2024年06月, 日本顕微鏡学会, 瀬藤賞　　　　　　　　　　　　　　　
活動依存的シナプス回路発達の分子細胞基盤の解明

2023年06月, 秋山記念生命科学振興財団, 秋山財団賞　　　　　　　　　　　　　　　
グルタミン酸情報伝達系によるシナプス回路発達の分子解剖学的基盤
渡辺雅彦

2019年10月, 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年07月, 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」
渡辺 雅彦

2015年03月, 北海道大学, 北海道大学総長研究賞（優秀賞）　　　　　　　　　　　　　　　
渡辺 雅彦

2006年03月, 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」
渡辺 雅彦

1996年04月, 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」
渡辺 雅彦

1992年01月, 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」
渡辺 雅彦

A pontine-medullary loop crucial for REM sleep and its deficit in Parkinson’s disease
Mitsuaki Kashiwagi, Goichi Beck, Mika Kanuka, Yoshifumi Arai, Kaeko Tanaka, Chika Tatsuzawa, Yumiko Koga, Yuki C. Saito, Marina Takagi, Yo Oishi, Masanori Sakaguchi, Kousuke Baba, Masashi Ikuno, Hodaka Yamakado, Ryosuke Takahashi, Masashi Yanagisawa, Shigeo Murayama, Takeshi Sakurai, Kazuya Sakai, Yoshimi Nakagawa, Masahiko Watanabe, Hideki Mochizuki, Yu Hayashi
Cell, Elsevier BV, 2024年09月, ［査読有り］
研究論文（学術雑誌）

Direct and indirect pathways for heterosynaptic interaction underlying developmental synapse elimination in the mouse cerebellum.
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年07月03日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Remodeling of the postsynaptic proteome in male mice and marmosets during synapse development
Takeshi Kaizuka, Takehiro Suzuki, Noriyuki Kishi, Kota Tamada, Manfred W. Kilimann, Takehiko Ueyama, Masahiko Watanabe, Tomomi Shimogori, Hideyuki Okano, Naoshi Dohmae, Toru Takumi
Nature Communications, 15, 1, Springer Science and Business Media LLC, 2024年03月28日
研究論文（学術雑誌）, Abstract

Postsynaptic proteins play crucial roles in synaptic function and plasticity. During brain development, alterations in synaptic number, shape, and stability occur, known as synapse maturation. However, the postsynaptic protein composition changes during development are not fully understood. Here, we show the trajectory of the postsynaptic proteome in developing male mice and common marmosets. Proteomic analysis of mice at 2, 3, 6, and 12 weeks of age shows that proteins involved in synaptogenesis are differentially expressed during this period. Analysis of published transcriptome datasets shows that the changes in postsynaptic protein composition in the mouse brain after 2 weeks of age correlate with gene expression changes. Proteomic analysis of marmosets at 0, 2, 3, 6, and 24 months of age show that the changes in the marmoset brain can be categorized into two parts: the first 2 months and after that. The changes observed in the first 2 months are similar to those in the mouse brain between 2 and 12 weeks of age. The changes observed in marmoset after 2 months old include differential expression of synaptogenesis-related molecules, which hardly overlap with that in mice. Our results provide a comprehensive proteomic resource that underlies developmental synapse maturation in rodents and primates.

Enhancement of Haloperidol-Induced Catalepsy by GPR143, an L-Dopa Receptor, in Striatal Cholinergic Interneurons.
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年03月13日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Behavioral analysis of kainate receptor KO mice and the role of GluK3 subunit in anxiety.
Izumi Iida, Kohtarou Konno, Rie Natsume, Manabu Abe, Masahiko Watanabe, Kenji Sakimura, Miho Terunuma
Scientific reports, 14, 1, 4521, 4521, 2024年02月24日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Behavioral analysis of kainate receptor KO mice and the role of GluK3 subunit in anxiety
Izumi Iida, Kohtarou Konno, Rie Natsume, Manabu Abe, Masahiko Watanabe, Kenji Sakimura, Miho Terunuma
Scientific Reports, 14, 1, Springer Science and Business Media LLC, 2024年02月24日
研究論文（学術雑誌）, 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.

Brain-enriched guanylate kinase-associated protein, a component of the post-synaptic density protein complexes, contributes to learning and memory.
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年12月12日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Shared GABA transmission pathology in dopamine agonist- and antagonist-induced dyskinesia.
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年10月17日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

All-synchronized picosecond pulses and time-gated detection improve the spatial resolution of two-photon STED microscopy in brain tissue imaging
Hirokazu Ishii, Kohei Otomo, Ching-Pu Chang, Miwako Yamasaki, Masahiko Watanabe, Hiroyuki Yokoyama, Tomomi Nemoto
PLOS ONE, 2023年08月24日, ［査読有り］
研究論文（学術雑誌）

Glyoxal fixation: An approach to solve immunohistochemical problem in neuroscience research.
Kohtarou Konno, Miwako Yamasaki, Taisuke Miyazaki, Masahiko Watanabe
Science advances, 9, 28, eadf7084, 2023年07月14日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

PTPδ is a presynaptic organizer for the formation and maintenance of climbing fiber to Purkinje cell synapses in the developing cerebellum
Yuto Okuno, Kazuto Sakoori, Kyoko Matsuyama, Miwako Yamasaki, Masahiko Watanabe, Kouichi Hashimoto, Takaki Watanabe, Masanobu Kano
Frontiers in Molecular Neuroscience, 16, Frontiers Media SA, 2023年06月22日
研究論文（学術雑誌）, 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.

Imbalanced expression of clustered protocadherins in pre- and post-synaptic compartments of CA1 pyramidal cells during hippocampal development
Etsuko Tarusawa, Saki Hasegawa, Daisuke Noda, Nanami Kawamura, Yugo Fukazawa, Masahiko Watanabe, Takahiro Hirabayashi, Takeshi Yagi
Cold Spring Harbor Laboratory, 2023年04月15日
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.

Characterisation of NPFF-expressing neurons in the superficial dorsal horn of the mouse spinal cord.
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年04月11日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Visualization of tans homophilic interaction of clustered protocadherin in neurons
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, Cold Spring Harbor Laboratory, 2023年04月
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.

Quantitative analysis of NMDA receptor subunits proteins in mouse brain.
Yasuhiro Suzuki, Chihiro Nakamoto, Izumi Watanabe-Iida, Masahiko Watanabe, Tomonori Takeuchi, Toshikuni Sasaoka, Manabu Abe, Kenji Sakimura
Neurochemistry international, 165, 105517, 105517, 2023年03月11日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Discrete localization of phospholipase Cβ3 and diacylglycerol kinase ι along the renal proximal tubules of normal rat kidney and gentamicin-induced changes in their expression.
Premrudee Hemha, Surang Chomphoo, Yada Polsan, Kaoru Goto, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
Histochemistry and cell biology, 159, 3, 293, 307, 2023年03月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Discrete localization patterns of PIP5Kγ and PLCβ3 working sequentially in phosphoinositide-cycle within mouse sensory neuron somata.
Sawetree Pakkarato, Hiroyuki Sakagami, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo, Surang Chomphoo
Microscopy research and technique, 86, 3, 351, 358, 2023年03月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Antibodies Against the Gastrin-releasing Peptide Precursor Pro-Gastrin-releasing Peptide Reveal Its Expression in the Mouse Spinal Dorsal Horn.
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年02月01日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Neurexins in serotonergic neurons regulate neuronal survival, serotonin transmission, and complex mouse behaviors.
Amy Cheung, Kotaro Konno, Yuka Imamura, Aya Matsui, Manabu Abe, Kenji Sakimura, Toshikuni Sasaoka, Takeshi Uemura, Masahiko Watanabe, Kensuke Futai
eLife, 12, 2023年01月25日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Regulation of NETosis and Inflammation by Cyclophilin D in Myeloperoxidase-Positive Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.
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年01月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression patterns of NKCC1 in neurons and non-neuronal cells during cortico-hippocampal development.
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年12月27日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Single-scan volumetric imaging throughout thick tissue specimens by one-touch installable light-needle creating device
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, Springer Science and Business Media LLC, 2022年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Behavioral characteristics of dopamine D5 receptor knockout mice
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, Springer Science and Business Media LLC, 2022年12月
研究論文（学術雑誌）, 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.

Preferential arborization of dendrites and axons of parvalbumin- and somatostatin-positive GABAergic neurons within subregions of the mouse claustrum.
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年11月26日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Tmem45b is essential for inflammation- and tissue injury-induced mechanical pain hypersensitivity.
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年11月08日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Nna1, Essential for Purkinje Cell Survival, Is also Associated with Emotion and Memory
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, MDPI AG, 2022年10月26日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma.
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年10月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Localization of phosphatidylinositol phosphate 5 kinase γ, phospholipase β3 and diacylglycerol kinase ζ in corneal epithelium in comparison with conjunctival epithelium of mice.
Sawetree Pakkarato, Hiroyuki Sakagami, Kaoru Goto, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo, Surang Chomphoo
Experimental eye research, 223, 109205, 109205, 2022年10月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Nna1, Essential for Purkinje Cell Survival, Is also Associated with Emotion and Memory. 2022 Oct 26;23(21):12961. doi: 10.3390/ijms232112961. PMID: 36361749.
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年10月, ［査読有り］
英語, 研究論文（学術雑誌）

Fluorochromized tyramide-glucose oxidase as a multiplex fluorescent tyramide signal amplification system for histochemical analysis.
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年09月12日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Pathogenic neuropsychiatric effect of stress-induced microglial interleukin 12/23 axis in systemic lupus erythematosus.
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年07月11日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Targeted proteoform mapping uncovers specific Neurexin-3 variants required for dendritic inhibition.
David Hauser, Katharina Behr, Kohtarou Konno, Dietmar Schreiner, Alexander Schmidt, Masahiko Watanabe, Josef Bischofberger, Peter Scheiffele
Neuron, 110, 13, 2094, 2109, 2022年07月06日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Ubiquitin-Specific Protease 2 in the Ventromedial Hypothalamus Modifies Blood Glucose Levels by Controlling Sympathetic Nervous Activation.
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年06月08日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

ATP spreads inflammation to other limbs through crosstalk between sensory neurons and interneurons.
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年06月06日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum.
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年05月16日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Histochemical characterization of the dorsal raphe-periaqueductal grey dopamine transporter neurons projecting to the extended amygdala.
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年05月13日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Characterisation of deep dorsal horn projection neurons in the spinal cord of the Phox2a::Cre mouse line.
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年04月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Neurexins play a crucial role in cerebellar granule cell survival by organizing autocrine machinery for neurotrophins
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, Elsevier BV, 2022年04月
研究論文（学術雑誌）

L-DOPA-Induced Neurogenesis in the Hippocampus Is Mediated Through GPR143, a Distinct Mechanism of Dopamine.
Yuka Kasahara, Daiki Masukawa, Kenta Kobayashi, Miwako Yamasaki, Masahiko Watanabe, Yoshio Goshima
Stem cells (Dayton, Ohio), 40, 2, 215, 226, 2022年03月16日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Loss of calsyntenin paralogs disrupts interneuron stability and mouse behavior.
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年03月12日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Coexpression of calcineurin A and B subunits in various subcellular and synaptic compartments of cerebellar neurons and glia with particular abundance at parallel fiber-Purkinje cell synapses.
Eriko Miura, Masahiko Watanabe
Neuroscience research, 2022年03月02日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Mitochondrial Localization of CB1 in Progesterone-producing Cells of Ovarian Interstitial Glands of Adult Mice.
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年03月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Activation of extrasynaptic kainate receptors drives hilar mossy cell activity.
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年02月22日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

SIPA1L1/SPAR1 interacts with the neurabin family of proteins and is involved in GPCR signaling.
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年02月04日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Intravenous transplantation of amnion-derived mesenchymal stem cells promotes functional recovery and alleviates intestinal dysfunction after spinal cord injury.
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年, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

GAD67-mediated GABA Synthesis and Signaling Impinges on Directing Basket Cell Axonal Projections Toward Purkinje Cells in the Cerebellum.
Hideki Miwa, Ken Kobayashi, Shinobu Hirai, Mitsuhiko Yamada, Masahiko Watanabe, Haruo Okado, Yuchio Yanagawa
Cerebellum (London, England), 2021年10月21日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Excitatory and inhibitory receptors utilize distinct post- and trans-synaptic mechanisms in vivo.
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年10月18日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Characterisation of lamina I anterolateral system neurons that express Cre in a Phox2a-Cre mouse line.
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年09月09日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression of type one cannabinoid receptor in different subpopulation of kisspeptin neurons and kisspeptin afferents to GnRH neurons in female mice.
Tamás Wilheim, Krisztina Nagy, Mahendravarman Mohanraj, Kamil Ziarniak, Masahiko Watanabe, Joanna Sliwowska, Imre Kalló
Brain structure & function, 226, 7, 2387, 2399, 2021年09月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Cell differentiation is disrupted by MYO5B loss through Wnt/Notch imbalance.
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年08月23日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

mGluR1 signaling in cerebellar Purkinje cells: Subcellular organization and involvement in cerebellar function and disease.
Miwako Yamasaki, Atsu Aiba, Masanobu Kano, Masahiko Watanabe
Neuropharmacology, 194, 108629, 108629, 2021年08月15日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

A comparative analysis of kainate receptor GluK2 and GluK5 knockout mice in a pure genetic background.
Izumi Iida, Kohtarou Konno, Rie Natsume, Manabu Abe, Masahiko Watanabe, Kenji Sakimura, Miho Terunuma
Behavioural brain research, 405, 113194, 113194, 2021年05月07日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Homeostatic p62 levels and inclusion body formation in CHCHD2 knockout mice.
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年04月30日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Rats deficient in the GAD65 isoform exhibit epilepsy and premature lethality.
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年02月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Decreased striatal adenosine A2A-dopamine D2 receptor heteromerization in schizophrenia.
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年02月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Nectin-2α is localized at cholinergic neuron dendrites and regulates synapse formation in the medial habenula.
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年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Global knockdown of glutamate decarboxylase 67 elicits emotional abnormality in mice.
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年01月07日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

In situ localization of diacylglycerol lipase α and β producing an endocannabinoid 2-arachidonoylglycerol and of cannabinoid receptor 1 in the primary oocytes of postnatal mice.
Anussara Kamnate, Juthathip Sirisin, Yada Polsan, Surang Chomphoo, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
Journal of anatomy, 238, 6, 1330, 1340, 2021年01月04日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Immunohistochemistry for Ion Channels and Their Interacting Molecules: Tips for Improving Antibody Accessibility
Kohtarou Konno, Masahiko Watanabe
Neuromethods, 169, 191, 199, Humana Press Inc., 2021年
英語, 論文集(書籍)内論文, 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.

