and endoneurial macrophages. One week after crush injury, 3PGDH was upregulated in the distal segment of injured nerves, where 3PGDH was intensified in activated Schwann cells and fibroblasts. 3PGDH was still negative in activated macrophages, which were instead associated or surrounded by activated Schwann cells with intensified 3PGDH. These results suggest that in the peripheral nervous system, these non-neuronal cells synthesize and may supply L-serine to satisfy metabolic demands for maintenance and regeneration of peripheral nerves and for proliferation and activation of macrophages upon nerve injury.
  • Noboru Yamashita, Kazuhisa Sakai, Shigeki Furuya, Masahiko Watanabe
    Archives of Histology and Cytology 66 (5) 429 - 436 0914-9465 2003/12 [Refereed][Not invited]
     
    Non-essential amino acid L-serine functions as a highly potent, glia-derived neurotrophic factor, because it is a precursor for syntheses of proteins, other amino acids, membrane lipids, and nucleotides, and also because its biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH) is preferentially expressed in particular glial cells within the brain. Here we pursued 3PGDH expression in peripheral nerves and its change after crush injury. In the pathway of rat sciatic nerves, 3PGDH was selectively expressed in non-neuronal elements: Schwann sheaths and endoneurial fibroblasts in sciatic nerves, satellite cells in dorsal root ganglia, and astrocytes and oligodendrocytes in the spinal ventral horn. In contrast, 3PGDH was immunonegative in axons, somata of spinal motoneurons and ganglion cells, and endoneurial macrophages. One week after crush injury, 3PGDH was upregulated in the distal segment of injured nerves, where 3PGDH was intensified in activated Schwann cells and fibroblasts. 3PGDH was still negative in activated macrophages, which were instead associated or surrounded by activated Schwann cells with intensified 3PGDH. These results suggest that in the peripheral nervous system, these non-neuronal cells synthesize and may supply L-serine to satisfy metabolic demands for maintenance and regeneration of peripheral nerves and for proliferation and activation of macrophages upon nerve injury.
  • N Kitanaka, Y Owada, SA Abdelwahab, H Iwasa, H Sakagami, M Watanabe, F Spener, H Kondo
    HISTOCHEMISTRY AND CELL BIOLOGY 120 (6) 465 - 473 0948-6143 2003/12 [Refereed][Not invited]
     
    Dendritic cells in the splenic white pulp of mice were intensely immunoreactive for epidermal-type fatty acid binding protein (E-FABP). This specific immunostaining revealed a clear difference in morphology between the dendritic cells in the periarterial lymphoid sheath (PALS) and follicular dendritic cells in the follicles in terms of cell sizes and process branching. No immunoreactivity was detected in dendritic cells in the marginal zones and the red pulp, although endothelial cells of almost all capillaries in the red pulp were immunoreactive for E-FABP. After peritoneal injection of lipopolysaccharide, the immunoreactive cells in PALS progressively enlarged and became rounded in shape with a peak in size at 24 h postinjection and they eventually resumed the dendritic form at 48 h postinjection. Within each of the enlarged immunoreactive cell perikarya were included small immunonegative apoptotic cells, presumptive lymphocytes. Taken together, E-FABP is useful as a marker for dendritic cells in the splenic white pulp, and may be involved through combination with fatty acids in antigen presentation and retention as well as in cytokine production.
  • H Jourdi, Y Iwakura, M Narisawa-Saito, K Ibaraki, HB Xiong, M Watanabe, Y Hayashi, N Takei, H Nawa
    DEVELOPMENTAL BIOLOGY 263 (2) 216 - 230 0012-1606 2003/11 [Refereed][Not invited]
     
    Postsynaptic molecules with PDZ domains (PDZ proteins) interact with various glutamate receptors and regulate their subcellular trafficking and stability. In rat neocortical development, the protein expression of AMPA-type glutamate receptor GluR1 lagged behind its mRNA expression and rather paralleled an increase in PDZ protein levels. One of the neurotrophins, brain-derived neurotrophic factor (BDNF), appeared to contribute to this process, regulating the PDZ protein expression. In neocortical cultures, BDNF treatment upregulated SAP97, GRIP1, and Pick1 PDZ proteins. Conversely, BDNF gene targeting downregulated these same PDZ molecules. The BDNF-triggered increases in PDZ proteins resulted in the elevation of their total association with the AMPA receptors GluR1 and GluR2/3, which led to the increase in AMPA receptor proteins. When Sindbis viruses carrying GluR1 or GluR2 C-terminal decoys disrupted their interactions, GluR2 C-terminal decoys inhibited both BDNF-triggered GluR1 and GluR2/3 increases, whereas GluR1 C-terminal decoys blocked only the BDNF-triggered GluR1 increase. In agreement, coexpression of SAP97 and GluR1 in nonneuronal HEK293 cells increased both proteins compared with their single transfection, implying mutual stabilization. This work reveals a novel function of BDNF in postsynaptic development by regulating the PDZ protein expression. (C) 2003 Elsevier Inc. All rights reserved.
  • Y Takahashi, H Mori, M Mishina, M Watanabe, T Fujiwara, J Shimomura, H Aiba, T Miyajima, Y Saito, A Nezu, H Nishida, K Imai, N Sakaguchi, N Kondo
    NEUROLOGY 61 (7) 891 - 896 0028-3878 2003/10 [Refereed][Not invited]
     
    Background: Antibody-mediated and cytotoxic T cell-mediated pathogenicity have been implicated as the autoimmune pathophysiologic mechanisms in Rasmussen's encephalitis. Methods: The authors investigated autoantibodies against the NMDA glutamate receptor (GluR) epsilon2 subunit and their epitopes in serum and CSF samples from 15 patients with chronic epilepsia partialis continua (EPC), 17 with West syndrome, 10 with Lennox-Gastaut syndrome, and 11 control subjects. Results: In 15 patients with chronic EPC, we detected NMDA-type GluR epsilon2 autoantibodies in histologically proven Rasmussen's encephalitis (3/3 patients), clinical Rasmussen's encephalitis (6/7 patients), acute encephalitis/encephalopathy (2/3 patients), and nonprogressive EPC (2/2 patients). Serum IgM autoantibodies were found in the early phase of EPC and became negative later in four patients. The autoantibodies were not detected in West syndrome, Lennox-Gastaut syndrome, or controls. Among 10 patients with histologically proven or clinical Rasmussen's encephalitis, epitope analyses showed that the autoantibodies were predominantly against C-terminal epitopes and rarely against N-terminal epitope, with inconsistency in profile during the courses of disease. Epitope recognition spectrum of autoantibodies was broader in CSF than in serum, and the serum or CSF profile showed an increase in number of epitopes as disease progressed in some patients. Conclusions: The presence of autoantibodies against NMDA GluR epsilon2 suggests autoimmune pathologic mechanisms but is not a hallmark of Rasmussen's encephalitis. Patients with Rasmussen's encephalitis may have autoantibodies against several neural molecules, and these autoantibodies may be produced in the CNS after cytotoxic T cell-mediated neuronal damage.
  • M Miyata, H Kashiwadani, M Fukaya, T Hayashi, DQ Wu, T Suzuki, M Watanabe, Y Kawakami
    JOURNAL OF NEUROSCIENCE 23 (22) 8098 - 8108 0270-6474 2003/09 [Refereed][Not invited]
     
    Phospholipase C (PLC) beta4, one of the four isoforms of PLCbetas, is the sole isoform expressed in the mouse ventral posterolateral thalamic nucleus (VPL), a key station in pain processing. The mouse thalamus also has been shown to express a high level of metabotropic glutamate receptor type 1 (mGluR1), which stimulates PLCbetas through activation of Galphaq/11 protein. It is therefore expected that the thalamic mGluR1-PLCbeta4 cascade may play a functional role in nociceptive transmission. To test this hypothesis, we first studied behavioral responses to various nociceptive stimuli in PLCbeta4 knock-out mice. We performed the formalin test and found no difference in the pain behavior in the first phase of the formalin test, which is attributed to acute nociception, between PLCbeta4 knock-out and wild-type mice. Consistent with this result, acute pain responses in the hot plate and tail flick tests were also unaffected in the PLCbeta4 knock-out mice. However, the nociceptive behavior in the second phase of the formalin test, resulting from the tissue inflammation, was attenuated in PLCbeta4 knock-out mice. In the dorsal horn of the spinal cord where PLCbeta1 and PLCbeta4 mRNAs are expressed, no difference was found between the wild-type and knock-out mice in the number of Fos-like immunoreactive neurons, which represent neuronal activity in the second phase in the formalin test. Thus, it is unlikely that spinal PLCbeta4 is involved in the formalin-induced inflammatory pain. Next, we found that pretreatment with PLC inhibitors, mGluR1 antagonists, or both, by either intracerebroventricular or intrathalamic injection, attenuated the formalin-induced pain behavior in the second phase in wild-type mice. Furthermore, activation of mGluR1 at the VPL enhanced pain behavior in the second phase in the wild-type mice. In contrast, PLCbeta4 knock-out mice did not show such enhancement, indicating that mGluR1 is connected to PLCbeta4 in the VPL. Finally, in parallel with the behavioral results, we showed in an electrophysiological study that the time course of firing discharges in VPL corresponds well to that of pain behavior in the formalin test in both wild-type and PLCbeta4 knock-out mice. These findings indicate that the thalamic mGluR1-PLCbeta4 cascade is indispensable for the formalin-induced inflammatory pain by regulating the response of VPL neurons.
  • M Sassoe-Pognetto, JK Utvik, P Camoletto, M Watanabe, FA Stephenson, DS Bredt, OP Ottersen
    JOURNAL OF COMPARATIVE NEUROLOGY 463 (3) 237 - 248 0021-9967 2003/08 [Refereed][Not invited]
     
    Glutamate neurotransmission in the olfactory bulb involves both axodendritic synapses and dendrodendritic reciprocal synapses and possibly also extrasynaptic receptors. By using a sensitive immunogold procedure, we have investigated the organization of two synaptic scaffolding molecules, PSD-95 and PSD-93, as well as N-methyl-D-aspartate (NMDA) and ot-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors, at these heterogeneous glutamate signaling sites. Immunolabeling for PSD-95 and PSD-93 was present in all major types of putative glutamatergic synapse, suggesting that these proteins are essential components of the synaptic signaling apparatus. The linear density and the subsynaptic distribution of PSD-95/PSD-93 gold particles did not differ significantly between axodendritic and dendrodendritic synapses. Antibodies recognizing NMDA and AMPA receptor subunits also labeled asymmetric synapses throughout the olfactory bulb. Immunolabeling for the AMPA receptor subunits GluR2/3 was similar in all types of synapse. In contrast, immunogold signals for the NR1 subunit of NMDA receptors varied significantly among different synapse populations, with olfactory nerve synapses in the glomerular layer showing the lowest labeling intensity. Although the lateral dendrites of mitral and tufted cells have been reported to respond to glutamate, they did not display significant plasma membrane labeling for the NR1 subunit or for PSD-95, suggesting that the physiological effects of glutamate at these sites are mediated by NMDA autoreceptors that are not clustered and occur only at a low density on the dendritic surface. Our quantitative analysis of olfactory bulb synapses indicates that the density of NMDA receptors is not determined by the complement of PSD-95/PSD-93. The latter molecules appear to be expressed in an all-or-none fashion and may form a standard lattice common to different types of glutamatergic synapse.
  • P Somogyi, Y Dalezios, R Lujan, JDB Roberts, M Watanabe, R Shigemoto
    EUROPEAN JOURNAL OF NEUROSCIENCE 17 (12) 2503 - 2520 0953-816X 2003/06 [Refereed][Not invited]
     
    The release of neurotransmitters is modulated by presynaptic metabotropic glutamate receptors (mGluRs), which show a highly selective expression and subcellular location in glutamatergic terminals in the hippocampus. Using immunocytochemistry, we investigated whether one of the receptors, mGluR7, whose level of expression is governed by the postsynaptic target, was present in GABAergic terminals and whether such terminals targeted particular cells. A total of 165 interneuron dendritic profiles receiving 466 synapses (82% mGluR7a-positive) were analysed. The presynaptic active zones of most GAD-(77%) or GABA-positive (94%) synaptic boutons on interneurons innervated by mGluR7a-enriched glutamatergic terminals (mGluR7a-decorated) were immunopositive for mGluR7a. GABAergic terminals on pyramidal cells and most other interneurons in str. oriens were mGluR7a-immunonegative. The mGluR7a-decorated cells were mostly somatostatin- and mGluR1alpha-immunopositive neurons in str. oriens and the alveus. Their GABAergic input mainly originated from VIP-positive terminals, 90% of which expressed high levels of mGluR7a in the presynaptic active zone. Parvalbumin-positive synaptic terminals were rare on mGluR7a-decorated cells, but on these neurons 73% of them were mGluR7a-immunopositive. Some type II synapses innervating interneurons were immunopositive for mGluR7b, as were some type I synapses. Because not all target cells of VIP-positive neurons are known it has not been possible to determine whether mGluR7 is expressed in a target-cell-specific manner in the terminals of single GABAergic cells. The activation of mGluR7 may decrease GABA release to mGluR7-decorated cells at times of high pyramidal cell activity, which elevates extracellular glutamate levels. Alternatively, the presynaptic receptor may be activated by as yet unidentified endogenous ligands released by the GABAergic terminals or the postsynaptic dendrites.
  • T Miyazaki, M Fukaya, H Shimizu, M Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 17 (12) 2563 - 2572 0953-816X 2003/06 [Refereed][Not invited]
     
    Two subtypes of the vesicular glutamate transporter are expressed differentially in two excitatory afferents synapsing on to Purkinje cells: VGluT1 (BNPI) in axon terminals of cerebellar granule cells (i.e. parallel fibres; PFs) and VGluT2 (DNPI) in those of the inferior olivary neurons (climbing fibres; CFs). In the present study, we examined their expression in the developing mouse cerebellum. By in situ hybridization, the inferior olivary nucleus selectively expressed VGluT2 mRNA through postnatal life. In the cerebellum, both subtypes were transcribed in the external and internal granular layers during the first postnatal week. Thereafter, VGluT1 mRNA showed marked upregulation in the internal granular layer, whereas VGluT2 mRNA disappeared from the external and internal granular layers by the end of the third postnatal week. By immunohistochemistry, CF terminals consistently exhibited VGluT2 immunoreactivity in the postnatal cerebellum. By contrast, in the first 10 days of postnatal life, VGluT2 predominated in PF terminals, despite the transcription of both transporters in developing granule cells. During the second 10 days, VGluT2 in PF terminals was replaced with VGluT1 from deep regions of the molecular layer upwards, correlating with dendritic translocation of CFs. This replacement was accomplished by postnatal day 30. Taking that late-borne PFs are laid down successively on earlier ones in the molecular layer, the deep-to-superficial replacement represents maturation-linked switching from VGluT2 to VGluT1 in individual PFs, and is likely to be regulated at both the transcription and translation levels.
  • T Miyazaki, M Fukaya, H Shimizu, M Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 17 (12) 2563 - 2572 0953-816X 2003/06 [Refereed][Not invited]
     
    Two subtypes of the vesicular glutamate transporter are expressed differentially in two excitatory afferents synapsing on to Purkinje cells: VGluT1 (BNPI) in axon terminals of cerebellar granule cells (i.e. parallel fibres; PFs) and VGluT2 (DNPI) in those of the inferior olivary neurons (climbing fibres; CFs). In the present study, we examined their expression in the developing mouse cerebellum. By in situ hybridization, the inferior olivary nucleus selectively expressed VGluT2 mRNA through postnatal life. In the cerebellum, both subtypes were transcribed in the external and internal granular layers during the first postnatal week. Thereafter, VGluT1 mRNA showed marked upregulation in the internal granular layer, whereas VGluT2 mRNA disappeared from the external and internal granular layers by the end of the third postnatal week. By immunohistochemistry, CF terminals consistently exhibited VGluT2 immunoreactivity in the postnatal cerebellum. By contrast, in the first 10 days of postnatal life, VGluT2 predominated in PF terminals, despite the transcription of both transporters in developing granule cells. During the second 10 days, VGluT2 in PF terminals was replaced with VGluT1 from deep regions of the molecular layer upwards, correlating with dendritic translocation of CFs. This replacement was accomplished by postnatal day 30. Taking that late-borne PFs are laid down successively on earlier ones in the molecular layer, the deep-to-superficial replacement represents maturation-linked switching from VGluT2 to VGluT1 in individual PFs, and is likely to be regulated at both the transcription and translation levels.
  • H Nishimura, H Sakagami, A Uezu, K Fukunaga, M Watanabe, H Kondo
    JOURNAL OF NEUROCHEMISTRY 85 (5) 1216 - 1227 0022-3042 2003/06 [Refereed][Not invited]
     
    Ca2+ /calmodulin-dependent protein kinase I (CaMKI), originally identified as a protein kinase phoshorylating synapsin I, has been shown to constitute a family of closely related isoforms (alpha, beta and gamma). Here, we have isolated and determined the complete primary structures of two alternatively splicing isoforms of CaMKI termed CaMKIgamma1 and -gamma2. CaMKIgamma1 and -gamma2 contain an identical N-terminal catalytic domain with different C-terminal regions due to the deletion of the 425-bp nucleotide sequence of CaMKIgamma1 in CaMKIgamma2. In vitro kinase assay has demonstrated the marked enhancement of the Ca2+ /CaM-dependent activity of CaMKIgamma1 by the preincubation with Ca2+ /calmodulin-dependent protein kinase kinase (CaMKK), but no significant activation of CaMKIgamma2. Northern blot analysis has demonstrated the predominant expression of CaMKIgamma in the brain. RT-PCR analysis has revealed similar expression patterns between CaMKIgamma1 and CaMKIgamma2 in various brain regions. In situ hybridization analysis has demonstrated that CaMKIgamma mRNA is expressed in a distinct pattern from other isoforms of CaMKI with predominant expression in some restricted brain regions such as the olfactory bulb, hippocampal pyramidal cell layer of CA3, central amygdaloid nuclei, ventromedial hypothalamic nucleus and pineal gland. In the primary hippocampal neurons and NG108-15 cells, transfected CaMKIgamma1 and -gamma2 are localized primarily in the cytoplasm and neurites but not in the nucleus. These findings suggest that both isoforms of CaMKIgamma may be involved in Ca2+ signal transduction in the cytoplasmic compartment of certain neuronal population.
  • Shigeki Furuya, Masahiko Watanabe
    Archives of Histology and Cytology 66 (2) 109 - 121 0914-9465 2003/05 [Refereed][Not invited]
     
    L-Serine is a non-essential amino acid that can be synthesized in the body. It derives from an intermediate of the glycolytic pathway, 3-phosphoglycerate, and utilized for the syntheses of proteins, other amino acids, membrane lipids, heme, and nucleotides. Emerging evidence indicates that L-serine functions as a glia-derived trophic factor, which strongly promotes the survival and differentiation of cultured neurons. L-Serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH) and small neutral amino acid transporter ASCT1 have been revealed to be expressed preferentially in the radial glia-astrocyte lineage and olfactory ensheathing glia of both adult and developing rodent brains. In contrast, these biosynthetic and transporter molecules for L-serine are faint or undetectable in neurons and phagocytic cells. In this review, we summarize recent progress to propose that L-serine synthesis in these glial cells and its supply to nearby neurons and other glia constitute a novel metabolic unit in the brain. Based on these neuroglial and glioglial relationships, glucose in neurons and phogocytes can be strategically used for energy production, while a variety of L-serine-derived biomolecules required for their proliferaton, survival, differentiation, and function are synthesized in and supplied from the radial glia-astrocyte lineage and olfactory ensheathing glia. A transient capillary expression of ASCT1 in fetal and neonatal brains further suggests that, in addition to the glia-borne L-serine, an active transport of blood-borne L-serine would play an essential role in neural development.
  • M Fukaya, A Kato, C Lovett, S Tonegawa, M Watanabe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 (8) 4855 - 4860 0027-8424 2003/04 [Refereed][Not invited]
     
    Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system, and the N-methyl-D-aspartate-selective glutamate receptor (NR) consisting of the NR1 subunit and an NR2 or NR3 subunit plays crucial roles in synaptic transmission, plasticity, and learning and memory. By using a knockout mouse strain, in which the NR1 gene deletion is primarily targeted to the CA1 pyramidal cells of the hippocampus, we investigated the in vivo effect of the loss of the NR1 subunit on the cellular expression and intracellular distribution of the NR2 subunits. The NR1 gene deletion had no apparent effect on the levels of NR2A or NR2B mRNA but led to severe reductions of NR2A and NR2B protein in dendrites of CA1 pyramidal cells. This reduced dendritic distribution of the NR2 subunits accompanied their robust accumulation in perikarya, where they were condensed in the lumen of the endoplasmic reticulum as electron-dense granules. These granules were also observed in CA1 pyramidal cells of the control mice but they were much fewer and contained no detectable levels of the NR2 subunit. The effect of the NR1 knockout on intracellular localization of the NR2 subunits was specific in that no such effect was observed for the GluR1 and PSD-95, two other major postsynaptic proteins. These results suggest that the NR1 subunit plays a crucial role in the release of the NR2 subunit from the endoplasmic reticulum in hippocampal pyramidal cells in vivo, and when the NR1 subunit is unavailable, the NR2 subunits are retained and aggregate into intracisternal granules.
  • M Fukaya, A Kato, C Lovett, S Tonegawa, M Watanabe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 (8) 4855 - 4860 0027-8424 2003/04 [Refereed][Not invited]
     
    Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system, and the N-methyl-D-aspartate-selective glutamate receptor (NR) consisting of the NR1 subunit and an NR2 or NR3 subunit plays crucial roles in synaptic transmission, plasticity, and learning and memory. By using a knockout mouse strain, in which the NR1 gene deletion is primarily targeted to the CA1 pyramidal cells of the hippocampus, we investigated the in vivo effect of the loss of the NR1 subunit on the cellular expression and intracellular distribution of the NR2 subunits. The NR1 gene deletion had no apparent effect on the levels of NR2A or NR2B mRNA but led to severe reductions of NR2A and NR2B protein in dendrites of CA1 pyramidal cells. This reduced dendritic distribution of the NR2 subunits accompanied their robust accumulation in perikarya, where they were condensed in the lumen of the endoplasmic reticulum as electron-dense granules. These granules were also observed in CA1 pyramidal cells of the control mice but they were much fewer and contained no detectable levels of the NR2 subunit. The effect of the NR1 knockout on intracellular localization of the NR2 subunits was specific in that no such effect was observed for the GluR1 and PSD-95, two other major postsynaptic proteins. These results suggest that the NR1 subunit plays a crucial role in the release of the NR2 subunit from the endoplasmic reticulum in hippocampal pyramidal cells in vivo, and when the NR1 subunit is unavailable, the NR2 subunits are retained and aggregate into intracisternal granules.
  • T Shibata, M Watanabe, R Ichikawa, K Ameda, T Koyanagi
    DEVELOPMENTAL BRAIN RESEARCH 140 (2) 263 - 268 0165-3806 2003/02 [Refereed][Not invited]
     
    Physiological studies have shown that lower urinary tract function is regulated through glutamate receptors at the levels of spinal and supraspinal cord. Of the receptor family, N-methyl-D-aspartate (NMDA) receptors mediate activity-dependent changes of synaptic efficacy, underlying synaptic plasticity and synapse development. To know the ontogenic changes of NMDA receptor expression in the visceromotor system innervating pelvic organs, including the bladder, we employed double labeling technique of retrograde neuronal tracing and in situ hybridization for detecting NMDA subunit mRNAs in preganglionic neurons (PGNs) of the lumbosacral cord. Rats at postnatal day 7 (P7), 14 (P14), 21 (P21), and adult were used. In situ hybridization was conducted using S-35-labeled antisense oligonucleotides specific to mRNAs for NMDA receptor subunits. Hybridizing signals in PGNs were detected by a dark-field microscope equipped fluorescence detector. PGNs showed strong signals for NR1 subunit mRNA at each developmental stage examined. Moderate signals for the NR2B and NR2D subunit mRNAs were found in PGNs at P7. However, their expression levels decreased thereafter, reaching the minimal level in adults. No significant signals for NR2A and NR2C subunit mRNAs were detected at any stages. This temporal pattern of expression suggests a possible involvement of NMDA receptors in the development of micturitional neural circuit through activity-dependent mechanisms. (C) 2002 Elsevier Science B.V. All rights reserved.
  • S Kakizawa, K Yamada, M Iino, M Watanabe, M Kano
    EUROPEAN JOURNAL OF NEUROSCIENCE 17 (3) 545 - 554 0953-816X 2003/02 [Refereed][Not invited]
     
    Functional neural circuit formation includes the process by which redundant synaptic connections formed earlier during development are subsequently eliminated. We report that insulin-like growth factor I (IGF-I) is a candidate factor that influences the developmental transition from multiple to mono innervation of cerebellar Purkinje cells (PCs) by climbing fibres (CFs). Continuous local application of exogenous IGF-I to the mouse cerebellum by means of ethylene-vinyl acetate copolymer (Elvax) significantly increased the degree of multiple CF innervation, when the IGF-I containing Elvax was implanted at postnatal day 8 (P8). In contrast, the IGF-I application starting at P12 had no effect on CF innervation. Conversely, continuous local application of antisera against IGF-I and its receptor significantly decreased the degree of multiple CF innervation when the application started at P8. We found that chronic treatment of exogenous IGF-I from P8 significantly enhanced the CF-mediated excitatory postsynaptic currents (CF-EPSCs). This effect was manifest for the smaller CF-EPSCs but not for the largest CF-EPSC of the multiple-innervated PCs. Conversely, chronic application of antisera from P8 caused attenuation of the largest CF-EPSCs. Other parameters for basic synaptic functions and cerebellar morphology were largely normal after the IGF-I or antisera treatment. These results suggest that IGF-I enhances the strength of developing CF synapses and may promote their survival, whereas the shortage of IGF-I impairs the development of CF synapses and, as a result, may facilitate their elimination. Thus, IGF-I is a potentially important factor among various signalling molecules that can influence CF synapse elimination during cerebellar development.
  • T Shibata, M Watanabe, R Ichikawa, K Ameda, T Koyanagi
    DEVELOPMENTAL BRAIN RESEARCH 140 (2) 263 - 268 0165-3806 2003/02 [Refereed][Not invited]
     
    Physiological studies have shown that lower urinary tract function is regulated through glutamate receptors at the levels of spinal and supraspinal cord. Of the receptor family, N-methyl-D-aspartate (NMDA) receptors mediate activity-dependent changes of synaptic efficacy, underlying synaptic plasticity and synapse development. To know the ontogenic changes of NMDA receptor expression in the visceromotor system innervating pelvic organs, including the bladder, we employed double labeling technique of retrograde neuronal tracing and in situ hybridization for detecting NMDA subunit mRNAs in preganglionic neurons (PGNs) of the lumbosacral cord. Rats at postnatal day 7 (P7), 14 (P14), 21 (P21), and adult were used. In situ hybridization was conducted using S-35-labeled antisense oligonucleotides specific to mRNAs for NMDA receptor subunits. Hybridizing signals in PGNs were detected by a dark-field microscope equipped fluorescence detector. PGNs showed strong signals for NR1 subunit mRNA at each developmental stage examined. Moderate signals for the NR2B and NR2D subunit mRNAs were found in PGNs at P7. However, their expression levels decreased thereafter, reaching the minimal level in adults. No significant signals for NR2A and NR2C subunit mRNAs were detected at any stages. This temporal pattern of expression suggests a possible involvement of NMDA receptors in the development of micturitional neural circuit through activity-dependent mechanisms. (C) 2002 Elsevier Science B.V. All rights reserved.
  • Kazuhisa Sakai, Hidemi Shimizu, Tatsuro Koike, Shigeki Furuya, Masahiko Watanabe
    Journal of Neuroscience 23 (2) 550 - 560 0270-6474 2003/01/15 [Refereed][Not invited]
     
    Nonessential amino acid L-Ser plays an essential role in neuronal survival and differentiation, through preferential expression of the L-Ser biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH), in particular in glial cells but not in neurons. To seek the molecular candidates responsible for glia-borne L-Ser transport, we performed histochemical analyses on amino acid transporter ASCT1, which prefers small neutral amino acids, such as Ala, Ser, Cys, and Thr, and mediates their obligatory exchange. At early developmental stages, neuroepithelial cells constituting the ventricular zone expressed ASCT1 mRNA and protein ubiquitously. Thereafter, ASCT1 expression was gradually downregulated in neuronal populations during the late embryonic and neonatal periods, whereas its high expression was transmitted to radial glial cells and then to astrocytes. High levels of ASCT1 were also detected in the olfactory ensheathing glia. The preferential glial expression of ASCT1 was consistent with that of 3PGDH, and their extensive colocalization was demonstrated at the cellular level. Moreover, high cellular contents of L-Ser were revealed in these glial cells by using a specific antibody to L-Ser. These results strongly suggest that a large amount of L-Ser is synthesized and stored in these glial cells and is released through ASCT1 in exchange for other extracellular substrates. In addition, we observed prominent expression of ASCT1 in capillary endothelial cells of embryonic and neonatal brains. Therefore, ASCT1 appears to be regulated to meet metabolic demands by differentiating and mature neurons through the transport of glia- and blood-borne small neutral amino acids.
  • Kazuhisa Sakai, Hidemi Shimizu, Tatsuro Koike, Shigeki Furuya, Masahiko Watanabe
    Journal of Neuroscience 23 (2) 550 - 560 0270-6474 2003/01/15 [Refereed][Not invited]
     
    Nonessential amino acid L-Ser plays an essential role in neuronal survival and differentiation, through preferential expression of the L-Ser biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH), in particular in glial cells but not in neurons. To seek the molecular candidates responsible for glia-borne L-Ser transport, we performed histochemical analyses on amino acid transporter ASCT1, which prefers small neutral amino acids, such as Ala, Ser, Cys, and Thr, and mediates their obligatory exchange. At early developmental stages, neuroepithelial cells constituting the ventricular zone expressed ASCT1 mRNA and protein ubiquitously. Thereafter, ASCT1 expression was gradually downregulated in neuronal populations during the late embryonic and neonatal periods, whereas its high expression was transmitted to radial glial cells and then to astrocytes. High levels of ASCT1 were also detected in the olfactory ensheathing glia. The preferential glial expression of ASCT1 was consistent with that of 3PGDH, and their extensive colocalization was demonstrated at the cellular level. Moreover, high cellular contents of L-Ser were revealed in these glial cells by using a specific antibody to L-Ser. These results strongly suggest that a large amount of L-Ser is synthesized and stored in these glial cells and is released through ASCT1 in exchange for other extracellular substrates. In addition, we observed prominent expression of ASCT1 in capillary endothelial cells of embryonic and neonatal brains. Therefore, ASCT1 appears to be regulated to meet metabolic demands by differentiating and mature neurons through the transport of glia- and blood-borne small neutral amino acids.
  • Contribution of Thalamic Phospholipase C  mediated by Metabotropic Glutamate Receptor Type 1 to Inflammatory Pain in Mice.
    Miyata M (CA, Kashiwadani H, Fukaya M, Hayashi T, Wu D, Suzuki T, Watanabe M, Kawakami Y
    J. Neurosci. 23 (22) 8089 - 8109 2003 [Refereed][Not invited]
  • T Saito, W Fujimoto, H Yanase, M Watanabe, T Iwanaga
    BIOMEDICAL RESEARCH-TOKYO 23 (6) 277 - 286 0388-6107 2002/12 [Refereed][Not invited]
     
    Since the source and disposition of serotonin is still unclear in non-neuronal cells, we investigated the cellular expression of serotonin transporter (SERT) in mouse adrenal chromaffin cells, mast cells and enterochromaffin cells, all of which contain serotonin in their secretory granules. Immunohistochemistry using SERT antibody in the adrenal medulla demonstrated an intense immunoreactivity in the plasma membrane of adrenalin (A) cells, but not in that of noradrenalin (NA) cells. Electron-microscopically, the expression of SERT appeared to occur mainly on the lateral plasma membranes of A cells facing adjacent A cells. Peritoneal mast cells also displayed a selective immunoreactivity for SERT along the plasma membrane, although the intensity varied cell to cell. In the tongue, stomach, nasal mucosa and trachea, connective tissue-type mast cells were immunoreactive, but not mucosal-type mast cells. Gastrointestinal epithelial cells including enterochromaffin cells were free of the immunoreactivity. These findings suggest that SERT is involved in the uptake of serotonin in adrenal A cells and connective tissue-type mast cells.
  • K Toyooka, S Iritani, T Makifuchi, O Shirakawa, N Kitamura, K Maeda, R Nakamura, K Niizato, M Watanabe, A Kakita, H Takahashi, T Someya, H Nawa
    JOURNAL OF NEUROCHEMISTRY 83 (4) 797 - 806 0022-3042 2002/11 [Refereed][Not invited]
     
