2021年, エクセレントティーチャー最優秀賞（講義、実習）　　　　　　　　　　　　　　　

2019年09月, 日本解剖学会 東北・北海道連合支部, 日本解剖学会 第65回東北・北海道連合支部学術集会 学会賞　　　　　　　　　　　　　　　

2019年, 北海道大学医学部, エクセレントティーチャー最優秀賞（講義、実習）　　　　　　　　　　　　　　　

2014年12月, 日本解剖学会, 平成２６年度 日本解剖学会奨励賞　　　　　　　　　　　　　　　
入力経路・標的細胞依存的なグルタミン酸作動性シナプス制御機構の分子解剖学的基盤

2014年, 北海道大学, 北海道大学教育総長賞（奨励賞）　　　　　　　　　　　　　　　

2013年, 北海道大学医学部, 最優秀教員特別賞（連続受賞）　　　　　　　　　　　　　　　

2012年, 北海道大学医学部, 最優秀教員賞　　　　　　　　　　　　　　　

2011年, 北海道大学医学部, 最優秀教員賞　　　　　　　　　　　　　　　

MitoNEET reduces the mitochondrial oxidative phosphorylation during epithelial-mesenchymal transition
Haruka Handa, Yasuhito Onodera, Tsukasa Oikawa, Shingo Takada, Koji Ueda, Daiki Setoyama, Takashi Yokota, Miwako Yamasaki, Masahiko Watanabe, Yoshizuki Fumoto, Ari Hashimoto, Soichiro Hata, Masaaki Murakami, Hisataka Sabe
Cold Spring Harbor Laboratory, 2024年07月29日
Mitochondrial functions range from catabolic to anabolic, which are tightly coordinated to meet cellular demands for proliferation and motility. MitoNEET is a mitochondrial outer membrane protein with a CDGSH domain and is involved in mitochondrial function. Epithelial-to-mesenchymal transition (EMT) is the process in which cells lose their epithelial characteristics and acquire mesenchymal traits, such as motility, which is a vital step for organism development and wound-healing. Cellular motility is associated with high ATP consumption owing to lamellipodia formation, which is supported by upregulated oxidative phosphorylation (OXPHOS) capacity. However, how mitoNEET is involved in the regulation of OXPHOS capacity and subsequent cellular motility remains unclear. Here we show that loss of mitoNEET regulation during EMT impairs both OXPHOS enhancement and cell motility in non-transformed NMuMG mouse mammary gland epithelial cells. We found that mitoNEET is downregulated during EMT, and that the aberrant expression of mitoNEET abolishes the upregulation of OXPHOS, leading to the inhibition of cell motility. Furthermore, we found that mitoNEET topology may be crucial for the regulation of the mitochondrial electron transfer chain, suggesting an additional regulatory pathway for OXPHOS capacity. Our results demonstrate that mitochondrial OXPHOS capacity during EMT is partly regulated by the dynamics of the outer membrane protein. We believe that our findings are the first step towards understanding the mechanisms by which mitochondrial outer membrane protein topology affects organelle functions

Glyoxal fixation: An approach to solve immunohistochemical problem in neuroscience research.
Kohtarou Konno, Miwako Yamasaki, Taisuke Miyazaki, Masahiko Watanabe
Science advances, 9, 28, eadf7084, 2023年07月14日, ［国際誌］
英語, 研究論文（学術雑誌）, The gold-standard fixative for immunohistochemistry is 4% formaldehyde; however, it limits antibody access to target molecules that are buried within specialized neuronal components, such as ionotropic receptors at the postsynapse and voltage-gated ion channels at the axon initial segment, often requiring additional antigen-exposing techniques to detect their authentic signals. To solve this problem, we used glyoxal, a two-carbon atom di-aldehyde. We found that glyoxal fixation greatly improved antibody penetration and immunoreactivity, uncovering signals for buried molecules by conventional immunohistochemical procedures at light and electron microscopic levels. It also enhanced immunosignals of most other molecules, which are known to be detectable in formaldehyde-fixed sections. Furthermore, we unearthed several specific primary antibodies that were once judged to be unusable in formaldehyde-fixed tissues, allowing us to successfully localize so far controversial synaptic adhesion molecule Neuroligin 1. Thus, glyoxal is a highly effective fixative for immunostaining, and a side-by-side comparison of glyoxal and formaldehyde fixation is recommended for routine immunostaining in neuroscience research.

PTPδ is a presynaptic organizer for the formation and maintenance of climbing fiber to Purkinje cell synapses in the developing cerebellum
Yuto Okuno, Kazuto Sakoori, Kyoko Matsuyama, Miwako Yamasaki, Masahiko Watanabe, Kouichi Hashimoto, Takaki Watanabe, Masanobu Kano
Frontiers in Molecular Neuroscience, 16, Frontiers Media SA, 2023年06月22日
研究論文（学術雑誌）, Functionally mature neural circuits are shaped during postnatal development by eliminating redundant synapses formed during the perinatal period. In the cerebellum of neonatal rodents, each Purkinje cell (PC) receives synaptic inputs from multiple (more than 4) climbing fibers (CFs). During the first 3 postnatal weeks, synaptic inputs from a single CF become markedly larger and those from the other CFs are eliminated in each PC, leading to mono-innervation of each PC by a strong CF in adulthood. While molecules involved in the strengthening and elimination of CF synapses during postnatal development are being elucidated, much less is known about the molecular mechanisms underlying CF synapse formation during the early postnatal period. Here, we show experimental evidence that suggests that a synapse organizer, PTPδ, is required for early postnatal CF synapse formation and the subsequent establishment of CF to PC synaptic wiring. We showed that PTPδ was localized at CF-PC synapses from postnatal day 0 (P0) irrespective of the expression of Aldolase C (Aldoc), a major marker of PC that distinguishes the cerebellar compartments. We found that the extension of a single strong CF along PC dendrites (CF translocation) was impaired in global PTPδ knockout (KO) mice from P12 to P29-31 predominantly in PCs that did not express Aldoc [Aldoc (–) PCs]. We also demonstrated via morphological and electrophysiological analyses that the number of CFs innervating individual PCs in PTPδ KO mice were fewer than in wild-type (WT) mice from P3 to P13 with a significant decrease in the strength of CF synaptic inputs in cerebellar anterior lobules where most PCs are Aldoc (–). Furthermore, CF-specific PTPδ-knockdown (KD) caused a reduction in the number of CFs innervating PCs with decreased CF synaptic inputs at P10-13 in anterior lobules. We found a mild impairment of motor performance in adult PTPδ KO mice. These results indicate that PTPδ acts as a presynaptic organizer for CF-PC formation and is required for normal CF-PC synaptic transmission, CF translocation, and presumably CF synapse maintenance predominantly in Aldoc (–) PCs. Furthermore, this study suggests that the impaired CF-PC synapse formation and development by the lack of PTPδ causes mild impairment of motor performance.

Single-scan volumetric imaging throughout thick tissue specimens by one-touch installable light-needle creating device
Ching-Pu Chang, Kohei Otomo, Yuichi Kozawa, Hirokazu Ishii, Miwako Yamasaki, Masahiko Watanabe, Shunichi Sato, Ryosuke Enoki, Tomomi Nemoto
Scientific Reports, 12, 1, Springer Science and Business Media LLC, 2022年12月
研究論文（学術雑誌）, Abstract

Biological tissues and their networks frequently change dynamically across large volumes. Understanding network operations requires monitoring their activities in three dimensions (3D) with single-cell resolution. Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups, as well as deep expertise for microscopic technologies, resulting in a high threshold for biologists. In this study, we propose an easy-to-use light-needle creating device for conventional two-photon microscopy systems. By only installing the device in one position for a filter cube that conventional fluorescent microscopes have, single scanning of the excitation laser light beam excited fluorophores throughout over 200 μm thickness specimens simultaneously. Furthermore, the developed microscopy system successfully demonstrated single-scan visualization of the 3D structure of transparent YFP-expressing brain slices. Finally, in acute mouse cortical slices with a thickness of approximately 250 μm, we detected calcium activities with 7.5 Hz temporal resolution in the neuronal population.

Behavioral characteristics of dopamine D5 receptor knockout mice
Hitomi Sasamori, Toshiaki Asakura, Chiaki Sugiura, Youcef Bouchekioua, Naoya Nishitani, Masaaki Sato, Takayuki Yoshida, Miwako Yamasaki, Akira Terao, Masahiko Watanabe, Yu Ohmura, Mitsuhiro Yoshioka
Scientific Reports, 12, 1, Springer Science and Business Media LLC, 2022年12月
研究論文（学術雑誌）, Abstract

Major psychiatric disorders such as attention-deficit/hyperactivity disorder and schizophrenia are often accompanied by elevated impulsivity. However, anti-impulsive drug treatments are still limited. To explore a novel molecular target, we examined the role of dopamine D₅ receptors in impulse control using mice that completely lack D₅ receptors (D5KO mice). We also measured spontaneous activity and learning/memory ability because these deficits could confound the assessment of impulsivity. We found small but significant effects of D₅ receptor knockout on home cage activity only at specific times of the day. In addition, an analysis using the q-learning model revealed that D5KO mice displayed lower behavioral adjustment after impulsive actions. However, our results also showed that baseline impulsive actions and the effects of an anti-impulsive drug in D5KO mice were comparable to those in wild-type littermates. Moreover, unlike previous studies that used other D₅ receptor-deficient mouse lines, we did not observe reductions in locomotor activity, working memory deficits, or severe learning deficits in our line of D5KO mice. These findings demonstrate that D₅ receptors are dispensable for impulse control. Our results also indicate that time series analysis and detailed analysis of the learning process are necessary to clarify the behavioral functions of D₅ receptors.