Fluorescent In Situ Hybridization for Sensitive and Specific Labeling
Miwako Yamasaki, Masahiko Watanabe
Neuromethods, 169, 145, 160, Humana Press Inc., 2021年
英語, 論文集(書籍)内論文, 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, 44回, 117, 117, (一社)日本脳神経外傷学会, 2021年01月
日本語

Spike firing attenuation of serotonin neurons in learned helplessness rats is reversed by ketamine.
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年, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Mesenchymal Stem Cell Sheet Promotes Functional Recovery and Palliates Neuropathic Pain in a Subacute Spinal Cord Injury Model.
Kazuyoshi Yamazaki, Masahito Kawabori, Toshitaka Seki, Soichiro Takamiya, Kotaro Konno, Masahiko Watanabe, Kiyohiro Houkin, Miki Fujimura
Stem cells international, 2021, 9964877, 9964877, 2021年, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

An Autism-Associated Neuroligin-3 Mutation Affects Developmental Synapse Elimination in the Cerebellum.
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年, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Kv11 (ether-à-go-go-related gene) voltage-dependent K+ channels promote resonance and oscillation of subthreshold membrane potentials.
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年01月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Perisomatic innervation and neurochemical features of giant pyramidal neurons in both hemispheres of the human primary motor cortex.
Péter Szocsics, Péter Papp, László Havas, Masahiko Watanabe, Zsófia Maglóczky
Brain structure & function, 226, 1, 281, 296, 2021年01月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Specific Neuroligin3-αNeurexin1 signaling regulates GABAergic synaptic function in mouse hippocampus.
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年12月23日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

CRISPR/Cas9-engineered Gad1 elimination in rats leads to complex behavioral changes: implications for schizophrenia.
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年12月08日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Substance P-expressing Neurons in the Superficial Dorsal Horn of the Mouse Spinal Cord: Insights into Their Functions and their Roles in Synaptic Circuits.
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年12月01日, ［国際誌］
英語, 研究論文（学術雑誌）, 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, 32, 1, 82, 82, (一社)日本脳循環代謝学会, 2020年11月
日本語

Enhanced Retrieval of Taste Associative Memory by Chemogenetic Activation of Locus Coeruleus Norepinephrine Neurons.
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年10月21日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Autism spectrum disorder-like behavior caused by reduced excitatory synaptic transmission in pyramidal neurons of mouse prefrontal cortex.
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年10月12日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Lysophosphatidic Acid Increases Maturation of Brush Borders and SGLT1 Activity in MYO5B-deficient Mice, a Model of Microvillus Inclusion Disease.
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年10月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Functional maintenance of calcium store by ShcB adaptor protein in cerebellar Purkinje cells.
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年09月02日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

A synthetic synaptic organizer protein restores glutamatergic neuronal circuits.
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年08月28日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression of green fluorescent protein defines a specific population of lamina II excitatory interneurons in the GRP::eGFP mouse.
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年08月06日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

FTY720 Attenuates Neuropathic Pain after Spinal Cord Injury by Decreasing Systemic and Local Inflammation in a Rat Spinal Cord Compression Model.
Kazuyoshi Yamazaki, Masahito Kawabori, Toshitaka Seki, Soichiro Takamiya, Takahiro Tateno, Kotaro Konno, Masahiko Watanabe, Kiyohiro Houkin
Journal of neurotrauma, 37, 15, 1720, 1728, 2020年08月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Opposing Ventral Striatal Medium Spiny Neuron Activities Shaped by Striatal Parvalbumin-Expressing Interneurons during Goal-Directed Behaviors.
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年06月30日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Development of an L-type Ca2+ channel-dependent Ca2+ transient during the radial migration of cortical excitatory neurons.
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年06月26日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Neuroligin3 splice isoforms shape inhibitory synaptic function in the mouse hippocampus.
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年06月19日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3.
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年05月27日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Compartmentalized Input-Output Organization of Lugaro Cells in the Cerebellar Cortex.
Taisuke Miyazaki, Miwako Yamasaki, Kenji F Tanaka, Masahiko Watanabe
Neuroscience, 462, 89, 105, 2020年05月26日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Different expression and subcellular localization of vesicular inhibitory amino acid transporter in ducts of major salivary glands: An in situ study in mice.
Atthapon Pidsaya, Anussara Kamnate, Juthathip Sirisin, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
Archives of oral biology, 113, 104689, 104689, 2020年05月, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain.
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年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

A Glial-Neuronal Circuit in the Median Eminence Regulates Thyrotropin-Releasing Hormone-Release via the Endocannabinoid System.
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, Elsevier BV, 2020年03月27日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Neurotransmitters and Motoneuron Contacts of Multifunctional and Behaviorally Specialized Turtle Spinal Cord Interneurons.
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年03月25日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression and localization of endogenous phospholipase Cβ3 confined to basal cells in situ of immature ducts and adult excretory ducts of submandibular gland of mice.
Atsara Rawangwong, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
Acta histochemica, 122, 2, 151497, 151497, 2020年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

PSA-NCAM Colocalized with Cholecystokinin-Expressing Cells in the Hippocampus Is Involved in Mediating Antidepressant Efficacy.
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年01月22日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

GluD1 knockout mice with a pure C57BL/6N background show impaired fear memory, social interaction, and enhanced depressive-like behavior.
Chihiro Nakamoto, Meiko Kawamura, Ena Nakatsukasa, Rie Natsume, Keizo Takao, Masahiko Watanabe, Manabu Abe, Tomonori Takeuchi, Kenji Sakimura
PloS one, 15, 2, e0229288, 2020年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Polarized PtdIns(4,5)P2 distribution mediated by a voltage-sensing phosphatase (VSP) regulates sperm motility.
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年12月17日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Thyrotropin-Releasing-Hormone-Synthesizing Neurons of the Hypothalamic Paraventricular Nucleus Are Inhibited by Glycinergic Inputs.
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年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Calretinin positive neurons form an excitatory amplifier network in the spinal cord dorsal horn.
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年11月12日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression of Neuropeptide FF Defines a Population of Excitatory Interneurons in the Superficial Dorsal Horn of the Mouse Spinal Cord that Respond to Noxious and Pruritic Stimuli.
Maria Gutierrez-Mecinas, Andrew Bell, Erika Polgár, Masahiko Watanabe, Andrew J Todd
Neuroscience, 416, 281, 293, 2019年09月15日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Differential expression of neurexin genes in the mouse brain.
Motokazu Uchigashima, Amy Cheung, Julie Suh, Masahiko Watanabe, Kensuke Futai
The Journal of comparative neurology, 527, 12, 1940, 1965, 2019年08月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Hippocampus: Cannabidiol Blunts Δ9-Tetrahydrocannabinol-Induced Cognitive Impairment.
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年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression of cholecystokinin by neurons in mouse spinal dorsal horn.
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年08月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Revealing Adenosine A2A-Dopamine D2 Receptor Heteromers in Parkinson's Disease Post-Mortem Brain through a New AlphaScreen-Based Assay.
Víctor Fernández-Dueñas, Maricel Gómez-Soler, Marta Valle-León, Masahiko Watanabe, Isidre Ferrer, Francisco Ciruela
International journal of molecular sciences, 20, 14, 2019年07月23日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Defining a Spinal Microcircuit that Gates Myelinated Afferent Input: Implications for Tactile Allodynia.
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年07月09日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

mGluR1 in cerebellar Purkinje cells is essential for the formation but not expression of associative eyeblink memory.
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年05月14日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Localization of phospholipase C β3 in the major salivary glands of adult mice.
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年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Heterogeneous localization of muscarinic cholinoceptor M1 in the salivary ducts of adult mice.
Atsara Rawangwong, Suthankamon Khrongyut, Surang Chomphoo, Kohtaro Konno, Miwako Yamasaki, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
Archives of oral biology, 100, 14, 22, 2019年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression and localization of VIAAT in distal uriniferous tubular epithelium of mouse.
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年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Morphological and functional properties distinguish the substance P and gastrin-releasing peptide subsets of excitatory interneuron in the spinal cord dorsal horn.
Dickie AC, Bell AM, Iwagaki N, Polgár E, Gutierrez-Mecinas M, Kelly R, Lyon H, Turnbull K, West SJ, Etlin A, Braz J, Watanabe M, Bennett DLH, Basbaum AI, Riddell JS, Todd AJ
Pain, 160, 2, 442, 462, 2019年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Expression of Calretinin Among Different Neurochemical Classes of Interneuron in the Superficial Dorsal Horn of the Mouse Spinal Cord.
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年02月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Selective distribution of GLUT3-expressing nerve fibers in the lamina terminalis among the circumventricular organs of mice.
Toshihiko Iwanaga, Kohtarou Konno, Masahiko Watanabe
Biomedical research (Tokyo, Japan), 40, 5, 207, 214, 2019年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Endocannabinoid and nitric oxide systems of the hypothalamic paraventricular nucleus mediate effects of NPY on energy expenditure.
Péterfi Z, Farkas I, Denis RGP, Farkas E, Uchigashima M, Füzesi T, Watanabe M, Lechan RM, Liposits Z, Luquet S, Fekete C
Molecular metabolism, 18, 120, 133, 2018年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Deletion of exons encoding carboxypeptidase domain of Nna1 results in Purkinje cell degeneration (pcd) phenotype.
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, 2018年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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/.

Distribution of Caskin1 protein and phenotypic characterization of its knockout mice using a comprehensive behavioral test battery.
Tayo Katano, Keizo Takao, Manabu Abe, Maya Yamazaki, Masahiko Watanabe, Tsuyoshi Miyakawa, Kenji Sakimura, Seiji Ito
Molecular brain, 11, 1, 63, 63, 2018年10月25日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Metabotropic glutamate type 5 receptor requires contactin-associated protein 1 to control memory formation.
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, 2018年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

CDC42EP4, a perisynaptic scaffold protein in Bergmann glia, is required for glutamatergic tripartite synapse configuration.
Natsumi Ageta-Ishihara, Kohtarou Konno, Maya Yamazaki, Manabu Abe, Kenji Sakimura, Masahiko Watanabe, Makoto Kinoshita
Neurochemistry international, 119, 190, 198, 2018年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Structural insights into modulation and selectivity of transsynaptic neurexin-LRRTM interaction.
Yamagata A, Goto-Ito S, Sato Y, Shiroshima T, Maeda A, Watanabe M, Saitoh T, Maenaka K, Terada T, Yoshida T, Uemura T, Fukai S
Nature communications, 9, 1, 3964, 3964, 2018年09月27日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Monitoring and Updating of Action Selection for Goal-Directed Behavior through the Striatal Direct and Indirect Pathways.
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, 2018年09月19日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

mGlu1 Receptors Monopolize the Synaptic Control of Cerebellar Purkinje Cells by Epigenetically Down-Regulating mGlu5 Receptors.
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年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

ASCT1 (Slc1a4) transporter is a physiologic regulator of brain d-serine and neurodevelopment.
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, 2018年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Cell- and stage-specific localization of galectin-3, a β-galactoside-binding lectin, in a mouse model of experimental autoimmune encephalomyelitis.
Itabashi T, Arima Y, Kamimura D, Higuchi K, Bando Y, Takahashi-Iwanaga H, Murakami M, Watanabe M, Iwanaga T, Nio-Kobayashi J
Neurochemistry international, 118, 176, 184, Elsevier Ltd, 2018年09月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Ca2+-binding protein NECAB2 facilitates inflammatory pain hypersensitivity.
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, 2018年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Multiple Phases of Climbing Fiber Synapse Elimination in the Developing Cerebellum.
Kano M, Watanabe T, Uesaka N, Watanabe M
Cerebellum (London, England), 17, 6, 722, 734, 2018年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Localization of nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons and its regulatory role in the subcellular localization of the voltage-gated A-type K⁺ channel Kv4.2 in the medial habenula.
Shiotani H, Miyata M, Itoh Y, Wang S, Kaito A, Mizoguchi A, Yamasaki M, Watanabe M, Mandai K, Mochizuki H, Takai Y
The Journal of comparative neurology, 526, 9, 1527, 1549, Wiley-Liss Inc., 2018年06月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Activity-Induced Regulation of Synaptic Strength through the Chromatin Reader L3mbtl1
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, Elsevier B.V., 2018年06月12日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Ectopic positioning of Bergmann glia and impaired cerebellar wiring in Mlc1-over-expressing mice.
Kikuchihara S, Sugio S, Tanaka KF, Watanabe T, Kano M, Yamazaki Y, Watanabe M, Ikenaka K
Journal of neurochemistry, 147, 3, 344, 360, 2018年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Post mortem single-cell labeling with DiI and immunoelectron microscopy unveil the fine structure of kisspeptin neurons in humans
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, Springer Verlag, 2018年06月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Author Correction: Circuit dissection of the role of somatostatin in itch and pain.
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年06月, ［国際誌］
英語, 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.

Circuit dissection of the role of somatostatin in itch and pain
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, Nature Publishing Group, 2018年05月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Anti-Sez6l2 antibody detected in a patient with immune-mediated cerebellar ataxia inhibits complex formation of GluR1 and Sez6l2
Hiroaki Yaguchi, Ichiro Yabe, Hidehisa Takahashi, Masashi Watanabe, Taichi Nomura, Takahiro Kano, Masahiko Watanabe, Shigetsugu Hatakeyama
Journal of Neurology, 265, 4, 962, 965, Dr. Dietrich Steinkopff Verlag GmbH and Co. KG, 2018年04月01日, ［査読有り］, ［国際誌］
英語

Differential association of GABAB receptors with their effector ion channels in Purkinje cells
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, Springer Verlag, 2018年04月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Gene expression of A6-like subgroup of ATP-binding cassette transporters in mouse brain parenchyma and microvessels
Masanori Tachikawa, Hidetoh Toki, Masahiko Watanabe, Masatoshi Tomi, Ken-ichi Hosoya, Tetsuya Terasaki
Anatomical Science International, 93, 4, 1, 8, Springer Tokyo, 2018年03月08日, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Retrograde Signaling from Progranulin to Sort1 Counteracts Synapse Elimination in the Developing Cerebellum.
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, 2018年02月21日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Munc13-3 Is Required for the Developmental Localization of Ca2+ Channels to Active Zones and the Nanopositioning of Cav2.1 Near Release Sensors
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, Elsevier B.V., 2018年02月20日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Inhibitory neuron-specific Cre-dependent red fluorescent labeling using VGAT BAC-based transgenic mouse lines with identified transgene integration sites
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, Wiley-Liss Inc., 2018年02月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Elavl3 is essential for the maintenance of Purkinje neuron axons.
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年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Cell-Type-Specific Spatiotemporal Expression of Creatine Biosynthetic Enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase in Developing Mouse Brain
Masanori Tachikawa, Masahiko Watanabe, Masahiro Fukaya, Kazuhisa Sakai, Tetsuya Terasaki, Ken-ichi Hosoya
Neurochemical Research, 43, 2, 500, 510, Springer New York LLC, 2018年02月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Postsynaptic GABABRs Inhibit L-Type Calcium Channels and Abolish Long-Term Potentiation in Hippocampal Somatostatin Interneurons
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, Elsevier B.V., 2018年01月02日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

SK2 Channels Associate With mGlu_1α Receptors and Ca_V2.1 Channels in Purkinje Cells.
Luján R, Aguado C, Ciruela F, Arus XM, Martín-Belmonte A, Alfaro-Ruiz R, Martínez-Gómez J, de la Ossa L, Watanabe M, Adelman JP, Shigemoto R, Fukazawa Y
Frontiers in cellular neuroscience, 12, 311, 311, 2018年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Two isoforms of cyclic GMP-dependent kinase-I exhibit distinct expression patterns in the adult mouse dorsal root ganglion.
Uchida H, Matsumura S, Katano T, Watanabe M, Schlossmann J, Ito S
Molecular pain, 14, 1744806918796409, 2018年01月, ［査読有り］

Repeated fluvoxamine treatment recovers early postnatal stress-induced hypersociability-like behavior in adult rats
Haruko Kumamoto, Taku Yamaguchi, Kohtarou Konno, Takeshi Izumi, Takayuki Yoshida, Yu Ohmura, Masahiko Watanabe, Mitsuhiro Yoshioka
Journal of Pharmacological Sciences, 136, 1, 1, 8, Japanese Pharmacological Society, 2018年01月01日, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, 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.