    Many postsynaptic density proteins carrying postsynaptic density-95/discs large/zone occludens-1 (PDZ) domain(s) interact with glutamate receptors to control receptor dynamics and synaptic plasticity. Here we examined the expression of PDZ proteins, synapse-associated protein (SAP) 97, postsynaptic density (PSD)-95, chapsyn-110, GRIP1 and SAP102, in post-mortem brains of schizophrenic patients and control subjects, and evaluated their contribution to schizophrenic pathology. Among these PDZ proteins, SAP97 exhibited the most marked change: SAP97 protein levels were decreased to less than half that of the control levels specifically in the prefrontal cortex of schizophrenic patients. In parallel, its binding partner, GluR1, similarly decreased in the same brain region. The correlation between SAP97 and GluR1 levels in control subjects was, however, altered in schizophrenic patients. SAP102 levels were also significantly reduced in the hippocampus of schizophrenic patients, but this reduction was correlated with sample storage time and post-mortem interval. There were no changes in the levels of the other PDZ proteins in any of the regions examined. In addition, neuroleptic treatment failed to mimic the SAP97 change. These findings suggest that a phenotypic loss of SAP97 is associated with the postsynaptic impairment in prefrontal excitatory circuits of schizophrenic patients.
  • M Miura, M Watanabe, S Offermanns, MI Simon, M Kano
    JOURNAL OF NEUROSCIENCE 22 (19) 8379 - 8390 0270-6474 2002/10 [Refereed][Not invited]
     
    Heterotromeric G-proteins of the Gq family are thought to transduce signals from group I metabotropic glutamate receptors (mGluRs) in central neurons. We investigated roles of this cascade in hippocampal long-term potentiation (LTP) by using null-mutant mice lacking the alpha subunit of Gq (Galphaq) or G11 (Galpha11). We found no obvious abnormalities in the morphology, layer structure, expression of NMDA receptors, and basic parameters of excitatory synaptic transmission in the hippocampus of Galphaq mutant mice. We used theta burst stimulation (TBS) (3-10 burst trains at 5 Hz; each train consisted of five stimuli at 100 Hz) to induce LTP at Schaffer collateral to CA1 pyramidal cell synapses. Conventional TBS with 10 burst trains induced robust LTP in wild-type, Galphaq mutant, and Galpha11 mutant mice. Weak TBS with three burst trains consistently induced LTP in wild-type mice. In contrast, the same weak TBS was insufficient to induce LTP in Galphaq and Galpha11 mutant mice. In wild-type mice, the LTP by weak TBS was abolished by inhibiting group I mGluR or protein kinase C (PKC) but not by blocking muscarinic acetylcholine receptors. Prior activation of group I mGluR by an agonist significantly enhanced the LTP by weak TBS in wild-type mice. However, this priming effect was absent in Galphaq mutant mice. These results indicate that the signaling from group I mGluR to PKC involving Galphaq/Galpha11 does not constitute the main pathway for LTP, but it secures LTP induction by lowering its threshold in the hippocampal area CA1.
  • R Ichikawa, T Miyazaki, M Kano, T Hashikawa, H Tatsumi, K Sakimura, M Mishina, Y Inoue, M Watanabe
    JOURNAL OF NEUROSCIENCE 22 (19) 8487 - 8503 0270-6474 2002/10 [Refereed][Not invited]
     
    Organized synapse formation on to Purkinje cell (PC) dendrites by parallel fibers (PFs) and climbing fibers (CFs) is crucial for cerebellar function. In PCs lacking glutamate receptor delta2 (GluRdelta2), PF synapses are reduced in number, numerous free spines emerge, and multiple CF innervation persists to adulthood. In the present study, we conducted anterograde and immunohistochemical labelings to investigate how CFs innervate PC dendrites under weakened synaptogenesis by PFs. In the GluRdelta2 knock-out mouse, CFs were distributed in the molecular layer more closely to the pial surface compared with the wild-type mouse. Serial electron microscopy demonstrated that CFs in the knock-out mouse innervated all spines protruding from proximal dendrites of PCs, as did those in the wildtype mouse. In the knock-out mouse, however, CF innervation extended distally to spiny branchlets, where nearly half of the spines were free of innervation in contrast to complete synapse formation by PFs in the wild-type mouse. Furthermore, from the end point of innervation, CFs aberrantly jumped to form ectopic synapses on adjacent spiny branchlets, whose proximal portions were often innervated by different CFs. Without GluRdelta2, CFs are thus able to expand their territory along and beyond dendritic trees of the target PC, resulting in persistent surplus CFs by innervating the distal dendritic segment. We conclude that GluRdelta2 is essential to restrict CF innervation to the proximal dendritic segment, by which territorized innervation by PFs and CFs is properly structured and the formation of excess CF wiring to adjacent PCs is suppressed.
  • R Ichikawa, T Miyazaki, M Kano, T Hashikawa, H Tatsumi, K Sakimura, M Mishina, Y Inoue, M Watanabe
    JOURNAL OF NEUROSCIENCE 22 (19) 8487 - 8503 0270-6474 2002/10 [Refereed][Not invited]
     
    Organized synapse formation on to Purkinje cell (PC) dendrites by parallel fibers (PFs) and climbing fibers (CFs) is crucial for cerebellar function. In PCs lacking glutamate receptor delta2 (GluRdelta2), PF synapses are reduced in number, numerous free spines emerge, and multiple CF innervation persists to adulthood. In the present study, we conducted anterograde and immunohistochemical labelings to investigate how CFs innervate PC dendrites under weakened synaptogenesis by PFs. In the GluRdelta2 knock-out mouse, CFs were distributed in the molecular layer more closely to the pial surface compared with the wild-type mouse. Serial electron microscopy demonstrated that CFs in the knock-out mouse innervated all spines protruding from proximal dendrites of PCs, as did those in the wildtype mouse. In the knock-out mouse, however, CF innervation extended distally to spiny branchlets, where nearly half of the spines were free of innervation in contrast to complete synapse formation by PFs in the wild-type mouse. Furthermore, from the end point of innervation, CFs aberrantly jumped to form ectopic synapses on adjacent spiny branchlets, whose proximal portions were often innervated by different CFs. Without GluRdelta2, CFs are thus able to expand their territory along and beyond dendritic trees of the target PC, resulting in persistent surplus CFs by innervating the distal dendritic segment. We conclude that GluRdelta2 is essential to restrict CF innervation to the proximal dendritic segment, by which territorized innervation by PFs and CFs is properly structured and the formation of excess CF wiring to adjacent PCs is suppressed.
  • K Nakamura, K Tanoue, T Satoh, M Takekawa, M Watanabe, H Shima, K Kikuchi
    JOURNAL OF BIOCHEMISTRY 132 (3) 463 - 470 0021-924X 2002/09 [Refereed][Not invited]
     
    We have identified a novel dual-specificity phosphatase (DSP), called LDP-2 (low-molecular-mass DSP-2), composed of 220 amino acid residues showing high sequence homology to VHR and LDP-1/TMDP, which belong to a family of DSPs with low molecular masses. The LDP-2 gene is ubiquitously expressed, and LDP-2 is localized in the cytoplasm. The main structural feature of LDP-2 is that the serine-156 residue located in the common active site sequence motif, HCXXGXXRS, for DSP is naturally substituted with an alanine residue. The recombinant LDP-2 protein showed extremely low phosphatase activity towards p-nitrophenyl phosphate (pNPP). Back-mutation of Ala-156 in LDP-2 to a serine (A156S mutation) conferred significant phosphatase activity towards pNPP. However, both LDP-2 and LDP-2 (A156S) exhibited substantial phosphatase activities towards both phospho-seryl/threonyl and -tyrosyl residues of myelin basic protein, with similar specific activities. Ala-156 of LDP-2 might be crucially involved in the recognition of a physiological substrate. We analyzed the effect of VHR and LDP-2 on mitogen-activated protein kinases (MAPKs) in vivo. We first found that VHR inhibits the activation of p38 as well as ERK and JNK, with similar efficiency. Under the conditions ussed, LDP-2 specifically suppressed JNK activation.
  • M Kinoshita, R Ueda, S Kojima, K Sato, M Watanabe, A Urano, E Ito
    EUROPEAN JOURNAL OF NEUROSCIENCE 16 (5) 868 - 876 0953-816X 2002/09 [Refereed][Not invited]
     
    To map the functional synaptic organization over a wide area in the optic tectum, we directly monitored two-dimensional propagation of postsynaptic depolarization evoked by firing of retinotectal afferents in optic tectum slices prepared from rainbow trout (Oncorhynchus mykiss), using a voltage-sensitive dye and a photodiode array system. The postsynaptic responses to afferent stimulation first propagated in the stratum opticum and stratum fibrosum et griseum superficiale in an anterograde fashion in the afferents and then expanded vertically into the deep layers. This vertical propagation appeared to occur along a bundle-like structure that corresponded well with a cluster of neurons whose somata are located in the stratum periventriculare. Pharmacological studies showed that these postsynaptic responses were mediated by ionotropic glutamate receptors. On the other hand, the optical signals appeared to consist of at least two components (a transient signal and a slow signal). The second transient signal summated with the first slow signal by paired stimulation, suggesting that the transient and slow signals originated from different cell types. Taken together, these results showed that the functional synaptic organization of the teleost optic tectum comprises of two depolarization-signal propagating paths along a horizontal layer structure and a vertical bundle-like structure and that these synaptic responses occur via glutamatergic transmission.
  • Y Ogawa, K Sawamoto, T Miyata, S Miyao, M Watanabe, M Nakamura, BS Bregman, M Koike, Y Uchiyama, Y Toyama, H Okano
    JOURNAL OF NEUROSCIENCE RESEARCH 69 (6) 925 - 933 0360-4012 2002/09 [Refereed][Not invited]
     
    Neural progenitor cells, including neural stem cells, are a potential expandable source of graft material for transplantation aimed at repairing the damaged CNS. Here we present the first evidence that in vitro-expanded fetus-derived neurosphere cells were able to generate neurons in vivo and improve motor function upon transplantation into an adult rat spinal-cord-contusion injury model. As the source of graft material, we used a neural stem cell-enriched population that was derived from rat embryonic spinal cord (E14.5) and expanded in vitro by neurosphere formation. Nine days after contusion injury, these neurosphere cells were transplanted into adult rat spinal cord at the injury site. Histological analysis 5 weeks after the transplantation showed that mitotic neurogenesis occurred from the transplanted donor progenitor cells within the adult rat spinal cord, a nonneurogenic region; that these donor-derived neurons extended their processes into the host tissues; and that the neurites formed synaptic structures. Furthermore, analysis of motor behavior using a skilled reaching task indicated that the treated rats showed functional recovery. These results indicate that in vitro-expanded neurosphere cells derived from the fetal spinal cord are a potential source for transplantable material for treatment of spinal cord injury. (C) 2002 Wiley-Liss, Inc.
  • K Nakamura, K Tanoue, T Satoh, M Takekawa, M Watanabe, H Shima, K Kikuchi
    JOURNAL OF BIOCHEMISTRY 132 (3) 463 - 470 0021-924X 2002/09 [Refereed][Not invited]
     
    We have identified a novel dual-specificity phosphatase (DSP), called LDP-2 (low-molecular-mass DSP-2), composed of 220 amino acid residues showing high sequence homology to VHR and LDP-1/TMDP, which belong to a family of DSPs with low molecular masses. The LDP-2 gene is ubiquitously expressed, and LDP-2 is localized in the cytoplasm. The main structural feature of LDP-2 is that the serine-156 residue located in the common active site sequence motif, HCXXGXXRS, for DSP is naturally substituted with an alanine residue. The recombinant LDP-2 protein showed extremely low phosphatase activity towards p-nitrophenyl phosphate (pNPP). Back-mutation of Ala-156 in LDP-2 to a serine (A156S mutation) conferred significant phosphatase activity towards pNPP. However, both LDP-2 and LDP-2 (A156S) exhibited substantial phosphatase activities towards both phospho-seryl/threonyl and -tyrosyl residues of myelin basic protein, with similar specific activities. Ala-156 of LDP-2 might be crucially involved in the recognition of a physiological substrate. We analyzed the effect of VHR and LDP-2 on mitogen-activated protein kinases (MAPKs) in vivo. We first found that VHR inhibits the activation of p38 as well as ERK and JNK, with similar efficiency. Under the conditions ussed, LDP-2 specifically suppressed JNK activation.
  • M Kinoshita, R Ueda, S Kojima, K Sato, M Watanabe, A Urano, E Ito
    EUROPEAN JOURNAL OF NEUROSCIENCE 16 (5) 868 - 876 0953-816X 2002/09 [Refereed][Not invited]
     
    To map the functional synaptic organization over a wide area in the optic tectum, we directly monitored two-dimensional propagation of postsynaptic depolarization evoked by firing of retinotectal afferents in optic tectum slices prepared from rainbow trout (Oncorhynchus mykiss), using a voltage-sensitive dye and a photodiode array system. The postsynaptic responses to afferent stimulation first propagated in the stratum opticum and stratum fibrosum et griseum superficiale in an anterograde fashion in the afferents and then expanded vertically into the deep layers. This vertical propagation appeared to occur along a bundle-like structure that corresponded well with a cluster of neurons whose somata are located in the stratum periventriculare. Pharmacological studies showed that these postsynaptic responses were mediated by ionotropic glutamate receptors. On the other hand, the optical signals appeared to consist of at least two components (a transient signal and a slow signal). The second transient signal summated with the first slow signal by paired stimulation, suggesting that the transient and slow signals originated from different cell types. Taken together, these results showed that the functional synaptic organization of the teleost optic tectum comprises of two depolarization-signal propagating paths along a horizontal layer structure and a vertical bundle-like structure and that these synaptic responses occur via glutamatergic transmission.
  • S Oshima, M Fukaya, N Masabumi, T Shirakawa, H Oguchi, M Watanabe
    NEUROSCIENCE RESEARCH 43 (3) 239 - 250 0168-0102 2002/07 [Refereed][Not invited]
     
    Oro-facial sensorimotor function conducted by the brainstem is vital to newborn mammals, and N-methyl-D-aspartate (NMDA) receptors play an important role in the regulation. Here we examined the expression of NMDA receptor subunits in the mouse hypoglossal nucleus from embryonic day 13 (E13) through postnatal day 21 (P21). Compared with other brainstem regions, early onset of GluRepsilonl (NR2A) mRNA expression was conspicuous to the embryonic hypoglossal nucleus. The expression peaked at P1-P7, when other brainstem regions just started to express it. At P1, GluRepsilon1 subunit was localized to asymmetrical synapses on motoneuron dendrites, particularly, on the postsynaptic junction membrane. In developing motoneurons, expressions of GluRepsilon2 (NR2B), GluRepsilon4 (NR2D), and GluRzeta1 (NR1) mRNAs were accompanied. Until P21, however, all of these subunits were down-regulated with particular reduction for GluRepsilon2 and GhtRepsilon4 mRNAs. Similar patterns of temporal expressions were observed in motoneurons of other brainstem motor nuclei. Taking that high levels of GluRepsilon1, GluRepsilon2, and GuRzeta1 subunits are also found in the adult hippocampus and cerebral cortex, it can be assumed that NMDA receptors in developing motoneurons are highly potent and potentially involved in structural and functional development of the brainstem motor system. (C) 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • K Nakazawa, MC Quirk, RA Chitwood, M Watanabe, MF Yeckel, LD Sun, A Kato, CA Carr, D Johnston, MA Wilson, S Tonegawa
    SCIENCE 297 (5579) 211 - 218 0036-8075 2002/07 [Refereed][Not invited]
     
    Pattern completion, the ability to retrieve complete memories on the basis of incomplete sets of cues, is a crucial function of biological memory systems. The extensive recurrent connectivity of the CA3 area of hippocampus has led to suggestions that it might provide this function. We have tested this hypothesis by generating and analyzing a genetically engineered mouse strain in which the N-methyl-D-asparate ( NMDA) receptor gene is ablated specifically in the CA3 pyramidal cells of adult mice. The mutant mice normally acquired and retrieved spatial reference memory in the Morris water maze, but they were impaired in retrieving this memory when presented with a fraction of the original cues. Similarly, hippocampal CA1 pyramidal cells in mutant mice displayed normal place- related activity in a full- cue environment but showed a reduction in activity upon partial cue removal. These results provide direct evidence for CA3 NMDA receptor involvement in associative memory recall.
  • S Oshima, M Fukaya, N Masabumi, T Shirakawa, H Oguchi, M Watanabe
    NEUROSCIENCE RESEARCH 43 (3) 239 - 250 0168-0102 2002/07 [Refereed][Not invited]
     
    Oro-facial sensorimotor function conducted by the brainstem is vital to newborn mammals, and N-methyl-D-aspartate (NMDA) receptors play an important role in the regulation. Here we examined the expression of NMDA receptor subunits in the mouse hypoglossal nucleus from embryonic day 13 (E13) through postnatal day 21 (P21). Compared with other brainstem regions, early onset of GluRepsilonl (NR2A) mRNA expression was conspicuous to the embryonic hypoglossal nucleus. The expression peaked at P1-P7, when other brainstem regions just started to express it. At P1, GluRepsilon1 subunit was localized to asymmetrical synapses on motoneuron dendrites, particularly, on the postsynaptic junction membrane. In developing motoneurons, expressions of GluRepsilon2 (NR2B), GluRepsilon4 (NR2D), and GluRzeta1 (NR1) mRNAs were accompanied. Until P21, however, all of these subunits were down-regulated with particular reduction for GluRepsilon2 and GhtRepsilon4 mRNAs. Similar patterns of temporal expressions were observed in motoneurons of other brainstem motor nuclei. Taking that high levels of GluRepsilon1, GluRepsilon2, and GuRzeta1 subunits are also found in the adult hippocampus and cerebral cortex, it can be assumed that NMDA receptors in developing motoneurons are highly potent and potentially involved in structural and functional development of the brainstem motor system. (C) 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Yamada K, Watanabe M
    Anatomical science international / Japanese Association of Anatomists 2 77 (2) 94 - 108 1447-6959 2002/06 [Refereed][Not invited]
  • M Ohgoh, T Hanada, T Smith, T Hashimoto, M Ueno, Y Yamanishi, M Watanabe, Y Nishizawa
    JOURNAL OF NEUROIMMUNOLOGY 125 (1-2) 170 - 178 0165-5728 2002/04 [Refereed][Not invited]
     
    Amelioration of experimental autoimmune encephalomyelitis (EAE) by blockade of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), has been recently demonstrated [Nat. Med. 6 (2000) 67; Nat. Med. 6 (2000) 62]. However, the mechanisms underlying regulation of the extracellular glutamate concentration in EAE are unclear. To address this, we examined the expression of three distinct Na(+)-dependent glutamate transporters (GLT-1, GLAST and EAAC1) in the spinal cord of the Lewis rat EAE. EAE induced a dramatic increase in EAAC1 protein and mRNA levels, which cot-responded closely with the course of neurological symptoms. In contrast, the levels of GLT-1 and GLAST protein were down-regulated in the spinal cord at the peak of disease symptoms, and no recovery was observed after remission. Furthermore, these changes in GLT-1, GLAST and EAAC1 expression were suppressed by treatment with NBQX. These results suggest that AMPA receptor activation precedes the altered expression of glutamate transporters, and that the dysregulation of extracellular glutamate concentration might play a critical role in pathological changes and neuronal dysfunction in EAE. (C) 2002 Elsevier Science B.V. All rights reserved.
  • Y Miyagi, T Yamashita, M Fukaya, T Sonoda, T Okuno, K Yamada, M Watanabe, Y Nagashima, Aoki, I, K Okuda, M Mishina, S Kawamoto
    JOURNAL OF NEUROSCIENCE 22 (3) 803 - 814 0270-6474 2002/02 [Refereed][Not invited]
     
    The glutamate receptor delta2 (GluRdelta2) subunit is selectively expressed in cerebellar Purkinje cells and plays an important role in cerebellar long-term depression, motor learning, motor coordination, and synapse development. We identified a novel GluRdelta2-interacting protein, named Delphilin, that contains a single PDZ domain and formin homology (FH) domains FH1 and FH2 plus coiled-coil structure. As far as we know, this is the first reported protein that contains both PDZ and FH domains. Yeast two-hybrid and surface plasmon resonance (SPR) analyses indicated that Delphilin interacts with the GluRdelta2 C terminus via its PDZ domain. This was also supported by coimmunoprecipitation experiments using a heterologous expression system in mammalian cells. Yeast cell and SPR analyses also demonstrated the possibility that the FH1 proline-rich region of Delphilin interacts with profilin, an actin-binding protein, and with the Src homology 3 domain of neuronal Src protein tyrosine kinase. In situ hybridization demonstrated the highest expression of Delphilin mRNA in Purkinje cells. Delphilin polypeptide was highly enriched in the synaptosomal membrane fraction of the cerebellum and coimmunoprecipitated with the GluRdelta2 subunit. The post-embedding immunogold technique demonstrated that Delphilin is selectively localized at the postsynaptic junction site of the parallel fiber-Purkinje cell synapse and colocalized with GluRdelta2. Thus, Delphilin is a postsynaptic scaffolding protein at the parallel fiber-Purkinje cell synapse, where it may serve to link GluRdelta2 with actin cytoskeleton and various signaling molecules.
  • M Watanabe
    TRENDS IN NEUROSCIENCES 25 (1) 5 - 6 0166-2236 2002/01 [Refereed][Not invited]
     
    Bergmann glia in the cerebellum elaborately ensheathe Purkinje cell dendrites and synapses, and express high levels of Ca2+-permeable glutamate receptors (GluRs). Suppression of the Ca2+ permeability by virus-mediated GluR2 transfer has revealed that GluR-mediated Ca2+ signaling is essential to maintain both structural and functional connections between the glial cell and the glutamatergic synapses.
  • T Tsukada, M Watanabe, T Yamashima
    JOURNAL OF NEUROCHEMISTRY 79 (6) 1196 - 1206 0022-3042 2001/12 [Refereed][Not invited]
     
    The exact molecular mechanism of ischemic neuronal death still remains unclear from rodents to primates. A number of studies using lower species animals have suggested implication of apoptosis cascade, while using monkeys the authors recently claimed necrosis cascade by calpain-induced leakage of lysosomal cathepsins (calpain-cathepsin hypothesis). This paper is to study implications of apoptotic versus necrotic cascades for the development of hippocampal CAI neuronal death in the primate brain undergoing complete global ischemia. Here, we focused on two terminal cell death effectors; caspase-activated DNase (CAD) and lysosomal enzyme DNase II, in the monkey CAI sector undergoing 18 min ischemia. The expressions of their mRNA and proteins, and the subcellular localizations as well as ultrastructure and specific DNA gel electrophoresis were examined. Expression of CAD was much less in the normal brain, compared with the lymph node or heart tissues. On day 1 after ischemia, however, CAD mRNA and protein were significantly increased in the CAI sector, and then CAD protein immunohistochemically showed a translocation from the perikarya into the nucleus. Activated DNase II protein was significantly increased on days 2 and 3 after ischemia, and also showed a similar translocation indicating lysosomal leakage. Although the post-ischemic CAI neurons showed positive terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining on days 3-5, they showed eosinophilic coagulation necrosis on light microscopy, and frank membrane disruption and mild chromatin condensation on electron microscopy. Furthermore, DNA smear pattern typical for necrosis was observed instead of DNA laddering. These data altogether suggest that the post-ischemic CAI neuronal death of the monkey occurs not by apoptosis but by necrosis with participations of lysosomal enzymes DNase II and cathepsins as well as CAD. The interactions between apoptotic (caspase-3 and CAD) and necrotic (calpain, cathepsin and DNase II) cascades should be studied further.
  • K Hashimoto, R Ichikawa, H Takechi, Y Inoue, A Aiba, K Sakimura, M Mishina, T Hashikawa, A Konnerth, M Watanabe, M Kano
    JOURNAL OF NEUROSCIENCE 21 (24) 9701 - 9712 0270-6474 2001/12 [Refereed][Not invited]
     
    Climbing fiber (CF) synapse formation onto cerebellar Purkinje cells (PCs) is critically dependent on the synaptogenesis from parallel fibers (PFs), the other input to PCs. Previous studies revealed that deletion of the glutamate receptor delta2 subunit (GluR delta2) gene results in persistent multiple CF innervation of PCs with impaired PF synaptogenesis, whereas mutation of the metabotropic glutamate receptor subtype 1 (mGluR1) gene causes multiple CF innervation with normal PF synaptogenesis. We demonstrate that atypical CF-mediated EPSCs (CF-EPSCs) with slow rise times and small amplitudes coexisted with typical CF-EPSCs with fast rise times and large amplitudes in PCs from GluR delta2 mutant cerebellar slices. CF-EPSCs in mGluR1 mutant and wild-type PCs had fast rise times. Atypical slow CF responses of GluR delta2 mutant PCs were associated with voltage-dependent Ca2+ signals that were confined to PC distal dendrites. In the wild-type and mGluR1 mutant PCs, CF-induced Ca2+ signals involved both proximal and distal dendrites. Morphologically, CFs of GluR delta2 mutant mice extended to the superficial regions of the molecular layer, whereas those of wildtype and mGluR1 mutant mice did not innervate the superficial one-fifth of the molecular layer. It is therefore likely that surplus CFs of GluR delta2 mutant mice form ectopic synapses onto distal dendrites, whereas those of wild-type and mGluR1 mutant mice innervate proximal dendrites. These findings suggest that GluR delta2 is required for consolidating PF synapses and restricting CF synapses to the proximal dendrites, whereas the mGluR1-signaling pathway does not affect PF synaptogenesis but is involved in eliminating surplus CF synapses at the proximal dendrites.
  • Miwako Yamasaki, Keiko Yamada, Shigeki Furuya, Junya Mitoma, Yoshio Hirabayashi, Masahiko Watanabe
    Journal of Neuroscience 21 (19) 7691 - 7704 0270-6474 2001/10/01 [Refereed][Not invited]
     
    L-Serine is synthesized from glycolytic intermediate 3-phosphoglycerate and is an indispensable precursor for the synthesis of proteins, membrane lipids, nucleotides, and neuroactive amino acids D-serine and glycine. We have recently shown that L-serine and its interconvertible glycine act as Bergmann glia-derived trophic factors for cerebellar Purkinje cells. To investigate whether such a metabolic neuron-glial relationship is fundamental to the developing and adult brain, we examined by in situ hybridization and immunohistochemistry the cellular expression of 3-phosphoglycerate dehydrogenase (3PGDH), the initial step enzyme for de novo L-serine biosynthesis in animal cells. At early stages when the neural wall consists exclusively of the ventricular zone, neuroepithelial stem cells expressed 3PGDH strongly and homogeneously. Thereafter, 3PGDH expression was downregulated and eventually disappeared in neuronal populations, whereas its high expression was transmitted to the radial glia and later to astrocytes in the gray and white matters. In addition, 3PGDH was highly expressed throughout development in the olfactory ensheathing glia, a specialized supporting cell that thoroughly ensheathes olfactory nerves. These results establish a fundamental link of the radial glia/astrocyte lineage and olfactory ensheathing glia to L-serine biosynthesis in the brain. We discuss this finding in the context of the hypothesis that 3PGDH expression in these glia cells contributes to energy metabolism in differentiating and differentiated neurons and other glia cells, which are known to be vulnerable to energy loss.
  • K Masuda, H Shima, M Watanabe, K Kikuchi
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 (42) 39002 - 39011 0021-9258 2001/10 [Refereed][Not invited]
     
    Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) negatively regulate MAPK activity. In the present study, we have identified a novel MKP, designated MKP-7, and mapped it to human chromosome 12p12. MKP-7 possesses a long C-terminal stretch containing both a nuclear export signal and a nuclear localization signal, in addition to the rhodanese-like domain and the dual specificity phosphatase catalytic domain, both of which are conserved among MKP family members. When expressed in mammalian cells MKP-7 protein was localized exclusively in the cytoplasm, but this localization became exclusively nuclear following leptomycin B treatment or introduction of a mutation in the nuclear export signal. These findings indicate that MKP-7 is the first identified leptomycin B-sensitive shuttle MKP. Forced expression of MKP-7 suppressed activation of MAPKs in COS-7 cells in the order of selectivity, JNK >> p38 > ERK. Furthermore, a mutant form MKP-7 functioned as a dominant negative particularly against the dephosphorylation of JNK, suggesting that MKP-7 works as a JNK-specific phosphatase in vivo. Co-immunoprecipitation experiments and histological analysis suggested that MKP-7 determines the localization of MAPKs in the cytoplasm.
  • インシュリン様成長因子Iのマウス小脳登上線維-プルキンエ細胞シナプスの生後発達に及ぼす影響
    柿澤 昌, 山田 和之, 渡辺 雅彦, 狩野 方伸
    神経化学 日本神経化学会 40 (2-3) 340 - 340 0037-3796 2001/09
  • H Shiraishi, Y Watanabe, M Watanabe, Y Inoue, T Fujiwara, K Yagi
    EPILEPSIA 42 (7) 875 - 882 0013-9580 2001/07 [Refereed][Not invited]
     
    Purpose: To determine whether magnetoencephalography (MEG) has any clinical value for the analysis of seizure discharges in patients with medial frontal lobe epilepsy (FLE). Methods: Four patients were studied with 74-channel MEG. Interictal and ictal electroencephalographic (EEG) and MEG recordings were obtained. The equivalent current dipoles (ECDs) of the MEG spikes were calculated. Results: In two patients with postural seizures, interictal EEG spikes occurred at Cz or Fz. The ECDs of interictal MEG spikes were localized around the supplementary motor area. In the other two patients with focal motor or oculomotor seizures, interictal EEG spikes occurred at Fz or Cz. The ECDs of interictal MEG spikes were localized at the top of the medial frontal region. The ECDs detected at MEG ictal onset were also localized in the same area as those of the interictal discharges. Conclusions: In medial FLE patients, interictal and ictal MEG indicated consistent ECD localization that corresponded to the semiology of clinical seizures. Our findings demonstrate that MEG is a useful tool for detecting epileptogenic focus.
  • K Yamada, M Fukaya, H Shimizu, K Sakimura, M Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 13 (11) 2025 - 2036 0953-816X 2001/06 [Refereed][Not invited]
     
    Cerebellar N-methyl-D-aspartate (NMDA) receptors are concentrated in the granular layer and are involved in motor coordination and the induction of long-term potentiation at mossy fibre-granule cell synapses. In the present study, we used immunohistochemistry to examine the distribution of NMDA receptor subunits in the adult mouse cerebellum. We found that appropriate pepsin pretreatment of sections greatly enhanced the sensitivity and specificity of immunohistochemical detection. As a result, intense immunolabelling for GluR epsilon1 (NR2A), GluR epsilon3 (NR2C), and GluR zeta1 (NR1) all appeared in synaptic glomeruli of the granular layer. Double immunofluorescence showed that these subunits were colocalized in individual synaptic glomeruli. Within the glomerulus, NMDA receptor subunits were located between centrally-located huge mossy fibre terminals and peripherally-located tiny Golgi axon terminals. By immunoelectron microscopy, all three subunits were detected at the postsynaptic junction in granule cell dendrites, forming synapses with mossy fibre terminals. Consistent with the known functional localization, GluR epsilon1, GluR epsilon3, and GluR zeta1 are, thus, anatomically concentrated at the mossy fibre-granule cell synapse. By contrast, immunohistochemical signals were very low in Purkinje cell somata and dendrites in the molecular layer. The lack of GluR zeta1 immunolabelling in Purkinje cells was unexpected because the cells express GluR zeta1 mRNA at high levels and high levels of GluR zeta1 protein in the molecular layer were revealed by immunoblot. As Purkinje cells are exceptionally lacking GluR epsilon expression, the discrepant result may provide in vivo evidence suggesting the importance of accompanying GluR epsilon subunits in synaptic localization of GluR zeta1.
  • M Yamazaki, M Fukaya, M Abe, K Ikeno, T Kakizaki, M Watanabe, K Sakimura
    NEUROSCIENCE LETTERS 304 (1-2) 81 - 84 0304-3940 2001/05 [Refereed][Not invited]
     
    Glutamate receptor interacting protein (GRIP) is a member of the PDZ domain-containing protein family that is localized in the postsynaptic density area. This protein has been reported to interact specifically with the C-termini of AMPA-selective glutamate receptor channel subunits, GluR alpha2 and GluR alpha3 through its PDZ domains. To clarify the physiological functions of GRIP, we cloned mouse GRIP1, and found that there are three sites for alternative splicing and two putative translational start codons by characterizing GRIP1 cDNA clones and reverse transcription-polymerase chain reaction products. Metabolic labeling of COS-7 cells expressing two N-terminal GRIP1 proteins demonstrated that these proteins differed in their pattern of palmitoylation. These findings suggested that the molecular diversity of GRIP1 underlies the localization and functional heterogeneity of this protein. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.
  • M Shimuta, M Yoshikawa, M Fukaya, M Watanabe, H Takeshima, T Manabe
    MOLECULAR AND CELLULAR NEUROSCIENCE 17 (5) 921 - 930 1044-7431 2001/05 [Refereed][Not invited]
     