Nna1, Essential for Purkinje Cell Survival, Is also Associated with Emotion and Memory. 2022 Oct 26;23(21):12961. doi: 10.3390/ijms232112961. PMID: 36361749.
Zhou L, Konno K, Yamazaki M, Abe M, Natsume R, Watanabe M, Takebayashi H, Sakimura K
Int J Mol Sci., 23(21), 12961, 1, 16, 2022年10月, ［査読有り］
英語, 研究論文（学術雑誌）

Cyclophilin D regulates NETosis and inflammation in myeloperoxidase-antineutrophil cytoplasmic antibody-associated vasculitis.
Takashi Kudo, Daigo Nakazawa, Kanako Watanabe-Kusunoki, Masatoshi Kanda, Satoka Shiratori-Aso, Nobuya Abe, Saori Nishio, Jun-Ichiro Koga, Sari Iwasaki, Takahiro Tsuji, Yuichiro Fukasawa, Miwako Yamasaki, Masahiko Watanabe, Sakiko Masuda, Utano Tomaru, Masaaki Murakami, Yasuaki Aratani, Akihiro Ishizu, Tatsuya Atsumi
Arthritis & rheumatology (Hoboken, N.J.), 75, 1, 71, 83, 2022年07月29日, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVE: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis where ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial necrosis occurs. Cyclophilin D (CypD) plays an important role in mediating cell necrosis and inflammation via opening of mitochondrial permeability transition pores (mPTP). Here, we examined the role of CypD in AAV pathogenesis. METHODS: In vitro, the role and mechanism of CypD in ANCA-stimulated neutrophils were assessed by immunostaining and electron microscopy. A comprehensive RNA sequencing analysis was performed on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, an-anti-MPO IgG-transfer AAV model or spontaneous AAV model mice were induced in CypD knockout or wild-type mice. RESULTS: In vitro, pharmacological and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species/cytochrome c release from the mitochondria. The analysis of RNA sequencing in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, and CypD deficiency reduced ANCA-induced alterations in gene expression. Furthermore, the upstream regulator analysis revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that CypD-dependent mPTP opening is associated with ANCA-induced neutrophil activation and NETosis. In both AAV models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis. CONCLUSIONS: CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.

L-DOPA-Induced Neurogenesis in the Hippocampus Is Mediated Through GPR143, a Distinct Mechanism of Dopamine.
Yuka Kasahara, Daiki Masukawa, Kenta Kobayashi, Miwako Yamasaki, Masahiko Watanabe, Yoshio Goshima
Stem cells (Dayton, Ohio), 40, 2, 215, 226, 2022年03月16日, ［国際誌］
英語, 研究論文（学術雑誌）, Neurogenesis occurs in the hippocampus throughout life and is implicated in various physiological brain functions such as memory encoding and mood regulation. L-3,4-dihydroxyphenylalanine (L-DOPA) has long been believed to be an inert precursor of dopamine. Here, we show that L-DOPA and its receptor, GPR143, the gene product of ocular albinism 1, regulate neurogenesis in the dentate gyrus (DG) in a dopamine-independent manner. L-DOPA at concentrations far lower than that of dopamine promoted proliferation of neural stem and progenitor cells in wild-type mice under the inhibition of its conversion to dopamine; this effect was abolished in GPR143 gene-deficient (Gpr143-/y) mice. Hippocampal neurogenesis decreased during development and adulthood, and exacerbated depression-like behavior was observed in adult Gpr143-/y mice. Replenishment of GPR143 in the DG attenuated the impaired neurogenesis and depression-like behavior. Our findings suggest that L-DOPA through GPR143 modulates hippocampal neurogenesis, thereby playing a role in mood regulation in the hippocampus.

Activation of extrasynaptic kainate receptors drives hilar mossy cell activity.
Czarina Ramos, Stefano Lutzu, Miwako Yamasaki, Yuchio Yanagawa, Kenji Sakimura, Susumu Tomita, Masahiko Watanabe, Pablo E Castillo
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2022年02月22日, ［国際誌］
英語, 研究論文（学術雑誌）, Mossy cells (MCs) of the dentate gyrus (DG) are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place field encoding, pattern separation and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission, and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished in GluK2 KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus.SIGNIFICANCE STATEMENTHilar mossy cells (MCs) are an understudied population of hippocampal neurons that form an excitatory loop with dentate granule cells. MCs have been implicated in pattern separation, spatial navigation, and epilepsy. Despite their importance in hippocampal function and disease, little is known about how MC activity is recruited. Here, we show for the first time that MCs express extrasynaptic kainate receptors (KARs), a subtype of glutamate receptors critically involved in neuronal function and epilepsy. While we found no evidence for synaptic KARs in MCs, KAR activation induced strong action potential firing of MCs, raising the possibility that extracellular KARs regulate MC excitability in vivo and may also promote dentate gyrus hyperexcitability and epileptogenesis.

SIPA1L1/SPAR1 interacts with the neurabin family of proteins and is involved in GPCR signaling.
Ken Matsuura, Shizuka Kobayashi, Kohtarou Konno, Miwako Yamasaki, Takahiro Horiuchi, Takao Senda, Tomoatsu Hayashi, Kiyotoshi Satoh, Fumiko Arima-Yoshida, Kei Iwasaki, Lumi Negishi, Naomi Yasui-Shimizu, Kazuyoshi Kohu, Shigenori Kawahara, Yutaka Kirino, Tsutomu Nakamura, Masahiko Watanabe, Tadashi Yamamoto, Toshiya Manabe, Tetsu Akiyama
The Journal of neuroscience : the official journal of the Society for Neuroscience, 42, 12, 2448, 2473, 2022年02月04日, ［国際誌］
英語, 研究論文（学術雑誌）, SIPA1L1 (also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the PSD-95/NMDA-R-complex. However, its physiological role remains poorly understood. Here, we performed expression analyses using super-resolution microscopy in mouse brain and demonstrated that SIPA1L1 is mainly localized to general submembranous regions in neurons, but surprisingly, not to PSD. Our screening for physiological interactors of SIPA1L1 in mouse brain identified spinophilin and neurabin-1, regulators of GPCR signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1 -/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1 -/- mice showed aberrant responses to α2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation, and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Male mice were used for all experiments. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome.SIGNIFICANCE STATEMENTSIPA1L1 is thought to regulate essential synaptic functions as a component of the PSD-95/NMDA-R-complex. In our screening for physiological SIPA1L1-interactors, we identified GPCR-signaling regulators. Moreover, SIPA1L1 KO mice showed striking behavioral anomalies, which may be relevant to GPCR signaling. Our findings revealed an unexpected role of SIPA1L1, which may open new avenues for research on neuropsychiatric disorders that involve dysregulated GPCR signaling. Another important aspect of this paper is that we showed effective methods for checking PSD association and identifying native protein interactors that are difficult to solubilize. These results may serve as a caution for future claims about interacting proteins and PSD proteins, which could eventually save time and resources for researchers and avoid confusion in the field.

mGluR1 signaling in cerebellar Purkinje cells: Subcellular organization and involvement in cerebellar function and disease
Miwako Yamasaki, Atsu Aiba, Masanobu Kano, Masahiko Watanabe
Neuropharmacology, 194, 108629, 108629, Elsevier {BV}, 2021年08月, ［国際誌］
英語, 研究論文（学術雑誌）, The cerebellum is essential for the control, coordination, and learning of movements, and for certain aspects of cognitive function. Purkinje cells are the sole output neurons in the cerebellar cortex and therefore play crucial roles in the diverse functions of the cerebellum. The type 1 metabotropic glutamate receptor (mGluR1) is prominently enriched in Purkinje cells and triggers downstream signaling pathways that are required for functional and structural plasticity, and for synaptic responses. To understand how mGluR1 contributes to cerebellar functions, it is important to consider not only the operational properties of this receptor, but also its spatial organization and the molecular interactions that enable its proper functioning. In this review, we highlight how mGluR1 and its related signaling molecules are organized into tightly coupled microdomains to fulfill physiological functions. We also describe emerging evidence that altered mGluR1 signaling in Purkinje cells underlies cerebellar dysfunction in ataxias of human patients and mouse models.

Compartmentalized Input-Output Organization of Lugaro Cells in the Cerebellar Cortex.
Taisuke Miyazaki, Miwako Yamasaki, Kenji F Tanaka, Masahiko Watanabe
Neuroscience, 462, 89, 105, 2021年05月10日, ［国際誌］
英語, 研究論文（学術雑誌）, Purkinje cells (PCs) are principal cerebellar neurons, and several classes of interneurons modulate their activity. Lugaro cells (LCs) are one such inhibitory interneuron with distinctive cytology and location, but still most enigmatic among cerebellar neurons. Here we serendipitously produced a novel transgenic mouse line, where a half of Yellow Cameleon (YC)(+) cells in the cerebellar cortex were judged to be LCs, and YC(+) LCs were estimated to constitute one-third of the total LC populations. Neurochemically, two-thirds of YC(+) LCs were dually GABAergic/glycinergic, with the rest being GABAergic. Beneath the PC layer, they extended a sheet of somatodendritic meshwork interconnected with neighboring LCs by adherens junctions, and received various inputs from climbing fibers, mossy fibers, granule cell axons, recurrent PC axons, Golgi cell axons, LC axons, and serotonergic fibers. Intriguingly, somatodendritic elements of individual LCs preferentially extended within a given cerebellar compartment defined by aldolase C expression. In turn, YC(+) LCs projected a dense lattice of ascending and transverse axons to the molecular layer, and innervated molecular layer interneurons (basket and stellate cells) and Golgi cells, but not PCs. Of note, ascending axons profusely innervated individual targets within a cerebellar compartment, while transverse axons ran across several compartments and innervated targets sparsely. This unique circuit configuration highlights that LCs integrate various excitatory, inhibitory, and modulatory inputs coming to the belonging cerebellar compartment and that, as an interneuron-selective interneuron, LCs can effectively disinhibit cerebellar cortical activities in a compartment-dependent manner through inhibition of inhibitory interneurons selectively targeting PCs and granule cells.