Sez6l2 regulates phosphorylation of ADD and neuritogenesis.
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年12月09日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Differential surface density and modulatory effects of presynaptic GABAB receptors in hippocampal cholecystokinin and parvalbumin basket cells
Sam A. Booker, Daniel Althof, Claudius E. Degro, Masahiko Watanabe, Ákos Kulik, Imre Vida
Brain Structure and Function, 222, 8, 3677, 3690, Springer Verlag, 2017年11月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Maturation of Cerebellar Purkinje Cell Population Activity during Postnatal Refinement of Climbing Fiber Network
Jean-Marc Good, Michael Mahoney, Taisuke Miyazaki, Kenji F. Tanaka, Kenji Sakimura, Masahiko Watanabe, Kazuo Kitamura, Masanobu Kano
CELL REPORTS, 21, 8, 2066, 2073, CELL PRESS, 2017年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

A QUANTITATIVE STUDY OF NEUROCHEMICALLY DEFINED POPULATIONS OF INHIBITORY INTERNEURONS IN THE SUPERFICIAL DORSAL HORN OF THE MOUSE SPINAL CORD
Kieran A. Boyle, Maria Gutierrez-Mecinas, Erika Polgar, Nicole Mooney, Emily O'Connor, Takahiro Furuta, Masahiko Watanabe, Andrew J. Todd
NEUROSCIENCE, 363, 120, 133, PERGAMON-ELSEVIER SCIENCE LTD, 2017年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Glycinergic Input to the Mouse Basal Forebrain Cholinergic Neurons
Zsuzsanna Bardoczi, Balazs Pal, Aron Koszeghy, Tamas Wilheim, Masahiko Watanabe, Laszlo Zaborszky, Zsolt Liposits, Imre Kallo
JOURNAL OF NEUROSCIENCE, 37, 39, 9534, 9549, SOC NEUROSCIENCE, 2017年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Endocytosis following dopamine D2 receptor activation is critical for neuronal activity and dendritic spine formation via Rabex-5/PDGFRβ signaling in striatopallidal medium spiny neurons.
Shioda N, Yabuki Y, Wang Y, Uchigashima M, Hikida T, Sasaoka T, Mori H, Watanabe M, Sasahara M, Fukunaga K
Mol Psychiatry, 22, 8, 1205, 1222, NATURE PUBLISHING GROUP, 2017年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Alternative splicing in the C-terminal tail of Ca(v)2.1 is essential for preventing a neurological disease in 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, OXFORD UNIV PRESS, 2017年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Retrograde BDNF to TrkB signaling promotes synapse elimination in the developing cerebellum
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, NATURE PUBLISHING GROUP, 2017年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

SNAP-25 phosphorylation at Ser187 regulates synaptic facilitation and short-term plasticity in an age-dependent manner
Norikazu Katayama, Saori Yamamori, Masahiro Fukaya, Shizuka Kobayashi, Masahiko Watanabe, Masami Takahashi, Toshiya Manabe
SCIENTIFIC REPORTS, 7, 1, 7996, 7996, NATURE PUBLISHING GROUP, 2017年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Localization of photoperiod responsive circadian oscillators in the mouse suprachiasmatic nucleus
Tomoko Yoshikawa, Natsuko F. Inagaki, Seiji Takagi, Shigeru Kuroda, Miwako Yamasaki, Masahiko Watanabe, Sato Honma, Ken-ichi Honma
SCIENTIFIC REPORTS, 7, 1, 8210, 8210, NATURE PUBLISHING GROUP, 2017年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

The Parkinson's disease-associated GPR37 receptor interacts with striatal adenosine A(2A) receptor controlling its cell surface expression and function in vivo
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, NATURE PUBLISHING GROUP, 2017年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

LUsing a novel PV-Cre rat model to characterize pallidonigral cells and their terminations
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, SPRINGER HEIDELBERG, 2017年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Intrarenal signaling mediated by CCK plays a role in salt intake-induced natriuresis
Hiromi Takahashi-Iwanaga, Shunsuke Kimura, Kohtarou Konno, Masahiko Watanabe, Toshihiko Iwanaga
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY, 313, 1, F20, F29, AMER PHYSIOLOGICAL SOC, 2017年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells
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, NATL ACAD SCIENCES, 2017年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Diacylglycerol kinase ε localizes to subsurface cisterns of cerebellar Purkinje cells.
Hozumi Y, Fujiwara H, Kaneko K, Fujii S, Topham MK, Watanabe M, Goto K
Cell and tissue research, 368, 3, 441, 458, SPRINGER, 2017年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Numbers of presynaptic Ca2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses
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, NATL ACAD SCIENCES, 2017年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Serotonin rebalances cortical tuning and behavior linked to autism symptoms in 15q11-13 CNV mice
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, AMER ASSOC ADVANCEMENT SCIENCE, 2017年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Distribution of corticotropin-releasing factor neurons in the mouse brain: a study using corticotropin-releasing factor-modified yellow fluorescent protein knock-in mouse
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, SPRINGER HEIDELBERG, 2017年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

KCTD12 Auxiliary Proteins Modulate Kinetics of GABA(B) Receptor-Mediated Inhibition in CholecystokininContaining Interneurons
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, OXFORD UNIV PRESS INC, 2017年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Preprotachykinin A is expressed by a distinct population of excitatory neurons in the mouse superficial spinal dorsal horn including cells that respond to noxious and pruritic stimuli
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, LIPPINCOTT WILLIAMS & WILKINS, 2017年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Ventrolateral Striatal Medium Spiny Neurons Positively Regulate Food-Incentive, Goal-Directed Behavior Independently of D1 and D2 Selectivity
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, SOC NEUROSCIENCE, 2017年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Late postnatal shifts of parvalbumin and nitric oxide synthase expression within the GABAergic and glutamatergic phenotypes of inferior colliculus neurons
Hisataka Fujimoto, Kotaro Konno, Masahiko Watanabe, Shozo Jinno
JOURNAL OF COMPARATIVE NEUROLOGY, 525, 4, 868, 884, WILEY-BLACKWELL, 2017年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Dysfunction of ventrolateral striatal dopamine receptor type 2-expressing medium spiny neurons impairs instrumental motivation
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, NATURE PUBLISHING GROUP, 2017年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Anatomical and Molecular Properties of Long Descending Propriospinal Neurons in Mice
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, FRONTIERS MEDIA SA, 2017年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Intrarenal signaling mediated by CCK plays a role in salt intake-induced natriuresis
Hiromi Takahashi-Iwanaga, Shunsuke Kimura, Kohtarou Konno, Masahiko Watanabe, Toshihiko Iwanaga
American Journal of Physiology - Renal Physiology, 313, 1, F20, F29, American Physiological Society, 2017年, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Two distinct classes of muscarinic action on hippocampal inhibitory synapses: M2-mediated direct suppression and M1/M3-mediated indirect suppression through endocannabinoid signaling.
Fukudome Y, Ohno-Shosaku T, Matsui M, Omori Y, Watanabe M, Kano M
Eur J Nuerosci., 19, 10, 2682, 2692, 2017年, ［査読有り］

Astrocyte-mediated infantile-onset leukoencephalopathy mouse model.
Sugio S, Tohyama K, Oku S, Fujiyoshi K, Yoshimura T, Hikishima K, Yano R, Fukuda T, Nakamura M, Okano H, Watanabe M, Fukata M, Ikenaka K, Tanaka FK
Glia, 65, 1, 150, 168, 2017年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

The Cellular and Synaptic Architecture of the Mechanosensory Dorsal Horn
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, +, CELL PRESS, 2017年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Immunochemical characterization on pathological oligomers of mutant Cu/Znsuperoxide dismutase in amyotrophic lateral sclerosis
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, BIOMED CENTRAL LTD, 2017年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Distinct and Cooperative Functions for the Protocadherin-α, -β and -γ Clusters in Neuronal Survival and Axon Targeting.
Hasegawa S, Kumagai M, Hagihara M, Nishimaru H, Hirano K, Kaneko R, Okayama A, Hirayama T, Sanbo M, Hirabayashi M, Watanabe M, Hirabayashi T, Yagi T
Frontiers in molecular neuroscience, 9, 155, 155, FRONTIERS MEDIA SA, 2016年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling
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, Nature Publishing Group, 2016年11月11日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Roles of Cbln1 in Non-Motor Functions of Mice
Shintaro Otsuka, Kohtarou Konno, Manabu Abe, Junko Motohashi, Kazuhisa Kohda, Kenji Sakimura, Masahiko Watanabe, Michisuke Yuzaki
JOURNAL OF NEUROSCIENCE, 36, 46, 11801, 11816, SOC NEUROSCIENCE, 2016年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

QRFP-Deficient Mice Are Hypophagic, Lean, Hypoactive and Exhibit Increased Anxiety-Like Behavior
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, PUBLIC LIBRARY SCIENCE, 2016年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Erratum to: Comparative anatomical distribution of neuronal calcium-binding protein (NECAB) 1 and -2 in rodent and human spinal cord (Brain Struct Funct, 10.1007/s00429-016-1191-3)
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, Springer Verlag, 2016年09月01日, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Comparative anatomical distribution of neuronal calcium-binding protein (NECAB) 1 and-2 in rodent and human spinal cord
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, SPRINGER HEIDELBERG, 2016年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

The Metabotropic Glutamate Receptor Subtype 1 Mediates Experience-Dependent Maintenance of Mature Synaptic Connectivity in the Visual Thalamus
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, CELL PRESS, 2016年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Locus coeruleus and dopaminergic consolidation of everyday memory
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, +, NATURE PUBLISHING GROUP, 2016年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Involvement of Brain-Enriched guanylate Kinase-Associated protein (BEGAIN) in chronic pain after peripheral nerve injury
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, Society for Neuroscience, 2016年09月01日, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

The active zone protein CAST regulates synaptic vesicle recycling and quantal size in the mouse hippocampus
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, WILEY-BLACKWELL, 2016年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

IMMUNOSTAINING FOR HOMER REVEALS THE MAJORITY OF EXCITATORY SYNAPSES IN LAMINAE I-III OF THE MOUSE SPINAL DORSAL HORN
Maria Gutierrez-Mecinas, Emily D. Kuehn, Victoria E. Abraira, Erika Polgar, Masahiko Watanabe, Andrew J. Todd
NEUROSCIENCE, 329, 171, 181, PERGAMON-ELSEVIER SCIENCE LTD, 2016年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Crucial Roles of the Endocannabinoid 2-Arachidonoylglycerol in the Suppression of Epileptic Seizures
Yuki Sugaya, Maya Yamazaki, Motokazu Uchigashima, Kenta Kobayashi, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
CELL REPORTS, 16, 5, 1405, 1415, CELL PRESS, 2016年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Distinct Subunit Domains Govern Synaptic Stability and Specificity of the Kainate Receptor
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, CELL PRESS, 2016年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Ionic Basis for Membrane Potential Resonance in Neurons of the Inferior Olive
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, CELL PRESS, 2016年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Developmental Switch in Spike Timing-Dependent Plasticity and Cannabinoid-Dependent Reorganization of the Thalamocortical Projection in the Barrel Cortex
Chiaki Itami, Jui-Yen Huang, Miwako Yamasaki, Masahiko Watanabe, Hui-Chen Lu, Fumitaka Kimura
JOURNAL OF NEUROSCIENCE, 36, 26, 7039, 7054, SOC NEUROSCIENCE, 2016年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Neural FFA3 activation inversely regulates anion secretion evoked by nicotinic ACh receptor activation in rat proximal colon
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, WILEY-BLACKWELL, 2016年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Serotonin 5-HT7 Receptor in the Ventral Hippocampus Modulates the Retrieval of Fear Memory and Stress-Induced Defecation
Yu Ohmura, Takayuki Yoshida, Kohtarou Konno, Masabumi Minami, Masahiko Watanabe, Mitsuhiro Yoshioka
INTERNATIONAL JOURNAL OF NEUROPSYCHOPHARMACOLOGY, 19, 6, OXFORD UNIV PRESS, 2016年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Medial septal GABAergic projection neurons promote object exploration behavior and type 2 theta rhythm
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, NATL ACAD SCIENCES, 2016年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Transsynaptic Modulation of Kainate Receptor Functions by C1q-like Proteins
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, CELL PRESS, 2016年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders (vol 7, pg 10594, 2016)
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, NATURE PUBLISHING GROUP, 2016年04月, ［査読有り］
英語

TARP γ-2 and γ-8 Differentially Control AMPAR Density Across Schaffer Collateral/Commissural Synapses in the Hippocampal CA1 Area.
Yamasaki M, Fukaya M, Yamazaki M, Azechi H, Natsume R, Abe M, Sakimura K, Watanabe M
The Journal of neuroscience : the official journal of the Society for Neuroscience, 36, 15, 4296, 4312, SOC NEUROSCIENCE, 2016年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

GluD2 Endows Parallel Fiber-Purkinje Cell Synapses with a High Regenerative Capacity
Ryoichi Ichikawa, Kenji Sakimura, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 36, 17, 4846, 4858, SOC NEUROSCIENCE, 2016年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Dopamine synapse is a neuroligin-2-mediated contact between dopaminergic presynaptic and GABAergic postsynaptic structures
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, NATL ACAD SCIENCES, 2016年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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, (一社)日本生理学会, 2016年03月
英語

A combined electrophysiological and morphological study of neuropeptide Y-expressing inhibitory interneurons in the spinal dorsal horn of the mouse
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, LIPPINCOTT WILLIAMS & WILKINS, 2016年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

The ascending median raphe projections are mainly glutamatergic in the mouse forebrain
Andras Szonyi, Marton I. Mayer, Csaba Cserep, Virag T. Takacs, Masahiko Watanabe, Tamas F. Freund, Gabor Nyiri
BRAIN STRUCTURE & FUNCTION, 221, 2, 735, 751, SPRINGER HEIDELBERG, 2016年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

A quantitative study of neurochemically defined excitatory interneuron populations in laminae I-III of the mouse spinal cord
Maria Gutierrez-Mecinas, Takahiro Furuta, Masahiko Watanabe, Andrew J. Todd
MOLECULAR PAIN, 12, SAGE PUBLICATIONS INC, 2016年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Membrane palmitoylated protein 2 is a synaptic scaffold protein required for synaptic SK2-containing channel function
Gukhan Kim, Rafael Lujan, Jochen Schwenk, Melissa H. Kelley, Carolina Aguado, Masahiko Watanabe, Bernd Fakler, James Maylie, John P. Adelman
ELIFE, 5, ELIFE SCIENCES PUBLICATIONS LTD, 2016年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Involvement of hormones in olfactory imprinting and homing in chum salmon
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, NATURE PUBLISHING GROUP, 2016年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Territories of heterologous inputs onto Purkinje cell dendrites are segregated by mGluR1-dependent parallel fiber synapse elimination
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, NATL ACAD SCIENCES, 2016年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders
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, NATURE PUBLISHING GROUP, 2016年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Phosphoproteomics of the Dopamine Pathway Enables Discovery of Rap1 Activation as a Reward Signal In Vivo
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, CELL PRESS, 2016年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Basket cells
Masahiko Watanabe
Essentials of Cerebellum and Cerebellar Disorders: A Primer for Graduate Students, 195, 199, Springer International Publishing, 2016年01月01日, ［査読有り］
英語, 論文集(書籍)内論文, 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).

