基本情報

所属

• 保健科学研究院　保健科学部門　リハビリテーション科学分野

職名

• 准教授

学位

• 博士（医学）（北海道大学）

ホームページURL

http://kaken.nii.ac.jp/ja/r/90374230

J-Global ID

• 200901078949750580

研究キーワード

• 小脳   グルタミン酸受容体   プルキンエ細胞   登上線維   平行線維   神経標識法   ルガロ細胞   遺伝子改変動物   バスケット細胞   臨界期   カルシウムイオン   脳   シナプス刈込み   シナプス回路   発達   大脳皮質   シナプス   

研究分野

• ライフサイエンス / 神経形態学
• ライフサイエンス / 神経科学一般

担当教育組織

•  学士課程　医学部（保健学科）
•  修士課程　保健科学院
•  博士課程　保健科学院

職歴

• 2019年04月 - 現在 北海道大学 医学部保健学科 准教授
• 2018年03月 - 2019年03月 Yale University School of Medcine, Department of Physiology Visiting Researcher
• 2012年04月 - 2019年03月 北海道大学 医学(系)研究科(研究院) 助教

研究活動情報

論文

• Neuronal DSCAM regulates the peri-synaptic localization of GLAST in Bergmann glia for functional synapse formation

  Ken-ichi Dewa, Nariko Arimura, Wataru Kakegawa, Masayuki Itoh, Toma Adachi, Satoshi Miyashita, Yukiko U. Inoue, Kento Hizawa, Kei Hori, Natsumi Honjoya, Haruya Yagishita, Shinichiro Taya, Taisuke Miyazaki, Chika Usui, Shoji Tatsumoto, Akiko Tsuzuki, Hirotomo Uetake, Kazuhisa Sakai, Kazuhiro Yamakawa, Takuya Sasaki, Jun Nagai, Yoshiya Kawaguchi, Masaki Sone, Takayoshi Inoue, Yasuhiro Go, Noritaka Ichinohe, Kozo Kaibuchi, Masahiko Watanabe, Schuichi Koizumi, Michisuke Yuzaki, Mikio Hoshino

  Nature Communications 15 1 2024年02月01日 

  Abstract In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.
• 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 

  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.
• 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 

  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.
• 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 

  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.
• TMEM163 Regulates ATP-Gated P2X Receptor and Behavior.

  Salm EJ, Dunn PJ, Shan L, Yamasaki M, Malewicz NM, Miyazaki T, Park J, Sumioka A, Hamer RRL, He WW, Morimoto-Tomita M, LaMotte RH, Tomita S

  Cell Rep. 31 2 107704 - 107704 2020年06月 [査読有り]
   

  Fast purinergic signaling is mediated by ATP and ATP-gated ionotropic P2X receptors (P2XRs), and it is implicated in pain-related behaviors. The properties exhibited by P2XRs vary between those expressed in heterologous cells and in vivo. Several modulators of ligand-gated ion channels have recently been identified, suggesting that there are P2XR functional modulators in vivo. Here, we establish a genome-wide open reading frame (ORF) collection and perform functional screening to identify modulators of P2XR activity. We identify TMEM163, which specifically modulates the channel properties and pharmacology of P2XRs. We also find that TMEM163 is required for full function of the neuronal P2XR and a pain-related ATP-evoked behavior. These results establish TMEM163 as a critical modulator of P2XRs in vivo and a potential target for the discovery of drugs for treating pain.
• Compartmentalized input-output organization of Lugaro cells in the cerebellar cortex.

  Miyazaki T, Yamasaki M, Tanaka KF, Watanabe M

  Neuroscience. 10 462 89 - 105 2020年05月 [査読有り]
   

  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.
• 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][通常論文]
   

  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.
• 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][通常論文]
   

  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.
• 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 2017年11月 [査読有り][通常論文]
   

  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.
• 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 2017年08月 [査読有り][通常論文]
   

  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  2017年08月 [査読有り][通常論文]
   

  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.
• 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 2017年07月 [査読有り][通常論文]
   

  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.
• 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  2017年06月 [査読有り][通常論文]
   

  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.
• 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 2016年09月 [査読有り][通常論文]
   

  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.
• 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 2016年07月 [査読有り][通常論文]
   

  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.
• 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 2016年06月 [査読有り][通常論文]
   

  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.
• 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 2016年02月 [査読有り][通常論文]
   

  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.
• 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 2015年03月 [査読有り][通常論文]
   

  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 2015年02月24日 [査読有り][通常論文]
   

  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.
• 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  2015年01月 [査読有り][通常論文]
  
• 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 2015年01月 [査読有り][通常論文]
   

  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.
• 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 2015年01月 [査読有り][通常論文]
   

  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.
• 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 5586  2014年11月 [査読有り][通常論文]
   

  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.
• 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 2014年09月 [査読有り][通常論文]
   

  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  2014年09月 [査読有り][通常論文]
   

  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.
• 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 2014年08月 [査読有り][通常論文]
   

  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.
• 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 2014年05月 [査読有り][通常論文]
   

  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.
• 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  2013年08月 [査読有り][通常論文]
   

  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.
• 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 2013年02月 [査読有り][通常論文]
   

  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.
• 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 2013年 [査読有り][通常論文]
   

  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.
• Difference in synaptic strengths among competing inputs and absolute synaptic strengths contribute to distinct phase of climbing fiber synapse development in cerebellum

  Kawata Shinya, Hashimoto Kouichi, Yamazaki Maya, Miyazaki Taisuke, Yamasaki Miwako, Mikuni Takayasu, Watanabe Masahiko, Sakimura Kenji, Kano Masanobu

  JOURNAL OF PHYSIOLOGICAL SCIENCES 63 S188  2013年 [査読有り][通常論文]
  
• Three types of neurochemical projection from the bed nucleus of the stria terminalis to the ventral tegmental area in adult mice.

  Kudo T, Uchigashima M, Miyazaki T, Konno K, Yamasaki M, Yanagawa Y, Minami M, Watanabe M

  The Journal of neuroscience : the official journal of the Society for Neuroscience 32 50 18035 - 18046 50 2012年12月 [査読有り][通常論文]
   

  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 (∼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 α1 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 2012年11月 [査読有り][通常論文]
   

  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.
• 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 2012年07月 [査読有り][通常論文]
   

  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.
• 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 2012年04月 [査読有り][通常論文]
   

  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.
• 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 2012年01月 [査読有り][通常論文]
   

  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.
• 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 2011年11月 [査読有り][通常論文]
   

  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.
• 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 2011年06月 [査読有り][通常論文]
   

  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.
• 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][通常論文]
   

  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 GluRδ2 and Cav2.1 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. © 2010 Japanese Association of Anatomists.
• 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][通常論文]
   

  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.
• Cellular expression and subcellular localization of secretogranin II in the mouse hippocampus and cerebellum.

  Miyazaki T, Yamasaki M, Uchigashima M, Matsushima A, Watanabe M

  The European journal of neuroscience 33 1 82 - 94 1 2011年01月 [査読有り][通常論文]
  
• Neurochemical characterization of neurons in the bed nucleus of the stria terminalis projecting to the ventral tegmental area

  Kudo Takehiro, Uchigashima Motokazu, Miyazaki Taisuke, Yamasaki Miwako, Minami Masabumi, Watanabe Masahiko

  NEUROSCIENCE RESEARCH 71 E322  2011年 [査読有り][通常論文]
  
• Glutamate transporter GLAST is essential for cytodifferentiation of Bergmann glia and maintenance of excitatory synaptic wiring in the cerebellum

  Miyazaki Taisuke, Yamasaki Miwako, Hashimoto Kouichi, Shimamoto Keiko, Kohda Kazuhisa, Yuzaki Michisuke, Tanaka Kohichi, Kano Masanobu, Watanabe Masahiko

  NEUROSCIENCE RESEARCH 71 E63  2011年 [査読有り][通常論文]
  
• Developmental switching of perisomatic innervation from climbing fibers to basket cell fibers in developing cerebellar purkinje cells

  Ichikawa Ryoichi, Yamasaki Miwako, Miyazaki Taisuke, Tatsumi Haruyuki, Watanabe Masahiko

  NEUROSCIENCE RESEARCH 71 E215  2011年 [査読有り][通常論文]
  
• Glutamate receptor delta 2 is essential for input pathway-dependent regulation of synaptic AMPAR contents in cerebellar Purkinje cells

  Yamasaki Miwako, Miyazaki Taisuke, Azechi Hirotsugu, Abe Manabu, Natsume Rie, Hagiwara Teruki, Aiba Atsu, Mishina Masayoshi, Sakimura Kenji, Watanabe Masahiko

  NEUROSCIENCE RESEARCH 71 E93  2011年 [査読有り][通常論文]
  
• 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][通常論文]
   

  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.
• 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 2010年04月 [査読有り][通常論文]
   

  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.
• Ablation of glutamate receptor GluD2 in adult Purkinje cells causes multiple innervation of climbing fibers by ectopic innervation of transverse collaterals

  Miyazaki Taisuke, Yamasaki Miwako, Takeuchi Tomonori, Sakimura Kenji, Mishina Masayoshi, Watanabe Masahiko

  NEUROSCIENCE RESEARCH 68 E86  2010年 [査読有り][通常論文]
  
• P/Q type voltage dependent Ca2+ channel is crucial for early postnatal development of climbing fiber to Purkinje cell synapse

  Hashimoto Kouichi, Tsujita Mika, Kitamura Kazuo, Miyazaki Taisuke, Yamazaki Maya, Shin Hee-Sup, Watanabe Masahiko, Sakimura Kenji, Kano Masanobu

  NEUROSCIENCE RESEARCH 68 E37 - E38 2010年 [査読有り][通常論文]
  
• 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 2009年08月 [査読有り][通常論文]
   

  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.
• The N-Terminal Domain of GluD2 (GluR delta 2) Recruits Presynaptic Terminals and Regulates Synaptogenesis in the Cerebellum In Vivo

  Wataru Kakegawa, Taisuke Miyazaki, Kazuhisa Kohda, Keiko Matsuda, Kyoichi Emi, Junko Motohashi, Masahiko Watanabe, Michisuke Yuzaki

  JOURNAL OF NEUROSCIENCE 29 18 5738 - 5748 2009年05月 [査読有り][通常論文]
   

  The delta 2 glutamate receptor (GluR delta 2; GluD2), which is predominantly expressed on postsynaptic sites at parallel fiber (PF)-Purkinje cell synapses in the cerebellum, plays two crucial roles in the cerebellum: the formation of PF synapses and the regulation of long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Although the induction of LTD and motor learning absolutely require signaling via the cytoplasmic C-terminal domain of GluD2, the mechanisms by which GluD2 regulates PF synaptogenesis have remained unclear. Here, we examined the role of the extracellular N-terminal domain (NTD) of GluD2 on PF synaptogenesis by injecting Sindbis virus carrying wild-type (GluD2(wt)) or mutant GluD2 into the subarachnoid supracerebellar space of GluD2-null mice. Remarkably, the expression of GluD2(wt), but not of a mutant GluD2 lacking the NTD (GluD2(Delta NTD)), rapidly induced PF synapse formation and rescued gross motor dyscoordination in adult GluD2-null mice just 1 d after injection. In addition, although the kainate receptor GluR6 (GluK2) did not induce PF synaptogenesis, a chimeric GluK2 that contained the NTD of GluD2 (GluD2(NTD)-GluK2) did. Similarly, GluD2(wt) and GluD2(NTD)-GluK2, but not GluD2(Delta NTD), induced synaptogenesis in heterologous cells in vitro. In contrast, LTD was restored in GluD2-null Purkinje cells expressing a mutant GluD2 lacking the NTD. These results indicate that the NTD of GluD2 is necessary and sufficient for the function of GluD2 in the regulation of PF -Purkinje cell synaptogenesis. Furthermore, our results suggest that GluD2 differently regulates PF synaptogenesis and cerebellar LTD through the extracellular NTD and the cytoplasmic C-terminal end, respectively.
• GLAST IS ESSENTIAL FOR CYTOLOGICAL DIFFERENTIATION OF BERGMANN GLIA AND CLIMBING FIBER MONOINNERVATION BY SUPPRESSING ECTOPIC INNERVATION