Fluorescent In Situ Hybridization for Sensitive and Specific Labeling
Miwako Yamasaki, Masahiko Watanabe
Neuromethods, 169, 145, 160, Humana Press Inc., 2021年
英語, 論文集(書籍)内論文, We describe a simplified and efficient protocol for non-isotopic fluorescent in situ hybridization experiments, allowing the implementation of multiple probe analyses at a single-cell resolution. This protocol involves simultaneous and specific hybridization of hapten-labeled riboprobes for mRNAs, followed by probe visualization via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. This procedure can be used to detect virtually any combination of two or three mRNA populations and is therefore a powerful method to characterize neuronal populations expressing a target gene and to determine the expression of genes of interest in the same or distinct cells. A combination of fluorescent in situ hybridization with immunofluorescence and retrograde fluorescent tracer labeling further expands the benefit and utility of histology-based methods. We also provide representative results and troubleshooting for the important steps of this protocol. Once specific probes and tissue sections are obtained, the total length of the entire procedure is 2–3 days.

Spike firing attenuation of serotonin neurons in learned helplessness rats is reversed by ketamine.
Kouichi Hashimoto, Yosuke Yamawaki, Kenji Yamaoka, Takayuki Yoshida, Kana Okada, Wanqin Tan, Miwako Yamasaki, Yoshiko Matsumoto-Makidono, Reika Kubo, Hisako Nakayama, Tsutomu Kataoka, Takashi Kanematsu, Masahiko Watanabe, Yasumasa Okamoto, Shigeru Morinobu, Hidenori Aizawa, Shigeto Yamawaki
Brain communications, 3, 4, fcab285, 2021年, ［国際誌］
英語, 研究論文（学術雑誌）, Animals suffering from uncontrollable stress sometimes show low effort to escape stress (learned helplessness). Changes in serotonin (5-hydroxytryptamine) signalling are thought to underlie this behaviour. Although the release of 5-hydroxytryptamine is triggered by the action potential firing of dorsal raphe nuclei 5-hydroxytryptamine neurons, the electrophysiological changes induced by uncontrollable stress are largely unclear. Herein, we examined electrophysiological differences among 5-hydroxytryptamine neurons in naïve rats, learned helplessness rats and rats resistant to inescapable stress (non-learned helplessness). Five-week-old male Sprague Dawley rats were exposed to inescapable foot shocks. After an avoidance test session, rats were classified as learned helplessness or non-learned helplessness. Activity-dependent 5-hydroxytryptamine release induced by the administration of high-potassium solution was slower in free-moving learned helplessness rats. Subthreshold electrophysiological properties of 5-hydroxytryptamine neurons were identical among the three rat groups, but the depolarization-induced spike firing was significantly attenuated in learned helplessness rats. To clarify the underlying mechanisms, potassium (K+) channels regulating the spike firing were initially examined using naïve rats. K+ channels sensitive to 500 μM tetraethylammonium caused rapid repolarization of the action potential and the small conductance calcium-activated K+ channels produced afterhyperpolarization. Additionally, dendrotoxin-I, a blocker of Kv1.1 (encoded by Kcna1), Kv1.2 (encoded by Kcna2) and Kv1.6 (encoded by Kcna6) voltage-dependent K+ channels, weakly enhanced the spike firing frequency during depolarizing current injections without changes in individual spike waveforms in naïve rats. We found that dendrotoxin-I significantly enhanced the spike firing of 5-hydroxytryptamine neurons in learned helplessness rats. Consequently, the difference in spike firing among the three rat groups was abolished in the presence of dendrotoxin-I. These results suggest that the upregulation of dendrotoxin-I-sensitive Kv1 channels underlies the firing attenuation of 5-hydroxytryptamine neurons in learned helplessness rats. We also found that the antidepressant ketamine facilitated the spike firing of 5-hydroxytryptamine neurons and abolished the firing difference between learned helplessness and non-learned helplessness by suppressing dendrotoxin-I-sensitive Kv1 channels. The dendrotoxin-I-sensitive Kv1 channel may be a potential target for developing drugs to control activity of 5-hydroxytryptamine neurons.

Kv11 (ether-à-go-go-related gene) voltage-dependent K+ channels promote resonance and oscillation of subthreshold membrane potentials.
Toshinori Matsuoka, Miwako Yamasaki, Manabu Abe, Yukiko Matsuda, Hiroyuki Morino, Hideshi Kawakami, Kenji Sakimura, Masahiko Watanabe, Kouichi Hashimoto
The Journal of physiology, 599, 2, 547, 569, 2021年01月, ［国際誌］
英語, 研究論文（学術雑誌）, KEY POINTS: Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so-called 'resonant properties'). Here, we report that heterologous expression of mouse Kv11 voltage-dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties. Kv11.3-expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor. The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice. This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. ABSTRACT: The plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor-like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+ ) channels, encoded by ether-á-go-go-related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole-cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3-, Kv11.1(Merg1b)- and Kv11.1(Merg1a)-expressing cells were 2-6 Hz, 2-4 Hz, and 0.6-0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor-inductor-capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11-dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at -30 to -60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency-dependent responses in a subtype-dependent manner from resting to depolarized membrane potentials.

Development of an L-type Ca2+ channel-dependent Ca2+ transient during the radial migration of cortical excitatory neurons.
Shin-Ichiro Horigane, Shun Hamada, Satoshi Kamijo, Hirokazu Yamada, Miwako Yamasaki, Masahiko Watanabe, Haruhiko Bito, Toshihisa Ohtsuka, Sayaka Takemoto-Kimura
Neuroscience research, 169, 17, 26, 2020年06月26日, ［国際誌］
英語, 研究論文（学術雑誌）, Increasing evidence has shown that voltage-gated L-type Ca2+ channels (LTCCs) are crucial for neurodevelopmental events, including neuronal differentiation/migration and neurite morphogenesis/extension. However, the time course of their functional maturation during the development of excitatory neurons remains unknown. Using a combination of fluorescence in situ hybridization and in utero electroporation-based labeling, we found that the transcripts of Cacna1c and Cacna1d, which encode the LTCC pore-forming subunits, were upregulated in the intermediate zone (IZ) during radial migration. Ca2+ imaging using GCaMP6s in acute brain slices showed spontaneous Ca2+ transients in migrating neurons throughout the IZ. Neurons in the IZ upper layer, especially in the multipolar-to-bipolar transition layer (TL), exhibited more frequent Ca2+ transients than adjacent layers and responded to FPL64176, a potent activator of LTCC. Consistently, nimodipine, an LTCC blocker, inhibited spontaneous Ca2+ transients in neurons in the TL. Collectively, we showed a hitherto unknown increased prevalence of LTCC-dependent Ca2+ transients in the TL of the IZ upper layer during the radial migration of excitatory neurons, which could be essential for the regulation of Ca2+-dependent neurodevelopmental processes.

TMEM163 Regulates ATP-Gated P2X Receptor and Behavior.
Elizabeth J Salm, Patrick J Dunn, Lili Shan, Miwako Yamasaki, Nathalie M Malewicz, Taisuke Miyazaki, Joongkyu Park, Akio Sumioka, R Richard L Hamer, Wei-Wu He, Megumi Morimoto-Tomita, Robert H LaMotte, Susumu Tomita
Cell reports, 31, 9, 107704, 107704, 2020年06月02日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Nectin-2α is localized at cholinergic neuron dendrites and regulates synapse formation in the medial habenula.
Hajime Shiotani, Muneaki Miyata, Takeshi Kameyama, Kenji Mandai, Miwako Yamasaki, Masahiko Watanabe, Kiyohito Mizutani, Yoshimi Takai
The Journal of comparative neurology, 529, 2, 450, 477, 2020年05月26日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The medial habenula (MHb) receives afferents from the triangular septum and the medial septal complex, projects efferents to the interpeduncular nucleus (IPN) in the midbrain to regulate dopamine and serotonin levels, and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed that the cell adhesion molecule nectin-2α is localized at the boundary between adjacent somata of clustered cholinergic neurons and regulates the voltage-gated A-type K+ channel Kv4.2 localization at membrane specializations in the MHb. This adhesion apparatus, named nectin-2α spots, is not associated with the nectin-binding protein afadin or any classic cadherins and their binding proteins p120-catenin and β-catenin. We showed here that nectin-2α was additionally localized at cholinergic neuron dendrites in synaptic regions of the MHb. The genetic ablation of nectin-2 reduced the number of synapses in the MHb without affecting their morphology. Nectin-2α was associated with afadin, cadherin-8, p120-catenin, β-catenin, and αN-catenin, forming puncta adherentia junctions (PAJs). Nectin-2α was observed in the IPN, but not in the triangular septum or the medial septal complex. The genetic ablation of nectin-2 did not affect synapse formation in the IPN. These results indicate that nectin-2α forms two types of adhesion apparatus in the MHb, namely nectin-2α spots at neighboring somata and PAJs at neighboring dendrites, and that dendritic PAJs regulate synapse formation in the MHb. This article is protected by copyright. All rights reserved.

Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain.
Chihiro Nakamoto, Kohtarou Konno, Taisuke Miyazaki, Ena Nakatsukasa, Rie Natsume, Manabu Abe, Meiko Kawamura, Yugo Fukazawa, Ryuichi Shigemoto, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe
The Journal of comparative neurology, 528, 6, 1003, 1027, 2020年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD-95-expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS-digested freeze-fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular-anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions.