    The precise function of ryanodine receptors (RyRs) in synaptic transmission is unknown, but three of their subtypes are expressed in the brain. We examined the role of RyRs in excitatory synaptic transmission in hippocampal slices, using type 3 RyR (RyR3)-deficient mice. The alpha -amino-3-hydroxy-5-methyl-4-isoxo-zolepropionic acid (AMPA) receptor-mediated basal synaptic responses in the CA1 region of mutant mice were smaller than those of wild-type mice, while there was no difference in N-methyl-D-aspartate receptor-mediated responses, suggesting selective postsynaptic modification of AMPA receptors by RyR3. The expression of synaptic AMPA receptor subunits examined by Western blotting or immunohistochemistry was indistinguishable, suggesting that the smaller AMPA synaptic responses in mutant mice were not due to the reduced number of synaptic AMPA receptors. Although the initial potentiation following tetanic stimulation of afferent fibers was similar, long-term potentiation (LTP) was smaller in mutant mice. There were no differences in presynaptic electrophysiological properties. We thus conclude that RyR3 postsynaptically regulates the properties of AMPA receptors and LTP.
  • K Kitayama, M Abe, T Kakizaki, D Honma, R Natsume, M Fukaya, M Watanabe, J Miyazaki, M Mishina, K Sakimura
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 281 (5) 1134 - 1140 0006-291X 2001/03 [Refereed][Not invited]
     
    Spatiotemporally restricted gene targeting is needed for analyzing the functions of various molecules in a variety of biological phenomena. We have generated an inducible cerebellar Purkinje cell-specific gene targeting system. This was achieved by establishing a mutant mouse line (D2CPR) from a C57BL/6 mouse ES cell line, which expressed a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR). The Purkinje cell-specific expression of CrePR was attained by inserting CrePR into the glutamate receptor delta2 subunit (GluR delta2) gene, which was expressed specifically in the Purkinje cells. Using the transgenic mice carrying the Cre-mediated reporter gene, we showed that the antiprogesterone RU486 could induce recombinase activity of the CrePR protein specifically in the mature cerebellar Purkinje cells of the D2CPR line. Thus this mutant line will be a useful tool for studying the molecular function of mature Purkinje cells by manipulating gene expression in a temporally restricted manner. (C) 2001 Academic Press.
  • K Hashimoto, M Miyata, M Watanabe, M Kano
    MOLECULAR NEUROBIOLOGY 23 (1) 69 - 82 0893-7648 2001/02 [Refereed][Not invited]
     
    The P isoforms of phospholipase C (PLC betas) are thought to mediate signals from metabotropic glutamate receptor subtype 1 (mGluR1) that is crucial for the modulation of synaptic transmission and plasticity Among four PLC beta isoforms, PLC beta4 is one of the two major isoforms expressed in cerebellar Purkinje cells. The authors have studied the roles of PLC beta4 by analyzing PLC beta4 knockout mice, which are viable, but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple climbing fiber innervation is clearly impaired in the rostral portion of the cerebellar vermis, where PLC beta4 mRNA is predominantly expressed in the wild-type mice. In the adult, long-term depression is deficient at parallel fiber to Purkinje cell synapses in the rostral. cerebellum of the PLC beta4 knockout mice. The impairment of climbing fiber synapse elimination and the loss of long-term depression are similar to those seen in mice defective in mGluR1, G alphaq, or protein kinase C. Thus, the authors' results strongly suggest that PLC beta4 is part of a signaling pathway, including the mGluR1, Gaq and protein kinase C, which is crucial for both climbing fiber synapse elimination in the developing cerebellum and long-term depression induction in the mature cerebellum.
  • K Nakamura, T Manabe, M Watanabe, T Mamiya, R Ichikawa, Y Kiyama, M Sanbo, T Yagi, Y Inoue, T Nabeshima, H Mori, M Mishina
    EUROPEAN JOURNAL OF NEUROSCIENCE 13 (1) 179 - 189 0953-816X 2001/01 [Refereed][Not invited]
     
    Telencephalin (TLCN) is a cell adhesion molecule selectively expressed in the telencephalon of the mammalian brain. The mutant mice lacking TLCN had no detectable abnormalities in their neural development and synaptic structures. Ablation of TLCN increased the hippocampal long-term potentiation and its saturation level. The TLCN mutation selectively enhanced the performance of the radial maze and water-finding tasks, learning tasks with appetitive reinforcers, but not the contextual fear conditioning and Morris water maze tasks with aversive stimuli for conditioning. Furthermore, the TLCN mutant mice showed an increase of prepulse inhibition of the acoustic startle response. These results suggest that TLCN is a determinant of the dynamic range of synaptic plasticity and plays roles in reward-motivated learning and memory and sensorimotor gating.
  • T Kitazawa, K Hosoya, M Watanabe, T Takashima, S Ohtsuki, H Takanaga, M Ueda, N Yanai, M Obinata, T Terasaki
    PHARMACEUTICAL RESEARCH 18 (1) 16 - 22 0724-8741 2001/01 [Refereed][Not invited]
     
    Purpose. To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat). Methods. Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37 degreesC during the first period of 3 days. Cells were subsequently cultured at 33 degreesC to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup. Results. Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1-5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33 degreesC with a doubling-time of 35-40 hr. [H-3]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by alpha -methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [H-3]-L- proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was five-fold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells. Conclusions. Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.
  • Shigeki Furuya, Toshihide Tabata, Junya Mitoma, Keiko Yamada, Miwako Yamasaki, Asami Makino, Toshifumi Yamamoto, Masahiko Watanabe, Masanobu Kano, Yoshio Hirabayashi
    Proceedings of the National Academy of Sciences of the United States of America 97 (21) 11528 - 11533 0027-8424 2000/10/10 [Refereed][Not invited]
     
    Glial cells support the survival and development of central neurons through the supply of trophic factors. Here we demonstrate that L-serine (L-Ser) and glycine (Gly) also are glia-derived trophic factors. These amino acids are released by astroglial cells and promote the survival, dendritogenesis, and electrophysiological development of cultured cerebellar Purkinje neurons. Although L-Ser and Gly are generally classified as nonessential amino acids, 3-phosphoglycerate dehydrogenase (3PGDH), a key enzyme for their biosynthesis, is not expressed in Purkinje neurons. By contrast, the Bergman glia, a native astroglia in the cerebellar cortex, highly expresses 3PGDH. These data suggest that L-Ser and Gly mediate the trophic actions of glial cells on Purkinje neurons.
  • M Fukaya, M Watanabe
    JOURNAL OF COMPARATIVE NEUROLOGY 426 (4) 572 - 586 0021-9967 2000/10 [Refereed][Not invited]
     
    Postsynaptic density (PSD)-95, SAP102, and Chapsyn-110 are members of the PSD-95/ SAP90 protein family, which interact with the C-terminus of N-methyl-D-aspartate (NMDA) receptor and shaker-type potassium channel subunits. Here we report; that appropriate section pretreatment with pepsin has led to qualitative and quantitative changes in light microscopic immunohistochemical detection of the protein family. First, pepsin pretreatment lowered the concentration of affinity-purified primary antibodies, while it greatly increased the intensity of immunoreactions. Second, the resulting overall distributions of PSD-95, SAP102, and Chapsyn-110 in the adult mouse brain were consistent with their mRNA distributions. Third, instead of the reported patterns of somatodendritic labeling, tiny punctate staining in the neuropil became overwhelming. Fourth, many PSD-95-immunopositive puncta were apposed closely to synaptophysin-positive nerve terminals and overlapped with NMDA receptor subunits. By postembedding immunogold, the PSD-95 antibody was shown to label exclusively the postsynaptic density at asymmetrical synapses. Based on these results, we conclude that antibody access and binding to the postsynaptically located PSD95/SAP90 protein family are hindered when conventional immunohistochemistry is adopted, and that pepsin pretreatment effectively unmasks the postsynaptic epitopes. On the other hand, PSD-95 in axon terminals of cerebellar basket cells, where high levels of potassium channels are present, was detectable irrespective of pepsin pretreatment, suggesting that PSD-95 antibody is readily accessible to the presynaptic epitopes. Consequently, the present immunohistochemical results have provided light microscopic evidence supporting the prevailing notion that the PSD-95/SAP90 protein family interacts with NMDA receptor subunits and potassium channel subunits. J. Comp. Neurol. 426:572-586, 2000. (C) 2000 Wiley-Liss, Inc.
  • T Miyazaki, M Watanabe, A Yamagishi, M Takahashi
    NEUROSCIENCE RESEARCH 37 (4) 299 - 306 0168-0102 2000/08 [Refereed][Not invited]
     
    Two isoforms of nonmuscle myosin heavy chain IIB (MHC-IIB) are generated by alternative splicing; MHC-IIB(B2) differs from MHC-IIB(Delta B2) by the insertion of B2 exon cassette near the actin binding region. Here we examined expressions of the two splice variants in developing and adult rat brains by in situ hybridization with isoform-specific oligonucleotide probes. In adult, MHC-IIB(Delta B2) mRNA was highly expressed in neurons of the cerebral cortex, hippocampus, and cerebellum, whereas MHC-IIB(B2) mRNA was mainly distributed in the brainstem and cerebellum, with the highest level in Purkinje cells. During development, MHC-IIB(Delta B2) mRNA was predominantly expressed in various regions of embryonic and neonatal brains, whereas MHC-IIB(B2) mRNA was low during embryonic stages. Up-regulation of MHC-IIB(B2) started in the cerebellum during early postnatal stages when dendritogenesis and synaptogenesis occur actively in Purkinje cells. We further employed immunofluorescence using two antibodies (one recognizing both splicing variants and another specific to MHC-IIB(B2)), and found similar and dense localization in cell bodies and dendrites of Purkinje cells. Therefore, splicing of the B2 exon cassette undergoes distinct temporal and spatial regulations in the brain in vivo, and the different exon usage seems unlikely to affect the somato-dendritic localization of MHC-IIB. (C) 2000 Elsevier Science Ireland Ltd and Japan Neuroscience Society. All rights reserved.
  • S Kakizawa, M Yamasaki, M Watanabe, M Kano
    JOURNAL OF NEUROSCIENCE 20 (13) 4954 - 4961 0270-6474 2000/07 [Refereed][Not invited]
     
    Synapse elimination is considered to be the final step in neural circuit formation, by causing refinement of redundant connections formed at earlier developmental stages. The developmental loss of climbing fiber innervation from cerebellar Purkinje cells is an example of such synapse elimination. It has been suggested that NMDA receptors are involved in the elimination of climbing fiber synapses. In the present study, we probed the NMDA receptor-dependent period of climbing fiber synapse elimination by using daily intraperitoneal injections of the NMDA receptor antagonist MK-801. We found that blockade of NMDA receptors during postnatal day 15 (P15) and P16, but not before or after this period, resulted in a higher incidence of multiple climbing fiber innervation and caused a mild but persistent loss of motor coordination. Neither basic synaptic functions nor cerebellar morphology were affected by this manipulation. Chronic local application of MK-801 to the cerebellum during P15 and P16 also yielded a higher incidence of multiple climbing fiber innervation. During P15-P16, large NMDA receptor-mediated EPSCs were detected at the mossy fiber-granule cell synapse, but not at the parallel fiber-Purkinje cell or climbing fiber-Purkinje cell synapse. It is therefore likely that the NMDA receptors located at the mossy fiber-granule cell synapse mediate signals leading to the elimination of surplus climbing fibers. These results suggest that an NMDA receptor-dependent phase of climbing fiber synapse elimination lasts 2 d at most. During this phase, the final refinement of climbing fiber synapses occurs, and disruption of this process leads to permanent impairment of cerebellar function.
  • F Mori, M Fukaya, H Abe, K Wakabayashi, M Watanabe
    NEUROSCIENCE LETTERS 285 (1) 57 - 60 0304-3940 2000/05 [Refereed][Not invited]
     
    Ryanodine receptors (RyR) are Ca2+-induced Ca2+ release channels located on the endoplasmic reticulum, and consist of three isoforms, termed RyR1-3. We examined their expression in developing mouse brains by in situ hybridization. During the embryonic stage, RyR1 mRNA levels were highest in the rostral cortical plate, whereas RyR3 mRNA was most prominent in the caudal cortical plate and hippocampus. Initially,, low levels of RyR2 mRNA were distributed in the diencephalon and brainstem. However, from postnatal day 7 onward, RyR2 mRNA became the major isoform in many brain regions, while RyR1 mRNA became prominent in the dentate gyrus and Purkinje cell layer. Postnatal downregulation in the caudal cerebral cortex restricted RyR3 mRNA expression to the hippocampus, particularly the CA1 region. Therefore, RyR expression undergoes dynamic changes during the early postnatal period, when neurons are undergoing structural and functional differentiation. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.
  • J Tanaka, S Nakagawa, E Kushiya, M Yamasaki, M Fukaya, T Iwanaga, MI Simon, K Sakimura, M Kano, M Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 12 (3) 781 - 792 0953-816X 2000/03 [Refereed][Not invited]
     
    Following cell surface receptor activation, the alpha subunit of the Gq subclass of GTP-binding proteins activates the phosphoinositide signalling pathway. Here we examined the expression and localization of Gq protein alpha subunits in the adult mouse brain by in situ hybridization and immunohistochemistry. Of the four members of the Gq protein alpha subunits, G alpha q and G alpha 11 were transcribed predominantly in the brain. The highest transcriptional level of G alpha q was observed in cerebellar Purkinje cells (PCs) and hippocampal pyramidal cells, while that of G alpha 11 was noted in hippocampal pyramidal cells. Antibody against the C-terminal peptide common to G alpha q and G alpha 11 strongly labelled the cerebellar molecular layer and hippocampal neuropil layers. In these regions, immunogold preferentially labelled the cytoplasmic face of postsynaptic cell membrane of PCs and pyramidal cells. Immunoparticles were distributed along the extra-junctional cell membrane of spines, dendrites and somata, but were almost excluded from the junctional membrane. By double immunofluorescence, G alpha q/G alpha 11 was extensively colocalized with metabotropic glutamate receptor mGluR1 alpha in dendritic spines of PCs and with mGluR5 in those of hippocampal pyramidal cells. Together with concentrated localization of mGluR1 alpha and mGluR5 in a peri-junctional annulus on PC and pyramidal cell synapses (Baude et al. 1993, Neuron, 11, 771-787; Lujan et al. 1996, Eur. J. Neurosci., 8, 1488-1500), the present molecular-anatomical findings suggest that peri-junctional stimulation of the group I metabotropic glutamate receptors is mediated by G alpha q and/or G alpha 11, leading to the activation of the intracellular effector, phospholipase C beta.
  • K Yamada, M Fukaya, T Shibata, H Kurihara, K Tanaka, Y Inoue, M Watanabe
    JOURNAL OF COMPARATIVE NEUROLOGY 418 (1) 106 - 120 0021-9967 2000/02 [Refereed][Not invited]
     
    Bergmann glia (BG) are unipolar cerebellar astrocytes, whose radial (or Bergmann) fibers associate with developing granule cells and mature Purkinje cells (PCs), In the present study, we investigated the morphodifferentiation of BG by immunohistochemistry for glutamate transporter GLAST and electron microscopy. GLAST was expressed widely in cerebellar radial. glia/astrocytes during fetal and neonatal periods and became concentrated in BG postnatally. During the second postnatal week when PC dendrites grow actively, GLAST immunostaining revealed dynamic cytologic changes in Bergmann fibers in a deep-to-superficial gradient; Bergmann fibers traversing the external granular layer were stained as rod-like fibers, whereas in the molecular layer, the rod-like pattern was gradually replaced with a reticular meshwork. At postnatal day 10, the superficial rod-like domain was composed of glial fibrillary acidic protein (GFAP)-positive/GLAST-positive straight fibers, forming cytoplasmic swellings and short filopodia. Along this domain, the tip of growing PC dendrites ascended vertically and entered the base of the external granular layer. The deeper reticular domain of Bergmann fibers was characterized by active expansion of GFAP-negative/GLAST-positive lamellate processes, which surrounded PC synapses almost completely. Therefore, the transformation of Bergmann fibers proceeds in correlation with dendritic differentiation of PCs. The intimate PC-BG relationships during cerebellar development raise the possibility that a preexisting glial shaft could serve as a structural substrate that directs dendritic outgrowth toward the pial surface, whereas the successive formation of a reticular glial meshwork should lead to structural maturation of newly formed PC synapses. (C) 2000 Wiley-Liss, Inc.
  • T Minami, E Okuda-Ashitaka, H Mori, K Sakimura, M Watanabe, M Mishina, S Ito
    NEUROSCIENCE 97 (1) 133 - 142 0306-4522 2000 [Refereed][Not invited]
     
    Activation of primary afferent C fibers gives rise to spinal release of substance P and glutamate, and these mediators facilitate the cascade of nociceptive processing. We recently showed that intrathecal administration of nociceptin or orphanin FQ thereafter called nociceptin) induced hyperalgesia to noxious thermal stimuli and allodynia to innocuous tactile stimuli applied to conscious mice. In the present study, we designed experiments to elucidate the pathways and mediators of nociceptin-evoked pain responses. Neonatal capsaicin treatment eliminated the induction of hyperalgesia and allodynia by nociceptin. Whereas this treatment markedly reduced the content of substance P in the spinal cord, it did not affect the nociceptin content or the expression of nociceptin receptors and GluR epsilon and GluR xi subunits of N-methyl-D-aspartate receptors in it. The substance P antagonists CP96,345 and CP99,994 blocked the nociceptin-induced hyperalgesia, but not the allodynia. In contrast, the nociceptin-evoked allodynia, but not hyperalgesia, disappeared in N-methyl-D-aspaaate receptor GluR epsilon 1 subunit knockout mice. Both nociceptin-evoked hyperalgesia and allodynia were attenuated by morphine in a dose-dependent manner. Taken together, these results demonstrate that capsaicin-sensitive primary afferent fibers are involved not only in thermal hyperalgesia but also in tactile allodynia induced by nociceptin, but in different pathways; the former is mediated by substance P and the latter is mediated by glutamate through the N-methyl-D-aspartate receptor comprising the GluR epsilon 1 subunit. (C) 2000 IBRO. Published by Elsevier Science Ltd.
  • K Hashimoto, M Watanabe, H Kurihara, S Offermanns, H Jiang, Y Wu, K Jun, HS Shin, Y Inoue, D Wu, MI Simon, M Kano
    CEREBELLAR MODULES: MOLECULES, MORPHOLOGY, AND FUNCTION 124 31 - 48 0079-6123 2000 [Refereed][Not invited]
  • M Tsujita, H Mori, M Watanabe, M Suzuki, J Miyazaki, M Mishina
    JOURNAL OF NEUROSCIENCE 19 (23) 10318 - 10323 0270-6474 1999/12 [Refereed][Not invited]
     
    To develop a cell type-specific and temporal regulation system of gene targeting in the cerebellum, we used the NMDA-type glutamate receptor GluR epsilon 3 subunit gene and Cre recombinase-progesterone receptor fusion (CrePR) gene in combination. Injection of the CrePR gene placed under the control of the 10 kb 5' region of the GluRe3 gene into C57BL/6 eggs yielded the ECP25 line that strongly expressed the CrePR mRNA selectively in the granule cells of the cerebellum. Using a transgenic mouse carrying a reporter gene for Cre-mediated recombination, we showed that antiprogestins could induce the recombinase activity of CrePR protein in the cerebellar granule cells of the ECP25 line. Thus, the established mouse line will provide a valuable tool to investigate the mechanism of cerebellar function by manipulating molecules in the temporally regulated and granule cell-specific manner.
  • K Nakamura, H Shima, M Watanabe, T Haneji, K Kikuchi
    BIOCHEMICAL JOURNAL 344 819 - 825 0264-6021 1999/12 [Refereed][Not invited]
     
    Dual-specificity protein phosphatases (DSPs) play roles in the regulation of mitogenic signal transduction for extracellular stimulation and the cell cycle. In the present study, we identified a novel DSP, termed TMDP (testis- and skeletal-muscle-specific DSP). Nucleotide sequence analysis of TMDP cDNA indicated that the open reading frame of 597 bp encodes a protein of 198 amino acid residues with a predicted molecular mass of 22.5 kDa. The deduced amino acid sequence contains a motif for a conserved catalytic domain of DSPs and shows highest similarity to human Vaccinia HI-related phosphatase (45.5 % identity) but low homology to the mitogen-activated protein kinase phosphatase and CDC25 subfamilies of DSPs. Recombinant TMDP protein exhibited intrinsic phosphatase activity towards both phospho-seryl/threonyl and -tyrosyl residues of myelin basic protein, with similar specific activities in vitro. Northern-blot analysis revealed that TMDP is most abundantly expressed in the testis. The expression in the testis is characterized as follows: (i) TMDP mRNA first appeared 3 weeks after birth, corresponding to the time that meiosis begins; (ii) TMDP mRNA was abundant in fractionated spermatocytes and round spermatids; and (iii) hybridization in situ showed that the TMDP mRNA is localized in spermatocytes and/or spermatids in seminiferous tubules. These data demonstrate that TMDP is a novel DSP abundantly expressed in the testis and suggest that TMDP may be involved in the regulation of meiosis and/or differentiation of testicular germ cells during spermatogenesis.
  • M Fukaya, K Yamada, M Nagashima, K Tanaka, M Watanabe
    NEUROSCIENCE RESEARCH 34 (3) 165 - 175 0168-0102 1999/08 [Refereed][Not invited]
     
    The glutamate transporter plays an important role in rapid removal of glutamate from the synaptic cleft. Glutamate transporter GLAST is highly expressed in the Bergmann glia (BG), a unipolar cerebellar astrocyte associated structurally and functionally with Purkinje cells (PCs). Here we investigated the expression and localization in the reeler and weaver mutant cerebella with disorganized cytoarchitecture and disrupted synaptic circuitry. In the cortex of both cerebella, GLAST-expressing cells were astrocytes associating PCs; they were located around PC somata and primary dendrites, and extended glial fibrillary acidic protein (GFAP)-immunspositive processes surrounding PC somata and dendrites. Additional signals were detected in astrocytes of the reeler subcortex: they were dispersed among ectopic PCs and had GFAP-positive processes apposing to PC somata and stunted dendrites. Therefore, GLAST expression in PC-associated astrocytes was conserved in these mutants. Compared to the wild-type BG, however, the transcription level in individual mutant astrocytes was significantly reduced to about one-third level in the reeler and weaver cortex or one-sixth level in the reeler subcortex. Taking previous results on remarkable up-regulation during dendritogenic/synaptogenic stages and down-regulation following experimental glutamatergic denervation, it is suggested that GLAST expression in cerebellar astrocytes is regulated correlatively with cytological and/or synaptic differentiation of neighboring PCs. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.
  • K Hashimoto, M Fukaya, XX Qiao, K Sakimura, M Watanabe, M Kano
    JOURNAL OF NEUROSCIENCE 19 (14) 6027 - 6036 0270-6474 1999/07 [Refereed][Not invited]
     
    The spontaneous recessive mutant mouse stargazer (stg) begins to show ataxia around postnatal day 14 and display a severe impairment in the acquisition of classical eyeblink conditioning in adulthood. These abnormalities have been attributed to the specific reduction in brain-derived neurotrophic factor (BDNF) and the subsequent defect in TrkB receptor signaling in cerebellar granule cells (GCs). In the stg mutant cerebellum, we found that EPSCs at mossy fiber (MF) to GC synapses are devoid of the fast component mediated by AMPA-type glutamate receptors despite the normal slow component mediated by NMDA receptors. The sensitivity of stg mutant GCs to exogenously applied AMPA was greatly reduced, whereas that to NMDA was unchanged. Glutamate release from MF terminals during synaptic transmission to GCs appeared normal. By contrast, AMPA receptor-mediated EP-SCs were normal in CA1 pyramidal cells of the stg mutant hippocampus. Thus, postsynaptic AMPA receptor function was selectively impaired in stg mutant GCs, although the transcription of four AMPA receptor subunit: genes in the stg GC was comparable to the wild-type GC. We also examined the cerebellum of BDNF knockout mice and found that their MF-GC synapses had a normal AMPA receptor-mediated EPSC component. Thus, the impaired AMPA receptor function in the stg mutant GC is not likely to result from the reduced BDNF-TrkB signaling. These results suggest that the defect in MF to GC synaptic transmission is a major factor that causes the cerebellar dysfunction in the stg mutant mouse.
  • C Knouff, ME Hinsdale, H Mezdour, MK Altenburg, M Watanabe, SH Quarfordt, PM Sullivan, N Maeda
    JOURNAL OF CLINICAL INVESTIGATION 103 (11) 1579 - 1586 0021-9738 1999/06 [Refereed][Not invited]
     
    We have generated mice expressing the human apo E4 isoform in place of the endogenous murine apo E protein and have compared them with mice expressing the human apo E3 isoform, Plasma lipid and apolipoprotein levels in the mice expressing only the apo E4 isoform (4/4) did not differ significantly from those in mice with the apo E3 isoform (3/3) on chow and were equally elevated in response to increased lipid and cholesterol in their diet. However, on all diets tested, the 4/4 mice had approximately twice the amount of cholesterol, apo E, and apo B-48 in their VLDL as did 3/3 mice. The 4/4 VLDL competed with human LDL for binding to the human LDL receptor slightly better than 3/3 VLDL, but the VLDL clearance rate in 4/4 mice was half that in 3/3 mice. On an atherogenic diet, there was a trend toward greater atherosclerotic plaque size in 4/4 mice compared with 3/3 mice. These data, together with our earlier observations in wild-type and human APOE*2-replacement mice, demonstrate a direct and highly significant correlation between VLDL clearance rate and mean atherosclerotic plaque size. Therefore, differences solely in apo E protein structure are sufficient to cause alterations in VLDL residence time and atherosclerosis risk in mice.
  • T Shibata, M Watanabe, R Ichikawa, Y Inoue, T Koyanagi
    JOURNAL OF COMPARATIVE NEUROLOGY 404 (2) 172 - 182 0021-9967 1999/02 [Refereed][Not invited]
     
    The glutamatergic transmission system plays a key role in afferent and efferent pathways involved in micturition. By in situ hybridization combined with retrograde Fast Blue labeling, expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (GluR-A to -D) and N-methyl-D-aspartate (NMDA) receptor (NR1 and NR2A-D) subunit mRNAs were examined in visceromotor and somatomotor neurons of the rat lumbosacral spinal cord. Parasympathetic preganglionic neurons (PC;Ns) in the intermediolateral nucleus highly expressed GluR-A and GluR-B subunit mRNAs, with very low levels for GluR-C and GluR-D subunits. As for the NMDA receptor, PGNs were associated with abundant signals for NR1 subunit mRNA, but without any NR2 subunit mRNAs. On the other hand, somatomotor neurons in the ventral horn (dorsolateral nucleus) express all four AMPA receptor subunit mRNAs, showing relatively abundant expressions of GluR-C and GluR-D subunit mRNA compared with PGNs. In addition to high levels of NR1 subunit mRNA, dorsolateral nucleus neurons moderately expressed NR2A and NR2B subunit mRNAs. These results suggest that molecular organization of both AMPA and NMDA receptor channels are distinct between PGNs and dorsolateral nucleus neurons. Considering that native NMDA receptors are heteromeric channels composed of NR1 and NR2 subunits, it seems Likely that dorsolateral nucleus neurons, not PGNs, are provided with functional NMDA receptors, which could induce activity-dependent changes in synaptic transmission in the efferent pathway for the lower urinary tract. (C) 1999 Wiley-Liss, Inc.
  • M Fukaya, H Ueda, K Yamauchi, Y Inoue, M Watanabe
    NEUROSCIENCE RESEARCH 33 (2) 111 - 118 0168-0102 1999/02 [Refereed][Not invited]
     
    PSD-95 (SAP90), SAP102. and Chapsyn-110 (PSD-93) are members of the membrane-associated guanylate kinase family, and interact with N-methyl-D-aspartate (NMDA) receptor NR2A (GluR epsilon 1) and NR2B (GluR epsilon 2) subunits and with Shaker-type Kt channel subunits to cluster into a channel complex. In the present study, we examined their expression in developing and adult mouse brains by in situ hybridization with antisense oligonucleotide probes. PSD-95 and SAP102 mRNAs were prominently expressed at embryonic day 13 (E13) in the mantle zone of various brain regions, where NMDA receptor NR2B subunit mRNA is expressed at high levels, In the early postnatal period when active synaptogenesis takes place, both mRNAs became elevated and concentrated in the telencephalon and cerebellar granular layer, where NR2A and/or NR2B subunit mRNAs are abundantly expressed. Chapsyn-110 mRNA was, though at low levels, found over the mantle zone of embryonic brains, and the level was progressively increased in the telencephalon starting at perinatal stages. The spatial and temporal correlations in the brain in vivo suggest that the PSD-95/SAP90 protein family can interact with NMDA receptor subunits to cluster them into channel complex at both synaptic and non-synaptic sites before, during and after synaptogenic stages. (C) 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.
  • マウス小脳プルキンエ細胞シナプスにおけるGTP結合蛋白Gαq/11の局在
    田中 淳, 中川 伸, 井上 芳郎, 渡辺 雅彦
    解剖学雑誌 (一社)日本解剖学会 74 (1) 56 - 56 0022-7722 1999/02
  • S Toki, M Watanabe, R Ichikawa, T Shirakawa, H Oguchi, Y Inoue
    NEUROSCIENCE RESEARCH 33 (1) 9 - 15 0168-0102 1999/01 [Refereed][Not invited]
     
    Vibrissae are tactile sense organs on the face of non-human mammals, and build up topographical representations in the brainstem trigeminal sensory nucleus called barrelettes. In the present study, we examined postnatal development of barrelettes corresponding to upper lip vibrissae by cytochrome oxidase (CO) histochemistry. At nuclear regions corresponding to upper lip vibrissae, a few segregated barrelettes first appeared at postnatal day 2 (P2), and segregation became clear for most upper lip barrelettes at P4. Compared with major barrelettes corresponding to mystacial vibrissae on the snout, the development of segregated pattern formation for upper lip barrelettes was retarded by 1-2 days. When vibrissa-related patterns were examined 5 days after infraorbital nerve transection, upper lip barrelettes became obscure in all mice lesioned at P1 and P2. Lesion-insensitive upper lip barrelettes first emerged in a few mice lesioned at P3 (33%), and the percentage attained 100% at P6. This temporal transition from lesion-sensitive to lesion-insensitive barrelettes was 3 days ahead of mystacial barrelettes. Therefore, upper lip barrelettes achieve rapid development within a narrow time frame during the first postnatal week. The early and rapid establishment of lesion-insensitive, mature barrelettes can be interpreted as suggesting the importance of oral sensory function in neonatal life. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.
  • T Ogurusu, K Yanagi, M Watanabe, M Fukaya, R Shingai
    RECEPTORS & CHANNELS 6 (6) 463 - 475 1060-6823 1999 [Refereed][Not invited]
     
    Ionotropic GABA receptors that are composed of rho subunits act to gate bicuculline-insensitive Cl- currents. Reverse transcription-polymerase chain reaction analysis revealed that the expression of rho 2 mRNA in adult rat brain was approximately eight times higher than mRNA in the rat brain at embryonic day 16, while that of rho 3 in the embryonic brain was approximately six times higher than in the adult brain. In the adult rat brain the rho 3 mRNA was present in the mesencephalon, hippocampus, cerebellum, thalamus and basal ganglia. In situ hybridization has been used to demonstrate the presence of rho 2 mRNA in the hippocampal CA1 region of the 8-day-old rat, and in the CA1 region of the hippocampus, lateral geniculate nucleus, superficial gray layer of the superior colliculus and the pars compacta of the substantia nigra of the adult rat. When the homooligomeric rho 3 receptors were expressed in Xenopus oocytes, applications of agonists induced ionic currents, The order of potency of the agonists was muscimol > GABA is approximately equal to trans-4-aminocrotonic acid > cis-4-aminocrotonic acid. The ionic currents induced by GABA were blocked by picrotoxinin and Zn2+ in dose-dependent manner. In heterooligomeric rho 2 rho 3 receptors, picrotoxinin sensitivity was significantly reduced.
  • M Kano, K Hashimoto, M Watanabe, H Kurihara, S Offermanns, HP Jiang, YP Wu, K Jun, HS Shin, Y Inoue, MI Simon, DQ Wu
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 95 (26) 15724 - 15729 0027-8424 1998/12 [Refereed][Not invited]
     