Determination of kainate receptor subunit ratios in mouse brain using novel chimeric protein standards
Izumi Watanabe-Iida, Kohtarou Konno, Kaori Akashi, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura
JOURNAL OF NEUROCHEMISTRY, 136, 2, 295, 305, WILEY-BLACKWELL, 2016年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Release probability of hippocampal glutamatergic terminals scales with the size of the active zone (vol 15, pg 988, 2012)
Noemi Holderith, Andrea Lorincz, Gergely Katona, Balazs Rozsa, Akos Kulik, Masahiko Watanabe, Zoltan Nusser
NATURE NEUROSCIENCE, 19, 1, 172, 172, NATURE PUBLISHING GROUP, 2016年01月, ［査読有り］
英語

A mutation in the low voltage-gated calcium channel CACNA1G alters the physiological properties of the channel, causing spinocerebellar ataxia.
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, BIOMED CENTRAL LTD, 2015年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance
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, NATURE PUBLISHING GROUP, 2015年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Expression and localization of the diacylglycerol kinase family and of phosphoinositide signaling molecules in adrenal gland
Yasukazu Hozumi, Ryo Akimoto, Akihito Suzuki, Koichi Otani, Masahiko Watanabe, Kaoru Goto
CELL AND TISSUE RESEARCH, 362, 2, 295, 305, SPRINGER, 2015年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

The organisation of spinoparabrachial neurons in the mouse
Darren Cameron, Erika Polgar, Maria Gutierrez-Mecinas, Maria Gomez-Lima, Masahiko Watanabe, Andrew J. Todd
PAIN, 156, 10, 2061, 2071, ELSEVIER SCIENCE BV, 2015年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Inhibitory and excitatory axon terminals share a common nano-architecture of their Cav2.1 (P/Q-type) Ca2+ channels
Daniel Althof, David Baehrens, Masahiko Watanabe, Noboru Suzuki, Bernd Fakler, Ákos Kulik
Frontiers in Cellular Neuroscience, 9, AUGUST, Frontiers Media S.A., 2015年08月11日, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Novel splice variants in the 5 ' UTR of Gtf2i expressed in the rat brain: alternative 5 ' UTRs and differential expression in the neuronal dendrites
Yoshinori Shirai, Masahiko Watanabe, Hiroyuki Sakagami, Tatsuo Suzuki
JOURNAL OF NEUROCHEMISTRY, 134, 3, 578, 589, WILEY-BLACKWELL, 2015年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Repeated fluvoxamine treatment recovers juvenile stress-induced morphological changes and depressive-like behavior in rats
Kerise Lyttle, Yu Ohmura, Kohtarou Konno, Takayuki Yoshida, Takeshi Izumi, Masahiko Watanabe, Mitsuhiro Yoshioka
BRAIN RESEARCH, 1616, 88, 100, ELSEVIER SCIENCE BV, 2015年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Multiple Forms of Endocannabinoid and Endovanilloid Signaling Regulate the Tonic Control of GABA Release
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, SOC NEUROSCIENCE, 2015年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Localization of Cannabinoid Receptor 1 (CB1) in Submandibular and Sublingual Salivary Glands of Mice throughout Postnatal Development
Wiphawi Hipkaeo, Masahiko Watanabe, Hisatake Kondo
INTERNATIONAL JOURNAL OF MORPHOLOGY, 33, 2, 695, 700, SOC CHILENA ANATOMIA, 2015年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay
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, NATURE PUBLISHING GROUP, 2015年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Weeding out bad waves: towards selective cannabinoid circuit control in epilepsy (vol 16, pg 264, 2015)
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, NATURE PUBLISHING GROUP, 2015年06月
英語

Inhibitory Interneurons That Express GFP in the PrP-GFP Mouse Spinal Cord Are Morphologically Heterogeneous, Innervated by Several Classes of Primary Afferent and Include Lamina I Projection Neurons among Their Postsynaptic Targets
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, SOC NEUROSCIENCE, 2015年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Localization of Vesicular Inhibitory Amino Acid Transporter (VIAAT) in the Submandibular Salivary Gland Throughout the Postnatal Development of Mice
Yanyong Toomsan, Surang Chomphoo, Sawetree Pakkarato, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
INTERNATIONAL JOURNAL OF MORPHOLOGY, 33, 1, 113, 118, SOC CHILENA ANATOMIA, 2015年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cellular and subcellular localization of cholecystokinin (CCK)-1 receptors in the pancreas, gallbladder, and stomach of mice
Kohtarou Konno, Hiromi Takahashi-Iwanaga, Motokazu Uchigashima, Kyoko Miyasaka, Akihiro Funakoshi, Masahiko Watanabe, Toshihiko Iwanaga
HISTOCHEMISTRY AND CELL BIOLOGY, 143, 3, 301, 312, SPRINGER, 2015年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

VGluT3-Expressing CCK-Positive Basket Cells Construct Invaginating Synapses Enriched with Endocannabinoid Signaling Proteins in Particular Cortical and Cortex-Like Amygdaloid Regions of Mouse Brains
Yuki Omiya, Motokazu Uchigashima, Kohtarou Konno, Miwako Yamasaki, Taisuke Miyazaki, Takayuki Yoshida, Ichiro Kusumi, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 35, 10, 4215, 4228, SOC NEUROSCIENCE, 2015年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Combined immunocytochemistry and tracing of neural connections
Taisuke Miyazaki, Masahiko Watanabe
Immunocytochemistry and Related Techniques, 299, 311, Springer New York, 2015年02月24日, ［査読有り］
英語, 論文集(書籍)内論文, 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.

Developmental Tightening of Cerebellar Cortical Synaptic Influx-Release Coupling
David Baur, Grit Bornschein, Daniel Althof, Masahiko Watanabe, Akos Kulik, Jens Eilers, Hartmut Schmidt
JOURNAL OF NEUROSCIENCE, 35, 5, 1858, 1871, SOC NEUROSCIENCE, 2015年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Retrograde Signaling for Climbing Fiber Synapse Elimination
Naofumi Uesaka, Motokazu Uchigashima, Takayasu Mikuni, Hirokazu Hirai, Masahiko Watanabe, Masanobu Kano
CEREBELLUM, 14, 1, 4, 7, SPRINGER, 2015年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

SCFA transport in rat duodenum
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, AMER PHYSIOLOGICAL SOC, 2015年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cell-specific STORM super-resolution imaging reveals nanoscale organization of cannabinoid signaling
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, Nature Publishing Group, 2015年01月01日, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cholinergic Afferent Stimulation Induces Axonal Function Plasticity in Adult Hippocampal Granule Cells
Katiuscia Martinello, Zhuo Huang, Rafael Lujan, Baouyen Tran, Masahiko Watanabe, Edward C. Cooper, David A. Brown, Mala M. Shah
NEURON, 85, 2, 346, 363, CELL PRESS, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Untangling dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats
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, COMPANY OF BIOLOGISTS LTD, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cerebellar plasticity and motor learning deficits in a copy-number variation mouse model of autism
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, NATURE PUBLISHING GROUP, 2015年01月, ［査読有り］
英語

Distinct subsynaptic localization of type 1 metabotropic glutamate receptors at glutamatergic and GABAergic synapses in the rodent cerebellar cortex
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, WILEY-BLACKWELL, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Chemical corrector treatment ameliorates increased seizure susceptibility in a mouse model of familial epilepsy
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, NATURE PUBLISHING GROUP, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Specific regions display altered grey matter volume in μ-opioid receptor knockout mice: MRI voxel-based morphometry
Sasaki K, Sumiyoshi A, Nonaka H, Kasahara Y, Ikeda K, Hall FS, Uhl GR, Watanabe M, Kawashima R, Sora I
Br J Pharmacol, 172, 2, 654, 667, WILEY-BLACKWELL, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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

Is D-aspartate produced by glutamic-oxaloacetic transaminase-1 like 1 (Got1l1): a putative aspartate racemase?
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, SPRINGER WIEN, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Anterograde C1ql1 Signaling Is Required in Order to Determine and Maintain a Single-Winner Climbing Fiber in the Mouse Cerebellum
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, CELL PRESS, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Immunohistochemical localization of GLUT3, MCT1, and MCT2 in the testes of mice and rats: the use of different energy sources in spermatogenesis
Ayuko Kishimoto, Toshina Ishiguro-Oonuma, Ritei Takahashi, Mamiko Maekawa, Kiyotaka Toshimori, Masahiko Watanabe, Toshihiko Iwanaga
BIOMEDICAL RESEARCH-TOKYO, 36, 4, 225, U59, BIOMEDICAL RESEARCH PRESS LTD, 2015年, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Involvement of diacylglycerol kinase β in the spine formation at distal dendrites of striatal medium spiny neurons.
Hozumi Y, Kakefuda K, Yamasaki M, Watanabe M, Hara H, Goto K
Brain Research, 1594, 36, 45, ELSEVIER SCIENCE BV, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Morphologic characteristics of esophageal epithelium in a rat model of duodenoesophageal reflux and protective effect of lafutidine
Natsuya Katada, Jiro Shibuya, Takafumi Ichikawa, Kei Hosoda, Keishi Yamashita, Masahiko Watanabe
ESOPHAGUS, 12, 1, 65, 72, SPRINGER JAPAN KK, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Structure-Function Relationships between Aldolase C/Zebrin II Expression and Complex Spike Synchrony in the Cerebellum
Shinichiro Tsutsumi, Maya Yamazaki, Taisuke Miyazaki, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano, Kazuo Kitamura
JOURNAL OF NEUROSCIENCE, 35, 2, 843, 852, SOC NEUROSCIENCE, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Rab11a is required for apical protein localisation in the intestine
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, COMPANY OF BIOLOGISTS LTD, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Expression of gastrin-releasing peptide by excitatory interneurons in the mouse superficial dorsal horn
Maria Gutierrez-Mecinas, Masahiko Watanabe, Andrew J. Todd
MOLECULAR PAIN, 10, BIOMED CENTRAL LTD, 2014年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Localization of SK2 channels relative to excitatory synaptic sites in the mouse developing Purkinje cells
Carmen Ballesteros-Merino, Jose Martinez-Hernandez, Carolina Aguado, Masahiko Watanabe, John P. Adelman, Rafael Lujan
FRONTIERS IN NEUROANATOMY, 8, FRONTIERS RESEARCH FOUNDATION, 2014年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Uncovering caffeine's adenosine a<inf>2A</inf> receptor inverse agonism in experimental parkinsonism
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, American Chemical Society, 2014年11月21日, ［査読有り］
英語, 研究論文（学術雑誌）, 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<
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receptor (A<
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2A<
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R) neutral antagonist or an inverse agonist is unresolved. Here we show that caffeine is an A<
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2A<
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R inverse agonist in cell-based functional studies and in experimental parkinsonism. Thus, we observed that caffeine triggers a distinct mode, opposite to A<
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2A<
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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<
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2A<
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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).

Selective innervation of NK1 receptor-lacking lamina I spinoparabrachial neurons by presumed nonpeptidergic A delta nociceptors in the rat
Najma Baseer, Abdullah S. Al-Baloushi, Masahiko Watanabe, Safa A. S. Shehab, Andrew J. Todd
PAIN, 155, 11, 2291, 2300, ELSEVIER SCIENCE BV, 2014年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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/).

Cerebellar plasticity and motor learning deficits in a copy-number variation mouse model of autism
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, NATURE PUBLISHING GROUP, 2014年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

The glutamate receptor GluN2 subunit regulates synaptic trafficking of AMPA receptors in the neonatal mouse brain
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, WILEY-BLACKWELL, 2014年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Neuron type- and input pathway-dependent expression of Slc4a10 in adult mouse brains
Xiaohong Song, Miwako Yamasaki, Taisuke Miyazaki, Kohtarou Konno, Motokazu Uchigashima, Masahiko Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 40, 5, 2797, 2810, WILEY-BLACKWELL, 2014年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

A New Mouse Allele of Glutamate Receptor Delta 2 with Cerebellar Atrophy and Progressive Ataxia
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, PUBLIC LIBRARY SCIENCE, 2014年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Ca(v)1.3 channels control D2-autoreceptor responses via NCS-1 in substantia nigra dopamine neurons
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, OXFORD UNIV PRESS, 2014年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Opposing Role of NMDA Receptor GluN2B and GluN2D in Somatosensory Development and Maturation
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, SOC NEUROSCIENCE, 2014年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Global Scaling Down of Excitatory Postsynaptic Responses in Cerebellar Purkinje Cells Impairs Developmental Synapse Elimination
Shinya Kawata, Taisuke Miyazaki, Maya Yamazaki, Takayasu Mikuni, Miwako Yamasaki, Kouichi Hashimoto, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
CELL REPORTS, 8, 4, 1119, 1129, CELL PRESS, 2014年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Dynorphin Acts as a Neuromodulator to Inhibit Itch in the Dorsal Horn of the Spinal Cord
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, CELL PRESS, 2014年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Enriched Expression of GluD1 in Higher Brain Regions and Its Involvement in Parallel Fiber-Interneuron Synapse Formation in the Cerebellum
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, SOC NEUROSCIENCE, 2014年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Retrograde semaphorin signaling regulates synapse elimination in the developing mouse brain
Naofumi Uesaka, Motokazu Uchigashima, Takayasu Mikuni, Takanobu Nakazawa, Harumi Nakao, Hirokazu Hirai, Atsu Aiba, Masahiko Watanabe, Masanobu Kano
SCIENCE, 344, 6187, 1020, 1023, AMER ASSOC ADVANCEMENT SCIENCE, 2014年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Functional phylogenetic analysis of LGI proteins identifies an interaction motif crucial for myelination
Linde Kegel, Martine Jaegle, Siska Driegen, Eerik Aunin, Kris Leslie, Yuko Fukata, Masahiko Watanabe, Masaki Fukata, Dies Meijer
DEVELOPMENT, 141, 8, 1749, 1756, COMPANY OF BIOLOGISTS LTD, 2014年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Multiple Mechanistically Distinct Modes of Endocannabinoid Mobilization at Central Amygdala Glutamatergic Synapses
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, CELL PRESS, 2014年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury
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, NATL ACAD SCIENCES, 2014年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Differential subcellular localization of SK3-containing channels in the hippocampus
Carmen Ballesteros-Merino, Masahiko Watanabe, Ryuichi Shigemoto, Yugo Fukazawa, John P. Adelman, Rafael Lujan
EUROPEAN JOURNAL OF NEUROSCIENCE, 39, 6, 883, 892, WILEY, 2014年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Heterogeneous presynaptic distribution of monoacylglycerol lipase, a multipotent regulator of nociceptive circuits in the mouse spinal cord
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, WILEY-BLACKWELL, 2014年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

The Synaptic Targeting of mGluR1 by Its Carboxyl-Terminal Domain Is Crucial for Cerebellar Function
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, SOC NEUROSCIENCE, 2014年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Dlx1 transcription factor regulates dendritic growth and postsynaptic differentiation through inhibition of neuropilin-2 and PAK3 expression
Xiaojing Dai, Hirohide Iwasaki, Masahiko Watanabe, Shigeo Okabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 39, 4, 531, 547, WILEY-BLACKWELL, 2014年02月, ［査読有り］
英語, 研究論文（学術雑誌）

Localization of Serine Racemase and Its Role in the Skin.
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, 2014年01月, ［査読有り］

A putative relay circuit providing low-threshold mechanoreceptive input to lamina I projection neurons via vertical cells in lamina II of the rat dorsal horn
Toshiharu Yasaka, Sheena Y. X. Tiong, Erika Polgar, Masahiko Watanabe, Eiichi Kumamoto, John S. Riddell, Andrew J. Todd
MOLECULAR PAIN, 10, BIOMED CENTRAL LTD, 2014年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Functional differences between neurochemically defined populations of inhibitory interneurons in the rat spinal dorsal horn
Erika Polgar, Thomas C. P. Sardella, Sheena Y. X. Tiong, Samantha Locke, Masahiko Watanabe, Andrew J. Todd
PAIN, 154, 12, 2606, 2615, ELSEVIER SCIENCE BV, 2013年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Association of Rgs7/Gb5 Complexes With Girk Channels and GABA(B) Receptors in Hippocampal CA1 Pyramidal Neurons
Ana Fajardo-Serrano, Nicole Wydeven, Daniele Young, Masahiko Watanabe, Ryuichi Shigemoto, Kirill A. Martemyanov, Kevin Wickman, Rafael Lujan
HIPPOCAMPUS, 23, 12, 1231, 1245, WILEY-BLACKWELL, 2013年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Point Mutation in Syntaxin-1A Causes Abnormal Vesicle Recycling, Behaviors, and Short Term Plasticity
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, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2013年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Deletions in GRID2 lead to a recessive syndrome of cerebellar ataxia and tonic upgaze in humans
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, LIPPINCOTT WILLIAMS & WILKINS, 2013年10月
英語, 研究論文（学術雑誌）, 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.