Localization of phospholipase C β3 in the major salivary glands of adult mice.
Atsara Rawangwong, Atthapon Pidsaya, Wipawee Thoungseabyoun, Apussara Tachow, Tarinee Sawatpanich, Waraporn Sakaew, Miwako Yamasaki, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
Acta histochemica, 121, 4, 484, 490, 2019年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Phospholipase C (PLC)β has a role in saliva secretion by controlling intracellular Ca2+via its product, IP3. The present study was attempted to localize PLCβ isoforms in mouse salivary glands in situ. A single major band was detected for PLCβ3 in immunoblots of the parotid and sublingual glands (PG, SLG), while no such band was seen in the submandibular gland (SMG). No bands were detected for PLCβ1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCβ3 was seen in the cytoplasm including the plasmalemma of almost all ductal cells, while no distinct immunoreactivity was discerned in most acinar cells except for sublingual demilune cells. Numerous ductal cells exhibited higher immunoreactivity for PLCβ3 in their apical/supranuclear cell domain including the plasmalemma than in the basal/infranuclear domain, indicating an apico-basal polarity. In immuno-gold electron microscopy of PG ducts and SLG ducts and demilunes, most gold particles were found in association with plasma membranes as well as various intracellular membranes, most of which formed small oblong or flattened vesicles and vacuoles. A few particles were seen without association with any membranous structures. The present finding supports the previous physio-pharmacological result that Ca2+-signaling proteins as well as initial intracellular Ca2+ changes occur in the apical cell domain including the plasma membranes of the exocrine cells.

mGluR1 in cerebellar Purkinje cells is essential for the formation but not expression of associative eyeblink memory.
Nakao H, Kishimoto Y, Hashimoto K, Kitamura K, Yamasaki M, Nakao K, Watanabe M, Kano M, Kirino Y, Aiba A
Scientific reports, 9, 1, 7353, 7353, 2019年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Classical eyeblink conditioning is a representative associative motor learning that requires both the cerebellar cortex and the deep cerebellar nucleus (DCN). Metabotropic glutamate receptor subtype 1 (mGluR1) is richly expressed in Purkinje cells (PCs) of the cerebellar cortex. Global mGluR1 knock-out (KO) mice show a significantly lower percentage of conditioned response (CR%) than wild-type mice in eyeblink conditioning, and the impaired CR% is restored by the introduction of mGluR1 in PCs. However, the specific roles of mGluR1 in major memory processes, including formation, storage and expression have not yet been defined. We thus examined the role of mGluR1 in these processes of eyeblink conditioning, using mGluR1 conditional KO (cKO) mice harboring a selective and reversible expression of mGluR1 in PCs. We have found that eyeblink memory is not latently formed in the absence of mGluR1 in adult mouse PCs. However, once acquired, eyeblink memory is expressed even after the depletion of mGluR1 in PCs. We thus conclude that mGluR1 in PCs is indispensable for the formation of eyeblink memory, while it is not required for the expression of CR.

Heterogeneous localization of muscarinic cholinoceptor M1 in the salivary ducts of adult mice.
Atsara Rawangwong, Suthankamon Khrongyut, Surang Chomphoo, Kohtaro Konno, Miwako Yamasaki, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
Archives of oral biology, 100, 14, 22, 2019年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We hypothesize variation in expression and localization, along the course of the glandular tubule, of muscarinic cholinergic receptor M1 which plays as a distinct contribution, though minor in comparison with M3 receptor, in saliva secretion. Localization of the M1 receptor was examined using immunohistochemistry in three major salivary glands. Although all glandular cells were more or less M1-immunoreactive, acinar cells were weakly immunoreactive, while ductal cells exhibited substantial M1-immunoreactivity. Many ductal cells exhibited clear polarity with higher immunoreactivity in their apical/supra-nuclear domain. However, some exhibited indistinct polarity because of additional higher immunoreactivity in their basal/infra-nuclear domain. A small group of cells with intense immunoreactivity was found, mostly located in the intercalated ducts or in portions of the striated ducts close to the intercalated ducts. In immuno-electron microscopy, the immunoreactive materials were mainly in the cytoplasm including various vesicles and vacuoles. Unexpectedly, distinct immunoreactivity on apical and basal plasma membranes was infrequent in most ductal cells. The heterogeneous localization of M1-immunoreactivity along the gland tubular system is discussed in view of possible modulatory roles of the M1 receptor in saliva secretion.

Silent Learning.
Janine I Rossato, Andrea Moreno, Lisa Genzel, Miwako Yamasaki, Tomonori Takeuchi, Santiago Canals, Richard G M Morris
Current biology : CB, 28, 21, 3508, 3515, 2018年11月05日, ［国際誌］
英語, 研究論文（学術雑誌）, We introduce the concept of "silent learning"-the capacity to learn despite neuronal cell-firing being largely absent. This idea emerged from thinking about dendritic computation [1, 2] and examining whether the encoding, expression, and retrieval of hippocampal-dependent memory could be dissociated using the intrahippocampal infusion of pharmacological compounds. We observed that very modest enhancement of GABAergic inhibition with low-dose muscimol blocked both cell-firing and the retrieval of an already-formed memory but left induction of long-term potentiation (LTP) and new spatial memory encoding intact (silent learning). In contrast, blockade of hippocampal NMDA receptors by intrahippocampal D-AP5 impaired both the induction of LTP and encoding but had no effect on memory retrieval. Blockade of AMPA receptors by CNQX impaired excitatory synaptic transmission and cell-firing and both memory encoding and retrieval. Thus, in keeping with the synaptic plasticity and memory hypothesis [3], the hippocampal network can mediate new memory encoding when LTP induction is intact even under conditions in which somatic cell-firing is blocked.

Localization of nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons and its regulatory role in the subcellular localization of the voltage-gated A-type K⁺ channel Kv4.2 in the medial habenula.
Shiotani H, Miyata M, Itoh Y, Wang S, Kaito A, Mizoguchi A, Yamasaki M, Watanabe M, Mandai K, Mochizuki H, Takai Y
The Journal of comparative neurology, 526, 9, 1527, 1549, Wiley-Liss Inc., 2018年06月15日, ［査読有り］
英語, 研究論文（学術雑誌）, The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin-like cell adhesion molecules (CAMs) nectin-2α and nectin-2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin-2α, but not nectin-2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin-2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin-2α at this boundary was not colocalized with the nectin-2α-binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin-2α forms a cell adhesion apparatus different from the Kv4.2-associated MSs. Genetic ablation of nectin-2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin-2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.

Olig2-Lineage astrocytes: A distinct subtype of astrocytes that differs from GFAP astrocytes
Kouko Tatsumi, Ayami Isonishi, Miwako Yamasaki, Yoshie Kawabe, Shoko Morita-Takemura, Kazuki Nakahara, Yuki Terada, Takeaki Shinjo, Hiroaki Okuda, Tatsuhide Tanaka, Akio Wanaka
Frontiers in Neuroanatomy, 12, 8, 8, Frontiers Media S.A., 2018年02月14日, ［査読有り］
英語, 研究論文（学術雑誌）, Astrocytes are the most abundant glia cell type in the central nervous system (CNS), and are known to constitute heterogeneous populations that differ in their morphology, gene expression and function. Although glial fibrillary acidic protein (GFAP) is the cardinal cytological marker of CNS astrocytes, GFAP-negative astrocytes can easily be found in the adult CNS. Astrocytes are also allocated to spatially distinct regional domains during development. This regional heterogeneity suggests that they help to coordinate post-natal neural circuit formation and thereby to regulate eventual neuronal activity. Here, during lineage-tracing studies of cells expressing Olig2 using Olig2CreER
Rosa-CAG- LSL-eNpHR3.0-EYFP transgenic mice, we found Olig2-lineage mature astrocytes in the adult forebrain. Long-term administration of tamoxifen resulted in sufficient recombinant induction, and Olig2-lineage cells were found to be preferentially clustered in some adult brain nuclei. We then made distribution map of Olig2-lineage astrocytes in the adult mouse brain, and further compared the map with the distribution of GFAP- positive astrocytes visualized in GFAPCre
Rosa-CAG-LSL-eNpHR3.0-EYFP mice. Brain regions rich in Olig2-lineage astrocytes (e.g., basal forebrain, thalamic nuclei, and deep cerebellar nuclei) tended to lack GFAP-positive astrocytes, and vice versa. Even within a single brain nucleus, Olig2-lineage astrocytes and GFAP astrocytes frequently occupied mutually exclusive territories. These findings strongly suggest that there is a subpopulation of astrocytes (Olig2-lineage astrocytes) in the adult brain, and that it differs from GFAP-positive astrocytes in its distribution pattern and perhaps also in its function. Interestingly, the brain nuclei rich in Olig2-lineage astrocytes strongly expressed GABA- transporter 3 in astrocytes and vesicular GABA transporter in neurons, suggesting that Olig2-lineage astrocytes are involved in inhibitory neuronal transmission.

Serotonergic Input to Orexin Neurons Plays a Role in Maintaining Wakefulness and REM Sleep Architecture.
Saito YC, Tsujino N, Abe M, Yamazaki M, Sakimura K, Sakurai T
Frontiers in neuroscience, 12, 892, 2018年, ［査読有り］

Localization of photoperiod responsive circadian oscillators in the mouse suprachiasmatic nucleus
Tomoko Yoshikawa, Natsuko F. Inagaki, Seiji Takagi, Shigeru Kuroda, Miwako Yamasaki, Masahiko Watanabe, Sato Honma, Ken-ichi Honma
SCIENTIFIC REPORTS, 7, 1, 8210, NATURE PUBLISHING GROUP, 2017年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The circadian pacemaker in the suprachiasmatic nucleus (SCN) yields photoperiodic response to transfer seasonal information to physiology and behavior. To identify the precise location involved in photoperiodic response in the SCN, we analyzed circadian Period1 and PERIOD2 rhythms in horizontally sectioned SCN of mice exposed to a long or short day. Statistical analyses of bioluminescence images with respective luciferase reporters on pixel level enabled us to identify the distinct localization of three oscillating regions; a large open-ring-shape region, the region at the posterior end and a sharply demarcated oval region at the center of the SCN. The first two regions are the respective sites for the so-called evening and morning oscillators, and the third region is possibly a site for mediating photic signals to the former oscillators. In these regions, there are two classes of oscillating cells in which Per1 and Per2 could play differential roles in photoperiodic responses.

Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells
Taisuke Miyazaki, Miwako Yamasaki, Kouichi Hashimoto, Kazuhisa Kohda, Michisuke Yuzaki, Keiko Shimamoto, Kohichi Tanaka, Masanobu Kano, Masahiko Watanabe
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 114, 28, 7438, 7443, NATL ACAD SCIENCES, 2017年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Astrocytes regulate synaptic transmission through controlling neurotransmitter concentrations around synapses. Little is known, however, about their roles in neural circuit development. Here we report that Bergmann glia (BG), specialized cerebellar astrocytes that thoroughly enwrap Purkinje cells (PCs), are essential for synaptic organization in PCs through the action of the L-glutamate/L-aspartate transporter (GLAST). In GLAST-knockout mice, dendritic innervation by the main ascending climbing fiber (CF) branch was significantly weakened, whereas the transverse branch, which is thin and non-synaptogenic in control mice, was transformed into thick and synaptogenic branches. Both types of CF branches frequently produced aberrant wiring to proximal and distal dendrites, causing multiple CF-PC innervation. Our electrophysiological analysis revealed that slow and small CF-evoked excitatory postsynaptic currents (EPSCs) were recorded from almost all PCs in GLAST-knockout mice. These atypical CF-EPSCs were far more numerous and had significantly faster 10-90% rise time than those elicited by glutamate spillover under pharmacological blockade of glial glutamate transporters. Innervation by parallel fibers (PFs) was also affected. PF synapses were robustly increased in the entire dendritic trees, leading to impaired segregation of CF and PF territories. Furthermore, lamellate BG processes were retracted from PC dendrites and synapses, leading to the exposure of these neuronal elements to the extracellular milieus. These synaptic and glial phenotypes were reproduced in wild-type mice after functional blockade of glial glutamate transporters. These findings highlight that glutamate transporter function by GLAST on BG plays important roles in development and maintenance of proper synaptic wiring and wrapping in PCs.

Locus Coeruleus and Dopamine-Dependent Memory Consolidation
Miwako Yamasaki, Tomonori Takeuchi
NEURAL PLASTICITY, 2017, 8602690, HINDAWI LTD, 2017年, ［査読有り］
英語, Most everyday memories including many episodic-like memories that we may form automatically in the hippocampus (HPC) are forgotten, while some of them are retained for a long time by a memory stabilization process, called initial memory consolidation. Specifically, the retention of everyday memory is enhanced, in humans and animals, when something novel happens shortly before or after the time of encoding. Converging evidence has indicated that dopamine (DA) signaling via D1/D5 receptors in HPC is required for persistence of synaptic plasticity and memory, thereby playing an important role in the novelty-associated memory enhancement. In this review paper, we aim to provide an overview of the key findings related to D1/D5 receptor-dependent persistence of synaptic plasticity and memory in HPC, especially focusing on the emerging evidence for a role of the locus coeruleus (LC) in DA-dependent memory consolidation. We then refer to candidate brain areas and circuits that might be responsible for detection and transmission of the environmental novelty signal and molecular and anatomical evidence for the LC-DA system. We also discuss molecular mechanisms that might mediate the environmental novelty-associated memory enhancement, including plasticity-related proteins that are involved in initial memory consolidation processes in HPC.

QRFP-Deficient Mice Are Hypophagic, Lean, Hypoactive and Exhibit Increased Anxiety-Like Behavior
Kitaro Okamoto, Miwako Yamasaki, Keizo Takao, Shingo Soya, Monica Iwasaki, Koh Sasaki, Kenta Magoori, Iori Sakakibara, Tsuyoshi Miyakawa, Michihiro Mieda, Masahiko Watanabe, Juro Sakai, Masashi Yanagisawa, Takeshi Sakurai
PLOS ONE, 11, 11, e0164716, PUBLIC LIBRARY SCIENCE, 2016年11月, ［査読有り］
英語, 研究論文（学術雑誌）, How the hypothalamus transmits hunger information to other brain regions to govern whole brain function to orchestrate feeding behavior has remained largely unknown. Our present study suggests the importance of a recently found lateral hypothalamic neuropeptide, QRFP, in this signaling. Qrfp(-/-) mice were hypophagic and lean, and exhibited increased anxiety-like behavior, and were hypoactive in novel circumstances as compared with wild type littermates. They also showed decreased wakefulness time in the early hours of the dark period. Histological studies suggested that QRFP neurons receive rich innervations from neurons in the arcuate nucleus which is a primary region for sensing the body's metabolic state by detecting levels of leptin, ghrelin and glucose. These observations suggest that QRFP is an important mediator that acts as a downstream mediator of the arcuate nucleus and regulates feeding behavior, mood, wakefulness and activity.

The active zone protein CAST regulates synaptic vesicle recycling and quantal size in the mouse hippocampus
Shizuka Kobayashi, Yamato Hida, Hiroyoshi Ishizaki, Eiji Inoue, Miki Tanaka-Okamoto, Miwako Yamasaki, Taisuke Miyazaki, Masahiro Fukaya, Isao Kitajima, Yoshimi Takai, Masahiko Watanabe, Toshihisa Ohtsuka, Toshiya Manabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 44, 5, 2272, 2284, WILEY-BLACKWELL, 2016年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size, and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system.

Locus coeruleus and dopaminergic consolidation of everyday memory
Tomonori Takeuchi, Adrian J. Duszkiewicz, Alex Sonneborn, Patrick A. Spooner, Miwako Yamasaki, Masahiko Watanabe, Caroline C. Smith, Guillen Fernandez, Karl Deisseroth, Robert W. Greene, Richard G. M. Morris
NATURE, 537, 7620, 357, +, NATURE PUBLISHING GROUP, 2016年09月, ［査読有り］
英語, 研究論文（学術雑誌）, The retention of episodic-like memory is enhanced, in humans and animals, when something novel happens shortly before or after encoding. Using an everyday memory task in mice, we sought the neurons mediating this dopamine-dependent novelty effect, previously thought to originate exclusively from the tyrosine-hydroxylase-expressing (TH+) neurons in the ventral tegmental area. Here we report that neuronal firing in the locus coeruleus is especially sensitive to environmental novelty, locus coeruleus TH+ neurons project more profusely than ventral tegmental area TH+ neurons to the hippocampus, optogenetic activation of locus coeruleus TH+ neurons mimics the novelty effect, and this novelty-associated memory enhancement is unaffected by ventral tegmental area inactivation. Surprisingly, two effects of locus coeruleus TH+ photoactivation are sensitive to hippocampal D-1/D-5 receptor blockade and resistant to adrenoceptor blockade: memory enhancement and long-lasting potentiation of synaptic transmission in CA1 ex vivo. Thus, locus coeruleus TH+ neurons can mediate post-encoding memory enhancement in a manner consistent with possible co-release of dopamine in the hippocampus.

Crucial Roles of the Endocannabinoid 2-Arachidonoylglycerol in the Suppression of Epileptic Seizures.
Sugaya Y, Yamazaki M, Uchigashima M, Kobayashi K, Watanabe M, Sakimura K, Kano M
Cell reports, 16, 5, 1405, 1415, 2016年08月, ［査読有り］

Distinct Subunit Domains Govern Synaptic Stability and Specificity of the Kainate Receptor
Christoph Straub, Yoav Noam, Toshihiro Nomura, Miwako Yamasaki, Dan Yan, Herman B. Fernandes, Ping Zhang, James R. Howe, Masahiko Watanabe, Anis Contractor, Susumu Tomita
CELL REPORTS, 16, 2, 531, 544, CELL PRESS, 2016年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.

Ionic Basis for Membrane Potential Resonance in Neurons of the Inferior Olive
Yoshiko Matsumoto-Makidono, Hisako Nakayama, Miwako Yamasaki, Taisuke Miyazaki, Kazuto Kobayashi, Masahiko Watanabe, Masanobu Kano, Kenji Sakimura, Kouichi Hashimoto
CELL REPORTS, 16, 4, 994, 1004, CELL PRESS, 2016年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Some neurons have the ability to enhance output voltage to input current with a preferred frequency, which is called resonance. Resonance is thought to be a basis for membrane potential oscillation. Although ion channels responsible for resonance have been reported, the precise mechanisms by which these channels work remain poorly understood. We have found that resonance is reduced but clearly present in the inferior olivary neurons of Cav3.1 T-type voltage-dependent Ca2+ channel knockout (KO) mice. The activation of Cav3.1 channels is strongly membrane potential dependent, but less frequency dependent. Residual resonance in Cav3.1 KO mice is abolished by a hyper-polarization-activated cyclic nucleotide-gated (HCN) channel blocker, ZD7288, and is partially suppressed by voltage-dependent K+ channel blockers. Resonance is inhibited by ZD7288 in wild-type mice and impaired in HCN1 KO mice, suggesting that the HCN1 channel is essential for resonance. The ZD7288-sensitive current is nearly sinusoidal and strongly frequency dependent. These results suggest that Cav3.1 and HCN1 channels act as amplifying and resonating conductances, respectively.

Developmental Switch in Spike Timing-Dependent Plasticity and Cannabinoid-Dependent Reorganization of the Thalamocortical Projection in the Barrel Cortex
Chiaki Itami, Jui-Yen Huang, Miwako Yamasaki, Masahiko Watanabe, Hui-Chen Lu, Fumitaka Kimura
JOURNAL OF NEUROSCIENCE, 36, 26, 7039, 7054, SOC NEUROSCIENCE, 2016年06月, ［査読有り］
英語, 研究論文（学術雑誌）, The formation and refinement of thalamocortical axons (TCAs) is an activity-dependent process (Katz and Shatz, 1996), but its mechanism and nature of activity are elusive. We studied the role of spike timing-dependent plasticity (STDP) in TCA formation and refinement in mice. At birth (postnatal day 0, P0), TCAs invade the cortical plate, from which layers 4 (L4) and L2/3 differentiate at P3-P4. A portion of TCAs transiently reach toward the pia surface around P2-P4 (Senft and Woolsey, 1991; Rebsam et al., 2002) but are eventually confined below the border between L2/3 and L4. We previously showed that L4-L2/3 synapses exhibit STDP with only potentiation (timing-dependent long-term potentiation [t-LTP]) during synapse formation, then switch to a Hebbian form of STDP. Here we show that TCA-cortical plate synapses exhibit robust t-LTP in neonates, whose magnitude decreased gradually after P4-P5. After L2/3 is differentiated, TCA-L2/3 gradually switched to STDP with only depression (t-LTD) after P7-P8, whereas TCA-L4 lost STDP. t-LTP was dependent on NMDA receptor and PKA, whereas t-LTD was mediated by Type 1 cannabinoid receptors (CB1Rs) probably located at TCA terminals, revealed by global and cortical excitatory cell-specific knock-out of CB1R. Moreover, we found that administration of CB1R agonists, including Delta(9)-tetrahydrocannabinol, caused substantial retraction of TCAs. Consistent with this, individual thalamocortical axons exuberantly innervated L2/3 at P12 in CB1R knock-outs, indicating that endogenous cannabinoid signaling shapes TCA projection. These results suggest that the developmental switch in STDP and associated appearance of CB1R play important roles in the formation and refinement of TCAs.