    Elimination of excess climbing fiber (CF)-Purkinje cell synapses during cerebellar development involves a signaling pathway that includes type 1 metabotropic glutamate receptor, G alpha q, and the gamma isoform of protein kinase C. To identify phospholipase C (PLC) isoforms involved in this process, we generated mice deficient in PLC beta 4, one of two major isoforms expressed in Purkinje cells. PLC beta 4 mutant mice are viable but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple CF innervation clearly is impaired in the rostral portion of the cerebellar vermis, in which PLC beta 4 mRNA is predominantly expressed. By contrast, CF synapse elimination is normal in the caudal cerebellum, in which low levels of PLC beta 4 mRNA but reciprocally high levels of PLC beta 3 mRNA are found. These results indicate that PLC beta 4 transduces signals that are required for CF synapse elimination in the rostral cerebellum.
  • A Mitani, M Watanabe, K Kataoka
    JOURNAL OF NEUROSCIENCE 18 (19) 7941 - 7952 0270-6474 1998/10 [Refereed][Not invited]
     
    The developing neurons have been reported I:, be extremely susceptible to toxicity of NMDA during a restricted developmental period. Pontosubicular neuronal necrosis is a typical type of perinatal human brain lesion and often coexists with other forms of cerebral hypoxic and ischemic injuries. To determine whether functional changes of NMDA receptors related to the susceptibility to NMDA toxicity are involved in developing neurons in the pontine nucleus, we have examined the lesion produced by in vivo direct injection of NMDA into the pontine nucleus of rats at postnatal days 1-30, recorded NMDA-induced whole-cell currents from neurons in the pontine nucleus in the developing rat brainstem slices, and performed in situ hybridization for NMDA receptor subunit mRNAs in the pontine nucleus. The susceptibility to NMDA neurotoxicity peaked near postnatal day 15, and the NMDA-induced currents showed prominent reduction of the voltage-dependent block by Mg2+ near postnatal day 15. The pontine nucleus near postnatal day 15 showed distinct expression of the NMDA receptor subunit NR2C mRNA. These results suggest that the susceptibility to NMDA neurotoxicity that is enhanced in the rat pontine nucleus near postnatal day 15 is mediated by the NMDA receptor channels that are relatively insensitive to Mg2+ and that the reduction in the sensitivity of NMDA receptors to Mg2+ correlates with the expression of the NR2C. We present the possibility that functional changes in the NMDA receptor channels play a crucial role in the occurrence of developmentally specific neuronal injury.
  • H Mori, T Manabe, M Watanabe, Y Satoh, N Suzuki, S Toki, K Nakamura, T Yagi, E Kushiya, T Takahashi, Y Inoue, K Sakimura, M Mishina
    NEURON 21 (3) 571 - 580 0896-6273 1998/09 [Refereed][Not invited]
     
    The synaptic localization of the N-methyl-D-aspartate (NMDA) type glutamate receptor (GluR) channel is a prerequisite for synaptic plasticity in the brain. We generated mutant mice carrying the carboxy-terminal truncated GluR epsilon 2 subunit of the NMDA receptor channel. The mutant mice died neonatally and failed to form barrelette structures in the brainstem. The mutation greatly decreased the NMDA receptor-mediated component of hippocampal excitatory postsynaptic potentials and punctate immunofluorescent labelings of GluR epsilon 2 protein in the neuropil regions, while GluR epsilon 2 protein expression was comparable. Immunostaining of cultured cerebral neurons showed the reduced punctate staining of the truncated GluR epsilon 2 protein at synapses. These results suggest that the carboxy-terminal region of the GluR epsilon 2 subunit is important for efficient clustering and synaptic localization of the NMDA receptor channel.
  • K Ikeda, M Watanabe, T Ichikawa, T Kobayashi, R Yano, T Kumanishi
    JOURNAL OF COMPARATIVE NEUROLOGY 399 (1) 139 - 151 0021-9967 1998/09 [Refereed][Not invited]
     
    Nociceptin/orphanin FQ (N/OFQ) and its receptor share similarities to opioids and their receptors in terms of the molecular structure and signaling pathway, but the two systems exhibit different actions in vivo. To understand the mechanism of N/OFQ-system actions, we examined, by in situ hybridization analysis, the distribution of preproN/OFQ and N/OFQ receptor mRNAs in the developing and adult mouse central nervous systems (CNS). In most neural regions, preproN/OFQ mRNA was mainly expressed in a small population of middle-sized neurons. These neurons were scattered between large projection-type neurons or within the neuropil, suggestive of interneurons. In some other nuclei (lateral septum, bed nucleus of the stria terminalis, reticular thalamic nucleus, inferior colliculus, and rostral periolivery nucleus), preproN/OFQ mRNA was expressed in a number of large projection-type neurons. By contrast, N/OFQ receptor mRNA was evenly expressed in most neurons of the adult CNS. Considering the inhibitory actions of N/OFQ, the distinct cellular expression pattern of the N/OFQ system suggests that the release of N/OFQ from interneurons may lower neuronal and synaptic activities of neighboring neurons, leading to integration or modulation of local circuits. Furthermore, the cellular expression pattern, distinct from that of the opioid system, may provide a possible molecular/cellular basis for the different in vivo actions of N/OFQ and opioids. In embryonic stages, both preproN/OFQ and N/OFQ receptor mRNAs were highly and widely expressed in the mantle zone, suggesting the possible importance of N/OFQ signaling in CNS development. J. Comp. Neurol. 399:139-151, 1998. (C) 1998 Wiley-Liss, Inc.
  • イオン性シグナル伝達の解明に挑む イオンチャネル構造機能研究の新展開 NMDA受容体チャネルの生理機能
    森 寿, 真鍋 俊也, 渡辺 雅彦, 崎村 建司, 中村 和裕, 城山 優治, 佐藤 泰司, 三品 昌美
    神経化学 (一社)日本神経化学会 37 (3) 260 - 260 0037-3796 1998/09
  • テレンセファリン遺伝子にCreリコンビナーゼ遺伝子を挿入した変異マウスの作製と解析
    中村 和裕, 森 寿, 城山 優治, 八木 健, 真鍋 俊也, 高橋 智幸, 渡辺 雅彦, 市川 量一, 井上 芳郎, 三品 昌美
    神経化学 (一社)日本神経化学会 37 (3) 555 - 555 0037-3796 1998/09
  • マウス小脳登上線維の生後発達の神経活動依存性と臨界期
    柿澤 昌, 渡辺 雅彦, 井上 芳郎, 狩野 方伸
    神経化学 日本神経化学会 37 (3) 563 - 563 0037-3796 1998/09
  • K Yamada, M Watanabe, T Shibata, M Nagashima, K Tanaka, Y Inoue
    JOURNAL OF NEUROSCIENCE 18 (15) 5706 - 5713 0270-6474 1998/08 [Refereed][Not invited]
     
    The glutamate transporter GLT-1 is expressed in astrocytes of the mature brain and spinal cord. In the present study, we examined its expression in the developing mouse spinal cord. By in situ hybridization, S-35-labeled antisense oligonucleotide probes for GLT-1 mRNA consistently labeled the mantle zone/ gray matter from embryonic day 11 through the adult stage. However, immunohistochemistry with a specific antibody visualized distinct regional and cellular localizations during the time between the fetal and postnatal stages. At fetal stages, GLT-1 immunoreactivity predominated in the marginal zone/white matter, observed as tiny puncta in cross-sections and as thin fibers in longitudinal sections. The GLT-1-immunopositive structures were also labeled for neuron-specific enolase, a glycolytic enzyme specific to postmitotic neurons and endocrine cells. By electron microscopy, GLT-1 immunoreactivity was detected in axons forming frequent enlargements and was focally localized on a small portion of the axolemma, particularly that facing adjacent axons. At early postnatal stages, GLT-1 disappeared from axons in white matter tracts and, instead, appeared in astrocytic processes surrounding various neuronal elements in the gray matter. Therefore, before switching to astrocytic expression, GLT-1 is transiently expressed in neurons and localized in differentiating axons. Together with our previous finding on the localization of glutamate transporter GLAST in radial glial fibers, GLT-1 and GLAST are thus localized during development on distinct directional cellular elements along which young neurons elongate their axons or move their cell bodies, respectively.
  • S Tanaka, K Suzuki, M Watanabe, A Matsuda, S Tone, T Koike
    JOURNAL OF NEUROSCIENCE 18 (16) 6358 - 6369 0270-6474 1998/08 [Refereed][Not invited]
     
    Cerebellar granule neurons isolated from postnatal day 7 (P7) rats and grown in normal K(+)medium begin to degenerate at approximately 4 d in vitro (DIV) and die. To search for genes upregulated in the process of neuronal cell death, differential hybridization was performed with subtracted cDNA probes and a cDNA library from 5 DIV. One of the genes isolated was microglial response factor-1 (mrf-1), which encoded a sequence of 177 amino acids with a single EF-hand calcium-binding motif. By Northern blots, the transcript was upregulated in cerebellar culture at 4 DIV, peaked at 6 DIV, and decreased at 7 DIV. Upregulation was also found when the apoptosis of granule cells was induced by replacing high K+ medium with normal K+ medium. However, when non-neuronal cells were thoroughly eliminated with aphidicolin, an antimitotic agent, the upregulation at 4-7 DIV did not occur. By immunocytochemistry, MRF-I was detected at 5 DIV in OX-42-positive cells (microglia), and it exhibited an increase in response to granule cell death. MRF-1 levels in microglia purified from cerebral cortex also upregulated in the presence of 5 DIV granule cells. In the developing cerebellum in vivo, levels of mrf-1 mRNA transiently increased in the early postnatal stages, reaching a peak at P7 when cerebellar neurons and astrocytes undergo extensive apoptosis. In adult brain sections, MRF-1 was detected in the perikarya and processes of ramified/resting microglia, and peripheral motor nerve dissection prominently increased the expression in activated microglia surrounding injured central motoneurons. Therefore, mrf-1 appears to be one of the microglial genes that respond to neuronal cell death and degeneration.
  • Mizuta, I, M Katayama, M Watanabe, M Mishina, K Ishii
    CELLULAR AND MOLECULAR LIFE SCIENCES 54 (7) 721 - 725 1420-682X 1998/07 [Refereed][Not invited]
     
    Using primary cultures of murine cerebral cortices, we investigated the developmental expression of N-methyl-D-aspartate (NMDA) receptor subunits in relation to the appearance of NMDA receptor-mediated glutamate neurotoxicity. The cultures were not affected by glutamate exposure on culture days 7-9, but became sensitive to glutamate neurotoxicity on day II. The expression of NMDA receptor subunit messenger RNAs (mRNAs) was investigated by means of reverse transcription polymerase chain reaction (RT-PCR). The epsilon 3-NR2C and epsilon 4-NR2D transcripts could not be detected in the culture. The epsilon 2-NR2B and zeta 1-NR1 subunit mRNAs, on the other hand, could be detected clearly and continuously from the culture initiation, and the epsilon 1-NR2A subunit mRNA became clearly detectable on culture day 4. The expression of these three subunits' proteins in the glutamate-insensitive stage (culture day 8) and the sensitive stage (day 11) were studied by means of Western blotting. The epsilon 2-NR2B and zeta 1-NR1 subunit proteins were clearly expressed on culture days 8 and 11, but the epsilon 1-NR2A subunit protein could hardly be detected on either day 8 or day 11. These results suggest that the glutamate neurotoxicity in the primary culture was mediated mainly by epsilon 2/zeta 1 NMDA receptors. The time lag between the protein expression of the epsilon 2-NR2B and zeta 1-NR1 subunits and the emergence of glutamate neurotoxicity may be necessary for the maturation of functional NMDA receptor systems, including heteromeric receptor formation, increase in receptor density and maturation of the postreceptor signal transduction system.
  • H Manya, J Aoki, M Watanabe, T Adachi, H Asou, Y Inoue, H Arai, K Inoue
    JOURNAL OF BIOLOGICAL CHEMISTRY 273 (29) 18567 - 18572 0021-9258 1998/07 [Refereed][Not invited]
     
    In a previous study, we demonstrated that Platelet-activating Factor (PAF) acetylhydrolase purified from bovine brain cortical cytosol consists of two mutually homologous catalytic subunits (alpha 1 and alpha 2) and one putative regulatory beta subunit, The latter is a product of the LIS1 gene, which is defective in the Miller-Dieker syndrome, a form of lissencephaly. In this study, we examined the expression patterns of these three subunits in the developing rat brain. All three subunits were expressed in embryonic brain, whereas only alpha 2 and beta subunit were detected in the adult brain by Western blotting, Biochemical analyses revealed that the alpha 1/alpha 2 heterodimer and alpha 2/alpha 2 homodimer are major catalytic units of embryonic and adult brain PAF acetylhydrolases, respectively. The alpha 1 transcript and protein were detected predominantly in embryonic and postnatal neural tissues, such as the brain and spinal cord. Furthermore, we found using primary cultured cells isolated from neonatal rat brain that alpha 1 protein were expressed only in neurons but not in glial cells and fibroblasts. In contrast, alpha 2 and beta transcripts and proteins were detected both in neural and non-neural tissues, and their expression level was almost constant from fetal stages through adulthood, These results indicate that alpha 1 expression is restricted to actively migrating neurons in rats and that switching of catalytic subunits from the alpha 1/alpha 2 heterodimer to the alpha 2/alpha 2 homodimer occurred in these cells during brain development, suggesting that PAF acetylhydrolase plays a role(s) in neuronal migration.
  • M Watanabe, M Nakamura, K Sato, M Kano, MI Simon, Y Inoue
    EUROPEAN JOURNAL OF NEUROSCIENCE 10 (6) 2016 - 2025 0953-816X 1998/06 [Refereed][Not invited]
     
    Ligand binding to neurotransmitter and hormone receptors which couple to the Go subclass of GTP-binding protein leads to the activation of phospholipase C beta (PLC beta) which hydrolyses phosphatidyl-inositol 4,5-bisphosphate, yielding a pair of second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP3), The expression of PLC beta 1-4 mRNAs was comparatively examined by in situ hybridization in the mouse brain. in adults, PLC beta 1 mRNA was expressed predominantly in the telencephalon, including the cerebral cortex, hippocampus, amygdala, lateral septum and olfactory bulb, with little expression in most thalamic nuclei, PLC beta 2 mRNA was distributed in the white matter, suggesting its expression in non-neuronal cells, most likely oligodendrocytes. PLC beta 3 mRNA was specifically expressed in cerebellar Purkinje cells, The highest levels of PLC beta 4 mRNA were detected in Purkinje cells. High levels of PLC beta 4 mRNA were also found in the thalamus and medial septum, whereas weak signals were detected in most telencephalic regions, thus showing an expression pattern almost reciprocal to that of PLC beta 1 mRNA. During development, such characteristic regional expression of PLC beta 1 and PLC beta 4 were observed starting in late foetal stages, while specific expression of PLC beta 2 and PLC beta 3 appeared in early postnatal stages. We conclude that despite the existence of four PLC beta isoforms, only one or two of them is expressed in individual neurons and glial cells, The distinct expression of PLC beta s provides a molecular basis for analysing the nature of the specific signal transduction pathway leading to the production of diacylglycerol and IP3 in distinct cell types and in different regions of the brain.
  • N Kohmura, K Senzaki, S Hamada, N Kai, R Yasuda, M Watanabe, H Ishii, M Yasuda, M Mishina, T Yagi
    NEURON 20 (6) 1137 - 1151 0896-6273 1998/06 [Refereed][Not invited]
     
    In mammals, neurons are highly differentiated and play distinctive functions even in the same brain region. We found a novel cadherin-related neuronal receptor (Cnr) gene family by studying Fyn-binding activity in mouse brain. CNR1 protein is located in the synaptic junction and forms a complex with Fyn. Sequence analysis of eight Cnr products of similar to 20 genes indicates that these comprise a novel cadherin family of the cadherin superfamily. The expression patterns of each member of this novel family were grossly similar to each other but restricted to subpopulations of neurons of the same type. The diversity of the Cnr family genes suggests that there are molecular mechanisms that govern highly differentiated neural networks in the mammalian CNS.
  • S Nakagama, M Watanabe, T Isobe, H Kondo, Y Inoue
    JOURNAL OF COMPARATIVE NEUROLOGY 395 (1) 112 - 120 0021-9967 1998/05 [Refereed][Not invited]
     
    The staggerer mouse carries a deletion in a gene encoding the nuclear hormone receptor ROR alpha, which leads to severe impairments in phenotypic differentiation of cerebellar Purkinje cells. We previously found parasagittal compartments in the mature staggerer cerebellum, as defined by different transcription levels of Purkinje cell-specific molecules including calbindin. In the present study, we developed a hightiter anti-calbindin antibody to examine morphological features of the staggerer Purkinje cells. Immunohistochemistry for calbindin revealed compartmentalized Purkinje cell populations with different cell sizes, alignments, cell densities, and dendritic arborization, as well as different immunoreactivities, corresponding to the "transcriptional" compartments. Based on these immunohistochemical and cytological characteristics, the rostral cerebellum was clearly subdivided into three to seven parasagittal zones (Zones I-VII). Purkinje cells in Zones I and III were associated with the strongest calbindin immunoreactivities and exhibited morphological features reminiscent of the wild-type cells, i.e., large flask-shaped cell bodies, monolayer alignment, and arborized dendrites. Purkinje cells in Zone V were also labeled strongly, but they were small in cell size, ectopic and possessed long unbranched dendrites. On the other hand. Purkinje cells in Zones II, IV, and VI were very low in calbindin immunoreactivity and marked by small cell size, ectopia, poorly-developed dendrites and low cell density. Considering that this unique cytological compartmentalization emerges as the result of ROR alpha gene mutation, it is suggested that normal cytodifferentiation of Purkinje cells is governed by both ROR alpha-dependent and -independent mechanisms, and further that the latter mechanism might exert unevenly along the mediolateral cerebellar axis. (C) 1998 Wiley-Liss. Inc.
  • T Harada, C Harada, M Watanabe, Y Inoue, T Sakagawa, N Nakayama, S Sasaki, S Okuyama, K Watase, K Wada, K Tanaka
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 95 (8) 4663 - 4666 0027-8424 1998/04 [Refereed][Not invited]
     
    In the retina, the glutamate transporter GLAST is expressed in Muller cells, whereas the glutamate transporter GLT-1 is found only in cones and various types of bipolar cells. To investigate the functional role of this differential distribution of glutamate transporters, we have analyzed GLAST and GLT-1 mutant mice. In GLAST-deficient mice, the electroretinogram b-wave and oscillatory potentials are reduced and retinal damage after ischemia is exacerbated, whereas GLT-1-deficient mice show almost normal electroretinograms and mild increased retinal damage after ischemia, These results demonstrate that GLAST is required for normal signal transmission between photoreceptors and bipolar cells and that both GLAST and GLT-1 play a neuroprotective role during ischemia in the retina.
  • K Watase, K Hashimoto, M Kano, K Yamada, M Watanabe, Y Inoue, S Okuyama, T Sakagawa, S Ogawa, N Kawashima, S Hori, M Takimoto, K Wada, K Tanaka
    EUROPEAN JOURNAL OF NEUROSCIENCE 10 (3) 976 - 988 0953-816X 1998/03 [Refereed][Not invited]
     
    To study the function of GLAST, a glutamate transporter highly expressed in the cerebellar Bergmann astrocytes, the mouse GLAST gene was inactivated, GLAST-deficient mice developed normally and could manage simple coordinated tasks, such as staying on a stationary or a slowly rotating rod, but failed more challenging task such as staying on a quickly rotating rod. Electrophysiological examination revealed that Purkinje cells in the mutant mice remained to be multiply innervated by climbing fibres even at the adult stage, We also found that oedema volumes in the mutant mice increased significantly after cerebellar injury, These results indicate that GLAST plays active roles both in the cerebellar climbing fibre synapse formation and in preventing excitotoxic cerebellar damage after acute brain injury.
  • M Watanabe, M Fukaya, K Sakimura, T Manabe, M Mishina, Y Inoue
    EUROPEAN JOURNAL OF NEUROSCIENCE 10 (2) 478 - 487 0953-816X 1998/02 [Refereed][Not invited]
     
    Hippocampal synapses express two distinct forms of the long-term potentiation (LTP), i.e. NMDA receptor-dependent and -independent LTPs. To understand its molecular-anatomical basis, we produced affinity-purified antibodies against the GluR epsilon 1 (NR2A), GluR epsilon 2 (NR2B), and GluR zeta 1 (NR1) subunits of the N-methyl-D-aspartate (NMDA) receptor channel, and determined their distributions in the mouse hippocampus, Using NMDA receptor subunit-deficient mice as the specificity controls, section pretreatment with proteases (pepsin and proteinase K) was found to be very effective to detect authentic NMDA receptor subunits. As the result of modified immunohistochemistry, all three subunits were detected at the highest level in the strata oriens and radiatum of the CA1 subfield, and high levels were also seen in most other neuropil layers of the CA1 and CA3 subfields and of the dentate gyrus. However, the stratum lucidum, a mossy fibre-recipient layer of the CA3 subfield, contained low levels of the GluR epsilon 1 and GluR zeta 1 subunits and almost excluded the GluR epsilon 2 subunit. Double immunofluorescence with the AMPA receptor GluR alpha 1 (GluR1 or GluR-A) subunit further demonstrated that the GluR epsilon 1 subunit was colocalized in a subset, not all, of GluR alpha 1-immunopositive structures in the stratum lucidum. Therefore, the selective scarcity of these NMDA receptor subunits in the stratum lucidum suggests that a different synaptic targeting mechanism exerts within a single CA3 pyramidal neurone in vivo, which would explain contrasting significance of the NMDA receptor channel in LTP induction mechanisms between the mossy fibre-CA3 synapse and other hippocampal synapses.
  • M Watanabe, J Aoki, H Manya, H Arai, K Inoue
    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1401 (1) 73 - 79 0167-4889 1998/01 [Refereed][Not invited]
     
    Brain intracellular platelet-activating factor acetylhydrolase (PAF-AH(Ib)) is a tertiary G-protein-complex-like heterotrimeric enzyme which is composed of alpha 1, alpha 2, and beta subunits and is implicated in stages of brain development such as the formation of the brain cortex. We have isolated and sequenced cDNA clones encoding these three subunits of rat brain PAF-AH(Ib). The amino acid sequences of brain PAF-AH has shown an extremely high homology among mammalian species. The tissue distribution of the three subunits was examined by Northern blot analysis. Although the mRNAs were detected in various organs, the ratio of the level of mRNA expression for the three subunits differed among rat tissues, raising the possibility that isoform(s) other than the heterotrimeric isoform exist in certain tissues. (C) 1998 Published by Elsevier Science B.V.
  • T Shibata, K Yamada, M Watanabe, K Ikenaka, K Wada, K Tanaka, Y Inoue
    JOURNAL OF NEUROSCIENCE 17 (23) 9212 - 9219 0270-6474 1997/12 [Refereed][Not invited]
     
    The glutamate transporter GLAST is localized on the cell membrane of mature astrocytes and is also expressed in the ventricular zone of developing brains. To characterize and follow the GLAST-expressing cells during development, we examined the mouse spinal cord by in situ hybridization and immunohistochemistry. At embryonic day (E) 11 and E13, cells expressing GLAST mRNA were present only in the ventricular zone, where GLAST immunoreactivity was associated with most of the cell bodies of neuroepithelial cells. In addition, GLAST immunoreactivity was detected in radial processes running through the mantle and marginal zones. From this characteristic cytology, GLAST-expressing cells at early stages were judged to be radial glia cells. At E15, cells expressing GLAST mRNA first appeared in the mantle zone, and GLAST-immunopositive punctate or reticular protrusions were formed along the radial processes. From E18 to postnatal day (P) 7, GLAST mRNA or its immunoreactivity gradually decreased from the ventricular zone and disappeared from radial processes, whereas cells with GLAST mRNA spread all over the mantle zone and GLAST-immunopositive punctate/reticular protrusions predominated in the neuropils. At P7, GLAST-expressing cells were immunopositive for glial fibrillary acidic protein, an intermediate filament specific to astrocytes. Therefore, the glutamate transporter GLAST is expressed from radial glia through astrocytes during spinal cord development. Furthermore, the distinct changes in the cell position and morphology suggest that both the migration and transformation of radial glia cells begin in the spinal cord between E13 and E15, when the active stage of neuronal migration is over.
  • S Offermanns, K Hashimoto, M Watanabe, W Sun, H Kurihara, RF Thompson, Y Inoue, M Kano, MI Simon
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 94 (25) 14089 - 14094 0027-8424 1997/12 [Refereed][Not invited]
     
    Mice lacking the alpha-subunit of the heterotrimeric guanine nucleotide binding protein G(q) (G alpha(q)) are viable but suffer from ataxia with typical signs of motor discoordination. The anatomy of the cerebellum Is not overtly disturbed, and excitatory synaptic transmission from parallel fibers to cerebellar Purkinje cells (PCs) and from climbing fibers (CFs) to PCs is functional. However, about 40% of adult G alpha(q) mutant PCs remain multiply innervated by CFs because of a defect in regression of supernumerary CFs in the third postnatal week. Evidence Is provided suggesting that G alpha 7q is part of a signaling pathway that is involved in the elimination of multiple CF innervation during this period.
  • H Kurihara, K Hashimoto, M Kano, C Takayama, K Sakimura, M Mishina, Y Inoue, M Watanabe
    JOURNAL OF NEUROSCIENCE 17 (24) 9613 - 9623 0270-6474 1997/12 [Refereed][Not invited]
     
    The glutamate receptor delta 2 subunit (GluR delta 2) is specifically expressed in cerebellar Purkinje cells (PCs) from early developmental stages and is selectively localized at dendritic spines forming synapses with parallel fibers (PFs). Targeted disruption of the GluR delta 2 gene leads to a significant reduction of PF-->PC synapses. To address its role in the synaptogenesis, the mor phology and electrophysiology of PF-->PC synapses were comparatively examined in developing GluR delta 2 mutant and wildtype cerebella. PCs in GluR delta 2 mutant mice were normally produced, migrated, and formed spines, as did those in wildtype mice. At the end of the first postnatal week, 74-78% of PC spines in both mice formed immature synapses, which were characterized by small synaptic contact, few synaptic vesicles, and incomplete surrounding by astroglial processes, eliciting little electrophysiological response. During the second and third postnatal weeks when spines and terminals are actively gener ated, the percentage of PC spines forming synapses attained 98-99% in wild type but remained as low 55-60% in mutants, and the rest were unattached to any nerve terminals. As a result, the number of PF synapses per single-mutant PCs was reduced to nearly a half-level of wild-type PCs. Parallelly, PF stimulation less effectively elicited EPSCs in mutant PCs than in wild-type PCs during and after the second postnatal week. These results suggest that the GluR delta 2 is involved in the stabilization and strengthening of synaptic connectivity between PFs and PCs, leading to the association of all PC spines with PF terminals to form functionally mature synapses.
  • M Itoh, Y Watanabe, M Watanabe, K Tanaka, K Wada, S Takashima
    BRAIN RESEARCH 767 (2) 265 - 271 0006-8993 1997/09 [Refereed][Not invited]
     
    A glutamate transporter subtype, EAAT4, is closely related to removal of glutamate from the synaptic cleft. Immunohistochemistry for EAAT4 demonstrated the specific distribution and localization of its expression in the developing human cerebellum. Purkinje cells showed faint EAAT4 immunostaining at 17 gestational weeks (GW), which became increasingly intense from 23 GW to the infantile period. In the late fetal to early infantile periods, Purkinje cells showed marked immunoreactivity. After the late infantile period, EAAT4 immunoreactivity was the same in extent as in the adult pattern. Its intracellular localization also changed with development. EAAT4 immunoreactivity was demonstrated in the short processes of Purkinje cells in the early embryonic period, in the cell bodies and dendrites in the late fetal to early infantile periods, and then in the spines after the late infantile period. In the adult cerebellum, immunoreactivity was detected strongly in the spines of Purkinje cells and weakly in the cell bodies. No immunoreactivity was found in the axons or axon terminals of the cells. Thus, the glutamate transporter exhibits developmental changes in its distribution in the cerebellum and its localization in Purkinje cells. EAAT4 immunoreactivity may be related to the dendritic arborization of cells in the molecular layer. (C) 1997 Elsevier Science B.V.
  • Y MaenoHikichi, K Tanaka, T Shibata, M Watanabe, Y Inoue, Y Mukainaka, K Wada
    MOLECULAR BRAIN RESEARCH 48 (1) 176 - 180 0169-328X 1997/08 [Refereed][Not invited]
     
    A cDNA encoding a neuron-specific glutamate transporter was isolated from mouse cerebellum and characterized, Expression of this cDNA in HEK293 cells induced high-affinity uptake of L-glutamate that was dependent on external Na+, The deduced amino-acid sequence of the cDNA shares 96% identity with its human homologue. Northern blot analysis of mouse tissues revealed that the mRNA of this transporter is specific to the brain, In situ hybridization in mouse brain sections showed that the mRNA is expressed densely and selectively in cell. bodies of Purkinje cells. (C) 1997 Elsevier Science B.V.
  • J Tanaka, R Ichikawa, M Watanabe, K Tanaka, Y Inoue
    NEUROREPORT 8 (11) 2461 - 2464 0959-4965 1997/07 [Refereed][Not invited]
     
    WE used silver-enhanced immunogold electron microscopy to reveal synaptic localization of the glutamate transporter EAAT4 in mouse cerebellar Purkinje cells (PCs). Gold-silver particles representing the EAAT4 were densely localized on extra-junctional membrane, but not on junctional membrane of PC spines in contact with parallel fiber or climbing fiber terminals. No particle accumulations were observed at inhibitory synapses formed on cell body and dendritic shafts of PCs. Therefore, the EAAT4 is selectively targeted to the extra-junctional site of excitatory PC synapses. The finding suggests that the EAAT4 transports glutamate or its related amino acids from outside the synaptic cleft, which would facilitate glutamate diffusion from the synaptic cleft to the extrasynaptic space and restrict glutamate spillover to adjacent synapses.
  • K Tanaka, K Watase, T Manabe, K Yamada, M Watanabe, K Takahashi, H Iwama, T Nishikawa, N Ichihara, S Hori, M Takimoto, K Wada
    SCIENCE 276 (5319) 1699 - 1702 0036-8075 1997/06 [Refereed][Not invited]
     
    Extracellular levels of the excitatory neurotransmitter glutamate in the nervous system are maintained by transporters that actively remove glutamate from the extracellular space. Homozygous mice deficient in GLT-1, a widely distributed astrocytic glutamate transporter, show lethal spontaneous seizures and increased susceptibility to acute cortical injury. These effects can be attributed to elevated levels of residual glutamate in the brains of these mice.
  • S Nakagawa, M Watanabe, Y Inoue
    NEUROSCIENCE RESEARCH 28 (2) 177 - 184 0168-0102 1997/06 [Refereed][Not invited]
     
    The ROR alpha is a member of the nuclear hormone receptor gene superfamily, and its deletion causes the staggerer mutation in mice. In the staggerer mutant mouse, Purkinje cells (PCs) are severely affected in the cytology, synapse formation and gene expression. We previously found the presence of mediolateral compartments unique to the staggerer cerebellum, based on different degrees of abnormalities in the cytology and gene expression. In this paper we investigated expression of the ROR alpha mRNA in developing mouse cerebellum, with a particular interest in its regional difference. At embryonic day 15, the ROR alpha mRNA was expressed at the highest level in the PC plate. The prominent expression in PCs was maintained from late embryonic stage through mature stage. At any developmental stages, no apparent regional differences in the ROR alpha mRNA expression were detected in the mediolateral and rostrocaudal axes of the cerebellum. The high expression from early developmental stages provides a molecular-anatomical basis for its important role in phenotypic differentiation of PCs. However, the even distribution in the cerebellum suggests that the unique staggerer compartments are not directly related to the loss of ROR alpha function. (C) 1997 Elsevier Science Ireland Ltd.
  • A KohyamaKoganeya, M Watanabe, Y Hotta
    FEBS LETTERS 409 (2) 258 - 264 0014-5793 1997/06 [Refereed][Not invited]
     
    We have cloned and characterized a new diacylglycerol kinase (DGK) isozyme which is expressed in the retina and the brain of rat. The cDNA contains an open reading frame of 567 amino acid residues with a predicted protein of 64 kDa and shows very high homology to human DGK epsilon. The new DGK isozyme contains two distinctive zinc-finger structures and a putative catalytic domain. This DGK expressed predominantly in the inner and outer nuclear layers of retina. This expression pattern is different from those of the previously cloned DGKs including the human DGK epsilon, suggesting that this DGK isozyme has potential importance in visual functions as was the case in Drosophila retinal cells. (C) 1997 Federation of European Biochemical Societies.
  • Ito, I, K Futai, H Katagiri, M Watanabe, K Sakimura, M Mishina, H Sugiyama
    JOURNAL OF PHYSIOLOGY-LONDON 500 (2) 401 - 408 0022-3751 1997/04 [Refereed][Not invited]
     