Suppressing aberrant GluN3A expression rescues synaptic and behavioral impairments in Huntington's disease models
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, 2013年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Disruption of cerebellar microzonal organization in GluD2 (GluRδ2) knockout mouse.
Hashizume M, Miyazaki T, Sakimura K, Watanabe M, Kitamura K, Kano M
Frontiers in neural circuits, 7, 130, FRONTIERS RESEARCH FOUNDATION, 2013年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Calcium-dependent regulation of climbing fibre synapse elimination during postnatal cerebellar development
Masanobu Kano, Hisako Nakayama, Kouichi Hashimoto, Kazuo Kitamura, Kenji Sakimura, Masahiko Watanabe
JOURNAL OF PHYSIOLOGY-LONDON, 591, 13, 3151, 3158, WILEY-BLACKWELL, 2013年07月, ［査読有り］
英語, 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.

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice
Natsuko Tsujino, Tomomi Tsunematsu, Motokazu Uchigashima, Kohtarou Konno, Akihiro Yamanaka, Kazuto Kobayashi, Masahiko Watanabe, Yoshimasa Koyama, Takeshi Sakurai
PLOS ONE, 8, 7, PUBLIC LIBRARY SCIENCE, 2013年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Diacylglycerol lipase α manipulation reveals developmental roles for intercellular endocannabinoid signaling.
Keimpema E, Alpár A, Howell F, Malenczyk K, Hobbs C, Hurd YL, Watanabe M, Sakimura K, Kano M, Doherty P, Harkany T
Scientific reports, 3, 2093, Nature Publishing Group, 2013年06月28日, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Distinct Expression and Localization of Diacylglycerol Kinase Isozymes in Rat Retina
Yasukazu Hozumi, Hirooki Matsui, Fumio Sakane, Masahiko Watanabe, Kaoru Goto
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY, 61, 6, 462, 476, SAGE PUBLICATIONS LTD, 2013年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Protocadherin 17 Regulates Presynaptic Assembly in Topographic Corticobasal Ganglia Circuits
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, CELL PRESS, 2013年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Expression of Vesicular Glutamate Transporters in Sensory and Autonomic Neurons Innervating the Mouse Bladder
Pablo R. Brumovsky, Rebecca P. Seal, Kerstin H. Lundgren, Kim B. Seroogy, Masahiko Watanabe, G. F. Gebhart
JOURNAL OF UROLOGY, 189, 6, 2342, 2349, ELSEVIER SCIENCE INC, 2013年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Left-right asymmetry of the hippocampal synapses with differential subunit allocation of glutamate receptors (vol 105, pg 19498, 2008)
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, NATL ACAD SCIENCES, 2013年06月, ［査読有り］
英語

The interaction between serotonergic and cannabinoidergic modulations involved in the fear extinction
Takayuki Yoshida, Masanobu Kano, Masahiko Watanabe, Mitsuhiro Yoshiok
Japanese Journal of Neuropsychopharmacology, 33, 3, 95, 99, 2013年06月, ［査読有り］
日本語, 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.

Homeostatic Control of Synaptic Transmission by Distinct Glutamate Receptors
Dan Yan, Miwako Yamasaki, Christoph Straub, Masahiko Watanabe, Susumu Tomita
NEURON, 78, 4, 687, 699, CELL PRESS, 2013年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Distinct synaptic localization patterns of brefeldin A-resistant guanine nucleotide exchange factors BRAG2 and BRAG3 in the mouse retina
Hiroyuki Sakagami, Osamu Katsumata, Yoshinobu Hara, Hideaki Tamaki, Masahiko Watanabe, Robert J. Harvey, Masahiro Fukaya
Journal of Comparative Neurology, 521, 4, 860, 876, 2013年03月01日, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

ACTIVATION OF P38 MITOGEN-ACTIVATED PROTEIN KINASE IN THE DORSAL ROOT GANGLION CONTRIBUTES TO PAIN HYPERSENSITIVITY AFTER PLANTAR INCISION
K. Mizukoshi, M. Sasaki, Y. Izumi, M. Miura, M. Watanabe, F. Amaya
NEUROSCIENCE, 234, 77, 87, PERGAMON-ELSEVIER SCIENCE LTD, 2013年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

L-Serine deficiency caused by genetic Phgdh deletion leads to robust induction of 4E-BP1 and subsequent repression of translation initiation in the developing central nervous system
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, 2013年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Type 2K+Cl cotransporter is preferentially recruited to climbing fiber synapses during development and the stellate cell-targeting dendritic zone at adulthood in cerebellar Purkinje cells
Issei Kawakita, Motokazu Uchigashima, Kohtarou Konno, Taisuke Miyazaki, Miwako Yamasaki, Masahiko Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 37, 4, 532, 543, WILEY-BLACKWELL, 2013年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Quantitative Localization of Ca(v)2.1 (P/Q-Type) Voltage-Dependent Calcium Channels in Purkinje Cells: Somatodendritic Gradient and Distinct Somatic Coclustering with Calcium-Activated Potassium Channels
Dwi Wahyu Indriati, Naomi Kamasawa, Ko Matsui, Andrea L. Meredith, Masahiko Watanabe, Ryuichi Shigemoto
JOURNAL OF NEUROSCIENCE, 33, 8, 3668, +, SOC NEUROSCIENCE, 2013年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Developmental and Visual Input-Dependent Regulation of the CB1 Cannabinoid Receptor in the Mouse Visual Cortex
Taisuke Yoneda, Katsuro Kameyama, Kazusa Esumi, Yohei Daimyo, Masahiko Watanabe, Yoshio Hata
PLoS ONE, 8, 1, 2013年01月08日, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Coenzyme Q10 prevents peripheral neuropathy and attenuates neuron loss in the db(-)/db(-) mouse, a type 2 diabetes model
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, NATL ACAD SCIENCES, 2013年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Differential GABAB-receptor-mediated effects in perisomatic- and dendrite-targeting parvalbumin interneurons
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, 2013年, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

PRE- AND POST-SYNAPTIC SWITCHES OF GABA ACTIONS ASSOCIATED WITH CL- HOMEOSTATIC CHANGES ARE INDUCED IN THE SPINAL NUCLEUS OF THE TRIGEMINAL NERVE IN A RAT MODEL OF TRIGEMINAL NEUROPATHIC PAIN
B. Wei, T. Kumada, T. Furukawa, K. Inoue, M. Watanabe, K. Sato, A. Fukuda
NEUROSCIENCE, 228, 334, 348, PERGAMON-ELSEVIER SCIENCE LTD, 2013年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Difference in synaptic strengths among competing inputs and absolute synaptic strengths contribute to distinct phase of climbing fiber synapse development in cerebellum
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, SPRINGER JAPAN KK, 2013年, ［査読有り］
英語

Unique neural characteristics of GAD67-containing 5-HTergic neurons in the rat dorsal raphe nucleus
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, JAPANESE PHARMACOLOGICAL SOC, 2013年, ［査読有り］
英語

Autoantibodies to epilepsy-related LGI1 in limbic encephalitis neutralize LGI1-ADAM22 interaction and reduce synaptic AMPA receptors
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, 46, 2013年, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation.
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年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

The NMDA receptor subunit GluN3A protects against 3-nitroproprionic-induced striatal lesions via inhibition of calpain activation
Rebeca Martinez-Turrillas, Elena Puerta, Dhrubajyoti Chowdhury, Sonia Marco, Masahiko Watanabe, Norberto Aguirre, Isabel Perez-Otano
NEUROBIOLOGY OF DISEASE, 48, 3, 290, 298, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2012年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Three Types of Neurochemical Projection from the Bed Nucleus of the Stria Terminalis to the Ventral Tegmental Area in Adult Mice
Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Kohtarou Konno, Miwako Yamasaki, Yuchio Yanagawa, Masabumi Minami, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 32, 50, 18035, 18046, SOC NEUROSCIENCE, 2012年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Three Types of Neurochemical Projection from the Bed Nucleus of the Stria Terminalis to the Ventral Tegmental Area in Adult Mice
Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Kohtarou Konno, Miwako Yamasaki, Yuchio Yanagawa, Masabumi Minami, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 32, 50, 18035, 18046, SOC NEUROSCIENCE, 2012年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Presynaptically Released Cbln1 Induces Dynamic Axonal Structural Changes by Interacting with GluD2 during Cerebellar Synapse Formation
Aya Ito-Ishida, Taisuke Miyazaki, Eriko Miura, Keiko Matsuda, Masahiko Watanabe, Michisuke Yuzaki, Shigeo Okabe
NEURON, 76, 3, 549, 564, CELL PRESS, 2012年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cannabinoid Modulation of Midbrain Urocortin 1 Neurones During Acute and Chronic Stress
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, WILEY-BLACKWELL, 2012年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Distinct Neurochemical and Functional Properties of GAD67-Containing 5-HT Neurons in the Rat Dorsal Raphe Nucleus
Hiroki Shikanai, Takayuki Yoshida, Kohtarou Konno, Miwako Yamasaki, Takeshi Izumi, Yu Ohmura, Masahiko Watanabe, Mitsuhiro Yoshioka
JOURNAL OF NEUROSCIENCE, 32, 41, 14415, 14426, SOC NEUROSCIENCE, 2012年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Elevated expression of calcineurin subunits during active mineralization of developing mouse molar teeth
Shohei Oshima, Masahiko Watanabe
EUROPEAN JOURNAL OF ORAL SCIENCES, 120, 5, 386, 394, WILEY-BLACKWELL, 2012年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Striatal Indirect Pathway Contributes to Selection Accuracy of Learned Motor Actions
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, SOC NEUROSCIENCE, 2012年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Localization of Acetylcholine-Related Molecules in the Retina: Implication of the Communication from Photoreceptor to Retinal Pigment Epithelium
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, PUBLIC LIBRARY SCIENCE, 2012年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Projection Neurons in Lamina III of the Rat Spinal Cord Are Selectively Innervated by Local Dynorphin-Containing Excitatory Neurons
Najma Baseer, Erika Polgar, Masahiko Watanabe, Takahiro Furuta, Takeshi Kaneko, Andrew J. Todd
JOURNAL OF NEUROSCIENCE, 32, 34, 11854, 11863, SOC NEUROSCIENCE, 2012年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Morphological, neurochemical and electrophysiological features of parvalbumin-expressing cells: A likely source of axo-axonic inputs in the mouse spinal dorsal horn
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, 2012年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Projection Neurons in Lamina III of the Rat Spinal Cord Are Selectively Innervated by Local Dynorphin-Containing Excitatory Neurons
Najma Baseer, Erika Polgar, Masahiko Watanabe, Takahiro Furuta, Takeshi Kaneko, Andrew J. Todd
JOURNAL OF NEUROSCIENCE, 32, 34, 11854, 11863, SOC NEUROSCIENCE, 2012年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Synapse type-independent degradation of the endocannabinoid 2-arachidonoylglycerol after retrograde synaptic suppression
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, NATL ACAD SCIENCES, 2012年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Lack of Molecular-Anatomical Evidence for GABAergic Influence on Axon Initial Segment of Cerebellar Purkinje Cells by the Pinceau Formation
Atsushi Iwakura, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 32, 27, 9438, 9448, SOC NEUROSCIENCE, 2012年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Release probability of hippocampal glutamatergic terminals scales with the size of the active zone
Noemi Holderith, Andrea Lorincz, Gergely Katona, Balazs Rozsa, Akos Kulik, Masahiko Watanabe, Zoltan Nusser
NATURE NEUROSCIENCE, 15, 7, 988, 997, NATURE PUBLISHING GROUP, 2012年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Activation of Type 5 Metabotropic Glutamate Receptors and Diacylglycerol Lipase-alpha Initiates 2-Arachidonoylglycerol Formation and Endocannabinoid-Mediated Analgesia
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, SOC NEUROSCIENCE, 2012年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Epigenetic Silencing of HOPX Promotes Cancer Progression in Colorectal Cancer
Hiroshi Katoh, Keishi Yamashita, Mina Waraya, Ofer Margalit, Akira Ooki, Hideaki Tamaki, Hiroyuki Sakagami, Kenichi Kokubo, David Sidransky, Masahiko Watanabe
NEOPLASIA, 14, 7, 559, +, NEOPLASIA PRESS, 2012年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Glutamatergic and GABAergic Innervation of Human Gonadotropin-Releasing Hormone-I Neurons
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, ENDOCRINE SOC, 2012年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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)

Developmental profile of SK2 channel expression and function in CA1 neurons
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, WILEY-BLACKWELL, 2012年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Rewiring of Afferent Fibers in the Somatosensory Thalamus of Mice Caused by Peripheral Sensory Nerve Transection
Yuichi Takeuchi, Miwako Yamasaki, Yasuyuki Nagumo, Keiji Imoto, Masahiko Watanabe, Mariko Miyata
JOURNAL OF NEUROSCIENCE, 32, 20, 6917, 6930, SOC NEUROSCIENCE, 2012年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

NMDA Receptors in GABAergic Synapses during Postnatal Development
Csaba Cserep, Eszter Szabadits, Andras Szonyi, Masahiko Watanabe, Tamas F. Freund, Gabor Nyiri
PLOS ONE, 7, 5, PUBLIC LIBRARY SCIENCE, 2012年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

TRIM67 Protein Negatively Regulates Ras Activity through Degradation of 80K-H and Induces Neuritogenesis
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, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