Transsynaptic Modulation of Kainate Receptor Functions by C1q-like Proteins
Keiko Matsuda, Timotheus Budisantoso, Nikolaos Mitakidis, Yuki Sugaya, Eriko Miura, Wataru Kakegawa, Miwako Yamasaki, Kohtarou Konno, Motokazu Uchigashima, Manabu Abe, Izumi Watanabe, Masanobu Kano, Masahiko Watanabe, Kenji Sakimura, A. Radu Aricescu, Michisuke Yuzaki
NEURON, 90, 4, 752, 767, CELL PRESS, 2016年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Postsynaptic kainate-type glutamate receptors (KARs) regulate synaptic network activity through their slow channel kinetics, most prominently at mossy fiber (MF)-CA3 synapses in the hippocampus. Nevertheless, how KARs cluster and function at these synapses has been unclear. Here, we show that C1q-like proteins C1ql2 and C1ql3, produced by MFs, serve as extracellular organizers to recruit functional postsynaptic KAR complexes to the CA3 pyramidal neurons. C1ql2 and C1ql3 specifically bound the amino-terminal domains of postsynaptic GluK2 and GluK4 KAR subunits and the presynaptic neurexin 3 containing a specific sequence in vitro. In C1ql2/3 double-null mice, CA3 synaptic responses lost the slow, KAR-mediated components. Furthermore, despite induction of MF sprouting in a temporal lobe epilepsy model, KARs were not recruited to postsynaptic sites in C1ql2/3 double-null mice, leading to reduced recurrent circuit activities. C1q family proteins, broadly expressed, are likely to modulate KAR function throughout the brain and represent promising antiepileptic targets.

TARP γ-2 and γ-8 Differentially Control AMPAR Density Across Schaffer Collateral/Commissural Synapses in the Hippocampal CA1 Area.
Yamasaki M, Fukaya M, Yamazaki M, Azechi H, Natsume R, Abe M, Sakimura K, Watanabe M
The Journal of neuroscience : the official journal of the Society for Neuroscience, 36, 15, 4296, 4312, SOC NEUROSCIENCE, 2016年04月, ［査読有り］
英語, 研究論文（学術雑誌）, The number of AMPA-type glutamate receptors (AMPARs) at synapses is the major determinant of synaptic strength and varies from synapse to synapse. To clarify the underlying molecular mechanisms, the density of AMPARs, PSD-95, and transmembrane AMPAR regulatory proteins (TARPs) were compared at Schaffer collateral/commissural (SCC) synapses in the adult mouse hippocampal CA1 by quantitative immunogold electron microscopy using serial sections. We examined four types of SCC synapses: perforated and nonperforated synapses on pyramidal cells and axodendritic synapses on parvalbumin-positive (PV synapse) and pravalbumin-negative interneurons (non-PV synapse). SCC synapses were categorized into those expressing high-density (perforated and PV synapses) or low-density (nonperforated and non-PV synapses) AMPARs. Although the density of PSD-95 labeling was fairly constant, the density and composition of TARP isoforms was highly variable depending on the synapse type. Of the three TARPs expressed in hippocampal neurons, the disparity inTARP gamma-2 labeling was closely related to that of AMPAR labeling. Importantly, AMPAR density was significantly reduced at perforated and PV synapses in TARP gamma-2-knock-out (KO) mice, resulting in a virtual loss of AMPAR disparity among SCC synapses. In comparison, TARP gamma-8 was the only TARP expressed at nonperforated synapses, where AMPAR labeling further decreased to a background level in TARP gamma-8-KO mice. These results show that synaptic inclusion of TARP gamma-2 potently increases AMPAR expression and transforms low-density synapses into high-density ones, whereas TARP gamma-8 is essential for low-density or basal expression of AMPARs at nonperforated synapses. Therefore, these TARPs are critically involved in AMPAR density control at SCC synapses.

Territories of heterologous inputs onto Purkinje cell dendrites are segregated by mGluR1-dependent parallel fiber synapse elimination
Ryoichi Ichikawa, Kouichi Hashimoto, Taisuke Miyazaki, Motokazu Uchigashima, Miwako Yamasaki, Atsu Aiba, Masanobu Kano, Masahiko Watanabe
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 113, 8, 2282, 2287, NATL ACAD SCIENCES, 2016年02月, ［査読有り］
英語, 研究論文（学術雑誌）, In Purkinje cells (PCs) of the cerebellum, a single "winner" climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9-15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase C gamma (PKC gamma), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1 alpha into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKC gamma signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites.

Molecular and anatomical evidence for the input pathway- and target cell type-dependent regulation of glutamatergic synapses
Miwako Yamasaki
ANATOMICAL SCIENCE INTERNATIONAL, 91, 1, 8, 21, SPRINGER, 2016年01月, ［査読有り］
英語, Glutamate mediates most fast excitatory transmission in the central nervous system by activating primarily two types of ionotropic glutamate receptors: alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Differential subunit combinations generate great functional diversity in both categories of receptors, making them highly suitable for meeting complex functional requirements. Converging evidence has indicated that distinct AMPA and NMDA receptor subtypes are selectively targeted to functionally different synapses according to different factors, including presynaptic inputs, postsynaptic cell types, and synaptic configurations. This article provides an overview of recent progress in understanding the basic principles governing the synaptic allocation of AMPA and NMDA receptors, and discusses the underlying mechanisms and functional implications.

VGluT3-Expressing CCK-Positive Basket Cells Construct Invaginating Synapses Enriched with Endocannabinoid Signaling Proteins in Particular Cortical and Cortex-Like Amygdaloid Regions of Mouse Brains
Yuki Omiya, Motokazu Uchigashima, Kohtarou Konno, Miwako Yamasaki, Taisuke Miyazaki, Takayuki Yoshida, Ichiro Kusumi, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 35, 10, 4215, 4228, SOC NEUROSCIENCE, 2015年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Invaginating synapses in the basal amygdala are a unique type of GABAergic synapses equipped with molecular-anatomical organization specialized for 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid signaling. Cholecystokinin (CCK)-positive basket cell terminals protrude into pyramidal cell somata and form invaginating synapses, where apposing presynaptic and postsynaptic elements are highly loaded with cannabinoid receptor CB1 or 2-AG synthetic enzyme diacylglycerol lipase-alpha (DGL alpha), respectively. The present study scrutinized their neurochemical and neuroanatomical phenotypes in adult mouse telencephalon. In the basal amygdala, vesicular glutamate transporter-3 (VGluT3) was transcribed in one-fourth of CB1-expressing GABAergic interneurons. The majority of VGluT3-positive CB1-expressing basket cell terminals apposed DGL alpha clusters, whereas the majority of VGluT3-negative ones did not. Importantly, VGluT3-positive basket cell terminals selectively constructed invaginating synapses. GABA(A) receptors accumulated on the postsynaptic membrane of invaginating synapses, whereas metabotropic glutamate receptor-5 (mGluR5) was widely distributed on the somatodendritic surface of pyramidal cells. Moreover, CCK2 receptor (CCK2R) was highly transcribed in pyramidal cells. In cortical regions, pyramidal cells equipped with such VGluT3/CB1/DGL alpha-accumulated invaginating synapses were found at variable frequencies depending on the subregions. Therefore, in addition to extreme proximity of CB1- and DGL alpha-loaded presynaptic and postsynaptic elements, tripartite transmitter phenotype of GABA/glutamate/CCK is the common neurochemical feature of invaginating synapses, suggesting that glutamate, CCK, or both can promote 2-AG synthesis through activating G alpha(q/11) protein-coupled mGluR5 and CCK2R. These molecular configurations led us to hypothesize that invaginating synapses might be evolved to provide some specific mechanisms of induction, regulation, and cooperativity for 2-AG-mediated retrograde signaling in particular cortical and cortex-like amygdaloid regions.

Involvement of diacylglycerol kinase β in the spine formation at distal dendrites of striatal medium spiny neurons.
Hozumi Y, Kakefuda K, Yamasaki M, Watanabe M, Hara H, Goto K
Brain research, 1594, 36, 45, ELSEVIER SCIENCE BV, 2015年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Spine formation, a salient feature underlying neuronal plasticity to adapt to a changing environment, is regulated by complex machinery involving membrane signal transduction. The diacylglycerol kinase (DGK) family, which is involved in membrane lipid metabolism, catalyzes the phosphorylation of a lipid second messenger, diacylglycerol (DG). Of the DGKs, DGK beta is characterized by predominant expression in a specific brain region: the striatum. We previously demonstrated that DGK beta is expressed selectively in medium spiny neurons (MSNs) and that it is highly enriched in the perisynaptic membrane on dendritic spines contacted with excitatory terminals. Moreover, DGK beta regulates spinogenesis through actin-based remodeling in an activity-dependent manner. However, the detailed mechanisms of spinogenesis regulation and its functional significance remain unclear. To address these issues, we performed Golgi-Cox staining to examine morphological aspects of MSNs in the striatum of DGK beta-knockout (KO) mice. Results show that striatal MSNs of DGK beta-KO mice exhibited lower dendritic spine density at distal dendrites than wild-type mice did. We also sought protein targets that interact with DGK beta and identified the GluA2 AMPA receptor subunit as a novel DGK beta binding partner. In addition, DGK beta-deficient brain exhibits significant reduction of TARP gamma-8, which represents a transmembrane AMPA receptor regulatory protein. These findings suggest that DGK beta regulates the spine formation at distal dendrites in MSNs. (C) 2014 Elsevier B.V. All rights reserved.