  Miyazaki Taisuke, Yamasaki Miwako, Tanaka Kouichi, Watanabe Masahiko

  JOURNAL OF PHYSIOLOGICAL SCIENCES 59 198  2009年 [査読有り][通常論文]
  
• Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant Ca(v)2.1 channels

  Kei Watase, Curtis F. Barrett, Taisuke Miyazaki, Taro Ishiguro, Kinya Ishikawa, Yuanxin Hu, Toshinori Unno, Yaling Sun, Sayumi Kasai, Masahiko Watainabe, Christopher M. Gomez, Hidehiro Mizusawa, Richard W. Tsien, Huda Y. Zoghbi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 33 11987 - 11992 2008年08月 [査読有り][通常論文]
   

  Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (Ca(v)) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of Ca(v)2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein. We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine (Sca6(84Q)) developed progressive motor impairment and aggregation of mutant Ca(v)2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca2(+) channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca6(84Q) neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant Ca(v)2.1 channels.
• Cerebellar pathology in transgenic mice expressing the pseudorabies virus immediate-early protein IE180

  Yukiko Tomioka, Taisuke Miyazaki, Satoshi Taharaguchi, Saori Yoshino, Masami Morimatsu, Toshimitsu Uede, Etsuro Ono, Masahiko Watanabe

  EUROPEAN JOURNAL OF NEUROSCIENCE 27 8 2115 - 2132 2008年04月 [査読有り][通常論文]
   

  Pseudorabies virus is an alphaherpesvirus causing fatal neurological diseases in animals. Pseudorabies virus carries a gene encoding immediate-early (IE) protein IE180, which controls the transcription of other viral and host cell genes. Previously, we reported that transgenic expression of IE180 in mice causes severe ataxia and cerebellar deformity. Here we identified profound abnormalities in adult IE180 transgenic mice, including malpositioning of Purkinje cells (PCs), granule cells (GCs) and Bergmann glia (BG), impaired dendritogenesis and synaptogenesis in PCs, disoriented BG fibers, absence of molecular layer interneurons, and increased apoptosis of neurons and glia. In accordance with the cellular defects, we found the expression of IE180 in PCs, GCs and astrocytes during cerebellar development. We next examined transgenic mice expressing truncated IE180 mutants: dlN132 lacking the acidic transcriptional active domain, dlC629 lacking the nuclear localization signal and dlC1081 having all known domains but lacking the carboxyl-terminal sequence. Despite similar expression levels of the transgenes, ataxia and cerebellar defects were only manifested in the dlC1081 transgenic mice but their phenotypes were milder compared with the IE180 transgenic mice. In the dlC1081 transgenic mice, cerebellar neurons and glia were normally positioned but cerebellar size was severely reduced due to GC deficits. Interestingly, dlC1081 was mainly expressed in the GCs with low expression in a few BG. Taken together, the present findings clarified a causal relationship between cerebellar pathology and cellular expression of IE180, and further afforded an experimental insight into different symptomatic severity as a consequence of different cellular defects caused by such cytotoxic viral agents.
• Differential regulation of synaptic plasticity and cerebellar motor learning by the C-terminal PDZ-binding motif of GluR delta 2

  Wataru Kakegawa, Taisuke Miyazaki, Kyoichi Emi, Keiko Matsuda, Kazuhisa Kohda, Junko Motohashi, Masayoshi Mishina, Shigenori Kawahara, Masahiko Watanabe, Michisuke Yuzaki

  JOURNAL OF NEUROSCIENCE 28 6 1460 - 1468 2008年02月 [査読有り][通常論文]
   

  The delta 2 glutamate receptor (GluR delta 2) is predominantly expressed in Purkinje cells and plays crucial roles in cerebellar functions: GluR delta 2(-/-) mice display ataxia and impaired motor learning. In addition, long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is abrogated, and synapse formation with PFs and climbing fibers (CFs) is severely disturbed in GluR delta 2(-/-) Purkinje cells. Recently, we demonstrated that abrogated LTD was restored in GluR delta 2(-/-) Purkinje cells by the virus-mediated expression of the wild-type GluR delta 2 transgene (Tgwt) but not by that of mutant GluR delta 2 lacking the C-terminal seven residues to which several PDZ proteins bind (Tg(Delta CT7)). These results indicated that the C terminus of GluR delta 2 conveys the signal(s) necessary for LTD. In contrast, other phenotypes of GluR delta 2(-/-) cerebellum, especially morphological abnormalities at PF and CF synapses, could not be rescued by virus-mediated transient expression. Thus, whether these phenotypes are mediated by the same signaling pathway remains unclear. To address these issues and to further delineate the function of GluR delta 2 in vivo, we generated transgenic mice that expressed Tg(Delta CT7) on a GluR delta 2(-/-) background. Interestingly, although Tg(Delta CT7) restored abnormal PF and CF synapse formation almost completely, it could not rescue abrogated LTD in GluR Delta 2(-/-) Purkinje cells. Furthermore, although the gross motor discoordination of GluR Delta 2(-/-) mice was restored, the cerebellar motor learning underlying delayed eyeblink conditioning remained impaired. These results indicate that LTD induction and motor learning are regulated by signaling via the C-terminal end of GluR delta 2, whereas other functions may be differentially regulated by other regions of GluR delta 2.
• Effects of FAK ablation on cerebellar foliation, Bergmann glia positioning and climbing fiber territory on Purkinje cells

  Fumihiro Watanabe, Taisuke Miyazaki, Tomonori Takeuchi, Masahiro Fukaya, Takanori Nomura, Shigeru Noguchi, Hisashi Mori, Kenji Sakimura, Masahiko Watanabe, Masayoshi Mishina

  EUROPEAN JOURNAL OF NEUROSCIENCE 27 4 836 - 854 2008年02月 [査読有り][通常論文]
   

  Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is widely expressed in the brain, and plays key roles in various cellular processes in response to both extracellular and intracellular stimuli. Here, we explored the role of FAK in cerebellar development. In the mouse cerebellum, FAK was found to be distributed as tiny cytoplasmic aggregates in various neuronal and glial elements, including Purkinje cells (PCs), Bergmann glia (BG), parallel fiber (PF)-terminals and climbing fiber (CF)-terminals. The neuron/glia-specific ablation of FAK impaired cerebellar foliation, such as variable decreases in foliation sizes and the lack of intercrural and precentral fissures. Some of the BG cells became situated ectopically in the molecular layer. Furthermore, the FAK ablation altered the innervation territories of CFs and PFs on PCs. CF innervation regressed to the basal portion of proximal dendrites and somata, whereas ectopic spines protruded from proximal dendrites and PFs expanded their territory by innervating the ectopic spines. Furthermore, the persistence of surplus CFs innervating PC somata caused multiple innervation. When FAK was selectively ablated in PCs, diminished dendritic innervation and persistent somatic innervation by CFs were observed, whereas cerebellar foliation and cell positioning of BG were normally retained. These results suggest that FAK in various neuronal and glial elements is required for the formation of normal histoarchitecture and cytoarchitecture in the cerebellum, and for the construction of proper innervation territory and synaptic wiring in PCs.
• IQ-ArfGEF/BRAG1 is a guanine nucleotide exchange factor for Arf6 that interacts with PSD-95 at postsynaptic density of excitatory synapses

  Hiroyuki Sakagami, Masashi Sanda, Masahiro Fukaya, Taisuke Miyazaki, Jun Sukegawa, Teruyuki Yanagisawa, Tatsuo Suzuki, Kohji Fukunaga, Masahiko Watanabe, Hisatake Kondo

  NEUROSCIENCE RESEARCH 60 2 199 - 212 2008年02月 [査読有り][通常論文]
   

  ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates dendritic differentiation possibly through the organization of actin cytoskeleton and membrane traffic. Here, we characterized IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor (GEF) for Arf6, in the mouse brain. In vivo Arf pull down assay demonstrated that IQ-ArfGEF/BRAG1 activated Arf6 more potently than Arf1. IQ-ArfGEF/BRAG1 mRNA was abundantly expressed in the brain with higher levels in forebrain structures and cerebellar granule cells. In hippocampal neurons, IQ-ArfGEF/BRAG1 mRNA was localized not only at neuronal cell bodies but also at dendritic processes, indicating its dendritic transport and localization. Immunoprecipitation and in vitro binding experiments revealed that IQ-ArfGEF/BRAG1 formed a protein complex with N-methyl-D-aspartate (NMDA)-type glutamate receptors through the interaction with a postsynaptic density (PSD) scaffold protein, PSD-95. Immunohistochemical analysis demonstrated that IQ-ArfGEF/BRAG1 was localized preferentially at the postsynaptic density of asymmetrical synapses on dendritic spines, but was lacking at GABAA receptor-carrying inhibitory synapses. Taken together, IQ-ArfGEF/BRAG1 forms a postsynaptic protein complex containing PSD-95 and NMDA receptors at excitatory synapses, where it may function as a GEF for Arf6. (C) 2007 Published by Elsevier Ireland Ltd and the Japan Neuroscience Society.
• Modulation of inhibitory synaptic transmission in cerebellar Purkinje cells by CbIn1

  Ishida Aya, Matsuda Keiko, Miyazaki Taisuke, Miura Eriko, Iijima Takatoshi, Kondo Tetsuro, Watanabe Masahiko, Yuzaki Michisuke

  NEUROSCIENCE RESEARCH 61 S78  2008年 [査読有り][通常論文]
  
• Abnormal features in mutant cerebellar Purkinje cells lacking junctophilins

  Atsushi Ikeda, Taisuke Miyazaki, Sho Kakizawa, Yasushi Okuno, Soken Tsuchiya, Akira Myomoto, Shin-Ya Saito, Tetsuji Yamamoto, Tetsuo Yamazaki, Masamitsu Iino, Gozoh Tsujimoto, Masahiko Watanabe, Hiroshi Takeshima

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 363 3 835 - 839 2007年11月 [査読有り][通常論文]
   

  Junctional membrane complexes (JMCs) generated by junctophilins are required for Ca (2+) -Mediated communication between cell-surface and intracellular channels in excitable cells. Knockout mice lacking neural junctophilins (JP-DKO) show severe motor defects and irregular cerebellar plasticity due to abolished channel crosstalk in Purkinje cells (PCs). To precisely understand aberrations in JP-DKO mice, we further analyzed the mutant PCs. During the induction of cerebellar plasticity via electrical stimuli, JP-DKO PCs showed insufficient depolarizing responses. Immunochemistry detected mild impairment in synaptic maturation and hyperphosphorylation of protein kinase C gamma in JP-DKO PCs. Moreover, gene expression was slightly altered in the JP-DKO cerebellum. Therefore, the mutant PCs bear marginal but widespread abnormalities, all of which likely cause cerebellar motor defects in JP-DKO mice. (C) 2007 Elsevier Inc. All rights reserved.
• CD3 and immunoglobulin G Fc receptor regulate cerebellar functions

  Kazuhiro Nakamura, Hirokazu Hirai, Takashi Torashima, Taisuke Miyazaki, Hiromichi Tsurui, Yan Xiu, Mareki Ohtsuji, Qing Shun Lin, Kazuyuki Tsukamoto, Hiroyuki Nishimura, Masao Ono, Masahiko Watanabe, Sachiko Hirose