    1. We have explored the effects of targeted disruption of the N-methyl-D-aspartate (NMDA) receptor epsilon 1 or epsilon 2 subunit gene on NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) and long-term potentiations (LTPs) at the two types of synapse in mouse hippocampal CA3 pyramidal neurons: those formed by the commissural/associational (C/A) and fimbrial (Fim) inputs. 2. Electrophysiological experiments were performed in hippocampal slices prepared from both wild-type and epsilon 1- or epsilon 2-disrupted mice using extracellular and whole-cell patch recording techniques. To assess the epsilon 1, epsilon 2 and zeta 1 subunit expression at cellular levels, we performed non-isotopic in situ hybridization with digoxigenin-labelled cRNA probes. 3. 3. We could record EPSCs in response to the stimulations to either of the C/A and Fim afferents from a single CA3 pyramidal neuron. The epsilon 1, epsilon 2 and zeta 1 subunits were expressed together in individual CA3 neurons. 4. The epsilon 1 subunit disruption selectively reduced NMDA EPSCs and LTP in the C/A-CA3 synapse without significantly affecting those in the Fim-CA3 synapse, whereas the epsilon 2 subunit mutation diminished NMDA EPSCs and LTP in the Fim-CA3 synapse with no appreciable functional modifications in the C/A-CA3 synapse. 5. These results suggest that NMDA receptors with different subunit compositions function within a single CA3 pyramidal cell in a synapse-selective manner.
  • K Yamada, S Wada, M Watanabe, K Tanaka, K Wada, Y Inoue
    NEUROSCIENCE RESEARCH 27 (3) 191 - 198 0168-0102 1997/03 [Refereed][Not invited]
     
    EAAT4 is a Purkinje cell (PC)-specific, postsynaptic glutamate transporter in the adult mouse brain. Here, we performed developmental analyses to reveal its temporal expression in relation to PC differentiation. Using in situ hybridization, EAAT4 mRNA was specifically detected in the PC layer of the cerebellar primordium at embryonic day 13 (E13). During late fetal and neonatal periods, the transcripts were detected only in the PC layer in the caudal cerebellum. At postnatal day 7 (P7) and, thereafter, the prominent transcripts were found on monolayered PCs in the entire cerebellum. Using immunohistochemistry, low levels of EAAT4 immunoreactivity were first observed at E18 in the perikarya of PCs in the caudal cerebellum, and this pattern of immunostaining was maintained at P1 and P7. At P14 and, thereafter, the molecular layer in the entire cerebellum became immunopositive for EAAT4, and the intense immunoreactivity was detected preferentially in PC spines synapsing with parallel fiber terminals. Therefore, the present study has clarified that the transcription of EAAT4 begins in PCs from early embryonic stages, and that the synaptic localization of EAAT4 is established during the second postnatal week. When considered in the light of the synaptogenesis of parallel fiber-PC synapses which actively occurs in the rodent cerebellum during the second and third weeks of life, synaptic localization of the glutamate transporter EAAT4 may be closely associated with the synapse formation. (C) 1997 Elsevier Science Ireland Ltd.
  • C Takayama, S Nakagawa, M Watanabe, H Kurihara, M Mishina, Y Inoue
    BRAIN RESEARCH 745 (1-2) 231 - 242 0006-8993 1997/01 [Refereed][Not invited]
     
    The glutamate receptor (GluR) channel delta 2 subunit is expressed abundantly and specifically in cerebellar Purkinje cells. Our previous study demonstrated that the GluR delta 2 mRNA is expressed as early as embryonic day 15 prior to Purkinje cell synaptogenesis, and its protein product accumulates in dendritic spines during normal Purkinje cell maturation. In this study, we examined expression and distribution of the GluR delta 2 in the weaver and reeler mutant cerebelli, which show abnormal cytoarchitecture and neural circuitry. In situ hybridization analysis showed that GluR delta 2 mRNA was expressed in entire Purkinje cells in both mutant mice. Immunohistochemical analysis revealed that intracellular localization of GluR delta 2 was altered in some region of mutant cerebelli. In the cortical surface where Purkinje cells form synapses with parallel fibers, GluR delta 2-immunoreactivity was restricted to dendritic spines of Purkinje cells as observed in normal mice. In contrast, in the subcortical region where granule cells and parallel fibers are absent, the immunoreactivity was found widely in Purkinje dendrites. Thus, the GluR gamma 2 protein did not accumulate to the dendritic spines of Purkinje cells lacking synaptic contact with parallel fibers. These results suggest that the expression of both GluR delta 2 mRNA and protein is independent of abnormalities in the mutant cerebelli, but relocalization of the GluR delta 2 protein might depend on the formation of synapses between Purkinje cells and parallel fibers.
  • M Kano, K Hashimoto, H Kurihara, M Watanabe, Y Inoue, A Aiba, S Tonegawa
    NEURON 18 (1) 71 - 79 0896-6273 1997/01 [Refereed][Not invited]
     
    Most of the cerebellar Purkinje cells (PCs) of an adult animal are innervated individually by a single climbing fiber (CF) that forms strong excitatory synapses with the PCs. This one-to-one relationship between a PC and a CF is a consequence of a developmentally regulated regression of the innervation of PCs by CFs. We found that, in mice deficient in the type 1 metabotropic glutamate receptor (mGluR1), the regression of supernumerary CFs ceases by the end of the second postnatal week, which is about one week earlier than in normal mice. Consequently, about one third of PCs in the mGluR1 mutant mice are innervated by multiple CFs in adulthood. We conclude that the regression of CFs normally occurs in two developmental phases and that mGluR1 plays a crucial role in the second phase.
  • Masanobu Kano, Kouichi Hashimoto, Stefan Offermanns, Melvin I. Simon, Masahiko Watanabe, Hideo Kurihara, Yoshiro Inoue, Huiping Jiang, Yanping Wu, Ki-Su Jun, Dianqing Wu, Hee-SUP Shin
    Keio Journal of Medicine 46 A108 - A119 1880-1293 1997 [Refereed][Not invited]
  • M Watanabe, IM Moise, Y Inoue
    NEUROSCIENCE RESEARCH 26 (4) 335 - 343 0168-0102 1996/12 [Refereed][Not invited]
     
    In terms of gene expression for the N-methyl-D-aspartate (NMDA) receptor channel subunits, the adult reeler cerebellum contains two Purkinje cell populations, one expressing the zeta 1 subunit mRNA (wild-type expression) and another expressing the epsilon 1 and zeta 1 subunit mRNAs (modified expression). To clarify development of these two populations, in situ hybridization analysis was performed in the reeler cerebellum from birth to 1 year of age. The epsilon 1 subunit mRNA was not detected in any Purkinje cell clusters (PCCs) until postnatal day 21 (P21). Additional signals for the epsilon 1 subunit mRNA first appeared at P56 in a subset of PCCs, and persisted until 1 year of age. The epsilon 2 and epsilon 4 subunit mRNAs were transiently expressed in PCCs at birth, and disappeared thereafter. No signals for the epsilon 3 subunit mRNA was detected in PCCs at any postnatal stages examined. The zeta 1 subunit mRNA was expressed in all PCCs from birth through 1 year. When compared with the wild-type Purkinje cells, reeler Purkinje cells with modified expression are thus differentiated from those once accomplished the adult wild-type expression (the zeta 1 subunit alone) by down-regulation of the epsilon subunits. The present results further indicate that the onset of the modified expression is subsequent to the establishment of synaptic connectivities in the cerebellum. Copyright (C) 1996 Elsevier Science Ireland Ltd
  • M Watanabe, Y Miura, A Ido, M Sakai, S Nishi, Y Inoue, T Hashimoto, T Tamaoki
    MOLECULAR BRAIN RESEARCH 42 (2) 344 - 349 0169-328X 1996/12 [Refereed][Not invited]
     
    The expression of the ATBF1 gene in developing brain was analyzed in mice from 13 days of gestation to 28 days after birth using RNase protection and in situ hybridization methods. The level of ATBF1 transcripts was the highest on embryonic day 13-15 and then progressively decreased to a hardly detectable level on postnatal day 28. Throughout the period examined, ATBF1 mRNA was expressed consistently in the basal telencephalon, diencephalon, and mesencephalon, with the highest levels in the inferior colliculus and thalamus. On the other hand, no significant expression was observed in cerebellum, neocortex, hippocampus, and olfactory bulb. These results illustrate significant regional differences in the ATBF1 expression and suggest a role of the ATBF1 gene in the formation of some specific cell populations in developing central nervous system.
  • S Nakagawa, M Watanabe, Y Inoue
    EUROPEAN JOURNAL OF NEUROSCIENCE 8 (12) 2644 - 2651 0953-816X 1996/12 [Refereed][Not invited]
     
    The gene expression of five NMDA receptor channel subunits, the epsilon 1, epsilon 2, epsilon 3, epsilon 4 and zeta 1 subunits, was examined in cerebellar Purkinje cells of the staggerer mouse at postnatal day 21. In the midline region of the staggerer cerebellum, signals for the epsilon 1, epsilon 4 and zeta 1 subunit mRNAs were distributed in Purkinje cells, which have a large cell body aligned in a monolayer between the granular and molecular layers. In addition to the midline region, labelled neurons in the intermediate cerebellar region were, though at lower levels, aligned almost in a monolayer between the granular and molecular layers. In the hemisphere, most labelled neurons occurred in various locations in the granular layer and the cerebellar medulla. These regions, populated with Purkinje cells expressing the epsilon 1, epsilon 4 and zeta 1 subunit mRNAs, were separated from each other by narrow gap regions that contained neurons without any detectable NMDA receptor channel subunit mRNAs. These results suggest that there is discrete mediolateral heterogeneity in staggerer Purkinje cell populations, in terms of expression properties of the NMDA receptor channel subunits. When compared with wild-type Purkinje cells that express the zeta 1 subunit alone, additional expression of the epsilon subunits presumably explains the persistence of NMDA responses in adult staggerer Purkinje cells (Dupont at al., Neuroscience, 12, 613-619, 1984).
  • K Yamada, M Watanabe, T Shibata, K Tanaka, K Wada, Y Inoue
    NEUROREPORT 7 (12) 2013 - 2017 0959-4965 1996/08 [Refereed][Not invited]
     
    To study cellular and subcellular localizations of the glutamate transporter EAAT4, antibody was raised against the N-terminal peptide. On immunoblotting the antibody recognized a band in membrane extracts from the cerebellum, but not from the forebrain. Immunohistochemistry revealed that its distribution was restricted to the cerebellar molecular layer, where the immunoreactivity was observed as numerous punctate stainings. Electron microscopy showed the antibody to label dendritic spines of the Purkinje cells. EAAT4 is, therefore, a Purkinje cell-specific, postsynaptic transporter. Together with dense localization of other transporter subtypes in Bergmann astrocytic membranes, Purkinje cell synapses are thus provided with distinct glutamate transporter subtypes at discrete synaptic elements, which would play important roles in regulating excitability of the Purkinje cells and protecting against excitotoxicity.
  • S Nakagawa, M Watanabe, Y Inoue
    EUROPEAN JOURNAL OF NEUROSCIENCE 8 (7) 1401 - 1407 0953-816X 1996/07 [Refereed][Not invited]
     
    The Purkinje cells in the staggerer mutant mouse have various cellular abnormalities, including reduced cell number, ectopia, smaller size and absence of dendritic spines. It is also known that some of these abnormalities exhibit regional variations in the cerebellum. In this paper we have investigated expression in the staggerer Purkinje cells of the calbindin and inositol 1,4,5-trisphosphate receptor type 1 mRNAs by in situ hybridization. Although the transcription levels of both mRNAs were significantly reduced compared with the wild-type cells, the reduction among the Purkinje cell populations was not even, varying greatly from region to region. Purkinje cells with different transcription levels were distributed in discrete regions and arranged alternately in the mediolateral direction. Moreover, the cell bodies with higher transcription levels were larger in size and aligned in a monolayer between the granular and molecular layers, whereas those with lower levels were smaller in size, fewer in number and dispersed throughout the granular layer. These findings suggest that there is a distinct mediolateral heterogeneity in the staggerer cerebellum with respect to transcription levels of these Purkinje cell-specific molecules, which might correlate with some cytological phenotypes.
  • C Takayama, S Nakagawa, M Watanabe, M Mishina, Y Inoue
    DEVELOPMENTAL BRAIN RESEARCH 92 (2) 147 - 155 0165-3806 1996/04 [Refereed][Not invited]
     
    The glutamate receptor (GluR) channel delta 2 subunit is considered to be a functional molecule involved in motor coordination, Purkinje cell synapse formation and cerebellar long-term depression. We examined developmental changes in expression and distribution of the GluR delta 2 in the mouse cerebellum by in situ hybridization and immunohistochemistry. The GluR delta 2 mRNA was detected as early as embryonic day 15 (E15) in a cell mass consisting of Purkinje neuroblasts in the posterior cerebellum. During late embryonic and postnatal periods, the GluR delta 2 mRNA was expressed abundantly and specifically in Purkinje cells. By immunohistochemistry, immunoreactivity of the GluR delta 2 was found in both shafts and spines of Purkinje dendrites at early postnatal period. By P21, however, the intense immunoreactivity became restricted to the dendritic spines, especially along the postsynaptic membrane in contact with parallel fiber terminals. These findings suggested that the transcription of the GluR delta 2 subunit occurs in the Purkinje cells from fetal through adult stage, but the intracellular localization of the protein products undergoes an alteration from non-synaptic to synaptic site when active synaptogenesis takes place.
  • T Shibata, M Watanabe, K Tanaka, K Wada, Y Inoue
    NEUROREPORT 7 (3) 705 - 709 0959-4965 1996/02 [Refereed][Not invited]
     
    DEVELOPMENTAL changes in gene expression for three glutamate transporter subtypes in the mouse brain were analysed by in situ hybridization. During embryonic stages, GluT-1 and GLT-1 mRNAs were expressed at high levels in the ventricular zone, whereas EAAC1 mRNA was not detected in the zone. In the mantle zone, transcription levels of three transporter mRNAs mere low during embryonic stages, and these levels, especially those of the GluT-1 and GLT-1 mRNAs, displayed remarkable increases postnatally to reach maximal levels at 14 days of age. These dynamic developmental regulations suggest that the glutamate transporter not only regulates the excitatory synaptic transmission at mature stages, but might also be intimately involved in the brain development.
  • T Kutsuwada, K Sakimura, T Manabe, C Takayama, N Katakura, E Kushiya, R Natsume, M Watanabe, Y Inoue, T Yagi, S Aizawa, M Arakawa, T Takahashi, Y Nakamura, H Mori, M Mishina
    NEURON 16 (2) 333 - 344 0896-6273 1996/02 [Refereed][Not invited]
     
    Multiple epsilon subunits are major determinants of the NMDA receptor channel diversity. Based on their functional properties in vitro and distributions, we have proposed that the epsilon 1 and epsilon 2 subunits play a role in synaptic plasticity. To investigate the physiological significance of the NMDA receptor channel diversity, we generated mutant mice defective in the epsilon 2 subunit. These mice showed no suckling response and died shortly after birth but could survive by hand feeding. The mutation hindered the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brain-stem trigeminal nucleus. In the hippocampus of the mutant mice, synaptic NMDA responses and long-term depression were abolished. These results suggest that the epsilon 2 subunit plays an essential role in both neuronal pattern formation and synaptic plasticity.
  • A Ido, Y Miura, M Watanabe, M Sakai, Y Inoue, T Miki, T Hashimoto, T Morinaga, S Nishi, T Tamaoki
    GENE 168 (2) 227 - 231 0378-1119 1996/02 [Refereed][Not invited]
     
    We have isolated a mouse ATBF1 cDNA which is 12-kb long and capable of encoding a 406-kDa protein containing four homeodomains and 23 zinc-finger motifs. Mouse ATBF1 is 94% homologous to the human ATBF1-A transcription factor. Northern blot and RNase protection analysis showed that levels of ATBF1 transcripts were low in adult mouse tissues, but high in developing brain, consistent with a role for ATBF1 in neuronal differentiation.
  • M Kano, K Hashimoto, C Chen, A Abeliovich, A Aiba, H Kurihara, M Watanabe, Y Inoue, S Tonegawa
    CELL 83 (7) 1223 - 1231 0092-8674 1995/12 [Refereed][Not invited]
     
    PKC gamma is highly expressed in Purkinje cells (PCs) but not in other types of neurons in the cerebellum. The expression of PKC gamma changes markedly during cerebellar development, being very low at birth and reaching a peak around the third postnatal week. This temporal pattern of PKC gamma expression coincides with the developmental transition from multiple to single climbing fiber innervation onto each PC. In adult mutant mice deficient in PKC gamma, we found that 41% of PCs are still innervated by multiple climbing fibers, while other aspects of the cerebellum including the morphology and excitatory synaptic transmission of PCs appear normal. Thus, elimination of multiple climbing fiber innervation appears to be specifically impaired in the mutant cerebellum. We suggest that the developmental role of PKC gamma may be to act as a downstream element in the signal cascade necessary for the elimination of surplus climbing fiber synapses.
  • Tatsuyuki Muratake, Shigenobu Hayashi, Yuka Ichimura, Ken Morii, Ryozo Kuwano, Tomio Ichikawa, Toshiro Kumanishi, Toshiaki Isobe, Masahiko Watanabe, Hisatake Kondo, Yasuo Takahashi
    Molecular Neurobiology 11 (1-3) 223 - 230 0893-7648 1995/08 [Refereed][Not invited]
     
    14·3·3 protein, a brain-specific protein, is an activator of tyrosine and tryptophan hydroxylases, key enzymes for biosynthesis of dopamine and serotonin. In this article, we describe cloning of cDNA for human brain 14·3·3 ν chain and expression of 14·3·3 ν chain mRNA in some human cultured cells. The cloned cDNA is 1730 bp long and contains 191 bp of a 5′-noncoding region, the complete 738 bp of coding region, and 801 bp of a 3′-noncoding region, containing three polyadenylation signals. This cDNA encoded a polypeptide of 246 amino acids (M, 28,196). Furthermore, using in situ hybridization histochemistry, the expression of mRNA for this protein was examined in the rat central nervous system. In situ hybridization histochemistry indicated that 14·3·3 {left right arrow through small circle} chain mRNA is detected not only in the monoamine-synthetic neurons, but also in other neurons in the discrete nuclei, which synthesize neither cathecholamine nor serotonin. Northern blot analysis demonstrated that the addition of methamphetamine into the cultured medium increased the mRNA level for 14·3·3 {left right arrow through small circle} chain in U-251 cells, but did not increase that of GFAP. © 1995 Humana Press Inc.
  • M WATANABE, S NAKAGAWA, C TAKAYAMA, M NAGASHIMA, K INOUE, R ICHIKAWA, M MISHINA, Y INOUE
    NEUROSCIENCE RESEARCH 22 (3) 335 - 345 0168-0102 1995/06 [Refereed][Not invited]
     
    Recent studies have identified five NMDA receptor subunits, which exhibit distinct cellular expressions in the normal rodent brain. The purpose of this investigation is to clarify the molecular-anatomical organization in the cerebellum of the reeler mutant mouse, in which various categories of the Purkinje cells are present as to the cell position and synaptic connectivity. In comparison with the distribution of the inositol 1,4,5-trisphosphate receptor mRNA, a molecular marker specific to the Purkinje cells, the epsilon 1 subunit mRNA of the NMDA receptor channel was found in the adjacent sections to be expressed in a subset of the Purkinje cells. In the rostrocaudal extent, the Purkinje cells expressing the epsilon 1 subunit mRNA were distributed preferentially in the rostral cerebellum, irrespective of the normal and heterotopic positions. In the mediolateral extent, they formed segregated cell clusters, interposed by epsilon 1 subunit mRNA-negative clusters. Hybridizing signals for the zeta 1 subunit mRNA were found in all the Purkinje cell population, whereas those for the epsilon 2, epsilon 3, and epsilon 4 subunit mRNAs were not detected in the cells. These findings suggest that the reeler cerebellum is topographically compartmentalized by two subpopulations of the Purkinje cells, one expressing the epsilon 1 and zeta 1 subunit mRNAs, and the other expressing the zeta 1 subunit mRNA alone.
  • Y ONO, M WATANABE, Y INOUE, T OHMOTO, K AKIYAMA, K TSUTSUI, S SEKI
    DEVELOPMENTAL BRAIN RESEARCH 86 (1-2) 1 - 6 0165-3806 1995/05 [Refereed][Not invited]
     
    Expression of the mammalian major apurinic/apyrimidinic (AP) endonuclease (designated as APEX nuclease, or HAP1, APE or Ref-1 gene product) during mouse brain development was investigated by in situ and northern blot hybridizations. The enzyme is known to be a redox factor (Ref-1) stimulating DNA binding activity of AP-1 binding proteins such as Fos and Jun as well as a multifunctional DNA repair enzyme having 5' AP endonuclease, DNA 3' repair diesterase, 3'-5' exonuclease and DNA 3'-phosphatase activities. In the embryonic and postnatal development, APEX mRNA was expressed at high levels in the proliferative zone of various brain regions, with showing temporal and spatial changes. Its expression decreased in association with brain development to the basal expression level which was observed even in adulthood, with the exception of its expression in the hippocampal formation. The growth-dependent expression of APEX gene suggests that it has some roles on cell proliferation and/or differentiation in developmental brain. Its expression on the hippocampal formation became significant from postnatal day 7 and then increased. The pyramidal and granule cell layers expressed it at a higher level than most other brain regions at postnatal day 21. The developmental change of APEX gene expression was not necessarily associated with the changes of expression of c-fos and c-jun genes measured by northern blot hybridization. However, the present results suggested that APEX/Ref-1 gene product can interact with AP-1 binding proteins in brain, especially in the hippocampal formation, to regulate some brain functions by redox-activation.
  • C TAKAYAMA, S NAKAGAWA, M WATANABE, M MISHINA, Y INOUE
    NEUROSCIENCE LETTERS 188 (2) 89 - 92 0304-3940 1995/03 [Refereed][Not invited]
     
    The localization of the glutamate receptor channel delta 2 subunit was investigated by immunohistochemistry. The delta 2-immunoreactivity was observed exclusively in the molecular layer of the cerebellar cortex. The electron microscopic analysis showed that the delta 2 subunit was localized in the dendritic spines of the Purkinje cells. The immunopositive spines often formed synaptic contacts with parallel fiber varicosities. As for the Purkinje cells ectopically localized in the cerebellar nuclei and brain stem, the dendritic shafts and cell bodies were strongly labeled. These ectopic Purkinje cells also formed asymmetrical synapses at the delta 2-immunopositive dendritic spines. Considering the specific localization of the delta 2 subunit in the postsynaptic site of the Purkinje cells, the subunit is suggested to be involved in the excitatory synaptic transmission in the cells, as a component of the glutamate receptor channel.
  • M WATANABE, J OSADA, Y ARATANI, K KLUCKMAN, R REDDICK, MR MALINOW, N MAEDA
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 92 (5) 1585 - 1589 0027-8424 1995/02 [Refereed][Not invited]
     
    Studies by various investigators have indicated that elevated levels of plasma homocyst(e)ine are strongly associated with the occurrence of occlusive vascular diseases, With the eventual aim of determining whether or not elevated plasma homocyst(e)ine concentrations are directly causative of cardiovascular diseases, we have generated mice that are moderately and severely homocyst(e)inemic, Homologous recombination in mouse embryonic stem cells was used to inactivate the cystathionine beta-synthase [L-serine hydrolyase (adding homocysteine), EC 4.2.1.22] gene, Homozygous mutants completely lacking cystathionine beta-synthase were born at the expected frequency from matings of heterozygotes, but they suffered from severe growth retardation and a majority of them died within 5 weeks after birth, Histological examination showed that the hepatocytes of homozygotes were enlarged, multinucleated, and filled with microvesicular lipid droplets, Plasma homocyst(e)ine levels of the homozygotes were approximate to 40 times normal, These mice, therefore, represent a model for severe homocyst(e)inemia resulting from the complete lack of cystathionine beta-synthase, Heterozygous mutants have approximate to 50% reduction in cystathionine beta-synthase mRNA and enzyme activity in the liver and have twice normal plasma homocyst(e)ine levels, Thus, the heterozygous mutants are promising for studying the in vivo role of elevated levels of homocyst(e)ine in the etiology of cardiovascular diseases.
  • A FUKAMIZU, M WATANABE, Y INOUE, Y KON, S SHIMADA, N SHIOTA, F SUGIYAMA, K MURAKAMI
    KIDNEY INTERNATIONAL 46 (6) 1533 - 1535 0085-2538 1994/12 [Refereed][Not invited]
     
    Cortical expression of the human angiotensinogen gene in the kidney of transgenic mice. We have previously generated ''Tsukuba hypertensive mice'' with elevated blood pressure by cross-mating separate lines of transgenic animals carrying either 15 kb of the human renin gene including its native 3-kb promoter or 14 kb of the human angiotensinogen gene along with its 1.3-kb promoter, the former of which is expressed predominantly in the kidney and the latter of which is also expressed in the kidney to levels comparable to those found in the liver. To investigate whether the integrated human angiotensinogen gene is prominently expressed in the kidney of transgenic mice, we have analyzed a production region of the transgene mRNA by in situ hybridization technique. This analysis clearly demonstrated that human angiotensinogen mRNA is localized specifically to the cortex region of transgenic mouse kidney. The present finding indicates a possible involvement of the renal renin-angiotensin system in the pathogenesis of high blood pressure in transgenic mice.
  • M WATANABE, T ISOBE, T ICHIMURA, R KUWANO, Y TAKAHASHI, H KONDO, Y INOUE
    MOLECULAR BRAIN RESEARCH 25 (1-2) 113 - 121 0169-328X 1994/08 [Refereed][Not invited]
     
    We isolated from the rat brain two cDNA clones encoding the zeta and theta subtypes of the 14-3-3 protein. Both clones encoded 245 amino acid sequences, which share a high sequence homology with each other and also with other subtypes of the 14-3-3 protein. The distribution of their mRNAs was determined in the developing brain, by in situ hybridization with subtype-specific oligonucleotide probes. At embryonic day 18, the zeta and theta subtype mRNAs were expressed at high levels throughout the brain and the spinal cord. Distribution patterns of the two mRNAs were distinct in the brain at postnatal day 21. The zeta subtype mRNA was distributed widely in the brain gray matter, and high levels of the transcripts were detected in various brain regions, including the neocortex, hippocampus, caudate-putamen, thalamus, cerebellar cortex, and several brainstem nuclei. On the other hand, high signal levels of the theta subtype mRNA in the gray matter were restricted to the cerebellar cortex and the hippocampus. In addition, significant signals for the theta subtype mRNA were found over the white matter, where cell bodies of glial cells are populated. The wide gene expression of the zeta and theta subtypes suggests their fundamental and essential role in the brain function, but the degrees of functional involvement by the respective subtypes would be heterogeneous between neuron and glia, and also among neuron types.
  • M WATANABE, M MISHINA, Y INOUE
    JOURNAL OF COMPARATIVE NEUROLOGY 345 (2) 314 - 319 0021-9967 1994/07 [Refereed][Not invited]
     
    The distribution of five N-methyl-D-aspartate (NMDA) receptor channel subunit mRNAs in the mouse spinal cord from embryonic day 13 (E13) through postnatal day 56 (P56) was semiquantitatively examined at the cervical level via in situ hybridization with subunit-specific oligonucleotide probes. Signals for the epsilon 1 subunit mRNA were restricted to the most ventral portion of the spinal cord during embryonic stages. They extended to all laminae of the spinal cord except for the lamina 2 (substantia gelatinosa) during postnatal development. A wide expression of the epsilon 2 subunit mRNA was found in the spinal gray matter from E13 through neonatal stages, but the signals became restricted to the lamina 2 by P21. No significant signals for the epsilon 3 subunit mRNA were detected in the spinal cord at any developmental stages. The epsilon 4 subunit mRNA was distributed widely in the spinal cord during embryonic and early postnatal periods but decreased nearly to background levels by P21. In contrast to the differential distribution of the epsilon subunit mRNAs, the zeta 1 subunit mRNA was found ubiquitously at each developmental stage examined. These findings suggest that the molecular organization of the epsilon subunits may be different between the dorsal horn and the remaining regions in the mature spinal cord, which provides a molecular basis for functional heterogeneity of the NMDA receptor channel. Moreover, this spatial heterogeneity might be generated through drastic alterations in the subunit composition of the channel complex during spinal cord development. (C) 1994 Wiley-Liss, Inc.
  • M WATANABE, M MISHINA, Y INOUE
    JOURNAL OF COMPARATIVE NEUROLOGY 343 (4) 513 - 519 0021-9967 1994/05 [Refereed][Not invited]
     
    The distribution of five NMDA receptor channel subunit mRNAs was examined in the mouse cerebellum from embryonic day 13 through postnatal day 56, by in situ hybridization with subunit-specific oligonucleotide probes. At postnatal days 21 and 56, each cerebellar neuron displayed differential expressions of the epsilon subunit mRNAs. The granule cells showed hybridizing signals for the epsilon 1 and epsilon 3 subunit mRNAs, the molecular layer neurons for the epsilon 4 subunit mRNA, and the cerebellar nucleus neurons for the epsilon 1 and epsilon 4 subunit mRNAs, whereas the Purkinje cells did not express any epsilon subunit mRNAs. At early postmitotic stages of development, the epsilon 2 subunit mRNA appeared in each cerebellar neuron, including the Purkinje cells, and the epsilon 4 subunit mRNA appeared in neurons of the molecular layer and the cerebellar nuclei. The expression patterns in the cerebellum altered drastically during the first 2 postnatal weeks: the epsilon 1 and epsilon 3 subunit mRNAs appeared in the granule cells and the cerebellar nucleus neurons, whereas the epsilon 2 subunit mRNA disappeared from each neuron and the signal levels of the epsilon 4 subunit mRNA decreased remarkably. In contrast to the differential expressions of the four epsilon subunit mRNAs, intense signals for the zeta 1 subunit mRNA were observed in each cerebellar neuron from early postmitotic stages through the mature stage. These findings suggest that anatomical organization of the epsilon subunits is heterogeneous in the cerebellum both spatially and temporally, which would give rise to functional diversity of the NMDA receptor channel.
  • M WATANABE, M MISHINA, Y INOUE
    JOURNAL OF COMPARATIVE NEUROLOGY 343 (4) 520 - 531 0021-9967 1994/05 [Refereed][Not invited]
     
    The distribution of five NMDA receptor channel subunit mRNAs in the mouse brainstem at postnatal day 21 was semiquantitatively examined by in situ hybridization with subunit-specific oligonucleotide probes. The epsilon 1 subunit mRNA was observed in various brainstem nuclei. On the other hand, the epsilon 2 and epsilon 3 subunit mRNAs were restricted to particular nuclei, and the epsilon 4 subunit mRNA was detected in several nuclei at very low levels. The dorsal cochlear nucleus exhibited differential expressions of the E subunit mRNAs in distinct neuron types: the epsilon 2 subunit mRNA in the pyramidal cells, the epsilon 3 subunit mRNA in the granule cells, and the epsilon 1 subunit mRNA in other neurons. In the vestibular nuclei, the medial nucleus expressed the epsilon 1, epsilon 3, and epsilon 4 subunit mRNAs, whereas the lateral and superior nuclei expressed the epsilon 1 subunit mRNA. Such region-specific expressions were also discerned in the central gray, the superior and inferior colliculi, the medial accessory oculomotor nucleus, the locus ceruleus, the parabrachial nucleus, nucleus of the solitary tract, the caudal subnucleus of the trigeminal spinal tract nucleus, and the inferior olive. In contrast to the differential distributions of the four E subunit mRNAs, intense signals for the zeta 1 subunit mRNA were distributed throughout the brainstem. These findings suggest that anatomical organization of the E subunits is heterogeneous in various regions of the brainstem, which would give rise to functional diversity of the NMDA receptor channel in these regions. (C) 1994 Wiley-Liss, Inc.
  • M TAOKA, T ISOBE, T OKUYAMA, M WATANABE, H KONDO, Y YAMAKAWA, F OZAWA, F HISHINUMA, M KUBOTA, A MINEGISHI, SY SONG, T YAMAKUNI
    JOURNAL OF BIOLOGICAL CHEMISTRY 269 (13) 9946 - 9951 0021-9258 1994/04 [Refereed][Not invited]
     
    We cloned cDNAs of a novel protein (designated V-1) that has been identified from among the developmentally regulated proteins in the rat cerebellum. Protein sequencing analysis (Taoka, M., Yamakuni, T., Song, S.-Y., Yamakawa, Y., Seta, K., Okuyama, T., and Isobe, T. (1992) Eur. J. Biochem. 207, 615-620) and cDNA sequence analysis revealed that the V-1 protein consists of 117 amino acids and contains 2.5 contiguous repeats of the cdc10/SWI6 motif, which was originally found in the products of the cell cycle control genes of yeasts and the cell fate determination genes in Drosophila and Caenorhabditis elegans. In situ hybridization histochemistry revealed that the expression of the V-1 gene is transiently increased in postmigratory granule cells during postnatal rat cerebellar development and thereafter is markedly suppressed, whereas Purkinje cells constitutively express V-1 mRNA. In contrast, cerebellar granule cells of the staggerer mutant mouse continue to express the V-1 gene even when the granule cells of the normal mouse have ceased to express the V-1 gene, suggesting that the expression of the V-1 gene in granule cells is regulated through the interaction with Purkinje cells. On the basis of these results, we postulate that the V-1 protein has a potential role in the differentiation of granule cells.
  • M WATANABE, K TSUTSUI, K TSUTSUI, Y INOUE
    NEUROSCIENCE RESEARCH 19 (1) 51 - 57 0168-0102 1994/02 [Refereed][Not invited]
     