GABAergic Inhibition Regulates Developmental Synapse Elimination in the Cerebellum
Hisako Nakayama, Taisuke Miyazaki, Kazuo Kitamura, Kouichi Hashimoto, Yuchio Yanagawa, Kunihiko Obata, Kenji Sakimura, Masahiko Watanabe, Masanobu Kano
NEURON, 74, 2, 384, 396, CELL PRESS, 2012年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Tolerance of Anhydrobiotic Eggs of the Tardigrade Ramazzottius varieornatus to Extreme Environments
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, MARY ANN LIEBERT INC, 2012年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Methamphetamine-Evoked Depression of GABA(B) Receptor Signaling in GABA Neurons of the VTA
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, CELL PRESS, 2012年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Striatal direct pathway modulates response time in execution of visual discrimination
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, WILEY-BLACKWELL, 2012年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Unbalance of CB1 receptors expressed in GABAergic and glutamatergic neurons in a transgenic mouse model of Huntington's disease
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, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2012年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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, 2012年, ［査読有り］

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, 2012年, ［査読有り］

Ca(v)2.1 in Cerebellar Purkinje Cells Regulates Competitive Excitatory Synaptic Wiring, Cell Survival, and Cerebellar Biochemical Compartmentalization
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, SOC NEUROSCIENCE, 2012年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function
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, WILEY, 2012年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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

Role of the amygdalar serotonergic system in emotional regulation and disorders　　　　　　　　　　　　　　　
Takeshi Izumi, Takayuki Yoshida, Masahiko Watanabe, Mitsuhiro Yoshioka
Insights into the Amygdala: Structure, Functions and Implications for Disorders, 25, 62, Nova Science Publishers, Inc., 2012年
英語, 論文集(書籍)内論文, Many studies indicate a key role of the amygdala in the pathophysiology of psychiatric traits and disorders, such as anxiety disorders, depression and aggression. Serotonin (5-HT) inhibits the output of the amygdala upon exposure to negative emotional stimuli in healthy subjects. Moreover, serotonin reuptake inhibitors (SSRIs) are the first choice of drug in the treatment of anxiety disorders, and they also may be effective in treating depression and aggression. SSRIs attenuate anxiety-induced c-Fos expression in the basolateral nucleus of the amygdala (BLA), and local injection of SSRIs into this nucleus produces anxiolytic effects. The BLA further receives a strong 5-HTergic innervation from the raphe, and therefore it was regarded as a major target of SSRIs. Based on these observations, the goal of this chapter is to explore 5-HT receptor subtypes and potential mechanisms through which the SSRIs might exert their effects in the amygdala. The BLA has a cortex-like architecture with glutamatergic, spiny, pyramidal-shaped projection neurons showing a burst or regular firing pattern with adaptation following current injection. BLA interneurons are mostly GABAergic, spine-sparse or aspiny, often coexpress calcium-binding proteins like parvalbumin (PV) or calbindin (CB), and show a fast firing pattern with little adaptation. 5-HT1A and 5-HT2A receptors are expressed on both types of BLA neurons, and they are coupled to Gi/o and Gq/11, respectively. Thus, direct stimulation of 5-HT1A receptors on projection neurons inhibits them, whereas stimulation of 5-HT1A receptors on interneurons reduces the output of interneurons, resulting in disinhibition of projection neurons. In contrast, stimulation of 5-HT2A receptors has the opposite effect of exciting interneurons, which leads to a higher inhibition of projection neurons, although projection neurons also may be excited directly. Local injection of 5-HT ligands into the BLA showed that 5-HT1A receptors mediate anxiolytic effects, and 5-HT2C and 5-HT3 receptors mediate anxiogenic effects. Systemic administration of 5-HT4, 5-HT6 and 5-HT7 receptor antagonists also induced anxiolysis, but the role of amygdalar 5-HT1B, 5-HT4, 5-HT6 and 5-HT7 receptors is less clear. Thus, the amygdalar 5-HTergic system modulates physiological and pathological fear and anxiety through 5-HT1A, 5-HT2C and 5-HT3 receptors, and probably also the 5-HT2A receptor. We hypothesize that SSRIs increase the 5-HT concentration in the BLA, and the surplus of 5-HT preferentially activates 5-HT1A receptors on glutamatergic projection neurons rather than on GABAergic interneurons, resulting in a decreased amygdala output. Alternatively, increased 5-HT concentrations following SSRI application may preferentially stimulate 5-HT2A receptors on GABAergic interneurons in the BLA. These two mechanisms are by no means exclusive, and in fact may act together to reduce the overall excitability and output of the amygdala. © 2012 by Nova Science Publishers, Inc. All rights reserved.

SK2 channels are neuroprotective for ischemia-induced neuronal cell death
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, NATURE PUBLISHING GROUP, 2011年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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

Developmental regulation of G protein-gated inwardly-rectifying K+ (GIRK/Kir3) channel subunits in the brain
Laura Fernandez-Alacid, Masahiko Watanabe, Elek Molnar, Kevin Wickman, Rafael Lujan
EUROPEAN JOURNAL OF NEUROSCIENCE, 34, 11, 1724, 1736, WILEY-BLACKWELL, 2011年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

SK2 channels are neuroprotective for ischemia-induced neuronal cell death
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, NATURE PUBLISHING GROUP, 2011年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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

Climbing fiber synapse elimination in cerebellar Purkinje cells
Masahiko Watanabe, Masanobu Kano
EUROPEAN JOURNAL OF NEUROSCIENCE, 34, 10, 1697, 1710, WILEY-BLACKWELL, 2011年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Selective Neural Pathway Targeting Reveals Key Roles of Thalamostriatal Projection in the Control of Visual Discrimination
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, SOC NEUROSCIENCE, 2011年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Redistribution of CB1 Cannabinoid Receptors in the Acute and Chronic Phases of Pilocarpine-Induced Epilepsy
Maria R. Karlocai, Kinga Toth, Masahiko Watanabe, Catherine Ledent, Gabor Juhasz, Tamas F. Freund, Zsofia Magloczky
PLOS ONE, 6, 11, PUBLIC LIBRARY SCIENCE, 2011年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Developmental Switching of Perisomatic Innervation from Climbing Fibers to Basket Cell Fibers in Cerebellar Purkinje Cells
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, SOC NEUROSCIENCE, 2011年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Expression of Vesicular Glutamate Transporters Type 1 and 2 in Sensory and Autonomic Neurons Innervating the Mouse Colorectum
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, WILEY-BLACKWELL, 2011年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

A QUANTITATIVE STUDY OF NEURONAL NITRIC OXIDE SYNTHASE EXPRESSION IN LAMINAE I-III OF THE RAT SPINAL DORSAL HORN
T. C. P. Sardella, E. Polgar, M. Watanabe, A. J. Todd
NEUROSCIENCE, 192, 708, 720, PERGAMON-ELSEVIER SCIENCE LTD, 2011年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Dynorphin is expressed primarily by GABAergic neurons that contain galanin in the rat dorsal horn
Thomas C. P. Sardella, Erika Polgar, Francesca Garzillo, Takahiro Furuta, Takeshi Kaneko, Masahiko Watanabe, Andrew J. Todd
MOLECULAR PAIN, 7, BIOMED CENTRAL LTD, 2011年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Orexin Neurons Receive Glycinergic Innervations
Mari Hondo, Naoki Furutani, Miwako Yamasaki, Masahiko Watanabe, Takeshi Sakurai
PLOS ONE, 6, 9, PUBLIC LIBRARY SCIENCE, 2011年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Orexin Neurons Receive Glycinergic Innervations
Mari Hondo, Naoki Furutani, Miwako Yamasaki, Masahiko Watanabe, Takeshi Sakurai
PLOS ONE, 6, 9, PUBLIC LIBRARY SCIENCE, 2011年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Some lumbar sympathetic neurons develop a glutamatergic phenotype after peripheral axotomy with a note on VGLUT(2)-positive perineuronal baskets
Pablo R. Brumovsky, Kim B. Seroogy, Kerstin H. Lundgren, Masahiko Watanabe, Tomas Hokfelt, G. F. Gebhart
EXPERIMENTAL NEUROLOGY, 230, 2, 258, 272, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Some lumbar sympathetic neurons develop a glutamatergic phenotype after peripheral axotomy with a note on VGLUT(2)-positive perineuronal baskets
Pablo R. Brumovsky, Kim B. Seroogy, Kerstin H. Lundgren, Masahiko Watanabe, Tomas Hokfelt, G. F. Gebhart
EXPERIMENTAL NEUROLOGY, 230, 2, 258, 272, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Acute Cocaine Exposure Weakens GABA(B) Receptor-Dependent G-Protein-Gated Inwardly Rectifying K+ Signaling in Dopamine Neurons of the Ventral Tegmental Area
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, SOC NEUROSCIENCE, 2011年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Distinct functions of kainate receptors in the brain are determined by the auxiliary subunit Neto1
Christoph Straub, David L. Hunt, Miwako Yamasaki, Kwang S. Kim, Masahiko Watanabe, Pablo E. Castillo, Susumu Tomita
NATURE NEUROSCIENCE, 14, 7, 866, U83, NATURE PUBLISHING GROUP, 2011年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Prickle2 is localized in the postsynaptic density and interacts with PSD-95 and NMDA receptors in the brain
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, OXFORD UNIV PRESS, 2011年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

The SK2-long isoform directs synaptic localization and function of SK2-containing channels
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, NATURE PUBLISHING GROUP, 2011年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Postsynaptic P/Q-type Ca2+ channel in Purkinje cell mediates synaptic competition and elimination in developing cerebellum
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, NATL ACAD SCIENCES, 2011年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Prickle2 is localized in the postsynaptic density and interacts with PSD-95 and NMDA receptors in the brain
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, OXFORD UNIV PRESS, 2011年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Postsynaptic P/Q-type Ca2+ channel in Purkinje cell mediates synaptic competition and elimination in developing cerebellum
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, NATL ACAD SCIENCES, 2011年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Retrieval of Conditioned Fear Activates the Basolateral and Intercalated Nucleus of Amygdala
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, WILEY-BLACKWELL, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Characterization of NPY Y2 Receptor Protein Expression in the Mouse Brain. II. Coexistence with NPY, the Y1 Receptor, and Other Neurotransmitter-Related Molecules
Davor Stanic, Jan Mulder, Masahiko Watanabe, Tomas Hokfelt
JOURNAL OF COMPARATIVE NEUROLOGY, 519, 7, 1219, 1257, WILEY, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

AMSH is required to degrade ubiquitinated proteins in the central nervous system
Shunya Suzuki, Keiichi Tamai, Masahiko Watanabe, Masanao Kyuuma, Masao Ono, Kazuo Sugamura, Nobuyuki Tanaka
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 408, 4, 582, 588, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Molecular and Morphological Configuration for 2-Arachidonoylglycerol-Mediated Retrograde Signaling at Mossy Cell-Granule Cell Synapses in the Dentate Gyrus
Motokazu Uchigashima, Maya Yamazaki, Miwako Yamasaki, Asami Tanimura, Kenji Sakimura, Masanobu Kano, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 31, 21, 7700, 7714, SOC NEUROSCIENCE, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Molecular and Morphological Configuration for 2-Arachidonoylglycerol-Mediated Retrograde Signaling at Mossy Cell-Granule Cell Synapses in the Dentate Gyrus
Motokazu Uchigashima, Maya Yamazaki, Miwako Yamasaki, Asami Tanimura, Kenji Sakimura, Masanobu Kano, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 31, 21, 7700, 7714, SOC NEUROSCIENCE, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Galanin-immunoreactivity identifies a distinct population of inhibitory interneurons in laminae I-III of the rat spinal cord
Sheena Y. X. Tiong, Erika Polgar, Josie C. van Kralingen, Masahiko Watanabe, Andrew J. Todd
MOLECULAR PAIN, 7, BIOMED CENTRAL LTD, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

AMSH is required to degrade ubiquitinated proteins in the central nervous system
Shunya Suzuki, Keiichi Tamai, Masahiko Watanabe, Masanao Kyuuma, Masao Ono, Kazuo Sugamura, Nobuyuki Tanaka
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 408, 4, 582, 588, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Characterization of NPY Y2 Receptor Protein Expression in the Mouse Brain. II. Coexistence with NPY, the Y1 Receptor, and Other Neurotransmitter-Related Molecules
Davor Stanic, Jan Mulder, Masahiko Watanabe, Tomas Hokfelt
JOURNAL OF COMPARATIVE NEUROLOGY, 519, 7, 1219, 1257, WILEY, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Retrieval of Conditioned Fear Activates the Basolateral and Intercalated Nucleus of Amygdala
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, WILEY-BLACKWELL, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Quantitative Study of NPY-Expressing GABAergic Neurons and Axons in Rat Spinal Dorsal Horn
Erika Polgar, Thomas C. P. Sardella, Masahiko Watanabe, Andrew J. Todd
JOURNAL OF COMPARATIVE NEUROLOGY, 519, 6, 1007, 1023, WILEY-BLACKWELL, 2011年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Molecular reorganization of endocannabinoid signalling in Alzheimer's disease
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, OXFORD UNIV PRESS, 2011年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

NMDA Receptors in Hippocampal GABAergic Synapses and Their Role in Nitric Oxide Signaling
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, SOC NEUROSCIENCE, 2011年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

NMDA Receptors in Hippocampal GABAergic Synapses and Their Role in Nitric Oxide Signaling
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, SOC NEUROSCIENCE, 2011年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Quantitative Study of NPY-Expressing GABAergic Neurons and Axons in Rat Spinal Dorsal Horn
Erika Polgar, Thomas C. P. Sardella, Masahiko Watanabe, Andrew J. Todd
JOURNAL OF COMPARATIVE NEUROLOGY, 519, 6, 1007, 1023, WILEY-BLACKWELL, 2011年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Molecular reorganization of endocannabinoid signalling in Alzheimer's disease
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, OXFORD UNIV PRESS, 2011年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Development of an anatomical technique for visualizing the mode of climbing fiber innervation in Purkinje cells and its application to mutant mice lacking GluRδ2 and Ca(v)2.1.
Miyazaki T, Watanabe M
Anatomical science international, 86, 1, 10, 18, 1, 2011年03月, ［査読有り］
英語, 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.

Involvement of spinal phosphorylation cascade of Tyr1472-NR2B, Thr286-CaMKII, and Ser831-GluR1 in neuropathic pain
Tayo Katano, Takanobu Nakazawa, Terumasa Nakatsuka, Masahiko Watanabe, Tadashi Yamamoto, Seiji Ito
NEUROPHARMACOLOGY, 60, 4, 609, 616, PERGAMON-ELSEVIER SCIENCE LTD, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses
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, WILEY-BLACKWELL, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

NR3A-containing NMDARs promote neurotransmitter release and spike timing-dependent plasticity
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, NATURE PUBLISHING GROUP, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Differential interactions of cerebellin precursor protein (Cbln) subtypes and neurexin variants for synapse formation of cortical neurons
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, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Glutamate receptor δ2 is essential for input pathway-dependent regulation of synaptic AMPAR contents in cerebellar Purkinje cells.
Yamasaki M, Miyazaki T, Azechi H, Abe M, Natsume R, Hagiwara T, Aiba A, Mishina M, Sakimura K, Watanabe M
The Journal of neuroscience : the official journal of the Society for Neuroscience, 31, 9, 3362, 3374, 9, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Glutamate Receptor delta 2 Is Essential for Input Pathway-Dependent Regulation of Synaptic AMPAR Contents in Cerebellar Purkinje Cells
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, SOC NEUROSCIENCE, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

MOP Reduction During Long-Term Methamphetamine Withdrawal was Restored by Chronic Post-Treatment with Fluoxetine
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, BENTHAM SCIENCE PUBL LTD, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Development of an anatomical technique for visualizing the mode of climbing fiber innervation in Purkinje cells and its application to mutant mice lacking GluR delta 2 and Ca(v)2.1
Taisuke Miyazaki, Masahiko Watanabe
ANATOMICAL SCIENCE INTERNATIONAL, 86, 1, 10, 18, SPRINGER, 2011年03月, ［査読有り］
英語, 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.