  MOLECULAR AND CELLULAR BIOLOGY 27 14 5128 - 5134 2007年07月 [査読有り][通常論文]
   

  The immune and nervous systems display considerable overlap in their molecular repertoire. Molecules originally shown to be critical for immune responses also serve neuronal functions that include normal brain development, neuronal differentiation, synaptic plasticity, and behavior. We show here that Fc gamma RIIB, a low-affinity immunoglobulin G Fc receptor, and CD3 are involved in cerebellar functions. Although membranous CD3 and Fc gamma RIIB are crucial regulators on different cells in the immune system, both CD3 epsilon and Fc gamma RIIB; are expressed on Purkinje cells in the cerebellum. Both CD3 epsilon-deficient mice and Fc gamma RIIB-deficient mice showed an impaired development of Purkinje neurons. In the adult, rotarod performance of these mutant mice was impaired at high speed. In the two knockout mice, enhanced paired-pulse facilitation of parallel fiber-Purkinje cell synapses was shared. These results indicate that diverse immune molecules play critical roles in the functional establishment in the cerebellum.
• Junctophilin-mediated channel crosstalk essential for cerebellar synaptic plasticity

  Sho Kakizawa, Yasushi Kishimoto, Kouichi Hashimoto, Taisuke Miyazaki, Kazuharu Furutani, Hidemi Shimizu, Masahiro Fukaya, Miyuki Nishi, Hiroyuki Sakagami, Atsushi Ikeda, Hisatake Kondo, Masanobu Kano, Masahiko Watanabe, Masamitsu Iino, Hiroshi Takeshima

  EMBO JOURNAL 26 7 1924 - 1933 2007年04月 [査読有り][通常論文]
   

  Functional crosstalk between cell-surface and intracellular ion channels plays important roles in excitable cells and is structurally supported by junctophilins (JPs) in muscle cells. Here, we report a novel form of channel crosstalk in cerebellar Purkinje cells (PCs). The generation of slow afterhyperpolarization (sAHP) following complex spikes in PCs required ryanodine receptor (RyR)-mediated Ca2+ induced Ca2+ release and the subsequent opening of small-conductance Ca2+ activated K+ (SK) channels in somatodendritic regions. Despite the normal expression levels of these channels, sAHP was abolished in PCs from mutant mice lacking neural JP subtypes (JP-DKO), and this defect was restored by exogenously expressing JPs or enhancing SK channel activation. The stimulation paradigm for inducing long-term depression (LTD) at parallel fiber-PC synapses adversely established long-term potentiation in the JP-DKO cerebellum, primarily due to the sAHP deficiency. Furthermore, JP-DKO mice exhibited impairments of motor coordination and learning, although normal cerebellar histology was retained. Therefore, JPs support the Ca2+-mediated communication between voltage-gated Ca2+ channels, RyRs and SK channels, which modulates the excitability of PCs and is fundamental to cerebellar LTD and motor functions.
• Ca2+ permeability of the channel pore is not essential for the delta 2 glutamate receptor to regulate synaptic plasticity and motor coordination

  Wataru Kakegawa, Taisuke Miyazaki, Hirokazu Hirai, Junko Motohashi, Masayoshi Mishina, Masahiko Watanabe, Michisuke Yuzaki

  JOURNAL OF PHYSIOLOGY-LONDON 579 3 729 - 735 2007年03月 [査読有り][通常論文]
   

  The delta 2 glutamate receptor (GluR delta 2) plays a crucial role in cerebellar functions; mice with a disrupted GluR delta 2 gene (GluR delta 2(-/-)) display impaired synapse formation and abrogated long-term depression (LTD). However, the mechanisms by which GluR delta 2 functions have remained unclear. Because a GluR delta 2 mutation in lurcher mice causes channel activities characterized by Ca2+ permeability, GluR delta 2 was previously suggested to serve as a Ca2+-permeable channel in Purkinje cells. To test this hypothesis, we introduced a GluR delta 2 transgene, which had a mutation (Gln618Arg) in the putative channel pore, into GluR delta 2(-/-) mice. Interestingly, the mutant transgene rescued the major functional and morphological abnormalities of GluR delta 2(-/-) Purkinje cells, such as enhanced paired-pulse facilitation, impaired LTD at parallel fibre synapses, and sustained innervation by multiple climbing fibres. These results indicate that the conserved glutamine residue in the channel pore, which is crucial for all Ca2+-permeable glutamate receptors, is not essential for the function of GluR delta 2.
• GLAST is essential for cytological differentiation of Bergmann glia and climbing fiber monoinnervation by suppressing local ectopic innervation

  Miyazaki Taisuke, Tanaka Kouichi, Watanabe Masahiko

  NEUROSCIENCE RESEARCH 58 S132  2007年 [査読有り][通常論文]
  
• Functional uncoupling between Ca2+ release and afterhyperpolarization in mutant hippocampal neurons lacking junctophilins

  Shigeki Moriguchi, Miyuki Nishi, Shinji Komazaki, Hiroyuki Sakagami, Taisuke Miyazaki, Haruko Masumiya, Shin-ya Saito, Masahiko Watanabe, Hisatake Kondo, Hiromu Yawo, Kohji Fukunaga, Hiroshi Takeshima

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 28 10811 - 10816 2006年07月 [査読有り][通常論文]
   

  Junctional membrane complexes (JMCs) composed of the plasma membrane and endoplasmic/sarcoplasmic reticulum seem to be a structural platform for channel crosstalk. Junctophilins (JPs) contribute to JMC formation by spanning the sarcoplasmic reticulum membrane and binding with the plasma membrane in muscle cells. In this article, we report that mutant JP double-knockout (JP-DKO) mice lacking neural JP subtypes exhibited an irregular hindlimb reflex and impaired memory. Electrophysiological experiments indicated that the activation of small-conductance Ca2+-activated K+ channels responsible for afterhyperpolarization in hippocampal neurons requires endoplasmic reticulum Ca2+ release through ryanodine receptors, triggered by NMDA receptor-mediated Ca2+ influx. We propose that in JP-DKO neurons lacking afterhyperpolarization, the functional communications between NMDA receptors, ryanodine receptors, and small-conductance Ca2+-activated K+ channels are disconnected because of JMC disassembly. Moreover, JP-DKO neurons showed an impaired long-term potentiation and hyperactivation of Ca2+/calmodulin-dependent protein kinase II. Therefore, JPs seem to have an essential role in neural excitability fundamental to plasticity and integrated functions.
• Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family

  Taisuke Miyazaki, Kouichi Hashimoto, Atsushi Uda, Hiroyuki Sakagami, Yoshitaka Nakamura, Shin-ya Saito, Miyuki Nishi, Hideaki Kume, Akira Tohgo, Izumi Kaneko, Hisatake Kondo, Kohji Fukunaga, Masanobu Kano, Masahiko Watanabe, Hiroshi Takeshima

  FEBS LETTERS 580 17 4057 - 4064 2006年07月 [査読有り][通常論文]
   

  By DNA cloning, we have identified the BSRP (brain-specific receptor-like proteins) family of three members in mammalian genomes. BSRPs were predominantly expressed in the soma and dendrites of neurons and localized in the endoplasmic reticulum (ER). Expression levels of BSRPs seemed to fluctuate greatly during postnatal cerebellar maturation. Triple-knockout mice lacking BSRP members exhibited motor discoordination, and Purkinje cells (PCs) were often innervated by multiple climbing fibers with different neuronal origins in the mutant cerebellum. Moreover, the phosphorylation levels of protein kinase C alpha (PKC alpha) were significantly downregulated in the mutant cerebellum. Because cerebellar maturation and plasticity require metabotropic glutamate receptor signaling and resulting PKC activation, BSRPs are likely involved in ER functions supporting PKCa activation in PCs. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• Impaired cerebellar functions in mutant mice lacking DNER

  A Tohgo, M Eiraku, T Miyazaki, E Miura, S Kawaguchi, M Nishi, M Watanabe, T Hirano, M Kengaku, H Takeshima

  MOLECULAR AND CELLULAR NEUROSCIENCE 31 2 326 - 333 2006年02月 [査読有り][通常論文]
   

  DNER is a transmembrane protein carrying extracellular EGF repeats and is strongly expressed in Purkinje cells (PCs) in the cerebellum. Current study indicated that DNER functions as a new Notch ligand and mediates the functional communication via cell-cell interaction. By producing and analyzing knockout mice lacking DNER, we demonstrate its essential roles in functional and morphological maturation of the cerebellum. The knockout mice exhibited motor discoordination in the fixed bar and rota-rod tests. The cerebellum from the knockout mice showed significant retardation in morphogenesis and persistent abnormality in fissure organization. Histochemical and electrophysiological analyses detected that PCs retained multiple innervations from climbing fibers (CFs) in the mutant cerebellum. Synaptic transmission from parallel fibers (PFs) or CFs to PCs was apparently normal, while glutamate clearance at the PF-PC synapses was significantly impaired in the mutant mice. Moreover, the protein level of GLAST, the glutamate transporter predominantly expressed in Bergmann glia (BG), was reduced in the mutant cerebellum. Our results indicate that DNER takes part in stimulation of BG maturation via intercellular communication and is essential for precise cerebellar development. (c) 2005 Elsevier Inc. All rights reserved.
• Morphological analysis of multiple climbing fiber innervation in PKC gamma-deficient mouse

  Yamasaki Miwako, Hashimoto Kouichi, Miyazaki Taisuke, Watanabe Masahiko, Kano Masanobu

  NEUROSCIENCE RESEARCH 55 S174  2006年 [査読有り][通常論文]
  
• Maintenance of presynaptic function by AMPA receptor-mediated excitatory postsynaptic activity in adult brain

  S Kakizawa, T Miyazaki, D Yanagihara, M Iino, M Watanabe, M Kano

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 52 19180 - 19185 2005年12月 [査読有り][通常論文]
   

  Activity-dependent synaptic modification occurs in both developing and mature animals. For reliable information transfer and storage, however, once established, synapses must be maintained stably. We investigated how chronic blockade of neuronal activity or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors affects excitatory climbing fiber (CF) to Purkinje cell (PC) synapses in adult mouse cerebellum. Both treatments caused reduced glutamate concentration transient at the synaptic cleft, decreased frequency of quantal excitatory postsynaptic current, and diminished CF innervation of PC shaft dendrites but no change in CF's release probability. These results indicate that, in the mature cerebellum, AMPA receptor-mediated excitatory postsynaptic activity maintains CF's functional glutamate-release sites and its innervation of PC shaft dendrites.
• Cbln1 is essential for synaptic integrity and plasticity in the cerebellum

  H Hirai, Z Pang, DH Bao, T Miyazaki, LY Li, E Miura, J Parris, YQ Rong, M Watanabe, M Yuzaki, JI Morgan

  NATURE NEUROSCIENCE 8 11 1534 - 1541 2005年11月 [査読有り][通常論文]
   

  Cbln1 is a cerebellum-specific protein of previously unknown function that is structurally related to the C1q and tumor necrosis factor families of proteins. We show that Cbln1 is a glycoprotein secreted from cerebellar granule cells that is essential for three processes in cerebellar Purkinje cells: the matching and maintenance of pre- and postsynaptic elements at parallel fiber-Purkinje cell synapses, the establishment of the proper pattern of climbing fiber-Purkinje cell innervation, and induction of long-term depression at parallel fiber-Purkinje cell synapses. Notably, the phenotype of cbln1-null mice mimics loss-of-function mutations in the orphan glutamate receptor, GluR delta 2, a gene selectively expressed in Purkinje neurons. Therefore, Cbln1 secreted from presynaptic granule cells may be a component of a transneuronal signaling pathway that controls synaptic structure and plasticity.
• Differential roles of glial and neuronal glutamate transporters in Purkinje cell synapses