    Distributions of the topoisomerase II alpha and II beta mRNAs were examined in the developing rat brain, by in situ hybridization with isoform-specific oligonucleotide probes. Intense signals for the topoisomerase II alpha mRNA were detected in the ventricular zone of each brain region at embryonic day 13-15 (E13-E15), and in the external granular layer of the cerebellum at postnatal day 7-14 (P7-P14). Thereafter, the signals rapidly decreased in levels and eventually disappeared from respective regions. Administration of bromodeoxyuridine (BrdU) into embryos at E13 showed that the topoisomerase II alpha mRNA was expressed in the BrdU-incorporated region and its ventricular side of the neural wall, suggesting that transcription of this isoform occurs in neurons from S-phase through M-phase. On the other hand, the topoisomerase II beta mRNA was distributed throughout the brain from E13 through P21, irrespective of the ventricular and mantle zones. Signal levels of the topoisomerase II beta mRNA were much stronger during early developmental stages than at mature stages in various brain regions. The characteristic and differential spatio-temporal expressions suggest that the topoisomerase II alpha is involved in the proliferation, while the topoisomerase II beta is closely related to differentiation and maturation of neurons.
  • M WATANABE, M MISHINA, Y INOUE
    NEUROSCIENCE LETTERS 165 (1-2) 183 - 186 0304-3940 1994/01 [Refereed][Not invited]
     
    The distribution of five N-methyl-D-aspartate (NMDA) receptor channel subunit mRNAs was examined in the mouse peripheral nervous tissues by in situ hybridization. The trigeminal and dorsal root ganglion cells showed signals for the zeta 1 subunit mRNA from embryonic stages through postnatal day 21, but not for the epsilon 1, epsilon 2, epsilon 3, and epsilon 4 subunit mRNAs at any developmental stages examined. The adrenal medulla also expressed the zeta 1 subunit mRNA alone. These findings suggest that molecular organization of the NMDA receptor channel in peripheral neurons are distinct from those in central neurons, which may result in different functional properties of the channel between them.
  • M WATANABE, M MISHINA, Y INOUE
    BRAIN RESEARCH 634 (2) 328 - 332 0006-8993 1994/01 [Refereed][Not invited]
     
    In the retina, the epsilon 2 and zeta 1 subunit mRNAs of the NMDA receptor channel were expressed from embryonic stages and found in ganglion cell layer and whole layer of inner nuclear layer at postnatal day 21 (P21). The epsilon 1 subunit mRNA appeared postnatally and was distributed in ganglion cell layer and an inner third of inner nuclear layer at P21. These findings suggest that molecular organization of the NMDA receptor channel may alter during the retinal development.
  • M WATANABE, Y INOUE, K SAKIMURA, M MISHINA
    JOURNAL OF COMPARATIVE NEUROLOGY 338 (3) 377 - 390 0021-9967 1993/12 [Refereed][Not invited]
     
    The distributions of five NMDA receptor channel subunit mRNAs in the mouse forebrain at postnatal day 21 were semiquantitatively examined by in situ hybridization with subunit-specific oligonucleotide probes. In contrast to ubiquitous distribution of the zeta1 subunit mRNA throughout the forebrain, distributions of four epsilon subunit mRNAs were highly variable from nucleus to nucleus. The telencephalon (except for the septum) expressed the epsilon1 and epsilon2 subunit mRNAS. Various combinations of the epsilon1, epsilon2, epsilon3, and epsilon4 subunit mRNAs were present in different nuclei of the septum, the olfactory bulb, and the thalamus. In the hypothalamus, the suprachiasmatic nucleus expressed distinct signals for the epsilon3 subunit mRNA alone, whereas other nuclei showed faint signals for the epsilon1, epsilon2, and epsilon4 subunit mRNAs. Moreover, different signal levels of the epsilon subunit mRNAs were found in various regions. The hippocampal CA1 region expressed higher signals for the epsilon1 and epsilon2 subunit mRNAs than the CA3 region and the dentate gyrus. In the cerebral cortex, signal levels of the epsilon1 subunit mRNA were higher in the laminae II/III, IV, and VI than the lamina V, whereas those of the epsilon2 subunit mRNA were highest in laminae II/III and lowest in laminae IV and V. Different signal levels between the epsilon1 and E2 subunit mRNAs were also discerned in the amygdala, the caudate-putamen, and the thalamus. The distinct anatomical distributions and differential signal levels of the epsilon subunit mRNAs strongly suggest different subunit organizations of the NMDA receptor channel in different forebrain neurons, which may result in functional diversity of the channel in vivo. (C) 1993 Wiley-Liss, Inc.
  • K TSUTSUI, K TSUTSUI, S OKADA, M WATANABE, T SHOHMORI, S SEKI, Y INOUE
    JOURNAL OF BIOLOGICAL CHEMISTRY 268 (25) 19076 - 19083 0021-9258 1993/09 [Refereed][Not invited]
     
    cDNA segments for DNA topoisomerase II were amplified from rat brain RNA after reverse transcription by the polymerase chain reaction, using degenerate oligonucleotide primers deduced from the conserved regions of topoisomerase II of higher eukaryotes. The cDNA product from a successful amplification was homogeneous in length but heterogeneous in sequence. Restriction mapping of the cloned cDNA fragments revealed that they consisted of two distinct sequence groups. DNA sequencing of representative clones from each group, designated A and B, showed that they are highly homologous to cDNAs of human topoisomerase II isoforms, alpha and beta, respectively. Northern blot analysis indicated that the transcript level for rat topoisomerase IIalpha was high in embryonic brain and in the cerebellum of 2-day newborns, followed by rapid decrease to a undetectable level at 4 weeks after birth. In contrast, rat topoisomerase IIbeta transcript was present throughout the embryonic and postnatal stages. In the developing cerebellum, cells expressing topoisomerase IIalpha were confined exclusively to the outer mitotic zone of the external granular layer, whereas the transcript of topoisomerase IIbeta was detected over the entire cortical region. These results clearly indicate that the isoform alpha is expressed only in proliferating cells. The differential expression of topoisomerase II isozymes was also observed among developed tissues. Therefore, the isozymes are most likely to be involved in the following different physiological processes: topoisomerase IIalpha in cell proliferation, and topoisomerase IIbeta in some processes unrelated to cell proliferation.
  • M WATANABE, T ISOBE, T ICHIMURA, R KUWANO, Y TAKAHASHI, H KONDO
    DEVELOPMENTAL BRAIN RESEARCH 73 (2) 225 - 235 0165-3806 1993/06 [Refereed][Not invited]
     
    An in situ hybridization technique was applied to rat nervous tissues, to analyse the developmental changes in expression for the eta subtyPe mRNA of 14-3-3 protein, a putative regulatory protein for protein kinase C. Although signal levels of the eta subtype mRNA were low in mitotic cells in the ventricular zone, most neurons displayed a marked increase at their definitive location in the mantle zone. In general, neurons in the spinal ventral horn and peripheral ganglia showed this increase at E13-E15, those in the telencephalon, diencephalon, midbrain, pons and medulla oblongata at E18-P1, and the cerebellar Purkinje cells at P7-P14. It is at these developmental stages when neuronal differentiation including axonal and dendritic growth and ramification occurs actively. Subsequently high levels of the eta subtype mRNA were maintained until the adult stage in projection type neurons possessing larger cell bodies and highly developed dendritic fields, such as the olfactory mitral cells, hippocampal pyramidal cells, cerebellar Purkinje cells, and motor neurons in the brainstem and spinal cord. However, the signal levels decreased until the adult stage in smaller projection type neurons. On the other hand, the signal levels in local circuit type neurons were consistently low throughout development. These findings suggest that gene expression for the eta subtype mRNA of the 14-3-3 protein is regulated in close relation to both neuron types and their cytodifferentiation.
  • M WATANABE, T NAGAMINE, K SAKIMURA, Y TAKAHASHI, H KONDO
    JOURNAL OF COMPARATIVE NEUROLOGY 327 (3) 350 - 358 0021-9967 1993/01 [Refereed][Not invited]
     
    The gene expression for alpha and gamma subunits of enolase, a dimeric enzyme in the glycolytic pathway, was examined in the developing brain of rats by in situ hybridization. The expression for the gamma subunit of enolase was first detected in post-mitotic neurons settled in the mantle zone at E13, and it increased progressively until the adult stage. Expression signals for the alpha subunit were discerned in two discrete regions showing different developmental changes: the signals in the proliferative ventricular zone were intense at E13 and decreased and eventually disappeared around birth, whereas the signals in the mantle zone persisted until the adult stage. In the adult brain, mRNAs for the alpha and gamma subunits were expressed widely in neurons, resulting in almost similar temporal patterns in the brain except for the cerebellum. Expression levels of the alpha subunit in adult glial cells were below the detection threshold of the in situ hybridization analysis. These findings suggest that both alpha and gamma enolase subunits participate in energy production in neurons of the mature brain and that marked changes in the subunit composition of enolase occur according to both neuron type and maturation.
  • M WATANABE, T ISOBE, T ICHIMURA, R KUWANO, Y TAKAHASHI, H KONDO
    MOLECULAR BRAIN RESEARCH 17 (1-2) 135 - 146 0169-328X 1993/01 [Refereed][Not invited]
     
    We isolated cDNAs to beta and gamma subtypes of 14-3-3 protein, a putative regulatory protein for protein kinase C, from the brain and clarified a high homology in sequences of nucleotides and deduced amino acids between the two rat subtypes and the bovine counterparts and even reciprocally between the two rat subtypes. In Northern blot analysis, the gene expression of the two subtypes was detected weakly at E13, increased progressively after birth and reached a maximum at P7-P14. Thereafter it decreased slightly. In situ hybridization analysis allowed detection of the beta but not the gamma subtype in the matrix cells of the ventricular germinal zone of the neural wall. In post-mitotic neurons in the mantle zone and maturing brain loci, genes of the two subtypes were expressed in patterns similar to each other, and three neuron types were identified: type I neurons with high levels of expression throughout development; type II neurons showing high expression during the early developmental stages with a subsequent decrease in the expression at maturing and adult stages; and type III neurons showing consistently low levels of expression throughout development. The wider and more highly-patterned expression of the 14-3-3 protein family than expected suggests that this protein may be involved in the elaborate regulation of some fundamental cellular activities and differentiation of neurons.
  • M WATANABE, Y INOUE, K SAKIMURA, M MISHINA
    MOLECULAR BASIS OF ION CHANNELS AND RECEPTORS INVOLVED IN NERVE EXCITATION, SYNAPTIC TRANSMISSION AND MUSCLE CONTRACTION 707 463 - 466 0077-8923 1993 [Refereed][Not invited]
  • M WATANABE, Y INOUE, K SAKIMURA, M MISHINA
    NEUROREPORT 3 (12) 1138 - 1140 0959-4965 1992/12 [Refereed][Not invited]
     
    IN SITU hybridization analyses have revealed drastic changes in expression and distribution of five subunit mRNAs of the mouse NMDA receptor channel during brain development. The epsilon1 subunit mRNA is expressed postnatally and widely in the brain. On the other hand, the epsilon2 subunit mRNA is found throughout the entire embryonic brain, but its expression becomes restricted to the forebrain at postnatal stages. The epsilon3 subunit mRNA appears postnatally and predominantly in the cerebellum, whereas the epsilon4 subunit MRNA is abundantly expressed in the diencephalon and the brainstem at embryonic and neonatal stages. In contrast, the zeta1 subunit mRNA distributes ubiquitously in the brain throughout development. These findings suggest that changes in the subunit composition of the NMDA receptor channel take place during brain development.
  • H SAKAGAMI, M WATANABE, H KONDO
    MOLECULAR BRAIN RESEARCH 16 (1-2) 20 - 28 0169-328X 1992/11 [Refereed][Not invited]
     
    The localization and ontogenic changes of expression of the mRNA for Ca2+/calmodulin-dependent protein kinase of the cerebellar granule cell type or type IV (CaM kinase Gr or IV) in the rat brain were examined by in situ hybridization histochemistry. At the young adult stage, intense expression signals for this kinase mRNA were detected in the cerebellar granule cells, the hippocampal pyramidal cells, the dentate granule cells, and the piriform cortex. Moderate levels of the mRNA were expressed in the thalamic nuclei and the cerebral cortex. No distinct expression signals were detected in the Purkinje cells and most brainstem nuclei except for the pontine nuclei, locus ceruleus and inferior olive which showed weak expression. During development, two chronological patterns of changes in the gene expression for this kinase were discerned. The first was a high and persistent expression from the developing stages till the adult stage, which was observed in the cerebellar granule cells, the hippocampal pyramidal cells and the dentate granule cells. The other was a transiently high expression during limited developmental periods, which was observed in the Purkinje cells, neurons in the inferior olive, various brain stem nuclei, and the subventricular neuronal cells. These findings suggest that this Ca2+/calmodulin-dependent protein kinase is involved differentially in multiple Ca2+ signaling pathways in different developing and mature neurons.
  • K IKEDA, M NAGASAWA, H MORI, K ARAKI, K SAKIMURA, M WATANABE, Y INOUE, M MISHINA
    FEBS LETTERS 313 (1) 34 - 38 0014-5793 1992/11 [Refereed][Not invited]
     
    The primary structure of a novel subunit of the mouse NMDA (N-methyl-D-aspartate) receptor channel, designated epsilon4, has been revealed by cloning and sequencing the cDNA. The epsilon4 subunit shares high amino acid sequence identity with the epsilon1, epsilon2 and epsilon3 subunits of the mouse NMDA receptor channel, thus constituting the epsilon subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the epsilon4 subunit together with the zeta subunit in Xenopus oocytes yields functional NMDA receptor channels. The epsilon4/zeta1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The epsilon4 subunit is thus distinct in functional properties from the epsilon1, epsilon2 and epsilon3 subunits, and contributes further diversity of the NMDA receptor channel.
  • K GOTO, M WATANABE, H KONDO, H YUASA, F SAKANE, H KANOH
    MOLECULAR BRAIN RESEARCH 16 (1-2) 75 - 87 0169-328X 1992/11 [Refereed][Not invited]
     
    A 3.1 kbp cDNA clone encoding diacylglycerol (DG) kinase of 80 kDa (80K-DG kinase) was isolated from a rat brain cDNA library. The deduced amino acid sequence was 82% homologous to previously identified porcine 80K-DG kinase and contained zinc finger-like sequences, E-F hand motifs and ATP-binding sites similar to the porcine counterpart. By in situ hybridization histochemistry of rat brain at postnatal week 3, the expression signals for 80K-DG kinase mRNA appeared predominantly on somata of discrete cells in the white matter, and the expression pattern was similar to that of the myelin-specific proteins. In immunohistochemistry using the antibody against bacterially expressed DG kinase-fusion protein, numerous fibrous or dot-like structures exhibiting the immunoreactivity were concentrated in the white matter and they were arranged to radiate in the cerebral cortex and the cerebellar granular layer in a pattern almost identical to that of oligodendrocytes. No neuronal cells exhibited the immunoreactivity. The present finding thus strongly suggests that 80K-DG kinase is expressed specifically in the oligodendrocytes, but not neurons, and may be involved in the myelin formation and metabolism. In addition, the intense hybridization signals and the immunoreactivity for this protein were detected in the entire medulla of the thymus and the periarterial lymphatic area of the splenic white pulp both of which represent T-cell-dependent areas.
  • Y OWADA, M WATANABE, H KONDO
    NEUROSCIENCE LETTERS 144 (1-2) 9 - 13 0304-3940 1992/09 [Refereed][Not invited]
     
    A cDNA encoding rat beta-adrenergic receptor kinase (beta-ARK) was cloned and sequenced. By in situ hybridization histochemistry of adult brain, beta-ARK mRNA was expressed intensely in the cerebellar granule cell layer and moderately in the hippocampal pyramidal cells and dentate granule cells. The neocortex and piriform cortex expressed it moderately to weakly, whereas the thalamus and hypothalamus expressed it weakly to faintly. No significant expression of the mRNA was detected in the caudate-putamen. Weak expression of beta-ARK mRNA was detected in several nuclei of the brainstem and in the spinal gray matter.
  • H ABE, M WATANABE, H KONDO
    ACTA OTO-LARYNGOLOGICA 112 (5) 862 - 871 0001-6489 1992/09 [Refereed][Not invited]
     
    The detailed localization of spot 35-calbidin and its ontogenic change were studied in Nervus terminalis, the vomeronasal organ and the olfactory epithelium of the rat by immunohistochemistry. At the embryonic days 12 and 13 (E 12-13), calbindin-immunoreactive cells were found in the outermost layer of the presumptive olfactory bulb and within the olfactory placode. At E 14 to the postnatal day 1 (P 1), intense calbindin-immunoreactivity was localized in ganglionated fiber bundles of Nervus terminalis coursing through the mesenchymal spaces on both sides of the nasal septum. Nervus terminalis decreased the immunoreactivity abruptly after P 1 and it showed no distinct immunoreactivity for calbindin at P 7 and thereafter. On the other hand, numerous receptor cells in the olfactory epithelium and the thicker vomeronasal epithelium exhibited weak to moderate immunoreactivity for calbindin at E 18-P 1. Their immunoreactivity decreased in intensity progressively after P 7 and no distinct immunoreactivity for calbindin was detected in most of the receptor cells, whereas moderate immunoreactivity was detected in most of the vomeronasal and olfactory nerves at P 28 and P 63.
  • H ABE, M WATANABE, K GOTO, H SAKAGAMI, H KONDO
    MOLECULAR BRAIN RESEARCH 14 (4) 337 - 343 0169-328X 1992/08 [Refereed][Not invited]
     
    The localization of gene expression of calreticulin, a calcium-binding protein in the endoplasmic reticulum, was examined throughout the entire brain of adult mice by in situ hybridization. Calreticulin mRNA is expressed widely and heterogeneously in discrete neurons throughout the brain, but the white matters expressed it weekly or faintly. In the olfactory bulb, the mRNA is expresses moderately in the mitral cells. but weakly in the periglomerular cells and internal granule cells. In the cerebrum, the gene is expressed intensely in the piriform cortex. but weakly in neocortex, the entorhinal cortex and the amygdaloid nuclei. In the hippocampal formation, calreticulin mRNA is expressed intensely in the CA1-CA3 regions but less intensely in the granule cells of the dentate gyrus. The caudate-putamen, thalamic and hypothalamic nuclei, and mammillary nuclei express the mRNA weakly or faintly. In the mesencephalon. pons and medulla, moderate expression of the mRNA is detected in the pontine nuclei and the locus ceruleus. Weak expression of the mRNA is detected in several discrete nuclei and zones such as the substantia nigra, the superior colliculus and the central gray. Expression signals of calreticulin mRNA are faint in the inferior olive. In the cerebellum, calreticulin mRNA is expressed moderately in the Purkinje cells whereas no significant expression is detected in the granule cells. The plexus choroideus of the lateral, third and fourth ventriculi express calreticulin mRNA intensely although no distinct expression of the mRNA is discerned in the ependyma.
  • H KONDO, M YAMAMOTO, M WATANABE
    JOURNAL OF SUBMICROSCOPIC CYTOLOGY AND PATHOLOGY 24 (2) 241 - 250 1122-9497 1992/04 [Refereed][Not invited]
     
    Superior cervical ganglia of rats were centrifuged at 40,000 rpm (160,500 g) for 1 h at 4-degrees-C. Most neuronal somata exhibit a minor centripetal domain free of organelles and a major centrifugal domain rich in organelles. The former is occupied by numerous fine granules having low electron density unlike ribosomes in epoxy sections stained with uranium and lead, and is occupied by a meshwork of microtrabecular or filamentous elements similar to that of the centrifugal domain as well as that of the normal cells in PEG (polyethylene glycol)-processed embedment-free sections without staining. The latter centrifugal domain contains regular cell organelles except for neurofilaments without stratification. All the organelles are suspended in the meshwork of microtrabecular or filamentous elements. In immunolight microscopy, NFPs (neurofilament proteins) are confined to the centripetal domain. In immunoelectron microscopy using ultrathin cryosections and the protein A-gold labeling, numerous gold-particles for NFPs were deposited randomly in the centripetal cytoplasmic domain without long linear alignment. In the PEG sections the gold-labelings for NFPs are randomly deposited on portions of the microtrabecular strands in the centripetal domain. After incubation of the centrifuged ganglia in the anterior eve chamber overnight, NFPs-immunoreactivity appears again diffusely throughout the entire cytoplasm of all neuronal somata in immunolight microscopy. The organelle-free domain of the cytoplasm is no longer visible in electron microscopy. The present findings are discussed in relation to the state of the cytoplasmic soluble proteins and the reality of the microtrabecular or filamentous elements in the cytoplasm.
  • H ABE, M WATANABE, T YAMAKUNI, R KUWANO, Y TAKAHASHI, H KONDO
    NEUROSCIENCE LETTERS 138 (2) 211 - 215 0304-3940 1992/04 [Refereed][Not invited]
     
    Localization of gene expression of calbindin, a cytosolic calcium-binding protein, was examined throughout the adult rat brain by in situ hybridization with cDNA probes. The gene was expressed most intensely in the Purkinje cells in the cerebellum, intensely in the granule cells of the dentate gyrus, and moderately in the inferior olivary nucleus, in the nuclei of the trapezoid body, in the medial part of the lateral habenular nuclei, entorhinal cortex and in the mammillary nuclei. In addition, weak expression of the gene was widespread in the forebrain and brainstem gray matter, and also in small cells in the spinal posterior horn as well as the ependymal cells. The widespread and heterogeneous expression of the gene in the brain suggests that calbindin is differentially involved in calcium-regulated phenomena in different neurons.
  • M WATANABE, H KOMINAMI, N YANAIHARA, H KONDO
    HISTOCHEMISTRY 97 (3) 277 - 281 0301-5564 1992/03 [Refereed][Not invited]
     
    Cellular and subcellular localizations of gastrin-releasing peptide-like immunoreactivity (GRP-LI) were examined in the perikarya of trigeminal ganglion cells. By immunolight microscopy using semi-thin sections, GRP-LI was observed in almost all the neuronal somata with various intensity as granular profiles distributing widely in the cytoplasm. By immunoelectron microscopy using ultrathin frozen sections and protein A-gold, GRP-LI was found predominantly in rounded or oval membrane-bound structures which were 300-800 nm in diameter and displayed various electron-density and heterogenous contents. Double-labeling immunoelectron microscopy using antibodies for GRP and cathepsin L, a well-characterized lyosomal proteinase, clearly demonstrated that these GRP-immunoreactive intracytoplasmic structures were lysosomes. In contrast, GRP-LI was detected only occasionally in the large granular vesicles (100-200 nm in diameter). These findings strongly suggest that considerable amount of GRP or GRP-like peptide is subject to intracellular degradation in the lysosome rather than to the regulatory secretion pathway, and this is the reason why no fibers immunoreactive for GRP have been detected in the peripheral sensory field.
  • H ABE, M WATANABE, H KONDO
    HISTOCHEMISTRY 97 (2) 155 - 160 0301-5564 1992/02 [Refereed][Not invited]
     
    Tracheobronchial epithelium, thyroid organ, thymus, of the developing rats were examined by immunohistochemistry using anti-spot 35 calbindin-antiserum. At E 14, weak to moderate immunoreactivity for spot 35-calbindin was detected in the airway epithelia of the distal half of the trachea and the extrapulmonary bronchus. The immunoreactive cells increased in intensity at E 16-E 21, but decreased markedly after birth. These cells were non-ciliated cells and comprised a majority of the epithelial cells especially in the ventral/cartilaginous portion of the airway. They were characterized by microvilli, vacuoles, granular and agranular endoplasmic reticulum. Typical ciliated cells, which were much less numerous than the immunopositive non-ciliated cells, were immunonegative. In thyroid gland, calbindin-immunoreactive cells first appeared at E 18. They increased in number at E 20-P 1 and decreased gradually after P 7. These cells were the parafollicular cells characterized by numerous secretory granules and situated in close proximity to the basal surface of the follicular cells. In the thymus, immunoreactive cells appeared in the thymic medulla at E 20. They increased in number at P 1, but decreased gradually after P 7. They were stellate in shape and had vesicles, vacuoles, intermediate filaments and represented a subpopulation of thymic reticular epithelial cells. Such a transient appearance of spot 35-calbindin in these cells suggests that this protein may be involved in the regulation of differentiation or may be involved in the process of secretion during the limited developmental period.
  • H KONDO, H TAKAHASHIIWANAGA, H ABE, M WATANABE, Y TAKAHASHI
    ARCHIVES OF HISTOLOGY AND CYTOLOGY 54 (4) 437 - 445 0914-9465 1991/10 [Refereed][Not invited]
     
    By means of peroxidase-anti-peroxidase (PAP) immunohistochemistry with both the neurofilament (NF) triplet (small: 68 K, medium: 150 K, high: 200 K) antisera and the antiserum against spot 35-calbindin, developmental changes in expression of the immunoreactivity for the NF triplet proteins in different domains of the Purkinje cells was examined in the cerebella of postnatal rats. From birth till the postnatal day 6 (P6) the somata and dendrites exhibited moderately positive immunoreaction for small and medium NF subunits. In contrast, the incubation for the high NF subunit resulted in a negative immunoreaction for the somata and dendrites of the Purkinje cells at those stages. On P8 and P10 they were weakly immunoreactive for all NF triplet. Thereafter the intensity of their immunoreaction decreased progressively and the Purkinje cell somata and dendrites were immunonegative for all NF triplet proteins on P21, when the Purkinje cell attained an adult appearance in morphology. On the other hand, the Purkinje cell axons, which can be identified selectively by the positive immunoreaction for the spot 35-calbindin throughout the course of the postnatal development, exhibited positive immunoreactivity for all the NF triplet on the 9th, 21th and 50th postnatal days in the cerebellar white matter. The development of the slow axoplasmic transport and the posttranslational modification of the NF triplet proteins are discussed as possible mechanisms underlying the differential expression of the immunoreactivity for the NF triplet proteins in different domains of the Purkinje cells during postnatal development.
  • M WATANABE, T ISOBE, T OKUYAMA, T ICHIMURA, R KUWANO, Y TAKAHASHI, H KONDO
    MOLECULAR BRAIN RESEARCH 10 (2) 151 - 158 0169-328X 1991/05 [Refereed][Not invited]
     
    Activation of tyrosine and tryptophan hydroxylases, key enzymes for the catecholamine and serotonin biosynthesis, requires Ca2+/calmodulin-dependent protein kinase II and 14-3-3 protein which comprises a family of, at least, seven polypeptides in the bovine. Here we show that the amino acid sequence of the rat 14-3-3 eta chain deduced from the nucleotide sequence is completely identical to that of bovine counterpart. Using in situ hybridization the expression of mRNA for this protein is detected not only in the monoamine-synthetic neurons but also in many other discrete nuclei which synthesize neither catecholamine nor serotonin. The highly conservative structure between mammalian species and wider expression of this protein than expected in the central nervous system suggest that the 14-3-3 protein exerts some, though yet to be defined, functions fundamental to neuronal activities other than activation of the monoamine biosynthesis.
  • M WATANABE, N YANAIHARA, H KONDO
    DEVELOPMENTAL BRAIN RESEARCH 59 (1) 113 - 116 0165-3806 1991/03 [Refereed][Not invited]
     
    In rats at the postnatal week 3 (P3W), enkephalin-immunoreactivity was detected in subsets of principal ganglion cells in the superior cervical ganglion and nerve fibers within the submandibular gland, one of the ganglion, whereas it disappeared from them at P8W. Enkephalin-immunoreactive ganglion cells and intraglandular fibers were detected again after the colchicine-pretreatment at P8W. By in situ hybridization the population density of ganglion cells expressing mRNA for preproenkephalin and the expression intensity were similar in both ganglia at P3W and P8W. These findings suggest that the post-transcriptional down-regulation of gene expression for preproenkephalin is involved in the disappearance of enkephalin-immunoreactivity in the adult ganglion.
  • M WATANABE, T ONO, H KONDO
    JOURNAL OF ANATOMY 174 81 - 95 0021-8782 1991/02 [Refereed][Not invited]
     
    The localisation and ontogenic expression of heart fatty acid binding protein (H-FABP) were immunochemically examined in the entire body of the rat. In muscles, H-FABP immunoreactivity was observed not only in cardiac muscle cells but also in red skeletal muscles, where gold particles indicating the immunoreaction sites are distributed evenly in the sarcoplasm except for the interior of membranous organelles. In the urogenital system, the immunoreactivity was preferentially demonstrated in the distal tubular epithelial cells in the kidney, and in the superficial epithelial cells in the urinary bladder. The H-FABP immunoreactivity was further found in cells characterised by numerous lipid droplets, such as interstitial cells of the theca interna in the ovary and those of Leydig in the testis, and cell Element III in the placenta. In the digestive system, parietal cells in the stomach and pancreatic beta-cells were immunoreactive to H-FABP. In addition, capillary endothelial cells were immunostained in the cardiac and red skeletal muscles, the exocrine pancreas, digestive tract and thymus. From the rather wide distribution of H-FABP, it is suggested the H-FABP plays some fundamental roles in the active metabolism of fatty acids in the body. The present findings also indicate that H-FABP is a useful marker for the morphological study of interstitial cells in the ovary and testis and parietal cells in the stomach.
  • M WATANABE, K SAKIMURA, Y TAKAHASHI, H KONDO
    DEVELOPMENTAL BRAIN RESEARCH 53 (1) 89 - 96 0165-3806 1990/04 [Refereed][Not invited]
  • T WATANABE, S WAGURI, M WATANABE, Y ISHII, E KOMINAMI, Y UCHIYAMA
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 37 (12) 1899 - 1911 0022-1554 1989/12 [Refereed][Not invited]
  • H MATSUBA, T WATANABE, M WATANABE, Y ISHII, S WAGURI, E KOMINAMI, Y UCHIYAMA
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 37 (11) 1689 - 1697 0022-1554 1989/11 [Refereed][Not invited]
  • Y ISHII, T WATANABE, M WATANABE, S HASEGAWA, Y UCHIYAMA
    JOURNAL OF ANATOMY 166 85 - 95 0021-8782 1989/10 [Refereed][Not invited]
  • M TANAKA, Y IWAMURA, H AMANUMA, Y IRIE, M WATANABE, T WATANABE, Y UCHIYAMA, K YASURAOKA
    PARASITOLOGY 99 31 - 38 0031-1820 1989/08 [Refereed][Not invited]
  • Y UCHIYAMA, T WATANABE, M WATANABE, Y ISHII, H MATSUBA, S WAGURI, E KOMINAMI
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 37 (5) 691 - 696 0022-1554 1989/05 [Refereed][Not invited]
  • Y UCHIYAMA, M WATANABE, T WATANABE, Y ISHII, H MATSUBA, S WAGURI, E KOMINAMI
    CELL AND TISSUE RESEARCH 256 (2) 355 - 360 0302-766X 1989/05 [Refereed][Not invited]
  • T WATANABE, M WATANABE, Y ISHII, H MATSUBA, S KIMURA, T FUJITA, E KOMINAMI, N KATUNUMA, Y UCHIYAMA
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 37 (3) 347 - 351 0022-1554 1989/03 [Refereed][Not invited]
  • M WATANABE, T WATANABE, Y ISHII, H MATSUBA, S KIMURA, T FUJITA, E KOMINAMI, N KATUNUMA, Y UCHIYAMA
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 36 (7) 783 - 791 0022-1554 1988/07 [Refereed][Not invited]
  • M WATANABE, Y UCHIYAMA
    CELL AND TISSUE RESEARCH 253 (2) 337 - 345 0302-766X 1988 [Refereed][Not invited]
  • VM DARLEYUSMAR, M WATANABE, Y UCHIYAMA, KONDO, I, NG KENNAWAY, L GRONKE, H HAMAGUCHI
    CLINICA CHIMICA ACTA 158 (3) 253 - 261 0009-8981 1986/08 [Refereed][Not invited]
  • Y UCHIYAMA, M WATANABE
    CHRONOBIOLOGIA 12 (3) 280 - 280 0390-0037 1985 [Refereed][Not invited]
  • VM DARLEYUSMAR, M WATANABE
    JOURNAL OF BIOCHEMISTRY 97 (6) 1767 - 1775 0021-924X 1985 [Refereed][Not invited]
  • Y UCHIYAMA, M WATANABE
    CELL AND TISSUE RESEARCH 237 (1) 123 - 129 0302-766X 1984 [Refereed][Not invited]
  • Y UCHIYAMA, M WATANABE
    CELL AND TISSUE RESEARCH 237 (1) 131 - 138 0302-766X 1984 [Refereed][Not invited]
  • Y UCHIYAMA, M WATANABE
    CELL AND TISSUE RESEARCH 237 (1) 117 - 122 0302-766X 1984 [Refereed][Not invited]