The glutamate receptor GluN2 subunit regulates synaptic trafficking of AMPA receptors in the neonatal mouse brain
Shun Hamada, Itone Ogawa, Miwako Yamasaki, Yuji Kiyama, Hidetoshi Kassai, Ayako M. Watabe, Kazuki Nakao, Atsu Aiba, Masahiko Watanabe, Toshiya Manabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 40, 8, 3136, 3146, WILEY-BLACKWELL, 2014年10月, ［査読有り］
英語, 研究論文（学術雑誌）, The N-methyl-d-aspartate receptor (NMDAR) plays various physiological and pathological roles in neural development, synaptic plasticity and neuronal cell death. It is composed of two GluN1 and two GluN2 subunits and, in the neonatal hippocampus, most synaptic NMDARs are GluN2B-containing receptors, which are gradually replaced with GluN2A-containing receptors during development. Here, we examined whether GluN2A could be substituted for GluN2B in neural development and functions by analysing knock-in (KI) mice in which GluN2B is replaced with GluN2A. The KI mutation was neonatally lethal, although GluN2A-containing receptors were transported to the postsynaptic membrane even without GluN2B and functional at synapses of acute hippocampal slices of postnatal day 0, indicating that GluN2A-containing NMDARs could not be substituted for GluN2B-containing NMDARs. Importantly, the synaptic -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluA1 was increased, and the transmembrane AMPAR regulatory protein, which is involved in AMPAR synaptic trafficking, was increased in KI mice. Although the regulation of AMPARs by GluN2B has been reported in cultured neurons, we showed here that AMPAR-mediated synaptic responses were increased in acute KI slices, suggesting differential roles of GluN2A and GluN2B in AMPAR expression and trafficking in vivo. Taken together, our results suggest that GluN2B is essential for the survival of animals, and that the GluN2B-GluN2A switching plays a critical role in synaptic integration of AMPARs through regulation of GluA1 in the whole animal.

Neuron type- and input pathway-dependent expression of Slc4a10 in adult mouse brains
Xiaohong Song, Miwako Yamasaki, Taisuke Miyazaki, Kohtarou Konno, Motokazu Uchigashima, Masahiko Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 40, 5, 2797, 2810, WILEY-BLACKWELL, 2014年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Slc4a10 was originally identified as a Na+-driven Cl-/HCO3- exchanger NCBE that transports extracellular Na+ and HCO3- in exchange for intracellular Cl-, whereas other studies argue against a Cl--dependence for Na+-HCO3- transport, and thus named it the electroneutral Na+/HCO3- cotransporter NBCn2. Here we investigated Slc4a10 expression in adult mouse brains by in situ hybridization and immunohistochemistry. Slc4a10 mRNA was widely expressed, with higher levels in pyramidal cells in the hippocampus and cerebral cortex, parvalbumin-positive interneurons in the hippocampus, and Purkinje cells (PCs) in the cerebellum. Immunohistochemistry revealed an uneven distribution of Slc4a10 within the somatodendritic compartment of cerebellar neurons. In the cerebellar molecular layer, stellate cells and their innervation targets (i.e. PC dendrites in the superficial molecular layer) showed significantly higher labeling than basket cells and their targets (PC dendrites in the basal molecular layer and PC somata). Moreover, the distal dendritic trees of PCs (i.e. parallel fiber-targeted dendrites) had significantly greater labeling than the proximal dendrites (climbing fiber-targeted dendrites). These observations suggest that Slc4a10 expression is regulated in neuron type- and input pathway-dependent manners. Because such an elaborate regulation is also found for K+-Cl- cotransporter KCC2, a major neuronal Cl- extruder, we compared their expression. Slc4a10 and KCC2 overlapped in most somatodendritic elements. However, relative abundance was largely complementary in the cerebellar cortex, with particular enrichments of Slc4a10 in PC dendrites and KCC2 in molecular layer interneurons, granule cells and PC somata. These properties might reflect functional redundancy and distinction of these transporters, and their differential requirements by individual neurons and respective input domains.

Global Scaling Down of Excitatory Postsynaptic Responses in Cerebellar Purkinje Cells Impairs Developmental Synapse Elimination
Shinya Kawata, Taisuke Miyazaki, Maya Yamazaki, Takayasu Mikuni, Miwako Yamasaki, Kouichi Hashimoto, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
CELL REPORTS, 8, 4, 1119, 1129, CELL PRESS, 2014年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Synapse elimination is crucial for precise neural circuit formation during postnatal development. We examined how relative differences in synaptic strengths among competing inputs and/or absolute synaptic strengths contribute to climbing fiber (CF) to Purkinje cell (PC) synapse elimination in the cerebellum. We generated mice with PC-selective deletion of stargazin (TARP gamma-2), the major AMPA receptor auxiliary subunit in PCs (gamma-2 PC-KO mice). Whereas relative differences between "strong'' and "weak'' CF-mediated postsynaptic response are preserved, absolute strengths of CF inputs are scaled down globally in PCs of gamma-2 PC-KO mice. Although the early phase of CF elimination is normal, dendritic translocation of the strongest CF and the late phase of CF elimination that requires Ca2+-dependent activation of Arc/Arg3.1 in PCs are both impaired in gamma-2 PC-KO mice. We conclude that, although relative differences in CF synaptic inputs are initially essential, proper synaptic scaling is crucial for accomplishing CF synapse elimination.

Opposing Role of NMDA Receptor GluN2B and GluN2D in Somatosensory Development and Maturation
Miwako Yamasaki, Rieko Okada, Chihiro Takasaki, Shima Toki, Masahiro Fukaya, Rie Natsume, Kenji Sakimura, Masayoshi Mishina, Tetsuo Shirakawa, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 34, 35, 11534, 11548, SOC NEUROSCIENCE, 2014年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Development of correct topographical connections between peripheral receptors and central somatosensory stations requires activity-dependent synapse refinement, in which the NMDA type of glutamate receptors plays a key role. Here we compared functional roles of GluN2B (GluR epsilon 2 or NR2B) and GluN2D (GluR epsilon 4 or NR2D), two major regulatory subunits of neonatal NMDA receptors, in development of whisker-related patterning at trigeminal relay stations. Compared with control littermates, both the appearance of whisker-related patterning and the termination of the critical period, as assessed by unilateral infraorbital nerve transection, were delayed by nearly a day in the somatosensory cortex of GluN2B(+/-) mice but advanced by nearly a day in GluN2D(-/-) mice. Similar temporal shifts were found at subcortical relay stations in the thalamus and brainstem of GluN2B(+/-) and GluN2D(-/-) mice. In comparison, the magnitude of lesion-induced critical period plasticity in the somatosensory cortex, as assessed following row-C whisker removal, was normal in both mutants. Thus, GluN2B and GluN2D play counteractive roles in temporal development and maturation of somatosensory maps without affecting the magnitude of critical period plasticity. To understand the opposing action, we then examined neuronal and synaptic expressions of the two subunits along the trigeminal pathway. At each trigeminal station, GluN2B was predominant at asymmetrical synapses of non-GABAergic neurons, whereas GluN2D was selective to asymmetrical synapses of GABAergic neurons. Together, our findings suggest that GluN2B expressed at glutamatergic synapses on glutamatergic projection neurons facilitates refinement of ascending pathway synapses directly, whereas GluN2D expressed at glutamatergic synapses on GABAergic interneurons delays it indirectly.

Enriched Expression of GluD1 in Higher Brain Regions and Its Involvement in Parallel Fiber-Interneuron Synapse Formation in the Cerebellum
Kohtarou Konno, Keiko Matsuda, Chihiro Nakamoto, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Kenji Sakimura, Michisuke Yuzaki, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 34, 22, 7412, 7424, SOC NEUROSCIENCE, 2014年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Of the two members of the delta subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum and regulates their structural and functional connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. In the present study, we investigated this issue by producing specific and sensitive histochemical probes for GluD1 and analyzing cerebellar synaptic circuits in GluD1-knock-out mice. GluD1 was widely expressed in the adult mouse brain, with high levels in higher brain regions, including the cerebral cortex, striatum, limbic regions (hippocampus, nucleus accumbens, lateral septum, bed nucleus stria terminalis, lateral habenula, and central nucleus of the amygdala), and cerebellar cortex. In the cerebellar cortex, GluD1 mRNA was expressed at the highest level in molecular layer interneurons and its immunoreactivity was concentrated at PF synapses on interneuron somata. In GluD1-knock-out mice, the density of PF synapses on interneuron somata was significantly reduced and the size and number of interneurons were significantly diminished. Therefore, GluD1 is common to GluD2 in expression at PF synapses, but distinct from GluD2 in neuronal expression in the cerebellar cortex; that is, GluD1 in interneurons and GluD2 in PCs. Furthermore, GluD1 regulates the connectivity of PF-interneuron synapses and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function.

Protocadherin 17 Regulates Presynaptic Assembly in Topographic Corticobasal Ganglia Circuits
Naosuke Hoshina, Asami Tanimura, Miwako Yamasaki, Takeshi Inoue, Ryoji Fukabori, Teiko Kuroda, Kazumasa Yokoyama, Tohru Tezuka, Hiroshi Sagara, Shinji Hirano, Hiroshi Kiyonari, Masahiko Takada, Kazuto Kobayashi, Masahiko Watanabe, Masanobu Kano, Takanobu Nakazawa, Tadashi Yamamoto
NEURON, 78, 5, 839, 854, CELL PRESS, 2013年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Highly topographic organization of neural circuits exists for the regulation of various brain functions in corticobasal ganglia circuits. Although neural circuit-specific refinement during synapse development is essential for the execution of particular neural functions, the molecular and cellular mechanisms for synapse refinement are largely unknown. Here, we show that protocadherin 17 (PCDH17), one of the nonclustered delta 2-protocadherin family members, is enriched along corticobasal ganglia synapses in a zone-specific manner during synaptogenesis and regulates presynaptic assembly in these synapses. PCDH17 deficiency in mice causes facilitated presynaptic vesicle accumulation and enhanced synaptic transmission efficacy in corticobasal ganglia circuits. Furthermore, PCDH17(-/-) mice exhibit antidepressant-like phenotypes that are known to be regulated by corticobasal ganglia circuits. Our findings demonstrate a critical role for PCDH17 in the synaptic development of specific corticobasal ganglia circuits and suggest the involvement of PCDH17 in such circuits in depressive behaviors.