  Y Takayasu, M Iino, W Kakegawa, H Maeno, K Watase, K Wada, D Yanagihara, T Miyazaki, O Komine, M Watanabe, K Tanaka, S Ozawa

  JOURNAL OF NEUROSCIENCE 25 38 8788 - 8793 2005年09月 [査読有り][通常論文]
   

  Glutamate transporters are essential for terminating excitatory neurotransmission. Two distinct glutamate transporters, glutamate aspartate transporter ( GLAST) and excitatory amino acid transporter 4 ( EAAT4), are expressed most abundantly in the molecular layer of the cerebellar cortex. GLAST is expressed in Bergmann glial processes surrounding excitatory synapses on Purkinje cell dendritic spines, whereas EAAT4 is concentrated on the extrasynaptic regions of Purkinje cell spine membranes. To clarify the functional significance of the coexistence of these transporters, we analyzed the kinetics of EPSCs in Purkinje cells of mice lacking either GLAST or EAAT4. There was no difference in the amplitude or the kinetics of the rising and initial decay phase of EPSCs evoked by stimulations of climbing fibers and parallel fibers between wild-type and EAAT4-deficient mice. However, long-lasting tail currents of the EPSCs appeared age dependently in most of Purkinje cells in EAAT4-deficient mice. These tail currents were never seen in mice lacking GLAST. In the GLAST-deficient mice, however, the application of cyclothiazide that reduces desensitization of AMPA receptors increased the peak amplitude of the EPSC and prolonged its decay more markedly than in both wild-type and EAAT4-deficient mice. The results indicate that these transporters play differential roles in the removal of synaptically released glutamate. GLAST contributes mainly to uptake of glutamate that floods out of the synaptic cleft at early times after transmitter release. In contrast, the main role of EAAT4 is to remove low concentrations of glutamate that escape from the uptake by glial transporters at late times and thus prevents the transmitter from spilling over to neighboring synapses.
• Control of synaptic connection by glutamate receptor delta 2 in the adult cerebellum

  T Takeuchi, T Miyazaki, M Watanabe, H Mori, K Sakimura, M Mishina

  JOURNAL OF NEUROSCIENCE 25 8 2146 - 2156 2005年02月 [査読有り][通常論文]
   

  Precise topological matching of presynaptic and postsynaptic specializations is essential for efficient synaptic transmission. Furthermore, synaptic connections are subjected to rearrangements throughout life. Here we examined the role of glutamate receptor (GluR) delta2 in the adult brain by inducible and cerebellar Purkinje cell (PC)-specific gene targeting under the pure C57BL/6 genetic background. Concomitant with the decrease of postsynaptic GluRdelta2 proteins, presynaptic active zones shrank progressively and postsynaptic density (PSD) expanded, resulting in mismatching between presynaptic and postsynaptic specializations at parallel fiber-PC synapses. Furthermore, GluRdelta2 and PSD-93 proteins were concentrated at the contacted portion of mismatched synapses, whereas AMPA receptors were distributed in both the contacted and dissociated portions. When GluRdelta2 proteins were diminished, PC spines lost their synaptic contacts. We thus identified postsynaptic GluRdelta2 as a key regulator of the presynaptic active zone and PSD organization at parallel fiber-PC synapses in the adult brain.
• Rescue of abnormal phenotypes of the delta 2 glutamate receptor-null mice by mutant delta 2 transgenes

  H Hirai, T Miyazaki, W Kakegawa, S Matsuda, M Mushina, M Watanabe, M Yuzaki

  EMBO REPORTS 6 1 90 - 95 2005年01月 [査読有り][通常論文]
   

  The delta2 glutamate receptor (GluRdelta2) has a crucial role in cerebellar functions; disruption of GluRdelta2 alleles in mice (delta2(-/-)) impairs synapse formation and long-term depression, which is thought to underlie motor learning in the cerebellum, and consequently leads to motor discoordination. However, it has been unclear whether GluRdelta2 is activated by glutamate analogues. Here we introduced a GluRdelta2 transgene, which had a mutation (Arg514Lys) in the putative ligand-binding motif conserved in all mammalian ionotropic glutamate receptors (iGluRs) and their ancestral bacterial periplasmic amino-acid-binding proteins, into delta2(-/-) mice. Surprisingly, a mutant GluRdelta2 transgene, as well as a wild-type GluRdelta2 transgene, rescued all abnormal phenotypes of delta2(-/-) mice. Therefore, these results indicate that the conserved arginine residue, which is crucial for the binding of iGluRs to glutamate analogues, is not essential for the restoration of GluRdelta2 functions in delta2(-/-) mice.
• Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor

  F Nakatsu, M Okada, F Mori, N Kumazawa, H Iwasa, G Zhu, Y Kasagi, H Kamiya, A Harada, K Nishimura, A Takeuchi, T Miyazaki, M Watanabe, S Yuasa, T Manabe, K Wakabayashi, S Kaneko, T Saito, H Ohno

  JOURNAL OF CELL BIOLOGY 167 2 293 - 302 2004年10月 [査読有り][通常論文]
   

  A P-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B(-/-) mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B(-/-) mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.
• 成熟マウス小脳におけるシナプス後膜AMPA受容体の活動に依存したシナプス前終末の機能維持(Postsynaptic AMPA receptor activity maintains presynaptic function in the adult cerebellum)

  柿澤 昌, 宮崎 太輔, 飯野 正光, 渡辺 雅彦, 狩野 方伸

  神経化学 43 2-3 470 - 470 2004年08月
• P/Q-type Ca2+ channel alpha 1A regulates synaptic competition on developing cerebellar Purkinje cells

  T Miyazaki, K Hashimoto, HS Shin, M Kano, M Watanabe

  JOURNAL OF NEUROSCIENCE 24 7 1734 - 1743 2004年02月 [査読有り][通常論文]
   

  Synapse formation depends critically on the competition among inputs of multiple sources to individual neurons. Cerebellar Purkinje cells have highly organized synaptic wiring from two distinct sources of excitatory afferents. Single climbing fibers innervate proximal dendrites of Purkinje cells, whereas numerous parallel fibers converge on their distal dendrites. Here, we demonstrate that the P/Q-type Ca2+ channel alpha1A, a major Ca2+ channel subtype in Purkinje cells, is crucial for this organized synapse formation. In the alpha1A knock-out mouse, many ectopic spines were protruded from proximal dendrites and somata of Purkinje cells. Innervation territory of parallel fibers was expanded proximally to innervate the ectopic spines, whereas that of climbing fibers was regressed to the basal portion of proximal dendrites and somata. Furthermore, multiple climbing fibers consisting of a strong climbing fiber and one or a few weaker climbing fibers, persisted in the majority of Purkinje cells and were cowired to the same somata, proximal dendrites, or both. Therefore, the lack of alpha1A results in the persistence of parallel fibers and surplus climbing fibers, which should normally be expelled from the compartment innervated by the main climbing fiber. These results suggest that a P/Q-type Ca2+ channel alpha1A fuels heterosynaptic competition between climbing fibers and parallel fibers and also fuels homosynaptic competition among multiple climbing fibers. This molecular function facilitates the distal extension of climbing fiber innervation along the dendritic tree of the Purkinje cell and also establishes climbing fiber monoinnervation of individual Purkinje cells.
• Possible involvement of AP-3B in VGAT-associated inhibitory synaptic function

  Nakatsu, F, Okada, M, Mori, F, Kumazawa, N, Iwasa, H, Zhu, G, Kasagi, Y, Kamiya, H, Harada, A, Nishimura, K, Takeuchi, A, Miyazaki, T, Watanabe, M, Yuasa, S, Manabe, T, Wakabayashi, K, Kaneko, S, Saito, T, Ohno, H

  MOLECULAR BIOLOGY OF THE CELL 15 221A - 221A 2004年 [査読有り][通常論文]
  
• Subtype switching of vesicular glutamate transporters at parallel fibre-Purkinje cell synapses in developing mouse cerebellum

  T Miyazaki, M Fukaya, H Shimizu, M Watanabe

  EUROPEAN JOURNAL OF NEUROSCIENCE 17 12 2563 - 2572 2003年06月 [査読有り][通常論文]
   

  Two subtypes of the vesicular glutamate transporter are expressed differentially in two excitatory afferents synapsing on to Purkinje cells: VGluT1 (BNPI) in axon terminals of cerebellar granule cells (i.e. parallel fibres; PFs) and VGluT2 (DNPI) in those of the inferior olivary neurons (climbing fibres; CFs). In the present study, we examined their expression in the developing mouse cerebellum. By in situ hybridization, the inferior olivary nucleus selectively expressed VGluT2 mRNA through postnatal life. In the cerebellum, both subtypes were transcribed in the external and internal granular layers during the first postnatal week. Thereafter, VGluT1 mRNA showed marked upregulation in the internal granular layer, whereas VGluT2 mRNA disappeared from the external and internal granular layers by the end of the third postnatal week. By immunohistochemistry, CF terminals consistently exhibited VGluT2 immunoreactivity in the postnatal cerebellum. By contrast, in the first 10 days of postnatal life, VGluT2 predominated in PF terminals, despite the transcription of both transporters in developing granule cells. During the second 10 days, VGluT2 in PF terminals was replaced with VGluT1 from deep regions of the molecular layer upwards, correlating with dendritic translocation of CFs. This replacement was accomplished by postnatal day 30. Taking that late-borne PFs are laid down successively on earlier ones in the molecular layer, the deep-to-superficial replacement represents maturation-linked switching from VGluT2 to VGluT1 in individual PFs, and is likely to be regulated at both the transcription and translation levels.
• Distal extension of climbing fiber territory and multiple innervation caused by aberrant wiring to adjacent spiny branchlets in cerebellar Purkinje cells lacking glutamate receptor delta 2

  R Ichikawa, T Miyazaki, M Kano, T Hashikawa, H Tatsumi, K Sakimura, M Mishina, Y Inoue, M Watanabe

  JOURNAL OF NEUROSCIENCE 22 19 8487 - 8503 2002年10月 [査読有り][通常論文]
   

  Organized synapse formation on to Purkinje cell (PC) dendrites by parallel fibers (PFs) and climbing fibers (CFs) is crucial for cerebellar function. In PCs lacking glutamate receptor delta2 (GluRdelta2), PF synapses are reduced in number, numerous free spines emerge, and multiple CF innervation persists to adulthood. In the present study, we conducted anterograde and immunohistochemical labelings to investigate how CFs innervate PC dendrites under weakened synaptogenesis by PFs. In the GluRdelta2 knock-out mouse, CFs were distributed in the molecular layer more closely to the pial surface compared with the wild-type mouse. Serial electron microscopy demonstrated that CFs in the knock-out mouse innervated all spines protruding from proximal dendrites of PCs, as did those in the wildtype mouse. In the knock-out mouse, however, CF innervation extended distally to spiny branchlets, where nearly half of the spines were free of innervation in contrast to complete synapse formation by PFs in the wild-type mouse. Furthermore, from the end point of innervation, CFs aberrantly jumped to form ectopic synapses on adjacent spiny branchlets, whose proximal portions were often innervated by different CFs. Without GluRdelta2, CFs are thus able to expand their territory along and beyond dendritic trees of the target PC, resulting in persistent surplus CFs by innervating the distal dendritic segment. We conclude that GluRdelta2 is essential to restrict CF innervation to the proximal dendritic segment, by which territorized innervation by PFs and CFs is properly structured and the formation of excess CF wiring to adjacent PCs is suppressed.
• B2 exon splicing of nonmuscle myosin heavy chain IIB is differently regulated in developing and adult rat brain

  Miyazaki, T, Watanabe, M, Yamagishi, A, Takahashi, M

  Neuroscience Research 37 4 299 - 306 2000年 [査読有り][通常論文]
   

  Two isoforms of nonmuscle myosin heavy chain IIB (MHC-IIB) are generated by alternative splicing; MHC-IIB(B2) differs from MHC-IIB(Delta B2) by the insertion of B2 exon cassette near the actin binding region. Here we examined expressions of the two splice variants in developing and adult rat brains by in situ hybridization with isoform-specific oligonucleotide probes. In adult, MHC-IIB(Delta B2) mRNA was highly expressed in neurons of the cerebral cortex, hippocampus, and cerebellum, whereas MHC-IIB(B2) mRNA was mainly distributed in the brainstem and cerebellum, with the highest level in Purkinje cells. During development, MHC-IIB(Delta B2) mRNA was predominantly expressed in various regions of embryonic and neonatal brains, whereas MHC-IIB(B2) mRNA was low during embryonic stages. Up-regulation of MHC-IIB(B2) started in the cerebellum during early postnatal stages when dendritogenesis and synaptogenesis occur actively in Purkinje cells. We further employed immunofluorescence using two antibodies (one recognizing both splicing variants and another specific to MHC-IIB(B2)), and found similar and dense localization in cell bodies and dendrites of Purkinje cells. Therefore, splicing of the B2 exon cassette undergoes distinct temporal and spatial regulations in the brain in vivo, and the different exon usage seems unlikely to affect the somato-dendritic localization of MHC-IIB. (C) 2000 Elsevier Science Ireland Ltd and Japan Neuroscience Society. All rights reserved.