NR3A-containing NMDARs promote neurotransmitter release and spike timing-dependent plasticity
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, NATURE PUBLISHING GROUP, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Involvement of spinal phosphorylation cascade of Tyr1472-NR2B, Thr286-CaMKII, and Ser831-GluR1 in neuropathic pain
Tayo Katano, Takanobu Nakazawa, Terumasa Nakatsuka, Masahiko Watanabe, Tadashi Yamamoto, Seiji Ito
NEUROPHARMACOLOGY, 60, 4, 609, 616, PERGAMON-ELSEVIER SCIENCE LTD, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Quantitative Detection of mu Opioid Receptor: Western Blot Analyses Using mu Opioid Receptor Knockout Mice
Shinya Kasai, Hideko Yamamoto, Etsuko Kamegaya, George R. Uhl, Ichiro Sora, Masahiko Watanabe, Kazutaka Ikeda
CURRENT NEUROPHARMACOLOGY, 9, 1, 219, 222, BENTHAM SCIENCE PUBL LTD, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Differential interactions of cerebellin precursor protein (Cbln) subtypes and neurexin variants for synapse formation of cortical neurons
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, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses
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, WILEY-BLACKWELL, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Rho/Rho-kinase signaling pathway controls axon patterning of a specified subset of cranial motor neurons
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, WILEY-BLACKWELL, 2011年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Unique inhibitory synapse with particularly rich endocannabinoid signaling machinery on pyramidal neurons in basal amygdaloid nucleus
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, NATL ACAD SCIENCES, 2011年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

COMPLEMENTARY SYNAPTIC DISTRIBUTION OF ENZYMES RESPONSIBLE FOR SYNTHESIS AND INACTIVATION OF THE ENDOCANNABINOID 2-ARACHIDONOYLGLYCEROL IN THE HUMAN HIPPOCAMPUS
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, PERGAMON-ELSEVIER SCIENCE LTD, 2011年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cellular expression and subcellular localization of secretogranin II in the mouse hippocampus and cerebellum
Taisuke Miyazaki, Miwako Yamasaki, Motokazu Uchigashima, Ayano Matsushima, Masahiko Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 33, 1, 82, 94, WILEY-BLACKWELL PUBLISHING, INC, 2011年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cellular expression and subcellular localization of secretogranin II in the mouse hippocampus and cerebellum
Taisuke Miyazaki, Miwako Yamasaki, Motokazu Uchigashima, Ayano Matsushima, Masahiko Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 33, 1, 82, 94, WILEY-BLACKWELL PUBLISHING, INC, 2011年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Inositol 1,4,5-trisphosphate signaling maintains the activity of glutamate uptake in Bergmann glia
Yohei Okubo, Masato Mashimo, Toshiko Yamazawa, Miwako Yamasaki, Masahiko Watanabe, Toshihiko Murayama, Masamitsu Iino
NEUROSCIENCE RESEARCH, 71, E327, E327, ELSEVIER IRELAND LTD, 2011年, ［査読有り］
英語

Invaginating inhibitory synapse with particularly rich endocannabinoid signaling machinery in the basal nucleus of the amygdala
Takayuki Yoshida, Motokazu Uchigashima, Miwako Yamasaki, Istvan Katona, Maya Yamazaki, Kenji Sakimura, Masanobu Kano, Mitsuhiro Yoshioka, Masahiko Watanabe
NEUROSCIENCE RESEARCH, 71, E93, E94, ELSEVIER IRELAND LTD, 2011年, ［査読有り］
英語

Neurochemical characterization of neurons in the bed nucleus of the stria terminalis projecting to the ventral tegmental area
Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Masabumi Minami, Masahiko Watanabe
NEUROSCIENCE RESEARCH, 71, E322, E322, ELSEVIER IRELAND LTD, 2011年, ［査読有り］
英語

Glutamate transporter GLAST is essential for cytodifferentiation of Bergmann glia and maintenance of excitatory synaptic wiring in the cerebellum
Taisuke Miyazaki, Miwako Yamasaki, Kouichi Hashimoto, Keiko Shimamoto, Kazuhisa Kohda, Michisuke Yuzaki, Kohichi Tanaka, Masanobu Kano, Masahiko Watanabe
NEUROSCIENCE RESEARCH, 71, E63, E63, ELSEVIER IRELAND LTD, 2011年, ［査読有り］
英語

Glycinergic regulation of orexin neurons
Mari Hondo, Naoki Furutani, Miwako Yamasaki, Masahiko Watanabe, Takeshi Sakurai
NEUROSCIENCE RESEARCH, 71, E170, E170, ELSEVIER IRELAND LTD, 2011年, ［査読有り］
英語

Neurochemical characterization of neurons expressing mRNAs for the C1q-like (C1ql) family in the adult mouse brain
Kohtarou Konno, Miwako Yamasaki, Masahiko Watanabe
NEUROSCIENCE RESEARCH, 71, E213, E213, ELSEVIER IRELAND LTD, 2011年, ［査読有り］
英語

Developmental switching of perisomatic innervation from climbing fibers to basket cell fibers in developing cerebellar purkinje cells
Ryoichi Ichikawa, Miwako Yamasaki, Taisuke Miyazaki, Haruyuki Tatsumi, Masahiko Watanabe
NEUROSCIENCE RESEARCH, 71, E215, E215, ELSEVIER IRELAND LTD, 2011年, ［査読有り］
英語

Glutamate receptor delta 2 is essential for input pathway-dependent regulation of synaptic AMPAR contents in cerebellar Purkinje cells
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, ELSEVIER IRELAND LTD, 2011年, ［査読有り］
英語

Quantitative analysis of kainate receptor subunits in the mouse brain
Izumi Watanabe, Hirotsugu Azechi, Kaori Akashi, Maya Yamazaki, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura
NEUROSCIENCE RESEARCH, 71, E315, E316, ELSEVIER IRELAND LTD, 2011年, ［査読有り］
英語

Retrograde Endocannabinoid Signaling Reduces GABAergic Synaptic Transmission to Gonadotropin-Releasing Hormone Neurons
Imre Farkas, Imre Kallo, Levente Deli, Barbara Vida, Erik Hrabovszky, Csaba Fekete, Suzanne M. Moenter, Masahiko Watanabe, Zsolt Liposits
ENDOCRINOLOGY, 151, 12, 5818, 5829, ENDOCRINE SOC, 2010年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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)

Brain-specific Phgdh Deletion Reveals a Pivotal Role for L-Serine Biosynthesis in Controlling the Level of D-Serine, an N-methyl-D-aspartate Receptor Co-agonist, in Adult Brain
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, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Brain-specific Phgdh Deletion Reveals a Pivotal Role for L-Serine Biosynthesis in Controlling the Level of D-Serine, an N-methyl-D-aspartate Receptor Co-agonist, in Adult Brain
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, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Retrograde Endocannabinoid Signaling Reduces GABAergic Synaptic Transmission to Gonadotropin-Releasing Hormone Neurons
Imre Farkas, Imre Kallo, Levente Deli, Barbara Vida, Erik Hrabovszky, Csaba Fekete, Suzanne M. Moenter, Masahiko Watanabe, Zsolt Liposits
ENDOCRINOLOGY, 151, 12, 5818, 5829, ENDOCRINE SOC, 2010年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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)

Subcellular distribution of α1G subunit of T-type calcium channel in the mouse dorsal lateral geniculate nucleus.
Parajuli LK, Fukazawa Y, Watanabe M, Shigemoto R
The Journal of comparative neurology, 518, 21, 4362, 4374, 21, 2010年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Inositol 1,4,5-trisphosphate signaling maintains the activity of glutamate uptake in Bergmann glia
Masato Mashimo, Yohei Okubo, Toshiko Yamazawa, Miwako Yamasaki, Masahiko Watanabe, Toshihiko Murayama, Masamitsu Iino
EUROPEAN JOURNAL OF NEUROSCIENCE, 32, 10, 1668, 1677, WILEY-BLACKWELL PUBLISHING, INC, 2010年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Ablation of glutamate receptor GluRδ2 in adult Purkinje cells causes multiple innervation of climbing fibers by inducing aberrant invasion to parallel fiber innervation territory.
Miyazaki T, Yamasaki M, Takeuchi T, Sakimura K, Mishina M, Watanabe M
The Journal of neuroscience : the official journal of the Society for Neuroscience, 30, 45, 15196, 15209, 45, 2010年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Subcellular Distribution of alpha 1G Subunit of T-type Calcium Channel in the Mouse Dorsal Lateral Geniculate Nucleus
Laxmi Kumar Parajuli, Yugo Fukazawa, Masahiko Watanabe, Ryuichi Shigemoto
JOURNAL OF COMPARATIVE NEUROLOGY, 518, 21, 4362, 4374, WILEY-LISS, 2010年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Ablation of Glutamate Receptor GluR delta 2 in Adult Purkinje Cells Causes Multiple Innervation of Climbing Fibers by Inducing Aberrant Invasion to Parallel Fiber Innervation Territory
Taisuke Miyazaki, Miwako Yamasaki, Tomonori Takeuchi, Kenji Sakimura, Masayoshi Mishina, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 30, 45, 15196, 15209, SOC NEUROSCIENCE, 2010年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Inositol 1,4,5-trisphosphate signaling maintains the activity of glutamate uptake in Bergmann glia
Masato Mashimo, Yohei Okubo, Toshiko Yamazawa, Miwako Yamasaki, Masahiko Watanabe, Toshihiko Murayama, Masamitsu Iino
EUROPEAN JOURNAL OF NEUROSCIENCE, 32, 10, 1668, 1677, WILEY-BLACKWELL PUBLISHING, INC, 2010年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Evidence for oligomerization between GABA(B) receptors and GIRK channels containing the GIRK1 and GIRK3 subunits
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, WILEY-BLACKWELL, 2010年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cytochemical and cytological properties of perineuronal oligodendrocytes in the mouse cortex
Chihiro Takasaki, Miwako Yamasaki, Motokazu Uchigashima, Kohtarou Konno, Yuchio Yanagawa, Masahiko Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 32, 8, 1326, 1336, WILEY-BLACKWELL PUBLISHING, INC, 2010年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Differential Subcellular Recruitment of Monoacylglycerol Lipase Generates Spatial Specificity of 2-Arachidonoyl Glycerol Signaling during Axonal Pathfinding
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, SOC NEUROSCIENCE, 2010年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cytochemical and cytological properties of perineuronal oligodendrocytes in the mouse cortex
Chihiro Takasaki, Miwako Yamasaki, Motokazu Uchigashima, Kohtarou Konno, Yuchio Yanagawa, Masahiko Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 32, 8, 1326, 1336, WILEY-BLACKWELL PUBLISHING, INC, 2010年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Differential Subcellular Recruitment of Monoacylglycerol Lipase Generates Spatial Specificity of 2-Arachidonoyl Glycerol Signaling during Axonal Pathfinding
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, SOC NEUROSCIENCE, 2010年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Evidence for oligomerization between GABA(B) receptors and GIRK channels containing the GIRK1 and GIRK3 subunits
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, WILEY-BLACKWELL, 2010年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Impairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472
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, WILEY-BLACKWELL, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Coupled Activity-Dependent Trafficking of Synaptic SK2 Channels and AMPA Receptors
Mike T. Lin, Rafael Lujan, Masahiko Watanabe, Matthew Frerking, James Maylie, John P. Adelman
JOURNAL OF NEUROSCIENCE, 30, 35, 11726, 11734, SOC NEUROSCIENCE, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Impairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472
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, WILEY-BLACKWELL, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Coupled Activity-Dependent Trafficking of Synaptic SK2 Channels and AMPA Receptors
Mike T. Lin, Rafael Lujan, Masahiko Watanabe, Matthew Frerking, James Maylie, John P. Adelman
JOURNAL OF NEUROSCIENCE, 30, 35, 11726, 11734, SOC NEUROSCIENCE, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

WAG/Rij rats show a reduced expression of CB1 receptors in thalamic nuclei and respond to the CB1 receptor agonist, R(+)WIN55,212-2, with a reduced incidence of spike-wave discharges
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, WILEY-BLACKWELL, 2010年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Antigenic Compartmentation of the Cerebellar Cortex in the Chicken (Gallus domesticus)
Hassan Marzban, Seung-Hyuk Chung, Maryam Kherad Pezhouh, Hans Feirabend, Masahiko Watanabe, Jan Voogd, Richard Hawkes
JOURNAL OF COMPARATIVE NEUROLOGY, 518, 12, 2221, 2239, WILEY-LISS, 2010年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

TARPs gamma-2 and gamma-7 are essential for AMPA receptor expression in the cerebellum
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, WILEY-BLACKWELL, 2010年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Activity-dependent localization in spines of the F-actin capping protein CapZ screened in a rat model of dementia.
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, 7, 2010年06月, ［査読有り］, ［国際誌］
英語, 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.

TARPs gamma-2 and gamma-7 are essential for AMPA receptor expression in the cerebellum
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, WILEY-BLACKWELL, 2010年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Antigenic Compartmentation of the Cerebellar Cortex in the Chicken (Gallus domesticus)
Hassan Marzban, Seung-Hyuk Chung, Maryam Kherad Pezhouh, Hans Feirabend, Masahiko Watanabe, Jan Voogd, Richard Hawkes
JOURNAL OF COMPARATIVE NEUROLOGY, 518, 12, 2221, 2239, WILEY-LISS, 2010年06月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Activity-dependent localization in spines of the F-actin capping protein CapZ screened in a rat model of dementia.
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, 2010年06月, ［査読有り］, ［国際誌］
英語, 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.