MISC

Books etc

Association Memberships

  • Society for Neuroscience   THE JAPAN NEUROSCIENCE SOCIETY   THE JAPANESE ASSOCIATION OF ANATOMISTS   

Research Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2022/04 -2028/03 
    Author : 鍋倉 淳一, 根本 知己, 山中 めぐみ, 上野 直人, 真野 昌二, 大浪 修一, 藤森 俊彦, 野中 茂紀, 今村 健志, 平岡 泰, 甲本 真也, 松田 道行, 洲崎 悦生, 稲葉 一男, 菅谷 佑樹, 澤田 和明, 佐藤 良勝, 三上 秀治, 岡田 康志, 大野 伸彦, 安永 卓生, 太田 啓介, 小池 正人, 宮澤 淳夫, 深澤 有吾, 渡辺 雅彦, 豊岡 公徳, 片岡 洋祐, 定藤 規弘, 青木 茂樹, 岡田 直大, 林 拓也, 内田 誠一, 桧垣 匠, 舟橋 啓, 小田 祥久, 木森 義隆
     
    前年度までの新学術領域研究（学術研究支援基盤形成）「先端バイオイメージング支援プラットフォーム（ABiS）」の実績を元に、研究者のニーズを調査し、新たな支援内容も加えた体制を構築し、光学顕微鏡、電子顕微鏡、磁気共鳴機器による画像取得、およびそれらの機器より得られた画像の解析の支援を行った。2022年度は、光学顕微鏡支援として101件、電子顕微鏡支援として45件、磁気共鳴画像支援として17件、画像解析支援として17件行い、トレーニングを14回開催した。 新たな支援活動を行うにあたり、オフィシャルサイトをリニューアルした。新しいオフィシャルサイトでは、各支援内容を紹介する動画の掲載や連携している国際バイオイメージングコンソーシアム（GBI: Global BioImaging）の情報の自動アップデート機能などを実装した。公募の周知活動として、このオフィシャルサイトを随時更新して最新情報を発信するとともに、公募案内のポスターをリニューアルして、大学・研究機関に送付した（1,353部）。また、新しい体制を紹介するためのリーフレットも刷新し、周知活動に用いた。 本年度は、発生生物学会第55回大会、第74回日本細胞生物学会年会、日本植物学会第86回大会、第81回日本癌学会学術総会、第95回日本生化学会大会、第45回日本分子生物学会年会、日本生理学会第100回記念大会、日本薬学会143年会において、ブース出展やランチョンセミナー、シンポジウム共催を行い、大会参加者へ支援概要や応募方法の説明を行った。上記の大会のうち、第81回日本癌学会学術総会、第95回日本生化学会大会、第45回日本分子生物学会年会、日本生理学会第100回記念大会、日本薬学会143年会は、生命科学連携推進協議会のもと、4つのプラットフォームが合同で参加した。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2021/04 -2025/03 
    Author : 渡辺 雅彦, 宮崎 太輔
     
    興奮性シナプス活動により、イオンチャネル型グルタミン酸受容体iGluRと代謝型グルタミン酸受容体mGluRの活性化が起こる。iGluR活性化による脱分極は、小脳プルキンエ細胞ではP/Q型カルシウムチャネルの活性化を介して細胞外からCa2+流入を誘導する。受精の段階からこの遺伝子欠損を有するヌル欠損マウスでは、シナプス刈込みに障害が生じて、登上線維と平行線維のテリトリー不分離と登上線維の多重支配が残存することを示してきた。本研究課題では、シナプス回路完成後の成体期においてもシナプス回路維持機構として機能しているかを、成体期に遺伝子欠損誘導を行えるモデルを作成して明らかにすることである。 新潟大学脳研究所の崎村建司名誉教授らが以前開発した、P/Q型VDCC のチャネル本体をコードするCav2.1遺伝子のfloxマウスと、プロゲステロン誘導剤（RU-486）依存的に活性化するCreリコンビナーゼをプルキンエ細胞選択的に発現するGluD2-CrePRマウスを入手する。亮者を交配して、Cav2.1flox/CrePRマウスラインを得た。生後2ヶ月令の成体マウスにRU-486を腹腔投与したものを成体期欠損群（Cav2.1-/CrePR）、溶剤のみを腹腔投与したものをコントロール群（Cav2.1flox/CrePR）とした。研究代表者が開発したCav2.1特異抗体を用いて免疫染色法を用いて検討したところ、 Cav2.1-/CrePRのプルキンエ細胞における Cav2.1 タンパクの有意な減少が誘導後１週から確認された。一方、Cav2.1flox/CrePでは Cav2.1 タンパクの発現の減少は認められなかった。したがって、神経回路が完成した成体期においてCav2.1遺伝子を欠損させるモデルマウスが完成したことを確認できた。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Specially Promoted Research
    Date (from‐to) : 2020/07 -2025/03 
    Author : 柚崎 通介, 伊佐 正, 渡辺 雅彦, 武内 恒成, 坂内 博子, 溝口 明
     
    神経細胞は「シナプス」と呼ばれる接着構造によってお互いに結合してさまざまな神経回路を構成する。多くの精神・神経疾患ではシナプスに異常がみられることから、シナプス形成を担う分子群の解明は基礎・臨床神経科学における最重要課題の一つである。 近年、私たちは新しいシナプス形成分子として、細胞外足場タンパク質（Extracellular Scaffolding Protein: ESP）という概念を確立した。ESP は神経細胞やグリア細胞から分泌されて、シナプスにおいてシナプス前部や後部のさまざまな膜タンパク質と結合する足場として働く。ESPは従来のシナプス形成分子とは異なり、発達時のみでなく生涯にわたって、神経活動に応じたシナプスの再編や機能を制御する。さらに、神経細胞間や、神経細胞と非神経細胞の間にはシナプスとは異なった接着構造が存在し、ESPはこのような非シナプス性接着構造にも関与することがわかってきた。 そこで本研究では、シナプスおよび非シナプス性接着構造において機能するさまざまなESPのシグナル伝達機構の解明を進め、さらにESP の結晶構造を元にして人工的コネクターを開発することによって、神経回路網や非シナプス性接着構造の生理的機能を明らかにし、新しい観点から脳の動作原理および精神・神経疾患の病態の解明を進めることを目指す。 これまでにESPに属するシナプス形成分子として、補体ファミリー分子C1q, Cbln4, C1ql1、さらに神経ペントラキシン（NP）を中心にシグナル伝達機構の解明を進めてきた。特に正常発達時に起きる余分なシナプスの刈込み現象や、アルツハイマー病等における病的なシナプス減少において補体 C1q が関与するが、C1q がいったい何に結合するのかは不明であったが私たちは、これまでに中枢神経系における C1q 受容体の同定に初めて成功した（論文準備中）。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2021/04 -2024/03 
    Author : 七戸 俊明, 平野 聡, 渡辺 雅彦, 佐藤 典宏, 渡邊 祐介, 岡本 吉弘
     
    要約：本年度は下記の2つの研究を行った。来年度はこれらの内容をまとめて報告する予定である。１．医療機器開発における献体使用のパイロットスタディの実施（3回）２．有識者によるディスカッションの場としてカダバーワークショップの開催（2回） 概要：１．カダバーラボを使用した医療機器開発の共同研究（実施担当：北海道大学病院医療機器開発推進センター） 〇実施内容：①耳鼻科領域の経口的咽頭手術機器の医療機器開発を行った。研究では献体を使用した性能試験を行った。同時にダビンチとの比較試験を含む性能試験（ドライラボ）も実施した。②内視鏡を使用した心臓血管外科手術に使用する医療機器の性能試験を行った。同時にダビンチとの比較試験を含む性能試験（ドライラボ）も実施した。③呼吸器内科領域の医療機器開発として献体を使用した性能試験を行った。 ２．有識者によるワークショップとして「カダバーワークショップ」をwebで2回実施した。（共催：AMED次世代医療機器連携拠点整備等事業「国内唯一：カダバーラボで医療機器開発が可能な拠点」、文科省課題解決型高度医療人材養成プログラム「臨床医学の献体利用を促進する専門人材養成」）①2021年10月29日「医療機器開発における献体利用のあるべき姿：実例から考える」内容：北大での実施例の報告を行い、コメンテーターと参加者がカダバーラボの実施における課題を明らかにすべく、ディスカッションを行った。参加者：26名、参加企業：2社。②2022年1月14日「医療機器開発における献体利用のあるべき姿：理想を語る」内容：国内外のカダバーを使用した医療機器開発の動向の報告を行い、引き続き厚労省、経産省、PMDA等の行政と、企業関係者、臨床医、解剖学者、コーディネータ等が参加し、国内でカダバーラボを推進するためのディスカッションを行った。参加者：33名、参加企業：7社。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2021/04 -2024/03 
    Author : 安部 崇重, 渡辺 雅彦, 七戸 俊明, 篠原 信雄, 近野 敦
     
    鉗子動態に基づく手術技量評価法の開発 これまでのウエットラボ参加者70名の鉗子動態計測データを用いて、手術経験数を熟練度の指標として用い、サポートベクターマシン等を用いて、鉗子動態に基づく手術技量分析能を評価した。良好な弁別能を観察しており、現在論文投稿中である。また、これまでのウェットラボ記録ビデオに関して、腹腔鏡手術技術評価表であるGOALS評価表を使用し、2名の腹腔鏡技術認定医による技術評価を修了した。GOALS評価表は、1. Depth perception, 2. Bimanual dexterity, 3. Efficiency, 4.Tissu handling, 5. Autonomy の5項目を、それぞれ1-5段階で評価を行う形式で、5-25点の点数が割り付けられる。R3年度は、鉗子動態計測値とGOALSスコアの相関評価を行い、鉗子動態に基づく機械学習を用いたGOALSスコアの自動計算法を開発した。現在、論文投稿中である。
    カダバーを用いた腹腔鏡下腎摘除術トレーニングプログラムの開発と鉗子動態の計測・解析 使用するカダバーは、シール法と呼ばれる固定方法の献体で、生体と比較し良好な組織類似性が特徴である。2020年9月末より腹腔鏡下腎摘除術トレーニングにおいて、三眼カメラOptiTrack V 120 Trioを用いて鉗子動態計測を開始している。R3年度は、18名の参加者に関して、カダバー腹腔鏡下腎摘除術の鉗子動態の測定を行った。
  • Advanced Bioimaging Support
    Japan Society for the Promotion of Science：Grant-in-Aid for Scientific Research on Innovative Areas
    Date (from‐to) : 2016/04 -2022/03 
    Author : Masanobu Kano
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2018/06 -2020/03 
    Author : Shichinohe Toshiaki
     
    The purpose of this study was to develop an educational system to learn new advanced medical techniques for medical students and to examine the usefulness of clinical anatomy practice consisting of simulated surgery using donated cadavers. The contents of the research were a questionnaire survey of medical students and academics, and a pilot study of simulated endoscopic surgery. The simulated surgery consisted of surgical lectures, demonstrations by surgeons, and hands-on or observational practice by students. These studies showed that both conventional systematic anatomy and newly developed clinical anatomy were useful. It is desirable to introduce clinical anatomy as part of the clinical training curriculum other than conventional anatomy.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2013/06 -2018/03 
    Author : Okabe Shigeo, WATANABE Masahiko
     
    Learning and experience-dependent brain functions can be acquired at the postnatal critical period, when neural circuits receive remodeling and pruning of synapses. Imaging and electrophysiology were applied to study glial contributions to circuit remodeling in the critical period. In the cerebral cortex, the cortical volume is segmented into multiple zones defined by the distance from microglial cell bodies and these zones specify dendritic segments with higher or lower spine dynamics. On the other hand, a study of microglia-specific knockout mice of Csf1r revealed an important role of microglia in climbing fiber pruning in the cerebellum. Regulation of climbing fiber pruning was not dependent on phagocytotic activity, but was suggested to be mediated via inhibitory synapses.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014/04 -2017/03 
    Author : Hishimoto Akitoyo, Watanabe Masahiko, Kusumi Ichiro
     
    The genetic research of complete suicide is behind other mental problems because it is extremely difficult to obtain tissue samples of completed suicide. Under the difficult situation, we performed a GWAS using 500 samples of suicide completers, and found two candidates of suicide-related gene; PTCHD3 and RAB18. Here, we investigated the function of these candidate genes in chronic stressed rats and mouse neural stem cells. Stressed rats showed higher PTCHD3 expression in prefrontal cortex and hippocampus, and lower RAB18 expression in amygdala. In mouse neural stem cells with RAB18 knockdown, cell proliferation and survival were significantly increased compared to controls. Our results suggest aberrant expression of these suicide-related genes may be associated with brain abnormalities in stress conditons. Replication GWAS of suicide completers and further in vivo and in vitro analyses are needed in future studies.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014/04 -2017/03 
    Author : Hozumi Yasukazu, GOTO Kaoru, WATANABE Masahiko
     
    Results show that striatal medium spiny neurons (MSNs) of DGKb-KO mice exhibited lower dendritic spine density at distal dendrites than wild-type mice did. We identified the GluA2 AMPA receptor subunit as a novel DGKb binding partner. In addition, DGKb-deficient brain exhibits significant reduction of TARPg-8. These findings suggest that DGKb regulates the spine formation at distal dendrites in MSNs. Next, we investigated the cellular expression and subcellular localization of DGKe in the brain using specific DGKe antibody. In Purkinje cells, DGKe was localized to the subsurface cisterns. Behaviorally, DGKe-KO mice exhibited hyper-locomotive activities and impaired motor coordination and learning. These findings suggest that DGKe plays an important role in neuronal and brain functions through its distinct neuronal expression and subcellular localization, and also through coordinated arrangement with other molecules involving the phosphoinositide signaling pathway.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014/04 -2017/03 
    Author : KUBO Yoshihiro, YAMAMOTO Izumi, WATANABE Masahiro, Chen I-Shan, Yamamoto Tomomi, TATEYAMA Michihiro, TAKAO Keizo, MIYAKAWA Tsuyoshi, HATTORI Satoko, FUKATA Masaki, FUKATA Yuko, YOKOI Norihiko, SAKIMURA Kenji, SYUU Rei, NATSUME Rie
     
    Prrt3 is a putative G protein coupled receptor of family C, but its function is totally unknown. We aimed at the elucidation of the molecular function and physiological role of Prrt3, and observed the followings. (1) Prrt3 is partly cleaved at the end of N-terminal extracellular domain by Furin. (2) It binds to glutamate transporters EAAT1, 2 and Gi/o G protein. (3) It is expressed chiefly at the presynaptic terminal of excitatory neurons. (4) Conditional Prrt3 gene knock out homozygous mice in forebrain excitatory neurons showed impairment of long term retention of fear conditioning memory. (5) Prtt3 is activated by Oxo-M, a muscarinic Ach receptor agonist, but not by ACh.
  • 高次脳領域におけるシナプス伝達制御機構の分子形態学的研究
    日本学術振興会：科学研究費補助金 基盤研究（S)
    Date (from‐to) : 2012/04 -2017/03 
    Author : 渡辺 雅彦
  • マーモセット脳機能研究に最適化した経路選択的操作とその基盤となる 回路構造解析技術の開発
    AMED：Brain/MINDS
    Date (from‐to) : 2016 -2017 
    Author : Kazuto Kobayashi
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014/04 -2016/03 
    Author : Yoshida Takayuki, WATANABE Masahiko, OHMURA yu
     
    Endocannabinoids were produced by activation of postsynaptic serotonin (5-HT) receptor as well as decreased presynaptic 5-HT release by the activation of the CB1 receptor in the amygdala. We demonstrated that it was caused by calcium- and neuronal activity-dependent manner. In addition, it was revealed that excitability of the pyramidal neuron in the adult amygdala was enhanced by aversive stress at juvenile periods and by activations of 5-HTergic receptors. We also revealed that the 5-HT7 receptor of CA3 pyramidal cell in the ventral hippocampus participated in recalls of the fear memory through the activation of Ih current.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2013/04 -2016/03 
    Author : TAKASAKI Chihiro, UCHIGASHIMA Motokazu, WATANABE Masahiko
     
    Rapid transport of monocarboxylates is essential for carbohydrate, fat, and amino acid metabolisms. The transport is facilitated by proton-linked monocarboxylate transporters (MCTs). MCT1, MCT2 and MCT4 are highly expressed in the brain. In the present study, we examined cellular expression of MCT1 in the mouse brain by fluorescent in situ hybridization and immunohistochemistry. The results showed that dissociated transcription and translational control in neurons was found in pyramidal cells in the CA1 of hippocampus and cholinergic neurons in the dorsal motor nucleus of vagus nerve. Therefore, neuronal expression of MCT1 is transcriptionally active, but suppressed at the post-transcription levels. Through this mechanism, predominant MCT1 expression in astrocytes and capillary endothelial cells is constructed in the brain.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011/04 -2016/03 
    Author : UEDA Hiroshi, IIGO Masayuki, AMANO Masafumi
     
    Olfactory memory formation during juvenile salmon imprinting migration and retrieval during adult homing migration were investigated. In the brain of hatchery-reared underyearling juvenile chum salmon (Oncorhynchus keta), thyrotropin-releasing hormone (TRH) gene expression increased immediately after release from a hatchery into the natal steam, and the expression of the essential NR1 subunit of the N-methyl-D-aspartate receptor increased during downstream migration. Gene expression of salmon gonadotropin-releasing hormone (sGnRH) and NR1 increased in the adult chum salmon brain during homing from the Bering Sea to the natal hatchery. Thyroid hormone treatment in juveniles enhanced NR1 gene activation, and GnRHa treatment in adults improved stream odor discrimination. Olfactory memory formation during juvenile downstream migration and retrieval during adult homing migration of chum salmon might be controlled by endocrine hormones and could be clarified using NR1 as a molecular marker.
  • 文部科学省科学研究費補助金：新学術領域研究（研究領域提案型）
    Date (from‐to) : 2010/04 -2015/03 
    Author : 木村實
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2014/03 
    Author : YOSHIDA Takayuki, WATANABE Masahiko, TANAKA Kenji
     
    In this study, we found predominant neuronal projections of serotonergic, dopaminergic and cholinergic fibers in the basolateral amygdala (BLA), and CB1 receptor was frequently localized on serotonergic axon but not dopaminergic, noradrenergic or cholinergic fibers. Furthermore, extracellular release of serotonin was significantly reduced by WIN55212-2, CB agonist, whereas dopamine concentration was not altered, indicating presynaptic serotonin release is modulated by CB1 receptors. On the other hand, alpha-methyl-5-HT, 5-HT2 receptor agonist triggered postsynaptic endocannabinoid synthesis and induced the reduction of presynaptic GABA release. This suppression was completely blocked by MDL100907, 5-HT2A antagonist, or AM251, CB1 antagonist. These findings suggest that endocannabinoid is synthesized via activation of postsynaptic 5-HT2A receptors, and regulates on-demand local serotonergic transmission in the BLA, as well as neuronal activation of dorsal raphe nucleus.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011 -2013 
    Author : HOZUMI Yasukazu, GOTO Kaoru, WATANABE Masahiko
     
    Results show that stratal medium spiny neurons of DGKb-KO mice exhibit reduced dendritic spine density at the distal dendrites compared with wild-type mice. We also sought protein targets that interact with DGKb and identified the GluR2 AMPA receptor subunit as a novel DGKb binding partner. DGKe immunoreactivity was widely distributed in the cerebellulm. At higher magnifications, DGKe was detected as granular structure in Purkinje cells (PCs) dendrites and perikarya. Ultrastructural localization of DGKe was examined in PCs by pre-embedding immunoelectron microscopy, showing that DGKe-immunoreactivities were distributed in flattened or tubular smooth endoplasmic reticulum just under the cell membrane, which were presumed subsurface cisterns. In rat adrenal gland,RT-PCR analysis showed that the expression signals for DGKb and DGKe are intensely detected. At protein level, DGKb was not expressed in the adrenal gland, but DGKe was detected on the plasma membrane in the zone glomerulosa.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011 -2012 
    Author : HOSHINA Naosuke, YAMAMOTO Tadashi, NAKAZAWA Takanobu, KANO Masanobu, WATANABE Masahiko, KOBAYASHI Kazuto, TAKADA Masahiko, HIRANO Shinji
     
    Highly topographic organization of neural circuits exists for the regulation of various brain functions in cortico-basal ganglia circuits. We show that protocadherin 17 (PCDH17), one of the non-clustered δ2-protocadherin family members, is enriched along cortico-basal ganglia synapses in a topographic manner, and regulates synaptic development in these synapses. Furthermore, PCDH17 knockout mice exhibit antidepressant-like phenotypes.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2009 -2011 
    Author : SAKIMURA Kenji, NATSUME Rie, ABE Manabu, YAMAZAKI Maya, WATANABE Masahiko, KANO Masanobu
     
    The objective of this study is to verify a working hypothesis that the expression and stability of glutamate receptors, including(further) transition to synapses and removal, are responsible for(are involved in) different modes of neuronal regulation depending on each kind of brain cells and regions, which transforms simple inputs into various types of outputs, thus underlying complex neuronal expressions. For this purpose, we established floxed mice of 4 AMPA-type-and 4 NMDA-type-glutamate receptors, as well as several kinds of synaptic functional molecules floxed mice. We also generated Cre-driver mice such as GAD67-Cre mouse. By inter-crossing these mice, we have clarified that a GluN2B subunit is crucial for synaptic functional expression of NMDA-type receptors in a CA3 area of hippocampus, and that TARPγ-2 andγ-7 are essential for the expression of AMPA type receptors in cerebellum. We have also shown that at developing synapses GluN2A and GluN2B suppress AMPA receptors in their own mode, by using a single-cell NMDA receptor subunit deleted mouse.
  • 文部科学省科学研究費補助金：新学術領域研究（統合脳：代表丹治順）
    Date (from‐to) : 2005/04 -2010/03 
    Author : 渡辺 雅彦
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2007 -2010 
    Author : KAWAMATA Tomoyuki, WATANABE Masahiko, NARIMATSU Eichi
     
    This study was planned to clarify the mechanism of CB1 activation-induced analgesia the analgesic effects of CB1 on bone cancer pain. Immunohistochemical experiments showed that, although mu opioid receptor 1 expression was reduced in the superficial dorsal horn, CB1 expression was preserved. In addition, CB1 was mainly expressed in the axon terminals of spinal excitatory interneurons. Spinal CB1 activation reduced bone cancer-related pain behaviors, including behaviors related to spontaneous pain and movement-evoked pain. Spinal CB1 activation reduced bone cancer-related pain behavior. Presynaptic inhibition may contribute to the analgesic effects of spinal CB1 activation. These findings may lead to novel strategies for the treatment of bone cancer pain.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2004 -2010 
    Author : KANO Masanobu, IGARASHI Michihiro, MANABE Toshiya, OHMORI Harunori, WATANABE Wasahiko
     
    本研究は、脳の神経回路の機能解明を目指す特定領域研究(領域番号020)の総括班として、平成16年に開始された。平成16年度には、次年度に開始する計画研究及び公募研究の研究方針の決定、研究項目の策定を行い、平成17年度から21年度までは、計画研究と公募研究の円滑な進展のために、領域の年2回の領域班会議の開催、研究方針の策定、企画調整、評価、研究成果の発信等の活動を行った。脳の最大の特徴は神経細胞が単独で機能するのではなく、多くの神経細胞が連結した神経回路として機能することである。神経回路はからだが出来上がるにつれて「形成」され、成長・発達するにつれて機能的に「成熟」する。成体となるまでに、脳領域の特異性に応じて「発現」される個々の神経回路の独特な機能が完成する。これらのメカニズムを明らかにするため、本特定領域研究では、A01:神経回路の形成、A02:神経回路の機能的成熟、A03:神経回路の特異的機能発現、という3つの研究項目を設定し、12名の計画研究班員と、総勢79名の公募研究班員から構成した。班員の間で極めて活発に連携や共同研究がおこなわれ、5年間に、Nature, Science, Cell, Neuronなどの一流国際学術誌に900編を超える論文が発表された。また、新聞報道をはじめとして研究成果の一般への発信も盛んに行われた。本研究領域は平成21年度をもって終了したが、極めて多くの研究成果が得られており、共同研究をはじめとする人的交流が盛んに行われ、脳の神経回路の機能解明を目指す研究者間のネットワークが出来上がった。これらの有形・無形の財産を、今後の我が国の脳神経科学の発展のために役立てるため、平成22年度において、計画班員とこれまでに公募研究で参加した全ての公募班員の研究成果をまとめ、成果をわかりやすい形で冊子体にまとめ、本特定領域研究の集大成とした。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2007 -2008 
    Author : YAMADA Katsuya, WATANABE Masahiko
     
    脳血流は、神経活動に応じて局所的に調節されるが、この調節を司る脳内細血管のタイプと時間的な血流変化の関係などの詳細はわかっていない。血管平滑筋型ATP感受性カリウム(K_)チャネルのサブユニットKir6.1を欠失させたマウスの実験から、神経活動に対応する脳血流調節に脳内の特定の細動脈の拡張が関わることがわかり、更に議論のあったKir6.1の脳内局在についてもmRNAレベルでは一定の結論が得られた。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2005 -2006 
    Author : 渡辺 雅彦
     
    グルタミン酸は脳における主要な興奮性神経伝達物質で、シナプスにおけるグルタミン酸受容体を介してシグナルが伝達される。神経解剖学の重要な基本概念の一つに、「グルタミン酸作動性の興奮性シナプスはシナプス後部の肥厚が著しい非対称性シナプスで、GABAやグリシン作動性の抑制性シナプスは対称性シナプスである」がある。ところが、NMDA型グルタミン酸受容体のNR3サブユニットは、NR1サブユニットと会合してグリシン作動性の興奮性受容体を形成することが報告された。そこで、本研究では、「興奮性-非対称性シナプス/抑制性-対称性シナプス」の原則が正しいのか、「グルタミン酸-興奮性終末/グリシン-抑制性終末」の原則が正しいのかを明らかにする目的で、NR3サブユニットが分布するシナプスの形態学的特徴と神経回路における局在を検討した。この目的達成のため、NR3Aに対する特異抗体を作成したその結果、NR3Aは、小脳では抑制性ニューロンの樹状突起に選択的に発現し、登上線維との間の対称性シナプスと思われる部位に局在していた。一方、登上線維とプルキンエ細胞との間の非対称性シナプスや、平行線維と抑制性ニューロンとの間の非対称性シナプスには存在しなかった。従って、NR3Aはグルタミン酸を放出する神経終末の対称性シナプスに分布し、いずれの原則にも従わないユニークな発現局在をとることが明らかとなった。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2005 -2006 
    Author : WATANABE Masahiko
     
    In the present study, I studied on the molecular mechanisms regulating critical period development of the mouse somatosensory cortex using gene knockout mice. In particular, I focused on the roles played by NMDA-type glutamate receptor subunits GluRe2 (NR2B) and GLuRe4 (NR2D) and also by glutamate transporter GLAST and GLT1 in developmental appearance, critical period termination, critical period plasticity of somatosensory barrel structures. Here I found that, compared to wild-type control mice, heterozygous GlnRe2+/-mice showed one-day retardation in developmental appearance and critical period termination, whereas homozygous GluRe4-/-mice did on-day facilitation. No significant change was seen in the magnitude of critical period plasticity, as determined by row-C whisker cautery. In contrast, homozygous GLAST-/-and GLTI-/-mice displayed significant reduction in the magnitude of critical period plasticity, with normal developmental appearance and critical period termination. These results provide experimental evidence that N3IDA receptor activation regulates temporal properties of somatosensory development in a subunit-dependent manner, while transporters regulating extracellular glutamate concentration magnify critical period plasticity. Therefore, armed with the two mechanisms, synaptic refinement during development is shaped normally in a activity-dependent manner.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2003 -2004 
    Author : WATANABE Masahiko, FUKAYA Masahiro
     
    Synapse formation depends critically on the competition among inputs of multiple sources to individual neurons. Cerebellar Purkinje cells have highly organized synaptic wiring from two distinct sources of excitatory afferents. Single climbing fibers innervate proximal dendrites of Purkinje cells, whereas numerous parallel fibers converge on their distal dendrites. Here, we demonstrate that the P/Q-type Ca^<2+> channel a1A, a major Ca^<2+> channel subtype in Purkinje cells, is crucial for this organized synapse formation. In the alA knockout mouse, ectopic spine formation was frequently observed in proximal dendrites and somata of Purkinje cells. Innervation territory of parallel fibers was expanded proximally to innervate these ectopic spines, whereas that of climbing fibers was regressed to the basal portion of proximal dendrites and somata. Furthermore, multiple climbing fibers, consisting of a strong climbing fiber and one or a few weaker ones, persisted in the majority of Purkinje cells and were co-wired to the basal somatodendritic domain. The lack of alA, therefore, results in the survival of parallel fibers and surplus climbing fibers, which should normally be expelled from the compartment innervated by the main climbing fiber. We conclude that a P/Q-type Ca^<2+> channel alA fuels both heterosynaptic competition between climbing fibers and parallel fibers and homosynaptic competition among multiple climbing fibers. This molecular function facilitates the distal extension of climbing fiber innervation along Purkinje cell's dendritic tree and also establishes mono climbing fiber innervation of individual Purkinje cells.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2002 -2004 
    Author : WATANABE Masahiko, FUKUYA Masahiro, KANO Masanobu
     
    Innervation of cerebellar Purkinje cells (PCs) by parallel fibers (PFs) and climbing fibers (CFs) is one of the best model system for synaptic circuit development and maturation. Through our previous analysis on PF synapse-specific molecule GluRd2, we have shown that this molecule strengthens synaptogenesis by PFs. Without GluRd2, PF-PCPC synapses are markedly reduced in number, and many PC spines on distal dendrites became free of synaptic contact (Kurihara et al.,1997, J.Neurosci.17:9613-9623). In this research project, we aimed to clarify what happens for CF in nervation under weakened PF synaptogenesis. By employing anterograde tracer labeling and immunolabeling, distal extension of CF in nervation was shown to occur in GluRd2 knockout mice and innervated free spines on distal dendrites of the PC. The distal extension further occurred against distal dendrites on adjacent PCs, resulting in multiple CF in nervation (Hahimoto et al.,2001;J.Neurosci.21:9701-9712 ; Ichikawa et al.,2002, J.Neurosci.22:8487-8503). These results indicate that synaptogenesis on PC dendrites stands on active competition between PF and CF, in which GluRd2 strengthens PF innervation to distal dendrites. Furthermore, we further revealed that P/Q-type calcium channel alpha-1A, which is activated by CF and induces calcium influx to PC dendrites, is the molecular mechanism that strengthens in nervation by a single major CF at proximal dendrites and expels other lesser CFs and PFs from the dendrites (Miyazaki et al.,2004, J.Neurosci.24:1734-1743).
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2001 -2003 
    Author : WATANABE Masahiko
     
    L-Serine is a non-essential amino acid that can be synthesized in our body. It is synthesized from an intermediate of the glycolytic pathway, 3-phosphoglycerate, and utilized for syntheses of proteins, other amino acids, membrane lipids, heme, and nucleotides. Emerging evidence indicate that L-serine plays as a glia-derived trophic factor, which strongly promotes the survival and differentiation of cultured neurons. L-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH) and small neutral amino acid transporter ASCT1 have been revealed in the adult and developing brains of rodents to be expressed preferentially in the radial glia-astrocyte lineage and olfactory ensheathing glia. In contrast, these biosynthetic and transporter molecules for L-serine are low or undetectable in neurons and phagocytic cells. Based on these findings, we propose that L-serine synthesis in these glial cells and its supply to nearby neurons and other glia constitute the novel metabolic unit in the brain. Through the neuroglial and glioglial relationships, glucose in neurons and phagocytes can be strategically used for energy production, while a variety of L-Serine-derived biomolecules required for their proliferation, survival, differentiation, and function are synthesized in and supplied from the radial glia-astrocyte lineage and olfactory ensheathing glia. A transient capillary expression of ASCT1 in fetal and neonatal brains further suggests that, in addition to the glia-home L-serine, active transport of blood-borne L-serine would play an essential role in neural development.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2002 -2002 
    Author : 渡辺 雅彦, 深谷 昌弘
     