講演・口頭発表等

• Carbonic anhydrase related protein Car8 is essential for the establishment of cerebellar neuronal circuit, regulating precise matching of pre and postsynapse in Purkinje cell.

  Miyazaki T, Yamasaki M, Sakimura K, Watanabe M

  第126回 日本解剖学会総会･全国学術集会 第98回 日本生理学会大会合同大会 2021年03月
• 小脳抑制性介在ニューロンルガロ細胞の帯状構造特異的な入出力様式.  [招待講演]

  宮﨑太輔, 山崎美和子, 田中謙二, 渡辺雅彦

  生理学研究所研究会 自閉症、てんかんの病態原理に関与するシナプス制御・神経回路機構 2021年02月
• Principal rule of neurotransmitter receptor localization at synapse  [通常講演]

  宮崎太輔

  Current Trends and Future Directions of Synapse-Circuit Plasticity Research 2019年09月 口頭発表（一般）
• 小脳ルガロ細胞の帯状構造特異的な入力様式と介在ニューロンへの抑制性支配様式  [通常講演]

  宮崎太輔

  第４２回日本神経科学学会 2019年07月 口頭発表（一般）
• "Morphological classification of cerebellar interneurons and their connections"  [通常講演]

  宮崎太輔

  "Synaptic Transmission Synaptic Transmission Gordon Research Conference" 2018年08月 ポスター発表
• Calcineurin B1 subunit CNB1 in Purkinje cells regulates precise pre- and postsynaptic matching in inhibitory synapse formation  [通常講演]

  宮崎太輔

  第８回新潟大学脳研究所共同研究拠点国際シンポジウム 2018年02月 ポスター発表
• カルシウムチャネルCav2.1は成体期プルキンエ細胞の生存、バンド状発現、神経支配テリトリーの維持に不可欠である  [通常講演]

  宮崎太輔

  第63回東北・北海道連合支部学術集会 2017年09月 口頭発表（一般） 弘前大学大学院保健学研究科E棟6階（弘前大学医学部保健学科）
• Calcineurin B1 subunit is essential for climbing fiber mono-innervation and inhibitory synapse formation in Purkinje cells  [通常講演]

  MIYAZAKI Taisuke, SAKIMURA Kenji, WATANABE Masahiko

  日本解剖学会総会・全国学術集会講演プログラム・抄録集 2017年
• 小脳登上線維と抑制性介在細胞間における非シナプス性結合と選択的分子発現  [通常講演]

  山崎美和子, 深谷昌弘, 宋暁紅, 内ケ島基政, 今野幸太郎, 宮崎太輔, 渡辺雅彦

  日本解剖学会総会・全国学術集会講演プログラム・抄録集 2016年
• Neuroanatomical properties of cerebellar interneuron Lugaro cells using with GFP-expressing transgenic mice line  [通常講演]

  MIYAZAKI Taisuke, TANAKA Kenji F, WATANABE Masahiko

  日本解剖学会総会・全国学術集会講演プログラム・抄録集 2016年
• 新規カルシウム放出機構「一酸化窒素依存的カルシウム放出」と小脳依存性運動学習への関与  [通常講演]

  柿澤昌, 岸本泰司, 宮崎太輔, 田中碧, 村山尚, 渡辺雅彦, 飯野正光, 竹島浩

  日本NO学会学術集会プログラム抄録集 2015年06月
• Morphological analysis of Purkinje cell-specific calcineurin B1 subunit KO mice  [通常講演]

  MIYAZAKI Taisuke, SAKIMURA Kenji, WATANABE Masahiko

  J Physiol Sci 2015年
• プルキンエ細胞に発現するCNB1は小脳の興奮性および抑制性シナプス回路形成に重要である  [通常講演]

  Taisuke Miyazaki, Kenji Sakimura, Masahiko Watanabe

  第38回日本神経科学大会 2015年 神戸国際会議場 (兵庫県 神戸市)
• BDNF derived from Purkinje cell regulates synapse elimination in the developing cerebellum  [通常講演]

  CHOO Myeong Jeong, MIYAZAKI Taisuke, YAMASAKI Maya, TANIMURA Asami, UESAKA Naofumi, WATANABE Masahiko, SAKIMURA Kenji, KANO Masanobu

  J Physiol Sci 2014年
• Cav2.1カルボキシル末端細胞質内ドメインの病態生理学的意義の検討  [通常講演]

  相川知徳, 宮崎太輔, 三國貴康, 重本隆一, 若森実, 狩野方伸, 渡邊雅彦, 水澤英洋, 渡瀬啓

  日本神経学会学術大会プログラム・抄録集 2013年
• 蛍光タンパク発現モデルマウスを用いた小脳ルガロ細胞の形態学  [通常講演]

  宮崎 太輔, 山崎 美和子, 渡辺 雅彦

  第118回日本解剖学会総会・全国学術集会 2013年 サンポートホール高松 （香川県 高松市）
• 発達期小脳プルキンエ細胞でみられる細胞体シナプスの入力線維のスイッチング-登上線維から籠細胞線維へ  [通常講演]

  市川 量一, 山崎 美和子, 宮崎 太輔, 今野 幸太郎, 橋本 浩一, 辰巳 治之, 井上 芳郎, 狩野 正伸, 渡辺 雅彦

  北海道醫學雜誌 = Acta medica Hokkaidonensia 2012年04月
• 発達期小脳登上線維‐プルキンエ細胞間シナプス除去への抑制性シナプス伝達の関与  [通常講演]

  中山寿子, 宮崎太輔, 橋本浩一, 柳川右千夫, 小幡邦彦, 崎村建司, 渡辺雅彦, 狩野方伸

  日本生理学雑誌 2012年03月
• マウス小脳および海馬におけるセクレトグラニンII(SgII)の局在様式  [通常講演]

  宮崎太輔, 渡辺雅彦

  解剖学雑誌 2011年03月 旭川医科大学 （北海道 旭川市）
• 異種および同種入力線維間の競合を基盤とする小脳シナプス回路発達の分子機構に関する神経解剖学的研究  [通常講演]

  宮崎 太輔

  解剖學雜誌 2010年09月
• P/Q type voltage dependent Ca2+ channel is crucial for early postnatal development of climbing fiber to Purkinje cell synapse  [通常講演]

  HASHIMOTO Kouichi, TSUJITA Mika, KITAMURA Kazuo, MIYAZAKI Taisuke, YAMAZAKI Maya, SHIN Hee‐Sup, WATANABE Masahiko, SAKIMURA Kenji, KANO Masanobu

  神経化学 2010年08月
• グルタミン酸受容体GluD2は側方側枝による異所性支配を抑制し、登上線維単一支配の維持に関わっている  [通常講演]

  ○Taisuke Miyazaki, Miwako Yamasaki, Tomonori Takeuchi, Kenji Sakimura, Masayoshi Mishina, Masahiko Watanabe

  第33回日本神経科学大会 2010年08月 神戸コンベンションセンター（兵庫県 神戸市）
• グルタミン酸受容体GluD2は側方側枝による異所性支配を抑制し,登上線維単一支配の維持に関わっている  [通常講演]

  宮崎太輔, 山崎美和子, 竹内倫徳, 三品昌美, 渡辺雅彦

  解剖学雑誌 2010年03月
• グルタミン酸受容体GluD2は側方側枝による異所性支配を抑制し、登上線維単一支配の維持に関わっている  [通常講演]

  Taisuke Miyazaki, Miwako Yamasaki, Tomonori Takeuchi, Kenji Sakimura, Masayoshi Mishina, Masahiko Watanabe

  第115回日本解剖学会総会・全国学術集会 2010年 岩手県民会館 （岩手県 盛岡市）
• GLASTはバーグマングリアの形態分化および登上線維単一支配の維持に必要である。  [通常講演]

  宮崎太輔, 山崎美和子, 渡辺雅彦

  解剖学雑誌 2009年06月
• マウス小脳および海馬におけるセクレトグラニンII(SgII)の局在様式  [通常講演]

  宮崎 太輔, 渡辺 雅彦

  第56回東北・北海道連合支部学術集会 2009年03月 旭川医科大学 （北海道 旭川市）
• マウス小脳および海馬におけるセクレトグラニンII(SgII)の局在様式  [通常講演]

  宮崎 太輔, 渡辺 雅彦

  第114回 日本解剖学会総会・全国学術集会 2009年 岡山理科大学 （岡山県 岡山市）
• SCA6ノックインマウス表現型の経時的検討  [通常講演]

  渡瀬啓, BARRET Curtis, 宮崎太輔, 海野敏紀, 石川欽也, 笠井沙由美, 渡辺雅彦, 水澤英洋, TSIEN Richard, ZOGHBI Huda

  日本神経学会総会プログラム・抄録集 2009年
• GLASTは登上線維単一支配の維持とバーグマングリアによるプルキンエ細胞被覆形成に必須である  [通常講演]

  Taisuke Miyazaki, Kouichi Tanaka, Masahiko Watanabe

  第32回日本神経科学大会 2009年 名古屋国際会議場 （愛知県 名古屋市）
• 仮性狂犬病ウイルス前初期蛋白IE180発現マウスにおける小脳形成不全の病理組織学的解析  [通常講演]

  富岡幸子, 宮崎太輔, 田原口智士, 森松正美, 渡辺雅彦, 小野悦郎

  日本獣医学会学術集会講演要旨集 2008年03月
• グルタミン酸受容体GluRδ2は成体マウスにおける登上線維単一支配の維持に必須である  [通常講演]

  宮崎太輔, 渡辺雅彦

  解剖学雑誌 2008年03月
• グルタミン酸受容体GluRd2は成体マウスにおける登上線維単一支配の維持に必須である  [通常講演]

  第113回 日本解剖学会総会・全国学術集会 2008年 大分大学 （大分県 大分市）
• GLAST分子欠損マウスにおけるバーグマングリアの形態分化異常および形態学的登上線維多重支配  [通常講演]

  Taisuke Miyazaki, Kouichi Tanaka, Masahiko Watanabe

  第31回日本神経科学大会 2008年 東京国際フォーラム（東京）
• GLAST分子欠損マウスにおけるバーグマングリアの形態分化異常および局所的な異所性プルキンエ細胞支配による登上線維多重支配  [通常講演]