Distinct expression of C1q-like family mRNAs in mouse brain and biochemical characterization of their encoded proteins
Takatoshi Iijima, Eriko Miura, Masahiko Watanabe, Michisuke Yuzaki
EUROPEAN JOURNAL OF NEUROSCIENCE, 31, 9, 1606, 1615, WILEY-BLACKWELL, 2010年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Distinct expression of C1q-like family mRNAs in mouse brain and biochemical characterization of their encoded proteins
Takatoshi Iijima, Eriko Miura, Masahiko Watanabe, Michisuke Yuzaki
EUROPEAN JOURNAL OF NEUROSCIENCE, 31, 9, 1606, 1615, WILEY-BLACKWELL, 2010年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Technique of esophagojejunostomy using transoral placement of the pretilted anvil head after laparoscopic gastrectomy for gastric cancer
Shinichi Sakuramoto, Shiro Kikuchi, Nobue Futawatari, Hiromitsu Moriya, Natsuya Katada, Keishi Yamashita, Masahiko Watanabe
SURGERY, 147, 5, 742, 747, MOSBY-ELSEVIER, 2010年05月, ［査読有り］
英語, 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)

CB1 Signaling in Forebrain and Sympathetic Neurons Is a Key Determinant of Endocannabinoid Actions on Energy Balance
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, CELL PRESS, 2010年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cbln1 Is a Ligand for an Orphan Glutamate Receptor delta 2, a Bidirectional Synapse Organizer
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, AMER ASSOC ADVANCEMENT SCIENCE, 2010年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

CB1 Signaling in Forebrain and Sympathetic Neurons Is a Key Determinant of Endocannabinoid Actions on Energy Balance
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, CELL PRESS, 2010年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Imaging extrasynaptic glutamate dynamics in the brain
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, NATL ACAD SCIENCES, 2010年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cbln1 Is a Ligand for an Orphan Glutamate Receptor delta 2, a Bidirectional Synapse Organizer
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, AMER ASSOC ADVANCEMENT SCIENCE, 2010年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Cells from an anhydrobiotic chironomid survive almost complete desiccation
Yuichi Nakahara, Shigeo Imanishi, Kanako Mitsumasu, Yasushi Kanamori, Ken-ichi Iwata, Masahiko Watanabe, Takahiro Kikawada, Takashi Okuda
CRYOBIOLOGY, 60, 2, 138, 146, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2010年04月, ［査読有り］
英語, 研究論文（学術雑誌）, 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

Preferential Localization of Muscarinic M-1 Receptor on Dendritic Shaft and Spine of Cortical Pyramidal Cells and Its Anatomical Evidence for Volume Transmission
Miwako Yamasaki, Minoru Matsui, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 30, 12, 4408, 4418, SOC NEUROSCIENCE, 2010年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Development of the somatosensory cortex, the cerebellum, and the main olfactory system in Semaphorin 3F knockout mice
Ikuo Matsuda, Masahiro Fukaya, Harumi Nakao, Kazuki Nakao, Hideyuki Matsumoto, Kensaku Mori, Masahiko Watanabe, Atsu Aiba
NEUROSCIENCE RESEARCH, 66, 3, 321, 329, ELSEVIER IRELAND LTD, 2010年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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

COX-2 and Prostaglandin EP3/EP4 Signaling Regulate the Tumor Stromal Proangiogenic Microenvironment via CXCL12-CXCR4 Chemokine Systems
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, AMER SOC INVESTIGATIVE PATHOLOGY, INC, 2010年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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)

Preferential Localization of Muscarinic M-1 Receptor on Dendritic Shaft and Spine of Cortical Pyramidal Cells and Its Anatomical Evidence for Volume Transmission
Miwako Yamasaki, Minoru Matsui, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 30, 12, 4408, 4418, SOC NEUROSCIENCE, 2010年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

The Endocannabinoid 2-Arachidonoylglycerol Produced by Diacylglycerol Lipase alpha Mediates Retrograde Suppression of Synaptic Transmission
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, CELL PRESS, 2010年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Heart-Type Fatty Acid Binding Protein Regulates Dopamine D-2 Receptor Function in Mouse Brain
Norifumi Shioda, Yui Yamamoto, Masahiko Watanabe, Bert Binas, Yuji Owada, Kohji Fukunaga
JOURNAL OF NEUROSCIENCE, 30, 8, 3146, 3155, SOC NEUROSCIENCE, 2010年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Demonstration of vesicular glutamate transporter-1 in corticotroph cells in the anterior pituitary of the rat
Zsuzsa S. Kocsis, Csilla S. Molnar, Masahiko Watanabe, Guy Daneels, Dieder Moechars, Zsolt Liposits, Erik Hrabovszky
NEUROCHEMISTRY INTERNATIONAL, 56, 3, 479, 486, PERGAMON-ELSEVIER SCIENCE LTD, 2010年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Signaling Cascade of Diacylglycerol Kinase beta in the Pituitary Intermediate Lobe: Dopamine D2 Receptor/Phospholipase C beta 4/Diacylglycerol Kinase beta/Protein Kinase C alpha
Yasukazu Hozumi, Masahiko Watanabe, Kaoru Goto
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY, 58, 2, 119, 129, HISTOCHEMICAL SOC INC, 2010年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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)

VAV2 and VAV3 as Candidate Disease Genes for Spontaneous Glaucoma in Mice and Humans
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, PUBLIC LIBRARY SCIENCE, 2010年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

VAV2 and VAV3 as Candidate Disease Genes for Spontaneous Glaucoma in Mice and Humans
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, PUBLIC LIBRARY SCIENCE, 2010年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Host prostaglandin EP3 receptor signaling relevant to tumor-associated lymphangiogenesis
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, ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER, 2010年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Ablation of glutamate receptor GluD2 in adult Purkinje cells causes multiple innervation of climbing fibers by ectopic innervation of transverse collaterals
Taisuke Miyazaki, Miwako Yamasaki, Tomonori Takeuchi, Kenji Sakimura, Masayoshi Mishina, Masahiko Watanabe
NEUROSCIENCE RESEARCH, 68, E86, E86, ELSEVIER IRELAND LTD, 2010年, ［査読有り］
英語

TARPs gamma-2 and gamma-7 are functional components of cerebellar AMPA receptor
Maya Yamazaki, Masahiro Fukaya, Kouichi Hashimoto, Miwako Yamasaki, Makoto Itakura, Masami Takahashi, Masanobu Kano, Masahiko Watanabe, Kenji Sakimura
NEUROSCIENCE RESEARCH, 68, E223, E224, ELSEVIER IRELAND LTD, 2010年, ［査読有り］
英語

Spatiotemporal dynamics of glutamate spillover
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, JAPANESE PHARMACOLOGICAL SOC, 2010年, ［査読有り］
英語

Synaptic localization and content of four AMPA receptor subunits at excitatory hippocampal synapses
Miwako Yamasaki, Masahiro Fukaya, Manabu Abe, Kenji Sakimura, Masahiko Watanabe
NEUROSCIENCE RESEARCH, 68, E56, E56, ELSEVIER IRELAND LTD, 2010年, ［査読有り］
英語

Laparoscopy-assisted distal gastrectomy for early gastric cancer with complete situs inversus: Report of a case
Nobue Futawatari, Shiro Kikuchi, Hiromitsu Moriya, Natsuya Katada, Shinichi Sakuramoto, Masahiko Watanabe
SURGERY TODAY, 40, 1, 64, 67, SPRINGER, 2010年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Effect of bilateral lesion of the amygdala 5-HT terminals on the anxiety-related behaviors in rats
Takeshi Izumi, Yu Omura, Yukino Futami, Taku Yamaguchi, Takayuki Yoshida, Masahiko Watanabe, Mitsuhiro Yoshioka
JOURNAL OF PHARMACOLOGICAL SCIENCES, 112, 98P, 98P, JAPANESE PHARMACOLOGICAL SOC, 2010年, ［査読有り］
英語

Formal Analysis of STM Design with SAL Infinite Bounded Model Checker
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, IEEE, 2010年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）, 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.

P/Q type voltage dependent Ca2+ channel is crucial for early postnatal development of climbing fiber to Purkinje cell synapse
Kouichi Hashimoto, Mika Tsujita, Kazuo Kitamura, Taisuke Miyazaki, Maya Yamazaki, Hee-Sup Shin, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
NEUROSCIENCE RESEARCH, 68, E37, E38, ELSEVIER IRELAND LTD, 2010年, ［査読有り］
英語

Phospholipase C beta 4 in the Medial Septum Controls Cholinergic Theta Oscillations and Anxiety Behaviors
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, SOC NEUROSCIENCE, 2009年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Purkinje Cell Compartmentation of the Cerebellum of Microchiropteran Bats
Ji-Young Kim, Hassan Marzban, Seung-Hyuk Chung, Masahiko Watanabe, Leonard M. Eisenman, Richard Hawkes
JOURNAL OF COMPARATIVE NEUROLOGY, 517, 2, 193, 209, WILEY-LISS, 2009年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Psychological characteristics and subjective symptoms as determinants of psychological distress in patients prior to breast cancer diagnosis
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, SPRINGER, 2009年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Evidence against AMPA Receptor-Lacking Glutamatergic Synapses in the Superficial Dorsal Horn of the Rat Spinal Cord
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, SOC NEUROSCIENCE, 2009年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Input-Specific Intrasynaptic Arrangements of Ionotropic Glutamate Receptors and Their Impact on Postsynaptic Responses
Etsuko Tarusawa, Ko Matsui, Timotheus Budisantoso, Elek Molnar, Masahiko Watanabe, Minoru Matsui, Yugo Fukazawa, Ryuichi Shigemoto
JOURNAL OF NEUROSCIENCE, 29, 41, 12896, 12908, SOC NEUROSCIENCE, 2009年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

IQ-ArfGEF/BRAG1 is associated with synaptic ribbons in the mouse retina
Osamu Katsumata, Naoki Ohara, Hideaki Tamaki, Tomoko Niimura, Hideaki Naganuma, Masahiko Watanabe, Hiroyuki Sakagami
EUROPEAN JOURNAL OF NEUROSCIENCE, 30, 8, 1509, 1516, WILEY-BLACKWELL PUBLISHING, INC, 2009年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function
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, WILEY-BLACKWELL, 2009年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Signaling Cascade of Diacylglycerol Kinase {beta} in the Pituitary Intermediate Lobe: Dopamine D2 Receptor/ Phospholipase C{beta}4/ Diacylglycerol Kinase {beta}/ Protein Kinase C{alpha}
Hozumi Y, Watanabe M, Goto K
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 2009年10月01日, ［査読有り］

Input-Specific Intrasynaptic Arrangements of Ionotropic Glutamate Receptors and Their Impact on Postsynaptic Responses
Etsuko Tarusawa, Ko Matsui, Timotheus Budisantoso, Elek Molnar, Masahiko Watanabe, Minoru Matsui, Yugo Fukazawa, Ryuichi Shigemoto
JOURNAL OF NEUROSCIENCE, 29, 41, 12896, 12908, SOC NEUROSCIENCE, 2009年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Rapid Energy Transfer and Improved Performance of Organic Light-Emitting Diodes Using Composite Film Based on pi-Conjugated Polymers
Naoyuki Yamasaki, Masahiko Watanabe, Kimihiro Masuyama, Yasuo Miyake, Hitoshi Kubo, Akihiko Fujii, Masanori Ozaki
JAPANESE JOURNAL OF APPLIED PHYSICS, 48, 10, JAPAN SOCIETY APPLIED PHYSICS, 2009年10月, ［査読有り］
英語, 研究論文（学術雑誌）, 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

Lentiviral vector-mediated rescue of motor behavior in spontaneously occurring hereditary ataxic mice
Akira Iizuka, Kiyohiko Takayama, Takashi Torashima, Miwako Yamasaki, Chiho Koyama, Kazuhiro Mitsumura, Masahiko Watanabe, Hirozaku Hirai
NEUROBIOLOGY OF DISEASE, 35, 3, 457, 465, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2009年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

INFLUENCE OF PARALLEL FIBER-PURKINJE CELL SYNAPSE FORMATION ON POSTNATAL DEVELOPMENT OF CLIMBING FIBER-PURKINJE CELL SYNAPSES IN THE CEREBELLUM
K. Hashimoto, T. Yoshida, K. Sakimura, M. Mishina, M. Watanabe, M. Kano
NEUROSCIENCE, 162, 3, 601, 611, PERGAMON-ELSEVIER SCIENCE LTD, 2009年09月, ［査読有り］
英語, 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.

Phospholipase C beta 4 Expression Identifies a Novel Subset of Unipolar Brush Cells in the Adult Mouse Cerebellum
Seung-Hyuk Chung, Hassan Marzban, Masahiko Watanabe, Richard Hawkes
CEREBELLUM, 8, 3, 267, 276, SPRINGER, 2009年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

NMDA Receptor GluN2B (GluR epsilon 2/NR2B) Subunit Is Crucial for Channel Function, Postsynaptic Macromolecular Organization, and Actin Cytoskeleton at Hippocampal CA3 Synapses
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, SOC NEUROSCIENCE, 2009年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Longitudinal magnetic resonance imaging study on whiplash injury patients: minimum 10-year follow-up
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, SPRINGER TOKYO, 2009年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Rescue of abnormal phenotypes in delta 2 glutamate receptor-deficient mice by the extracellular N-terminal and intracellular C-terminal domains of the delta 2 glutamate receptor
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, WILEY-BLACKWELL PUBLISHING, INC, 2009年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Spinal Endocannabinoids and CB1 Receptors Mediate C-Fiber-Induced Heterosynaptic Pain Sensitization
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, AMER ASSOC ADVANCEMENT SCIENCE, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Early postnatal stress affects 5-HT1A receptor function in the medial prefrontal cortex in adult rats
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, ELSEVIER SCIENCE BV, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Diacylglycerol kinase beta promotes dendritic outgrowth and spine maturation in developing hippocampal neurons
Yasukazu Hozumi, Masahiko Watanabe, Koichi Otani, Kaoru Goto
BMC NEUROSCIENCE, 10, BIOMED CENTRAL LTD, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Protective effects of the free radical scavenger edaravone against glutamate neurotoxicity in nearly pure neuronal culture
Kenjiro Hisano, Masahiko Watanabe, Yuji Morimoto
JOURNAL OF ANESTHESIA, 23, 3, 363, 369, SPRINGER TOKYO, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Functional Coupling between mGluR1 and Ca(v)3.1 T-Type Calcium Channels Contributes to Parallel Fiber-Induced Fast Calcium Signaling within Purkinje Cell Dendritic Spines
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, SOC NEUROSCIENCE, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Subcellular compartment-specific molecular diversity of pre- and post-synaptic GABA(B)-activated GIRK channels in Purkinje cells
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, WILEY-BLACKWELL, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Early postnatal stress affects 5-HT1A receptor function in the medial prefrontal cortex in adult rats
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, ELSEVIER SCIENCE BV, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Protective effects of the free radical scavenger edaravone against glutamate neurotoxicity in nearly pure neuronal culture
Kenjiro Hisano, Masahiko Watanabe, Yuji Morimoto
JOURNAL OF ANESTHESIA, 23, 3, 363, 369, SPRINGER TOKYO, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Translocation of a "Winner" Climbing Fiber to the Purkinje Cell Dendrite and Subsequent Elimination of "Losers" from the Soma in Developing Cerebellum
Kouichi Hashimoto, Ryoichi Ichikawa, Kazuo Kitamura, Masahiko Watanabe, Masanobu Kano
NEURON, 63, 1, 106, 118, CELL PRESS, 2009年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Reduction of Bone Cancer Pain by Activation of Spinal Cannabinoid Receptor 1 and Its Expression in the Superficial Dorsal Horn of the Spinal Cord in a Murine Model of Bone Cancer Pain
Shingo Furuse, Tomoyuki Kawamata, Jun Yamamot, Yukitoshi Niiyama, Keiichi Omote, Masahiko Watanabe, Akiyoshi Namiki
ANESTHESIOLOGY, 111, 1, 173, 186, LIPPINCOTT WILLIAMS & WILKINS, 2009年07月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Reduction of Bone Cancer Pain by Activation of Spinal Cannabinoid Receptor 1 and Its Expression in the Superficial Dorsal Horn of the Spinal Cord in a Murine Model of Bone Cancer Pain
Shingo Furuse, Tomoyuki Kawamata, Jun Yamamot, Yukitoshi