    N-methyl-D-aspartate (NMDA)受容体は電位依存性のマグネシウム阻害と高いカルシウム透過性を背景として、神経活動依存的なシナプス回路の発達制御、シナプス可塑性の誘発、学習や記憶の基盤として重要な役割を果たしている。本研究では、NMDA受容体のシナプス局在制御機構を解明する目的で、海馬CA1特異的NR1ノックアウト(NR1-CA1-KO)マウスを用いて、NR1欠損状態におけるNR2サブユニットの細胞内局在変化を解析した。NR1-CA1-KOマウスでは、NR2サブユニットの転写レベルに変化は見られなかった。しかし、NR1欠損に伴い、NR2サブユニットのシナプス局在は完全に消失し、錐体細胞の細胞体に集積するようになった。その細胞内集積部位について包埋後免疫電顕法で解析した結果、NR2サブユニットは電子密度の高い顆粒(intracisternal granule)となって小胞体の腔内に貯留していた。一方、NMDA受容体と結合するシナプス後膜分子PSD-95やAMPA型グルタミン酸受容体の細胞内分布に変化は見られなかった。以上の結果は、NR2サブユニットの小胞体からゴルジ装置への輸送はNR1サブユニットに依存しており、その非存在下では小胞体腔内において凝集体となり処理されることを示している。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1999 -2002 
    Author : NAGASHIMA Masabumi, WATANABE Masahiko
     
    The central nervous system networks are established by the axon outgrowth within the white matter fascicles and the subsequent synaptogenesis between the projection fiber and target neurons. The growth cones at the axon's tips show extensive motility and seem exploring behavior within the cellular environment. Such pathfinding dynamism can be represented by in vitro experiment using dissociated cell culture or explant slice culture system. Two major hypotheses concerning the possible mechanism underlying the wiring of neuronal circuitry have been investigated; namely "diffusible chemoattractant" which is secreted by the target neurons and "cell-contact-mediated guidance." Our standpoint is the later. Interaction between differentiating neuronal and glial cell populations is possibly mediated by receptor or transporter proteins incorporated within the surface membrane. Our result of this project showed drastic influence of the developmentally regulated cues in the spinal cord membranes to the cortical growth cone behaviors. Another result revealed active contribution of the Bergman glia, which were visualized by immunochemistry with glutamate transporter, to the morphogenesis of the Purkinje cell's dendrites. For further extension of understanding the problem of the neurite outgrowth, developmental neurobiology using in vivo experiments should be coupled with cell biology techniques, e.g. dynamics of the fluorescently-labeled cytoskeleton, monitored by the high-resolution time-lapse video microscopy.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2001 -2001 
    Author : 井上 芳郎, 市川 量一, 渡辺 雅彦
     
    小脳プルキンエ細胞は平行線維と登上線維の2種類の興奮性入力を受け、前者はプルキンエ細胞の樹状突起の遠位領域に、後者は一本からなる線維が近位領域にシナプス結合していることを特徴としている。そのシナプス構築の形成には様々な分子が関与すると考えられるが、その中でグルタミン酸受容体δ2分子および代謝性グルタミン酸受容体1型分子に着目し、順行性標識法を用いてそれらの遺伝子欠損マウスの登上線維を選択的に発色させ登上線維と平行線維のプルキンエ細胞上におけるシナプス構築を調べ、そのデータをもとに各分子のシナプス構築形成に対する役割を検討した。 登上線維を選択的に発色させた小脳について、軟膜に平行な電子顕微鏡用超薄切片をプルキンエ細胞の細胞体レベルから樹状突起の終端レベルまで連続して作製し、プルキンエ細胞上における登上線維シナプスと平行線維シナプスについて解析した。その結果、グルタミン酸受容体δ2の遺伝子欠損マウスのプルキンエ細胞については、1)平行線維シナプスが分布する樹状突起の遠位部においては、スパイン数は変わらないものの平行線維シナプス数が半減し登上線維が侵入しシナプスを形成していた。2)一本の樹状突起に複数本の登上線維の入力を示すシナプス構築像が遠位部で観察された。以上からグルタミン酸受容体δ2は平行線維シナプスを安定化しそれによって余剰な登上線維の侵入を抑える働きをすることを示唆していた。一方、代謝性グルタミン酸受容体1型分子の遺伝子欠損マウスでは、平行線維シナプス、登上線維シナプスの数と分布には変化がみられなかった。しかし、他のプルキンエ細胞を伴走する登上線維から分枝した側枝が樹状突起近位部に伸び、その側枝由来の登上線維によって支配されるプルキンエ細胞が存在した。そのことから、代謝性グルタミン酸受容体1型分子は平行線維と登上線維の相互作用には関与せず、登上線維と登上線維間の相互作用、つまりプルキンエ細胞への一重支配を形成するうえで不要な登上線維を削除する際に大きな役割をはたすと推察される。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2000 -2001 
    Author : 渡辺 雅彦
     
    大脳皮質や小脳皮質では、ニューロンの樹状突起の主軸が軟膜方向へ伸展し、その周囲にシナプスを形成する末梢枝が多数派生するという共通した構築を示す。このような樹状突起構築の指向性がどのような細胞メカニズムにより形成されるのかを検討する目的で、発達段階の小脳に注目して分子形態学的解析を行った。昨年度は、プルキンエ細胞樹状突起をカルビンジン抗体を用いて、バーグマングリアの放射状突起をグルタミン酸トランスポーターGLAST抗体を用いて免疫組織化学を行い、樹状突起がグリア突起と密着して脳表に伸展していく事実を見い出した(Yamada et al.,2000)。本年度は、このグリア突起から樹状突起の伸展作用を有するセリンが分泌されていることを明らかにした。さらに、その分子機構を探索したところ、バーグマングリアには高濃度のセリン合成酵素3PGDHが発現しているが、プルキンエ細胞では全く発現陰性であった。この事実は、セリンを介するニューロングリア間の代謝的カップリングが、樹状突起の伸展誘導機構になっている可能性を示唆する。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1999 -2001 
    Author : WATANABE Masahiko, NAGASHIMA Masabumi, INOUE Kaoru
     
    In the research project, we studied metabotropic glutamate signaling to reveal the molecular anatomy and its role in synapse development and plasticity. We analyzed the following items; 1) GTP-binding protein and phospholipases that function at Purkinje cell synpases, 2) Purkinje cell synapse formation in knockout mice lacking GTP-binding protein and phospholipase, 3) the form of persistent multiple innervation in miceiacking mGluR1 and GluRδ2, and 4) NMDA-type glutamate receptors at granule cell synapses. Distinct synapse localization of these signaling molecules were clarified. Moreover, we found distinct roles played by mGluR1 and GluRδ2 in the elimination process of surplus climbing fibers. mGluR1 signaling is essential to homotypic competition among climbing fibers for the proximal dendritic segment of Purkinje cells, whereas GluRδ2 is important in heterotypic competition between parallel fibers and climbing fibers. With both functions, Purkinje cells can establish the territorized innervation underlying LTD induction and cerebellar motor learning.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1998 -2001 
    Author : WATANABE Masahiko
     
    The Bergmann glia (BG) is unipolar cerebellar astrocytes associating with migrating granule cells (GCs) and mature Purkinje cells (PCs). By taking cytochemical advantage of glutamate transporter GLAST localized on the plasmalemma of raidal glia/astrocyte lineage, we analyzed the morphodifferentiation of BG. Cells expressing GLAST mRNA were present only in the ventricular zone at embryonic day 13 (El3). At E14, they first appeared in the mantle zone, where they had oval to elongated cell bodies and radial fibers reaching the pial surface, suggesting the migration of cerebellar radial glia. From E18 to P7, GLAST-expressing cells in the cortex established characteristics of BG (i.e., monolayer alignment around PC bodies and expression of glial fibrillary acidic protein in Bergmann fibers) and further up-regulated GLAST expression, whereas those in other cerebellar regions were changed into multipolar astrocytes and down-regulated the expression. From P7 to P21, Bergmann fibers were changed from linear to reticular form in a deep-to-superficial gradient; the superficial linear portion was intimately contacted with the growing tips of PC dendrites, while the deeper reticular portion reflected active formation of membranous protrusions invading the neuropil elaborately to surround PC synapses. Following the accomplishment as unipolar cortical astrocytes, therefore, Bergmann fibers of BG undergo dynamic transformation in the active synaptogenic phase, leading to structural maturation of PC synapses. Intimate contacts with dendritic tips further suggest that Bergmann fibers could be a structural substrate for arborizing PC dendrites as well as for migrating GCs. To further pursue the molecular basis, neuron-glial relationship on serine metabolism was examined by producing antibody to serine synthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH). 3PGDH was highly and selectively expressed throughout development in astrocytes and olfactory ensheathing glia. In the cerebellum, the growing tips of 3PGDH-negative PC dendrites were contacted to 3PGDH-positive Bergmann fibers in developing cerebellum. These results suggest that serine biosynthesis by BG and its supply to PCs are one of the plausible reason for the intimate structural relationships between dendrites and glial fibers during development.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1999 -1999 
    Author : 井上 芳郎, 渡辺 雅彦
     
    脳虚血に伴うプルキンエ細胞の形態変化をひきおこす機構に対するグルタミン酸の関与の有無を明らかにするために、細胞外グルタミン酸濃度を上昇させた際の、プルキンエ細胞の変化を調べた。プルキンエ細胞の興奮性シナプスには、グルタミン酸トランスポーターGLASTとEAAT4の2種類があり、これらがシナプス間際に放出されたグルタミン酸を除去していると考えられる。そこで、これらのトランスポーター遺伝子を欠損させたマウスが虚血に陥った際の形態変化を調べた。なお、プルキンエ細胞は虚血をおこすと、細胞死に至らないまでも、樹状突起内の滑面小胞体が平板化し層状に配列するようになるスタッキングとよばれる構造変化を起こすことに着目し、その変化の程度を虚血のレベルの指標として用いた。 グルタミン酸のトランスポーターEATT4はプルキンエ細胞に特異的発現し、グルタミン酸トランスポーターGLASTは小脳のバーグマングリア細胞に特異的に発現する。和田、田中が作成したそれらの遺伝子のノックアウトマウスには、組織学的、超微形態的にも異常は見当たらなかった。また、両者の遺伝子をダブルノックアウトしたマウス、それはプルキンエ細胞シナプスに放出されたグルタミン酸の排除機構がほとんど消失することを示唆するが、組織学的にも超微形態的にも異常が見当たらなかった。しかし、数分間の無呼吸負荷を与えると、ダブルノックアウトマウスでは対照マウスに比べスタッキングが亢進しておこっていた。また、代謝性グルタミン酸受容体に対する拮抗薬を投与したマウスでは、虚血状態で引き起こされるスタッキングが起きにくくなるとの報告もあり、以上2つのことをあわせると、プルキンエ細胞の脳虚時には代謝性グルタミン酸受容体が過度に活性化されスタッキングが引き起こされている可能性が高いと考えられる。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1997 -1999 
    Author : KOIKE Tatsuro, TANAKA Shuuitsu, WATANABE Masahiko
     
    To searach for genes up-regulated during neuronal cell death, we have employed cerebellar cell cultures, in which glial cells respond to degeneration and cell death of granule neurons that begins to occur at 4DIV. Differential hybridization was performed with subtracted cDNA probes and a cDNA library from 5DIV. One of the genes up-regulated during cell death is a novel gene, mrf-1, in microglia. Another gene is highly homologous to the mouse genes with hematopoietic cell origin which may be involved in microglial activation associated with enhanced functions of lysosomes
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1998 -1998 
    Author : 渡辺 雅彦
     
    海馬と小脳では、一過性脳虚血や過度な神経活動が引金となってグルタミン酸毒性による神経細ね死が起こりやすい。本研究では、神経細胞死に関与するグルタミン酸シグナル伝達分子機構を解明することを目的とした。この目的遂行のため、グルタミン酸受容体およびそのシグナル伝達分子に対する特異抗体作製し、電顕および共焦点レーザー顕微鏡を用いた局在解析を行った。昨年度は海馬の結果について報告しているが、本年度は小脳プルキンエ細胞について解析した。 (1) NMDA型グルタミン酸受容体.小脳プルキンエ細胞には、GluR ζ 1サブユニットのみが発現し、GluR εの発現は検出できなかった。この事実は、ブルキンエ細胞では機能的なNMDA受容体は形成されないことを示唆しており、今後、non-NMDA受容体の活性化による細胞死メカニズムを追及する必要がある。 (2) 代謝型グルタミン酸受容体:プルキンエ細胞には1型代謝型グルタミン酸受容体(mGluR1)が優勢に発現していることが知られている。この受容体にはGq型GTP結合蛋白が共役し、フォスフォリバーゼCβ(PLCβ)を活性化する。in situ hybridzationにより、プルキンエ細胞において、GαqとPLCβ3/4が優勢に発現していることが判明した。特異抗体を作製して免疫組織化学法により検討した結果、これらの分子はプルキンエ細胞シナブスの周囲部においてmGluR1と共存しており、機能的なシグナルカスケードを構成していることが示唆される。 以上の結果は、プルキンエ細胞では海馬とは異なるグルタミン酸伝達機構を有しており、この分子機構を介して神経細胞死が誘発されるものと考えられる。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1998 -1998 
    Author : 渡辺 雅彦
     
    NMDA受容体はグルタミン酸受容体のサブタイプの一つで、その活性化はシナプス伝達効率の可塑的変化を誘発し、これが記憶や学習・脳の発達において重要な役割を果たしている。本研究では、終脳とくに大脳皮質領域の構造と機能が著しい分化を遂げた霊長類に注目し、この領域におけるNMDA受容体サブユニットの発現特性とシナプス局在を明らかにすることを目的とした。 マウスサブユニットのN末およびC末ペプチド領域を大腸菌にて発現させ、得られたペプチドを抗原として、大脳皮質に発現するGluRε1・GluRε2・GluRζ1の3種のサブユニットに対する特異抗体を作製した。イムノブロットにより、これらの抗体はサルのサブユニットをも認識することが判明した。そこで、これらの抗体を用いてサル終脳パラフィン切片を酵素抗体法にて免疫染色を行った。その結果、3種のサブユニットはいずれも海馬、大脳皮質、線条体などの各終脳領域において高レベルで分布していた。第1次運動野と第1次感覚野では、それぞれのサブユニットは比較的均一な層分布が認められた。一方、その周囲の大脳皮質では層により免疫反応の強度が異なっており、しかも、サブユニット間で層分布特性が異なっていた。今後、この異なる層分布(垂直分布)が、個々のニューロンにおける異なる転写調節によるものなのか、それとも蛋白分子の異なるシナプス局在化調節によるものなのかを追求する目的で、in situhybridization解析を行う必要がある。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1997 -1998 
    Author : WATANABE Masahiko
     
    The GluRdelta2 is a Purkinje cell (PC)-speciflc ionotropic glutamate receptor subunit, and the mGluR1 is a PC-predominant metabotropic glutamate receptor. Both molecules are known to be important in the induction of long-term depression (LTD), a form of synaptic plasticity underlying the motor learning. In the present research project, I analyzed the cerebellum of knockout mice deficient in the GluRdelta2 and mGluR1, to elucidate their roles in PC synapse development. Serial electron microscopic examination revealed that PC dendritic spines free of synaptic contact emerged in the GluRdelta2-KO mouse, resulting in the reduction of parallel fiber synapses to nearly a half level. By contrast, all PC spines examined in the wild-type control were contacted with parallel fiber terminals, In the mGluR1-KO mouse, parallel fiber synapses were normally formed on distal PC dendrites. However, multiple innervation by climbing fibers, axons from brainstem inferior olivary nucleus, persisted in the mGluR1-KO mouse. These results suggest that GluRdelta2 is important in the stabilization and strengthening of synaptic connectivity between parallel fibers and PCs, whereas mGluR1 is involved in the elimination of supernumerary climbing fibers. Therefore, the two glutamate receptors play distinct, but essential roles in the PC synapse development, as they do in the induction of LTD at mature stages.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1995 -1998 
    Author : MISHINA Masayoshi, WATANABE Masahiko, UMEMORI Hisashi, YAGI Takeshi, KANO Masanobu, MANABE Toshiya
     
    We investigated the relationship of synaptic plasticity to neural development, learning and memory by generating mutant mice lacking glutamate receptors (GluRs). The GluRε1 subunit mutant mice showed the decreased hippocampal LTP and impaired spatial learning in the Morris water maze. The GluRε2 subunit mutation hindered the formation of the whisker-related neuronal barrelette structure in the brainstem trigeminal nucleus and synaptic plasticity in the hippocampus. The GluRδ2 subunit selectively localized in cerebellar Purkinje cells was essential for cerebellarLTD, motor coordination, motor learning, stability of parallel fiber-Purkinje cell synapses and elimination of multiple climbing fibers (CF) during development. Metabotropic GluR1 mutant mice were defective in cerebellar LTD, associative eyeblink conditioning and CF elimination. Eyeblink conditioning was normal in PKCγ mutant mice with multiple CFs. These results suggest that synaptic plasticity is the cellular basis of a certain form of learning and memory. They also suggest that GluRs play important roles in neural network formation during brain development and in higher brain function, implying that these processes share common molecular mechanisms.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1997 -1997 
    Author : 渡辺 雅彦
     
    グルタミン酸興奮毒性の発現に関わる機能分子として、NMDA型グルタミン酸受容体とグルタミン酸トランスポーターに注目し、これらの遺伝子の組織発現局在解析と遺伝子欠損マウスの神経組織学的解析を行った。発現解析は、オリゴプローブによるin situ ハイブリダイゼーションと特異抗体作製・免疫組織学法により行った。その結果、グルタミン酸毒性に最も脆弱な脳領域として知られている海馬CA1領域において、NMDA受容体サブユニット(GluRε1,ε2,ζ1)が脳内で最も強い発現分布を示していた。また、4種のグルタミン酸トランスポーターの中で、GLT-1は海馬を含む終脳で高い発現レベルを示した。標的遺伝子組換え法により作製されたGLT-1欠損マウスは致死的なてんかん発作を引き起こし、組織学的に検索したところ海馬CA1領域の錐体細胞が細胞死に陥っていることが明らかとなった。これらの解析結果より、海馬CA1領域の脆弱性は、この領域に豊富に発現分布するNMDA型グルタミン酸受容体とGLT-1がその責任分子であることが判明した。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1996 -1997 
    Author : TANJI Jun, GRAYBIEL Ann M., WATANABE Masahiko, SHIMA Keisetsu, MUSHIAKE Hajime, KURATA Kiyoshi
     
    To understand functional organization of the motor areas in the frontal cortex, we planned and performed an integrative research project. Using primates as experimental animals, we conducted a series of experiments with a combined physiological and molecular approach. Researchers in Tohoku University visited MIT in Boston, and professors at MIT visited Tohoku University to advance mutual development of research works in each project, and joint works have done. As a result of intensive exchanges of ideas and collaborative works, the following results have been obtained. A novel aspect of functional organization of cortico-basal ganglia loops has been found. Although it has been established that the output from the basal ganglia is divided into two parts, direct and indirect pathway, neither their relationships with cortical motor areas nor their functional significance were known. ln the present study, we first mapped the primary motor cortex with physiological techniques, and then applied intracortical microstimulation (ICMS) for a period of 2 hrs. Immediately thereafter, brains were perfused, sectioned, and processed for immunohistochemistry to identify IEGs (c-fos and jun-B). At the same time, cells were immuno-stained for Enkephalin. IEG positive cells were found to be largely positive for Enkephalin, indicating that the cortico-striatal cells projecting from the primary motor cortex mostly belong to the indirect pathway. We subsequently found that a majority of IEG positive cells following stimulation of the supplementary motor area are not Enkephalin positive, indicating that the SMA projection to the striatum mainly belong to the direct pathway. These findings provide a new insight about functional roles of the cortico-basal ganglia pathway in motor control. In a separate project, we analyzed the distbution of subunits of NMDA glutamate receptors in cortical motor areas of primates. For this purpose, we first marked cortical areas after physiological identification. Subsequently, in situ hybridization method was applied to find subunits epsilon1, epsilon2, and xi of NMDA receptors. The subunit distribution differed greatly in different cortical layrs in the prefrontal, premotor, primary motor, and somatosensory cortex.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1995 -1997 
    Author : INOUE Yoshiro, ICHIKAWA Ryoichi, NAGASHIMA Masabumi, TAKAYAMA Chitoshi, WATANABE Masahiko
     
    1 The diversed oxpression of NMDA receptor subunits (epsilon1, epsilon2, epsilon3, epsilon4 and zeta1) and delta2 subunit were elucidated in the mouse cerebellum during development. In addition, expressions of NMDA receptor subunits in the adult normal mice were different from those of the reeler and straggerer mutants in combination of subunits expressed. Thus expression of NMDA receptor subunits might be influenced by the extrinsic factors like the afferent input. The delta2 subunit tended to accumulate in the dendritic spines of the Purkinje cells closely related with synaptic formation of parallel fibers. 2 The mice defective in glutamate receptor gene were generated. the epsilon1 mutant shows a moderate deficiency in spatical learning, significant reduction of the NMDA receptor channel current and LTP at the hippocumpus. The mutant mice defective in the epsilon1 subunit showed on suckling response and died shortly after birth. This disorder resulted in abnormal termination of the trigenimal afferent fibers. The epsilon4 mutant showed only a reduced spontaneous activity, bur not any morphological abnormality. By delta2 mutant this subunit is an important factor in strength and stability of synaptic connection between dendritic spines and parallel fibers. 3 The electrophysiological system for electric stimulation, electrophoretic injection and putch-clump was set up partially by this gland, and were successfully used for fine labeling of the climbing fibers from the olivary nucleus. The optical imaging system (Deltaron 1700) was also set up and the reaction of the Purkinje cells under stimulation of the parallel fibers was recorded, but the usage for analysis of the glutamate receptor functions was remained as a future problem.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1996 -1996 
    Author : 渡辺 雅彦
     
    小脳長期抑制は、登上線維/プルキンエ細胞間シナプスおよび平行線維/プルキンエ細胞巻シナプスにおける神経伝達活動に依存して生ずるシナプス可塑性現象で、運動学習の基盤と考えられている。本研究では、小脳長期抑制に関与している1)イオンチャネル型グルタミン酸受容体δ2サブユニット、2)代謝型グルタミン酸受容体mGluR1、3)蛋白リン酸化酵素PKCγの3種の機能分子に注目して、プルキンエ細胞におけるこれらの分子の発現局在と分子欠損に伴うシナプス形成異常を分子神経解剖学的観点から解析を行った。 その結果、δ2サブユニットは胎生期から成熟期に至るまで小脳プルキンエに特異的に発現する分子であることが判明した。その遺伝子ノックアウトは、平行線維シナプスの著明な減少をもたらし、登上線維の成熟が阻害され、小脳長期抑制現象が消失し、運動学習に障害が生じた。これに対して、プルキンエ細胞に高濃度に発現することが知られているmGluR1およびRKCγの分子欠損は、平行線維シナプスの形成には影響を及ぼさなかったが、登上線維シナプスの成熟が著しく阻害され、長期抑制現象が消失し、運動学習に障害が生じた。これらの事実は、イオンチャネル型のグルタミン酸と代謝型グルタミン酸カスケードがシナプスの成熟と可塑性に対して重要な役割を果たしているが、シナプス形成に対する寄与という点ではそれぞれの分子機構が異なる役割を担っていることを示唆している。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1996 -1996 
    Author : 田川 義継, 笹本 洋一, 渡辺 雅彦
     
    網膜のミクログリアについて、Wister Kingラットを用いて免疫組織学的に検討した。ミクログリアの同定には、一次抗体として抗サイモシン単クローン抗体を用い、酵素抗体法(ABC法)により免疫染色を行った。その結果、正常ラットの網膜内層に抗サイモシン抗体に強く染色される細胞が分布しているのが観察された。網膜血管との関連性は、特に見られなかった。網膜外層には陽性細胞は見られなかった。陽性細胞は、多数の長い突起を出し樹枝状の形態を示していた。陽性コントロールとして検討したラット脳内のミクログリアと同様の染色結果が得られ、多くがラミファイド型と呼ばれる静止状態のミクログリアの形態であった。網膜の他のグリア細胞であるMuller細胞のマーカーである抗Vimentin抗体と抗サイモシン抗体の二重染色をおこなったが、Muller細胞は抗サイモシン抗体では染色されなかった。以上の結果から、正常ラット網膜に脳と同様に静止状態のミクログリアが存在し、抗サイモシン抗体が細胞の同定に有用であることが明らかとなった。同時に行った他の抗IL-1などのサイトカイン抗体やMHCクラスII抗原の検索では、現在までの所ミクログリアには陽性所見は得られていない。次に、病的網膜におけるミクログリアの動態を知るため、視神経を切断し経時的にミクログリアを観察した。その結果、視神経切断後48〜72時間頃よりミクログリアの増加が観察された。 今年度の研究により、これまで不明な点の多かった網膜内ミクログリア同定のための新しいマーカーが得られた。今後は、病的状態におけるミクログリアの動態と機能についてさらに研究を継続する予定である。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1995 -1995 
    Author : 渡辺 雅彦
     
    神経細胞移動障害を特徴とするリーラーミュータントマウスでは、大量のプルキン工細胞が小脳深部に滞留し、正常とは異なる神経学的環境の中で細胞機能を営んでいる。この細胞におけるNMDA型グルタミン酸受容体サブユニットの発現をin situハイブリダイゼーション法により検索し、野生型マウスのそれと比較検討した。その結果、リーラーマウスの小脳には、1)ζ1サブユニットのみの野生型発現を示すプルキン工細胞と2)ε1およびζ1サブユニットによる変異型発現を示すプルキン工細胞が存在している事実が判明した。小脳の吻側では、変異型発現と野生型発現を持つプルキン工細胞が内外方向で交互に出現したのに対して、小脳尾側ではほとんどのプルキン工細胞は野生型発現を示した。この事実は、NMDA受容体サブユニットの発現様式がそれぞれの神経細胞種の固有なプログラムで完全に決定されているのではなく、ある種の状況下では変異しうることを示している。小脳求心性線維の中で脊髄に由来する苔状線維は主に小脳吻側に投射する事実や下オリーブ核に由来する登上線維が内外方向のバンド状配列をする事実を考え合わせると、今回観察された発現変異は異なるシナプス環境と連関して起きている可能性が示唆された。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1994 -1995 
    Author : NISHI Shinzo, MUNRO Neville, MORINAGA Tomonori, SHIMA Tomoko, FURUYAMA Junichi, WATANABE Masahiko, KOYAMA Yoshikazu, SAKAI Masaharu, TAMAOKI Taiki, ELLIOT Alpert
     
    The ATBF1(AT motif binding factor 1)cDNA was isolated based on the ability of its product to bind to an AT-rich enhancer element of the human alpha-fetoprotein(AFP)gene. This transcription factor is characterized by a large size(>300kd)and presence of many DNA-binding domains(4 homeotic-domain, 22 Zn-finger structure). This protein also contains many other functional domain structures, such as acidic-domain, glutamine-rich domain, ATP-ase-like domain and kination sequence. To understand the structure, expression and physiological functions of this interesting protein, we collaborate with members of different fields. The international collaboration was obtained the following results. 1.The mouse ATBF1 was cloned and shows 95% homologies with human. The homeotic domains were conserved at 100%. 2.Two isoform of the ATBF1(ATBF1-A and ATBF1-B)were expressed from the independent promoters and alternative splicing. The promoter specific for ATBF1-A controls the neural cell expression. 3.The results of in situ-hybridization and Northern blotting analysis of P19 cells indicate that the expression of ATBF1 is closely associate with neuronal differentiation. 4.ATBF1 genes of human and mouse were mapped on Chromosome 16p22.3-23.1 and 8E1, respectively. 5.ATBF1 suppresses the transcription of AFP gene. The required domains for the suppression were located on proline-rich domain and 4th homeotic-domain. 6.We have obtained many other interesting properties of ATBF1, i.e., ATBF1 functions as a dimer, ATBF1 might be processed in nucleus and ATBF1 worked as a suppressor of the cell growth. For keep the close contacts between collaborators and exchange the information, 12 researchers visited to Canada and 4 researchers invited to Japan within 2 years. This collaboration was very successfully functioned and made excellent results as described above.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1994 -1994 
    Author : 渡辺 雅彦
     
    マウス小脳におけるNMDA型グルタミン酸受容体を構成する5種のサブユニット遺伝子発現の発達変化を、in situハイブリダイゼーション法により解析した。小脳顆粒細胞では、生後発達に伴いε2サブユニットmRNAの顕著な発現が開始するが、生後14日を境にその発現に消失する。これに代って、生後14日までにε1およびε3サブユニットの遺伝子発現が起こり、成熟顆粒細胞に発現する代表的なεサブユニットとなる。一方、プルキンエ細胞では、胎生13日よりこの細胞の分布する小脳領域にε2サブユニットmRNAの発現が観察されるが、生後1日を境にこのサブユニットの遺伝子発現は消失する。小脳核細胞では、ε2およびε4サブユニットmRNAが胎生期より発現するが、生後発達に伴いε2サブユニットmRNAが消失し、これに代ってε1サブユニットmRNAが出現する。これに対して、ζ1サブユニットの遺伝子発現は、各発生段階のそれぞれの神経細胞において普遍的な発現が観察された。以上の所見は、生後発達に伴い小脳の各ニューロンに発現するεサブユニットの分子種がダイナミックに変動することを示しており、その結果、胎児型サブユニット構成を有する受容体チャネルが成熟型チャネルへと転換することを示唆している。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1994 -1994 
    Author : 三品 昌美, 遠山 正彌, 杉山 博之, 津本 忠治, 小澤 瀞司, 川合 述史, 渡辺 雅彦, 相沢 慎一, 勝木 元也
     
    本研究班は、中枢における神経情報伝達シナプス可塑性の鍵を握るグルタミン酸受容体を実体として明らかにするとともにその生理機能を分子構造と対応させて明確にすることを目標とした。研究計画が順調に進行していることから、本年度は生理機能を分子レベルで理解することに重点をおいて、以下の成果を挙げた。NMDA受容体チャネルのε1サブユニット遺伝子を不活化されたマウスを作成し、海馬シナプスにおける長期増強と空間学習の低下が起こることを見い出した。この成果は、記憶・学習のシナプス可塑性仮説を支持するものである(三品)。短時間虚血負荷後のスナネズミ海馬錘体細胞においてAMPA型グルタミン酸受容体チャネル電流の持続性やカルシウム透過性が変化することを単一チャネル電流の解析により明らかにした(川合)。海馬切片を対象として、スライスパッチクランプ法とRT-PCR法を組み合わせることにより、グルタミン酸受容体チャネルの電気生理学的特性とサブユニット構成との関連を明らかにした(小澤)。ラット視覚野から調整した培養神経細胞標本に穿孔パッチクランプ法を適用することにより、視覚野におけるシナプス長期増強の誘発にはシナプス後細胞の脱分極と同期したシナプス前部の賦括が必要であることを見い出した(津本)。代謝型グルタミン酸受容体のサブタイプ5にはスプライシングの違いによる2種類の型が存在し、発生の時期により調節されていることを見い出した(杉山)。AMPA型グルタミン酸名受容体チャネルmRNAがラットの腺細胞に発現していることならびに虚血条件下ではグルタミン酸トランスポーターの遺伝子発現と蛋白の細胞内局在が変化することを見い出した(遠山)。ホメオ蛋白Otx2遺伝子のヘテロ欠損マウスを作製し、Otx2が頭蓋においても神経系においても下顎セグメントの形態形成を特異的に制御することを明らかにした(相沢)。5種類のNMDA受容体チャネルサブユニットの遺伝子発現パターンは生後2週間の間に、領域差の小さい胎児型から顕著な空間的異質性を特徴とする成熟型へと変換することを明らかにした(渡辺)。バキュロウイルス発現系により大量発現させたAMPA型およびNMDA型グルタミン酸受容体チャネルを用いて、リガンド結合活性にN-グリコシル化が必須であることを明らかにした(川本)。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1993 -1993 
    Author : 渡辺 雅彦
     
    トポイソメラーゼは、DNA二重鎖を切断しその間を別のDNA鎖を通過させた後、断端を再接合することにより、DNA鎖間の空間的な位置関係変化を触媒する酵素である。この酵素には、Topo IIA-Topo IIBと名付けられた2種のアイソザイムが存在することが知られている。細胞の増殖と分化が空間的および時間的に厳密に制御されて神経組織の特性を利用して、それぞれのアイソザイム発現特性から機能特性を解明することを目指した。 まず、Polymerase Chain Reaction(PCR)法によりラットTopo IIA とTopo IIBをコードするそれぞれ683塩基長のcDNAをクローニングした。その塩基配列を基に、それぞれのmRNAに特異的な47塩基長のアンチセンスオリゴヌクレオチドプローブを合成し、^<35>S標識プローブを用いて、ラット脳の発生における発現分布をin situハイブリダイゼーション法により解析した。その結果、Topo IIA-mRNAの発現は分裂細胞の集積する脳室層や小脳外顆粒層に限局して観察された。これに対して、Topo IIA-mRNAの発現は、脳室層だけでなく最終分裂を終えた神経細胞が分化を遂げる外套層にも観察された。 以上の結果より、Topo IIAはDNAの複製や染色体の形成や分離に、一方Topo IIBはRNAの転写など、それぞれ特異な異なる細胞機能に関与していることが示唆された。
  • 活動依存的カルシウム流入による競合的シナプス回路発達の共通原理の解明
    日本学術振興会：科学研究費補助金 基盤研究（S)
    Author : 渡辺 雅彦

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