  Taisuke Miyazaki, Masahiko Watanabe

  第112回 日本解剖学会総会・全国学術集会 2007年 大阪国際会議場（大阪府 大阪市）
• 仮性狂犬病ウイルスの転写調節因子IE180の発現による神経病理発生メカニズムの解析  [通常講演]

  富岡幸子, 宮崎太輔, 田原口智士, 森松正美, 渡辺雅彦, 小野悦郎

  生化学 2007年
• GLAST分子欠損マウスにおけるバーグマングリアの形態分化異常および形態学的登上線維多重支配  [通常講演]

  Taisuke Miyazaki, Kouichi Tanaka, Masahiko Watanabe

  第30回日本神経科学大会 2007年 パシフィコ横浜（神奈川県 横浜市）
• オーエスキー病ウイルスC末端欠失変異型前初期蛋白IE180発現マウスの解析  [通常講演]

  富岡幸子, 宮崎太輔, 田原口智士, 吉野さおり, 森松正美, 渡辺雅彦, 上出利光, 小野悦郎

  日本獣医学会学術集会講演要旨集 2006年08月
• GLAST分子欠損マウスにおける登上線維多重支配様式の光学顕微鏡的解析  [通常講演]

  宮崎太輔, 渡辺雅彦

  解剖学雑誌 2006年06月
• GLAST欠損マウスにおける登上線維多重支配の形態学的解析  [通常講演]

  宮崎太輔, 渡辺雅彦

  解剖学雑誌 2006年03月
• GLAST分子欠損マウスにおける登上線維多重支配 の形態学的解析  [通常講演]

  Taisuke Miyazaki, Masahiko Watanabe

  第111回 日本解剖学会総会・全国学術集会 2006年 北里大学（神奈川県 相模原市）
• GLAST分子欠損マウスにおけるバーグマングリアの形態分化異常および局所的な異所性プルキンエ細胞支配による登上線維多重支配  [通常講演]

  Taisuke Miyazaki, Masahiko Watanabe

  第112回 日本解剖学会総会・全国学術集会 2006年 大阪国際会議場（大阪府 大阪市）
• Multiple climbing fiber innervation at proximal dendrite in GLAST-deficient mouse  [通常講演]

  Taisuke Miyazaki, Kouichi Tanaka, Masahiko Watanabe

  第28回日本神経科学大会 2005年 パシフィコ横浜（神奈川県 横浜市）
• カルシウムチャネルα1Aサブユニット欠損マウスにおけるプルキンエ細胞興奮性シナプス回路網の変化  [通常講演]

  宮崎太輔, 渡辺雅彦

  解剖学雑誌 2004年06月
• カルシウムチャネルα1Aサブユニット欠損マウスのプルキンエ細胞体における興奮性および抑制性入力  [通常講演]

  Taisuke Miyazaki, Kouichi Hashimoto, Hee-Sup Shin, Masanobu Kano, Masahiko Watanabe

  第27回日本神経科学大会 2004年 大阪国際会議場（大阪府 大阪市）
• 発達小脳における２種類のグルタミン酸トランスポーターVGluT1およびVGluT2の発現変化  [通常講演]

  宮崎 太輔, 渡辺 雅彦

  第108回 日本解剖学会総会・全国学術集会 2003年04月 福岡
• 発達小脳における２種類のグルタミン酸トランスポーターVGluT1およびVGluT2の発現変化  [通常講演]

  宮崎 太輔, 渡辺 雅彦

  第108回 日本解剖学会総会・全国学術集会 2003年 アクロス福岡（福岡県 福岡市）
• プルキンエ細胞への興奮性入力終末における２種類の小胞膜グルタミン酸トランスポーター発現の発達変化  [通常講演]

  宮崎 太輔, 深谷 昌弘, 清水 秀美, 渡辺 雅彦

  第25回日本神経科学大会 2002年07月 東京
• プルキンエ細胞への興奮性入力終末における２種類の小胞膜グルタミン酸トランスポーター発現の発達変化  [通常講演]

  宮崎 太輔, 深谷 昌弘, 清水 秀美, 渡辺 雅彦

  第25回日本神経科学大会 2002年 東京国際展示場 （東京）
• ラット脳における非筋細胞II型ミオシン重鎖Bの発現と局在  [通常講演]

  宮崎 太輔, 渡辺 雅彦

  第23回日本神経科学大会 2000年09月 パシフィコ横浜（神奈川県 横浜市）
• ラット脳における非筋細胞II型ミオシン重鎖Bの発現と局在  [通常講演]

  宮崎 太輔, 渡辺 雅彦

  第23回日本神経科学大会 2000年 パシフィコ横浜（神奈川県 横浜市）
• プルキンエ細胞への興奮性入力終末における２種類の小胞膜グルタミン酸トランスポーター発現の発達変化  [通常講演]

  宮崎 太輔, 深谷 昌弘, 清水 秀美, 渡辺 雅彦

  第47回東北・北海道連合支部学術集会 2000年 北海道大学 （北海道 札幌市）
• 中枢神経組織における非筋細胞ミオシンII‐B(B2)の局在  [通常講演]

  宮崎太輔, 高橋正行, 渡辺雅彦, 山岸あき彦

  日本分子生物学会年会プログラム・講演要旨集 1999年11月
• 中枢神経組織におけるミオシン II-B(B2)の局在  [通常講演]

  宮崎太輔, 高橋正行, 畠山大, 伊藤悦朗, 山岸晧彦

  日本分子生物学会年会プログラム・講演要旨集 1998年12月
• 中枢神経組織におけるミオシンII‐B(B2)の局在  [通常講演]

  宮崎太輔, 高橋正行, 畠山大, 伊藤悦朗, 山岸あき彦

  日本分子生物学会年会プログラム・講演要旨集 1998年11月
• カルシウムチャネルalpha1Aサブユニット欠損マウスにおけるプルキンエ細胞興奮性シナプス回路網の変化  [通常講演]

  宮崎 太輔, 渡辺 雅彦

  第49回東北・北海道連合支部学術集会 岩手医科大学（岩手県 盛岡市）
• GLAST分子欠損マウスにおける登上線維支配様式の光学顕微鏡的解析  [通常講演]

  宮崎 太輔, 渡辺 雅彦

  第51回東北・北海道連合支部学術集会 東北大学（宮城県 仙台市）
• GLASTは登上線維単一支配の維持に必要である  [通常講演]

  宮崎 太輔, 山崎 美和子, 渡辺 雅彦

  第54回東北・北海道連合支部学術集会 ビックアイ市民交流プラザ（福島県 郡山市）
• 小脳興奮性回路網維持とバーグマングリアの形態分化におけるグルタミン酸トランスポーターGLASTの機能的役割  [通常講演]

  Taisuke Miyazaki, Miwako Yamasaki, Kouichi Hashimoto, Keiko Shimamoto, Kazuhisa Kohda, Michisuke Yuzaki, Kohichi Tanaka, Masanobu Kano, Masahiko Watanabe

  第34回日本神経科学大会 パシフィコ横浜（神奈川県 横浜市）
• 蛍光タンパク発現モデルマウスを用いた小脳ルガロ細胞に関する形態学解析  [通常講演]

  Taisuke Miyazaki

  日本顕微鏡学会第70回記念学術講演会 幕張メッセ（千葉県 幕張市）
• プルキンエ細胞に発現するカルシニューリンCNB1サブユニットは小脳の興奮性および抑制性シナプス回路形成に重要である  [通常講演]

  宮崎 太輔, 崎村, 建司, 渡辺 雅彦

  第61回東北・北海道連合支部学術集会 盛岡市観光文化交流センター（岩手県 盛岡市）
• カルシニューリンB1サブユニットCNB1は登上線維−プルキンエ細胞単一支配化および抑制性シナプス回路形成に重要である  [通常講演]

  Taisuke Miyazaki, Kenji Sakimura, Masahiko Watanabe

  第122回日本解剖学会総会全国学術集会 長崎大学（長崎県 長崎市）
• カルシウムチャネルCav2.1は成体期プルキンエ細胞のシナプス回路、生存、バンド状発現の維持に不可欠である  [通常講演]

  Taisuke Miyazaki, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe

  第39回日本神経科学大会 パシフィコ横浜（神奈川県 横浜市）
• プルキンエ細胞に発現するカルシニューリンB1サブユニットは抑制性シナプス形成過程において適切なプレ−ポストシナプスの接着に関わっている  [通常講演]

  Taisuke Miyazaki, Kenji Sakimura, Masahiko Watanabe

  第４０回日本神経科学大会 幕張メッセ（千葉県 幕張市）

その他活動・業績

• 新規カルシウム放出機構「一酸化窒素依存的カルシウム放出」と小脳依存性運動学習への関与

  柿澤昌, 岸本泰司, 宮崎太輔, 田中碧, 村山尚, 渡辺雅彦, 飯野正光, 竹島浩 日本NO学会学術集会プログラム抄録集 15th 52 2015年06月12日 [査読無し][通常論文]
  
• Involvement of Nitric oxide-induced calcium release in extinction of cerebellum-dependent motor learning

  Sho Kakizawa, Yasushi Kishimoto, Taisuke Miyazaki, Midori Tanaka, Takashi Murayama, Masahiko Watanabe, Masamitsu Iino, Hiroshi Takeshima JOURNAL OF PHARMACOLOGICAL SCIENCES 124 100P -100P 2014年 [査読無し][通常論文]
  
• Cav2.1カルボキシル末端細胞質内ドメインの病態生理学的意義の検討

  相川 知徳, 宮崎 太輔, 三國 貴康, 重本 隆一, 若森 実, 狩野 方伸, 渡邊 雅彦, 水澤 英洋, 渡瀬 啓 臨床神経学 53 (12) 1495 -1495 2013年12月 [査読無し][通常論文]
  
• The active zone protein CAST regulates synaptic vesicle recycling and quantal size

  Kobayashi S, Hida Y, Ishizaki H, Inoue E, Tanaka-Okamoto M, Yamasaki M, Miyazaki T, Fukuya M, Kitajima I, Takai Y, Watanabe M, Ohtsuka T, Manabe T J Physiol Sci 63 S67 2013年 [査読無し][通常論文]
  
• 発達期小脳プルキンエ細胞でみられる細胞体シナプスの入力線維のスイッチング-登上線維から籠細胞線維へ

  市川 量一, 山崎 美和子, 宮崎 太輔, 今野 幸太郎, 橋本 浩一, 辰巳 治之, 井上 芳郎, 狩野 正伸, 渡辺 雅彦 北海道醫學雜誌 = Acta medica Hokkaidonensia 87 (2) 73 -73 2012年04月01日
• The maintenance of Purkinje axons by an RNA-binding protein HuC in adult cerebellum

  Kyoko Kakumoto, Taisuke Miyazaki, Masahiko Watanabe, Robert Darnell, Hirotaka Okano, Hideyuki Okano NEUROSCIENCE RESEARCH 71 E95 -E96 2011年 [査読無し][通常論文]
  
• Postsynaptic P/Q type voltage-dependent Ca2+ channel is involved in refinement of cerebellar climbing fiber to Purkinje cell synapses in early postnatal development

  Kouichi Hashimoto, Mika Tsujita, Taisuke Miyazaki, Kazuo Kitamura, Maya Yamazaki, Shin Hee-sup, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano NEUROSCIENCE RESEARCH 71 E322 -E322 2011年 [査読無し][通常論文]
  
• 異種および同種入力線維間の競合を基盤とする小脳シナプス回路発達の分子機構に関する神経解剖学的研究

  宮崎 太輔 解剖學雜誌 85 (3) 115 -116 2010年09月01日
• 小脳回路 発達と可塑性の最前線 登上線維の発達過程におけるP/Q型電位依存性カルシウムチャネルの役割(Cellebellar circuitry: Cutting-edge of development and plasticity P/Q type voltage dependent Ca2+ channel is crucial for early postnatal development of climbing fiber to Purkinje cell synapse)

  橋本 浩一, 辻田 実加, 喜多村 和郎, 宮崎 太輔, 山崎 真弥, Shin Hee-Sup, 渡辺 雅彦, 崎村 建司, 狩野 方伸 神経化学 49 (2-3) 461 -461 2010年08月 [査読無し][通常論文]
  
• The maintenance of Purkinje axons by HuC in adult cerebellum

  Kyoko Kakumoto, Taisuke Miyazaki, Masahiko Watanabe, Robert B. Darnell, Hirotaka James Okano, Hideyuki Okano NEUROSCIENCE RESEARCH 68 E61 -E61 2010年 [査読無し][通常論文]
  
• P/Q type voltage-dependent Ca2+ channel is required for refinement of climbing fiber to Purkinje cell synapse in early postnatal development

  Kouichi Hashimoto, Mika Tsujita, Taisuke Miyazaki, Maya Yamazaki, Kazuo Kitamura, Hee-sup Shin, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano JOURNAL OF PHYSIOLOGICAL SCIENCES 60 S83 -S83 2010年 [査読無し][通常論文]
  
• Analysis of physiological functions of cerebellar AMPA receptor subunits using a Purkinje cell-selective gene targeting system

  Manabu Abe, Kouichi Hashimoto, Taisuke Miyazaki, Hirotsugu Azechi, Rie Natsume, Masahiko Watanabe, Masanobu Kano, Kenji Sakimura NEUROSCIENCE RESEARCH 68 E231 -E231 2010年 [査読無し][通常論文]
  
• Cbln1 REGULATES INHIBITORY SYNAPTIC TRANSMISSION ON CEREBELLAR PURKINJE CELLS

  Aya Ito-Ishida, Eriko Miura, Taisuke Miyazaki, Tetsushi Sadakata, Ritsuko Katoh-Semba, Keiko Matsuda, Teiichi Furuichi, Masahiko Watanabe, Michisuke Yuzaki JOURNAL OF PHYSIOLOGICAL SCIENCES 59 145 -145 2009年 [査読無し][通常論文]
  
• GABAergic inhibition onto Purkinje cell soma is crucial for climbing fiber synapse elimination in developing cerebellum

  Hisako Nakayama, Taisuke Miyazaki, Kouichi Hashimoto, Yuchio Yanagawa, Kunihiko Obata, Masahiko Watanabe, Masanobu Kano NEUROSCIENCE RESEARCH 65 S52 -S52 2009年 [査読無し][通常論文]
  
• Loss of cerebellar granule cell AMPA receptors contributes to ataxia in stargazer mice

  May Yamazaki, Masahiro Fukaya, Taisuke Miyazaki, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura NEUROSCIENCE RESEARCH 65 S140 -S140 2009年 [査読無し][通常論文]
  
• DIFFERENTIAL REGULATION OF SYNAPSE FORMATION AND PLASTICITY BY GluR delta 2 IN THE CEREBELLUM

  Wataru Kakegawa, Taisuke Miyazaki, Kazuhisa Kohda, Keiko Matsuda, Kyoichi Emi, Junko Motohashi, Masahiko Watanabe, Michisuke Yuzaki JOURNAL OF PHYSIOLOGICAL SCIENCES 59 135 -135 2009年 [査読無し][通常論文]
  
• GLAST is essential to maintain climbing fiber monoinnervation and glial synapse enwrapping to cerebellar Purkinje cells

  Taisuke Miyazaki, Kohichi Tanaka, Masahiko Watanabe NEUROSCIENCE RESEARCH 65 S44 -S44 2009年 [査読無し][通常論文]
  
• Junctophilin-mediated functional crosstalk between ryanodine receptors and SK channels essential for long-term depression in the cerebellum

  S. Kakizawa, Y. Kishimoto, K. Hashimoto, T. Miyazaki, K. Furutani, H. Shimizu, M. Fukaya, M. Nishi, H. Sakagami, A. Ikeda, H. Kondo, M. Kano, M. Watanabe, M. Iino, H. Takeshima JOURNAL OF NEUROCHEMISTRY 106 50 -50 2008年07月 [査読無し][通常論文]
  
• Retardations in cerebellar foliation, the ectopic location of Bergmann glia, and altered climbing fiber territory on Purkinje cells by FAK ablation

  Fumihiro Watanabe, Taisuke Miyazaki, Masahiro Fukaya, Tomonori Takeuchi, Kenji Sakimura, Masahiko Watanabe, Masayoshi Mishina JOURNAL OF PHARMACOLOGICAL SCIENCES 106 82P -82P 2008年 [査読無し][通常論文]
  
• Strength of GABAergic transmission influences climbing fiber synapse elimination during cerebellar development

  Hisako Nakayama, Kouichi Hashimoto, Taisuke Miyazaki, Yuchio Yanagawa, Kunihiko Obata, Masahiko Watanabe, Masanobu Kano NEUROSCIENCE RESEARCH 58 S52 -S52 2007年 [査読無し][通常論文]
  
• Protocadherin-alpha family in the olivo-cerebellar system; their isoform expression and loss of function analysis

  You Kumode, Shun Hamada, Takashi Kitsukawa, Kouichi Hashimoto, Taisuke Miyazaki, Masanobu Kano, Masahiko Watanabe, Takeshi Yagi NEUROSCIENCE RESEARCH 58 S206 -S206 2007年 [査読無し][通常論文]
  
• Junctophilin-mediated functional coupling of channels essential for cerebellar synaptic plasticity

  Sho Kakizawa, Yasushi Kishimoto, Sin-ya Saito, Kouichi Hashimoto, Taisuke Miyazaki, Kazuharu Furutani, Miyuki Nishi, Hiroyuki Sakagami, Hisatake Kondo, Masanobu Kano, Masahiko Watanabe, Masamitsu Iino, Hiroshi Takeshima JOURNAL OF PHARMACOLOGICAL SCIENCES 103 68P -68P 2007年 [査読無し][通常論文]
  
• GLAST is essential for cytological differentiation of Bergmann glia and climbing fiber monoinnervation by suppressing local ectopic innervation

  Taisuke Miyazaki, Kohichi Tanaka, Masahiko Watanabe NEUROSCIENCE RESEARCH 55 S174 -S174 2006年 [査読無し][通常論文]
  
• Ca2+ influx through delta 2 glutamate receptors is not required for cerebellar functions

  Wataru Kakegawa, Taisuke Miyazaki, Hirokazu Hirai, Masayoshi Mishina, Masahiko Watanabe, Michisuke Yuzaki NEUROSCIENCE RESEARCH 55 S172 -S172 2006年 [査読無し][通常論文]
  
• Impaired cerebellar climbing fiber innervation in the nervous system-specific FAK deficient mice

  F Watanabe, T Miyazaki, M Fukaya, T Takeuchi, M Watanabe, M Mishina JOURNAL OF PHARMACOLOGICAL SCIENCES 100 246P -246P 2006年 [査読無し][通常論文]
  
• Control of synaptic connection by glutamate receptor delta 2 in the mature cerebellum

  T Takeuchi, T Miyazaki, M Watanabe, H Mori, K Sakimura, M Mishina JOURNAL OF PHARMACOLOGICAL SCIENCES 97 114P -114P 2005年 [査読無し][通常論文]
  
• RNA神経生物学 ニューロンにおける転写後の世界 IP3 receptor type 1 mRNAの3末端非翻訳領域に結合し,その樹状突起への局在化及び小脳運動学習に関わるRNA結合タンパク質Hzf(RNA neurobiology: Posttranscriptional world in neurons:Hzf binds to IP3R1 mRNA and is required for dendritic mRNA localization and the motor learning in ce

  飯島 崇利, 今井 貴雄, 高月 香菜子, 小倉 博雄, 宮崎 太輔, 岡野 洋尚, 川原 茂敬, 渡辺 雅彦, 桐野 豊, 岡野 栄之 神経化学 43 (2-3) 329 -329 2004年08月 [査読無し][通常論文]
  
• Conditional ablation of glutamate receptor delta 2 induces synaptic rearrangements in mature cerebellum

  T Takeuchi, T Miyazaki, M Watanabe, H Mori, K Sakimura, M Mishina JOURNAL OF PHARMACOLOGICAL SCIENCES 94 65P -65P 2004年 [査読無し][通常論文]
  
• Distal extension of climbing fiber territory and multiple innervation caused by aberrant wiring to adjacent spiny branchlets in cerebellar Purkinje cells lacking glutamate receptor GluRδ2

  Ichikawa Ryoichi, Miyazaki Taisuke, Kano Masanobu, Hashikawa Tsutomu, Tatsumi Haruyuki, Sakimura Kenji, Mishina Masayoshi, Inoue Yoshiro, Watanabe Masahiko The Journal of neuroscience : the official journal of the Society for Neuroscience 22 (19) 8487 -8503 2002年10月01日 

  Organized synapse formation on to Purkinje cell (PC) dendrites by parallel fibers (PFs) and climbing fibers (CFs) is crucial for cerebellar function. In PCs lacking glutamate receptor delta2 (GluRdelta2), PF synapses are reduced in number, numerous free spines emerge, and multiple CF innervation persists to adulthood. In the present study, we conducted anterograde and immunohistochemical labelings to investigate how CFs innervate PC dendrites under weakened synaptogenesis by PFs. In the GluRdelta2 knock-out mouse, CFs were distributed in the molecular layer more closely to the pial surface compared with the wild-type mouse. Serial electron microscopy demonstrated that CFs in the knock-out mouse innervated all spines protruding from proximal dendrites of PCs, as did those in the wild-type mouse. In the knock-out mouse, however, CF innervation extended distally to spiny branchlets, where nearly half of the spines were free of innervation in contrast to complete synapse formation by PFs in the wild-type mouse. Furthermore, from the end point of innervation, CFs aberrantly jumped to form ectopic synapses on adjacent spiny branchlets, whose proximal portions were often innervated by different CFs. Without GluRdelta2, CFs are thus able to expand their territory along and beyond dendritic trees of the target PC, resulting in persistent surplus CFs by innervating the distal dendritic segment. We conclude that GluRdelta2 is essential to restrict CF innervation to the proximal dendritic segment, by which territorized innervation by PFs and CFs is properly structured and the formation of excess CF wiring to adjacent PCs is suppressed.

受賞

• 2020年09月 日本解剖学会 東北・北海道連合支部会 学会賞
   小脳抑制性介在ニューロンルガロ細胞の帯状構造特異的な入出力様式 

  受賞者: 宮﨑太輔,山崎美和子、田中謙二, 渡辺雅彦
• 2017年 北海道大学 北海道大学医学部 優秀論文賞
   Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells 

  受賞者: 宮崎太輔
• 2009年 日本解剖学会 解剖学会奨励賞
   異種および同種入力線維間の競合を基盤とする小脳シナプス回路発達の分子機構に関する神経解剖学的研究 

  受賞者: 宮崎太輔

共同研究・競争的資金等の研究課題

• 小脳における興奮・抑制競合を基盤とした回路形成維持機構の解明

  日本学術振興会：科学研究費助成事業

  研究期間 : 2022年04月 -2025年03月 

  代表者 : 宮崎 太輔, 山崎 美和子
• 成体期におけるP/Q型カルシウムチャネルのシナプス刈込みへの機能的関与の検証

  日本学術振興会：科学研究費助成事業

  研究期間 : 2021年04月 -2025年03月 

  代表者 : 渡辺 雅彦, 宮崎 太輔

   

  興奮性シナプス活動により、イオンチャネル型グルタミン酸受容体iGluRと代謝型グルタミン酸受容体mGluRの活性化が起こる。iGluR活性化による脱分極は、小脳プルキンエ細胞ではP/Q型カルシウムチャネルの活性化を介して細胞外からCa2+流入を誘導する。受精の段階からこの遺伝子欠損を有するヌル欠損マウスでは、シナプス刈込みに障害が生じて、登上線維と平行線維のテリトリー不分離と登上線維の多重支配が残存することを示してきた。本研究課題では、シナプス回路完成後の成体期においてもシナプス回路維持機構として機能しているかを、成体期に遺伝子欠損誘導を行えるモデルを作成して明らかにすることである。 新潟大学脳研究所の崎村建司名誉教授らが以前開発した、P/Q型VDCC のチャネル本体をコードするCav2.1遺伝子のfloxマウスと、プロゲステロン誘導剤（RU-486）依存的に活性化するCreリコンビナーゼをプルキンエ細胞選択的に発現するGluD2-CrePRマウスを入手する。亮者を交配して、Cav2.1flox/CrePRマウスラインを得た。生後2ヶ月令の成体マウスにRU-486を腹腔投与したものを成体期欠損群（Cav2.1-/CrePR）、溶剤のみを腹腔投与したものをコントロール群（Cav2.1flox/CrePR）とした。研究代表者が開発したCav2.1特異抗体を用いて免疫染色法を用いて検討したところ、 Cav2.1-/CrePRのプルキンエ細胞における Cav2.1 タンパクの有意な減少が誘導後１週から確認された。一方、Cav2.1flox/CrePでは Cav2.1 タンパクの発現の減少は認められなかった。したがって、神経回路が完成した成体期においてCav2.1遺伝子を欠損させるモデルマウスが完成したことを確認できた。
• 発達期からの慢性ニコチン曝露がもたらす神経回路変容の統合的理解

  日本学術振興会：科学研究費助成事業

  研究期間 : 2020年04月 -2024年03月 

  代表者 : 山崎 美和子, 宮崎 太輔

   

  ニコチン性アセチルコリン受容体(nAChR)は、タバコの主成分であるニコチンと結合することにより様々な神経伝達修飾作用を及ぼす。よく知られたニコチン依存に加え、近年では発達期からのニコチン曝露がオピオイド系を始めとする薬物依存への道筋をつけるという新たな可能性が示唆されているが、その生物学的なメカニズムや脳内でnAChRの局在やその分子機構にも未だ不明な点が多い。本研究ではnAChRを介した神経伝達修飾の解剖学的な基盤を示し、発達期からのニコチン曝露が神経回路にどのような構造的・機能的な変容をもたらすかを明らかにする。今年度は以下の二項目で進捗があった。 1)a7サブユニットに対する特異抗体の開発:脳内の主要サブユニットであるa7サブユニットに関し、生化学解析に使用できる抗体は既に開発していたが、今年度、脳組織での免疫染色に使用可能な特異抗体の開発に成功した。野生型マウスでは、陽性シグナルが大脳皮質第１層や海馬の上昇層の抑制性介在ニューロンや、赤核、二丘体傍核に存在していた。これに対し、a7欠損マウスではシグナルが検出されず、特異性が確認された。また、a7サブユニットのアセンブリに必要なシャペロンTMEM35欠損マウスでもシグナルが検出されなかった。このことはa7の細胞膜への輸送ではなく、アセンブリにTMEM35Aが必須であることを示唆している。また、組織染色に有用というだけでなく、生化学解析での感度・特異度ともに高く今後の解析に非常に有用なツールとして期待できる。
  2) b4サブニットに対する特異抗体の開発:b4サブニットに対する特異抗体の開発に成功した。内側手綱核-脚間核投射系に選択的に局在していることが確認された。
• プルキンエ細胞シナプス後部におけるカルシニューリン依存的入力認識機構の解明

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2017年04月 -2020年03月 

  代表者 : 宮崎太輔
• 高次脳領域におけるシナプス伝達制御機構の分子形態学的研究

  日本学術振興会：科学研究費助成事業

  研究期間 : 2012年05月 -2017年03月 

  代表者 : 渡辺 雅彦, 崎村 建司, 山崎 美和子, 宮崎 太輔, 今野 幸太郎, 内ヶ島 基政, 狩野 方伸, 重本 隆一, 小林 和人

   

  シナプス伝達の「重み」は一様ではなく、入力選択的・標的選択的・活動依存的・状況依存的に制御されている。本研究課題では、この多層的伝達制御機構の分子解剖学的基盤を追求した。その結果、シナプス後部のTARPとGluDが入力・標的・活動依存的なAMPARの発現制御に関わり、シナプス前部にVGluT3を発現するCCK陽性の抑制性介在ニューロンがが特異な陥入型シナプスを形成し、そこにカンナビノイドを介する強力な逆行性脱抑制機構を構築していることを解明した。さらに、状況依存的行動制御に重要なドパミン投射が、GABA作動性シナプス後部分子を介して係留接着を形成するという、神経調節の新たな動作原理も発見した。
• 成体期プルキンエ細胞における異種シナプス間競合維持機構の解明

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2014年 -2016年 

  代表者 : 宮崎 太輔

   

  小脳のプルキンエ細胞は平行線維と登上線維による興奮性入力を受けている。発達期ではこの２種類の興奮性入力がそれぞれGluRδ2と電位依存性カルシウムチャネルCav2.1を強化分子として競合することが知られている。しかし、成体期におけるシナプス競合の存在については不明な点が多い。本研究課題では誘導型Cav2.1欠損マウスの形態学的・電気生理学的解析を行いCav2.1が成体小脳においても登上線維入力の強化分子機構として機能していることを明らかにした。この結果は「異種シナプス間競合関係が成体期においても存在し、この均衡維持が小脳の機能発現に重要である」という仮説を実験的に証明するものとなった。
• プルキンエ細胞体における登上線維シナプス除去機構の解明

  文部科学省：科学研究費補助金(若手研究(B))

  研究期間 : 2012年 -2013年 

  代表者 : 宮崎 太輔

   

  登上線維－小脳プルキンエ細胞投射系では細胞体での線維間競合を経て、単一の優勢線維による樹状突起支配が確立する。細胞体では登上線維シナプスの除去が進行する一方で、バスケット細胞による抑制性独占的支配が形成されるが、両者の機能的な因果関係については未だ不明であった。さらに発達期小脳ではプルキンエ細胞体直下の顆粒細胞層において、除去過程の登上線維終末が観察され、顆粒細胞層に存在する抑制性介在ニューロンの一つルガロ細胞と近接している様子が観察されている。以上の背景から本研究では発達期プルキンエ細胞体での抑制性ニューロンの関係に着目し、抑制性ニューロンの機能阻害下における登上線維シナプス除去過程の解析を行い、中枢神経回路における「特定の細胞内ドメインで起こる興奮性入力と抑制性入力による競合」の存在の実験的な証明を行なった。具体的にはレンチウイルスやGFP発現モデルマウスを用いた抑制性介在ニューロンの可視化、神経標識法を用いた登上線維の可視化を行い発達期小脳の観察を行い、以下の所見を得た。①発達期プルキンエ細胞体において、登上線維終末の排除とバスケット細胞由来の抑制性終末の増加が相補的に起こっていることを定量的に示した。②発達期小脳ではプルキンエ細胞体とルガロ細胞が密接している様子が観察され、この傾向は発達が進むにつれて減少していった。③発達期小脳で一過性に見られるプルキンエ細胞－ルガロ細胞密着領域では登上線維終末が高頻度に観察された。以上の所見はプルキンエ細胞体における登上線維終末がバスケット細胞軸索によって排除され、ルガロ細胞へと引き継がれることを示唆する結果であり、この研究成果は日本神経学会Neuro2012, Neuro2013、第118回日本解剖学会総会・全国学術集会で発表された。
• 活動依存的カルシウム流入による競合的シナプス回路発達の共通原理の解明

  日本学術振興会：科学研究費助成事業

  研究期間 : 2007年 -2011年 

  代表者 : 渡辺 雅彦, 崎村 建司, 狩野 方伸, 饗場 篤, 深谷 昌弘, 山崎 美和子, 宮崎 太輔, 狩野 方伸, 饗場 篤, 深谷 昌弘, 山崎 美和子, 宮崎 太輔

   

  生まれたばかりの個体のシナプス回路は、過剰で重複の多い混線回路である。生後早期の発達過程において、環境刺激や経験・訓練・学習に伴うシナプスの使用状況や神経活動の強弱に応じて、競合的なシナプス回路の強化と除去が起こる。その結果、未熟なシナプス回路は機能的で正確な投射関係を備えたシナプス回路へとリファインされ、正常な脳機能の発現基盤が完成する。この発達過程を経ることにより、ヒトでは臨界期と呼ばれる年少期において、ほとんどの高次神経機能ー認知、言語、楽器演奏、スポーツ、知能、思考、性格、社会性などーが飛躍的に発達する。現在、シナプスに発現するグルタミン酸受容体の活性化とそれによるシナプス後側ニューロンへのカルシウム流入が、この活動依存的シナプス選別過程を制御していることがわかっている。しかし、カルシウム濃度上昇が、一体どのような分子細胞メカニズムを通してシナプス回路の強化と除去を制御しているかについては、ほとんど不明であった。本研究では、カルシウム依存的な活動依存機構とこれに対抗する機構とが拮抗してシナプス回路発達を制御するという作業仮説を立て、これを神経解剖学・神経生理学・発生工学などの手法を用いて個体レベルで検証することを目的とした。本研究の推進により、小脳ではP/Q型カルシウムチャネルが登上線維によるプルキンエ細胞支配の形成・維持を制御し、大脳ではNMDA型グルタミン酸受容体やその制御調節に関わるグルタミン酸トランスポーターが体性感覚系シナプス回路発達を制御することを明らかにした。また、平行線維によるプルキンエ細胞支配をGluD2-Cbln1-ニューレキシンによる分子間相互作用が媒介し、これがカルシウム依存的な形成・維持機構により促進される登上線維支配と拮抗していることも判明した。
• 小脳成熟に伴う登上線維支配様式の可塑性減少およびその臨界期の特定

  文部科学省：科学研究費補助金(若手研究(B))

  研究期間 : 2006年 -2008年 

  代表者 : 宮崎 太輔

   

  小脳プルキンエ細胞 (PC)は、数十万本の平行線維 (PF)と1本の登上線維 (CF)による興奮性入力を受けている。幼若期のPCは複数のCFによる多重支配を受けているが、発達過程において余剰なCFは排除され、生後3週目までには一つのPCがただ1本のCFによって支配される単一支配が確立する。この単一支配化には、PFシナプス後部特異的に発現するグルタミン酸受容体GluRd2が重要であることが知られている。本研究では、誘導型GluRd2欠損マウスを用いて、CF-PC単一支配化の維持にGluRd2が関与しているかどうかを明らかにすべく行われた。誘導型GluRd2欠損マウスのCF-PC支配様式を、神経標識法および免疫組織化学法を用いて解析したところ、(1)分子欠損誘導後8週からCF多重支配が認められ、GluRd2欠損領域でCF支配領域の拡大が認められた、(2)分子欠損誘導後8週からtransverse branchと呼ばれるCF側枝が側方のPCに対して異所性支配を行い、CF-PC多重支配を引き起こしている様子が観察されたという2つの所見が得られた。以上の結果はGluRd2がCF側枝による側方支配を抑制することで成体期におけるCF-PC単一支配の維持に関わっていることを強く示唆するものであった。

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