Homeostatic Control of Synaptic Transmission by Distinct Glutamate Receptors
Dan Yan, Miwako Yamasaki, Christoph Straub, Masahiko Watanabe, Susumu Tomita
NEURON, 78, 4, 687, 699, CELL PRESS, 2013年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Glutamate is the most abundant excitatory neurotransmitter in the brain, and distinct classes of glutamate receptors coordinate synaptic transmission and spike generation upon various levels of neuronal activity. However, the mechanisms remain unclear. Here, we found that loss of synaptic AMPA receptors increased kainate receptor activity in cerebellar granule cells without changing NMDA receptors. The augmentation of kainate receptor-mediated currents in the absence of AMPA receptor activity is required for spike generation and is mediated by the increased expression of the GluK5 high-affinity kainate receptor subunit. Increase in GluK5 expression is sufficient to enhance kainate receptor activity by modulating receptor channel properties, but not localization. Furthermore, we demonstrate that the combined loss of the AMPA receptor auxiliary TARP gamma-2 subunit and the GluK5 subunit leads to early mouse lethality. Our findings reveal mechanisms mediated by distinct classes of postsynaptic glutamate receptors for the homeostatic maintenance of the neuronal activity.

Type 2K+Cl cotransporter is preferentially recruited to climbing fiber synapses during development and the stellate cell-targeting dendritic zone at adulthood in cerebellar Purkinje cells
Issei Kawakita, Motokazu Uchigashima, Kohtarou Konno, Taisuke Miyazaki, Miwako Yamasaki, Masahiko Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 37, 4, 532, 543, WILEY-BLACKWELL, 2013年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Postnatal expression of the type 2K+Cl cotransporter (KCC2) in neurons lowers the Cl equilibrium potential to values that are more negative than the resting potential, thereby converting the action of Cl-permeable GABAA and glycine receptors from excitatory to inhibitory. In the present study, we investigated the spatiotemporal expression of KCC2 in mouse cerebella, particularly focusing on Purkinje cells (PCs). First, we confirmed the fundamental expression profiles of KCC2 in the cerebellum, i.e. neuron-specific expression, somatodendritic distribution, and postnatal upregulation. We also found preferential recruitment to climbing fiber (CF) synapses during the second and third postnatal weeks, when perisomatic innervation in PCs switches from CFs to basket cell axons (BAs) and also when single winner CFs translocate from somata to dendrites. In parallel with this synaptic recruitment, the intracellular distribution shifted from a diffuse cytoplasmic to a predominantly cell surface pattern. In adult PCs, CF synapse-associated accumulation was obscured. Instead, significantly high expression was noted on the surface of PC dendrites in the superficial two-thirds of the molecular layer, in which stellate cells reside and project axons to innervate PC dendrites. Thus, the somatodendritic distribution in PCs is regulated in relation to particular inputs or input zones. During development, timed recruitment of KCC2 to CF synapses will augment inhibitory GABAergic actions by incoming BAs, promoting the CF-to-BA switchover in perisomatic PC innervation. In adulthood, enriched KCC2 expression at the stellate cell-targeting territory of PC dendrites might help in maintaining intracellular Cl homeostasis and the polarity of GABAA receptor-mediated responses upon sustained activity of this interneuron.

Autoantibodies to epilepsy-related LGI1 in limbic encephalitis neutralize LGI1-ADAM22 interaction and reduce synaptic AMPA receptors
Toshika Ohkawa, Yuko Fukata, Miwako Yamasaki, Taisuke Miyazaki, Norihiko Yokoi, Hiroshi Takashima, Masahiko Watanabe, Osamu Watanabe, Masaki Fukata
Journal of Neuroscience, 33, 46, 18161, 18174, 46, 2013年, ［査読有り］
英語, 研究論文（学術雑誌）, More than 30 mutations in LGI1, a secreted neuronal protein, have been reported with autosomal dominant lateral temporal lobe epilepsy (ADLTE). Although LGI1 haploinsufficiency is thought to cause ADLTE, the underlying molecular mechanism that results in abnormal brain excitability remains mysterious. Here, we focused on a mode of action of LGI1 autoantibodies associated with limbic encephalitis (LE), which is one of acquired epileptic disorders characterized by subacute onset of amnesia and seizures.Wecomprehensively screened human sera from patients with immune-mediated neurological disorders for LGI1 autoantibodies, which also uncovered novel autoantibodies against six cell surface antigens including DCC, DPP10, and ADAM23. Our developed ELISA arrays revealed a specific role for LGI1 antibodies in LE and concomitant involvement of multiple antibodies, including LGI1 antibodies in neuromyotonia, a peripheral nerve disorder. LGI1 antibodies associated with LE specifically inhibited the ligand-receptor interaction between LGI1 and ADAM22/23 by targeting the EPTP repeat domain of LGI1 and reversibly reduced synaptic AMPA receptor clusters in rat hippocampal neurons. Furthermore, we found that disruption of LGI1-ADAM22 interaction by soluble extracellular domain ofADAM22was sufficient to reduce synapticAMPAreceptors in rat hippocampal neurons and that levels ofAMPAreceptor were greatly reduced in the hippocampal dentate gyrus in the epileptic LGI1 knock-out mouse. Therefore, either genetic or acquired loss of the LGI1-ADAM22 interaction reduces theAMPAreceptor function, causing epileptic disorders. These results suggest that by finely regulating the synapticAMPAreceptors, the LGI1-ADAM22 interaction maintains physiological brain excitability throughout life. © 2013 the authors.

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］

Three Types of Neurochemical Projection from the Bed Nucleus of the Stria Terminalis to the Ventral Tegmental Area in Adult Mice
Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Kohtarou Konno, Miwako Yamasaki, Yuchio Yanagawa, Masabumi Minami, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 32, 50, 18035, 18046, SOC NEUROSCIENCE, 2012年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) play crucial roles in motivational control of behaviors, and their activity is regulated directly or indirectly via GABAergic neurons by extrinsic afferents from various sources, including the bed nucleus of the stria terminalis ( BST). Here, the neurochemical composition of VTA-projecting BST neurons and their outputs to the VTA were studied in adult mouse brains. By combining retrograde tracing with fluorescence in situ hybridization for 67 kDa glutamate decarboxylase (GAD67) and vesicular glutamate transporters (VGluTs), VTA-targeting BST neurons were classified into GAD67-positive (GAD67(+))/VGluT3-negative (VGluT3(-)), GAD67(+)/VGluT3(+), and VGluT2(+) neurons, of which GAD67(+)/VGluT3(-)neurons constituted the majority (similar to 90%) of VTA-projecting BST neurons. GABAergic efferents from the BST formed symmetrical synapses on VTA neurons, which were mostly GABAergic neurons, and expressed GABA(A) receptor alpha 1 subunit on their synaptic and extrasynaptic membranes. In the VTA, VGluT3 was detected in terminals expressing vesicular inhibitory amino acid transporter ( VIAAT), plasmalemmal serotonin transporter, or neither. Of these, VIAAT(+)/VGluT3(+) terminals, which should include those from GAD67(+)/VGluT3(+) BST neurons, formed symmetrical synapses. When single axons from VGluT3(+) BST neurons were examined, almost all terminals were labeled for VIAAT, whereas VGluT3 was often absent from terminals with high VIAAT loads. VGluT2(+) terminals in the VTA exclusively formed asymmetrical synapses, which expressed AMPA receptors on postsynaptic membrane. Therefore, the major mode of the BST-VTA projection is GABAergic, and its activation is predicted to disinhibit VTA DAergic neurons. VGluT2(+) and VGluT3(+) BST neurons further supply additional projections, which may principally convey excitatory or inhibitory inputs, respectively, to the VTA.

Distinct Neurochemical and Functional Properties of GAD67-Containing 5-HT Neurons in the Rat Dorsal Raphe Nucleus
Hiroki Shikanai, Takayuki Yoshida, Kohtarou Konno, Miwako Yamasaki, Takeshi Izumi, Yu Ohmura, Masahiko Watanabe, Mitsuhiro Yoshioka
JOURNAL OF NEUROSCIENCE, 32, 41, 14415, 14426, SOC NEUROSCIENCE, 2012年10月, ［査読有り］
英語, 研究論文（学術雑誌）, The serotonergic (5-HTergic) system arising from the dorsal raphe nucleus (DRN) is implicated in various physiological and behavioral processes, including stress responses. The DRN is comprised of several subnuclei, serving specific functions with distinct afferent and efferent connections. Furthermore, subsets of 5-HTergic neurons are known to coexpress other transmitters, including GABA, glutamate, or neuropeptides, thereby generating further heterogeneity. However, despite the growing evidence for functional variations among DRN subnuclei, relatively little is known about how they map onto neurochemical diversity of 5-HTergic neurons. In the present study, we characterized functional properties of GAD67-expressing 5-HTergic neurons (5-HT/GAD67 neurons) in the rat DRN, and compared with those of neurons expressing 5-HTergic molecules (5-HT neurons) or GAD67 alone. While 5-HT/GAD67 neurons were absent in the dorsomedial (DRD) or ventromedial (DRV) parts of the DRN, they were selectively distributed in the lateral wing of the DRN (DRL), constituting 12% of the total DRL neurons. They expressed plasmalemmal GABA transporter 1, but lacked vesicular inhibitory amino acid transporter. By using whole-cell patch-clamp recording, we found that 5-HT/GAD67 neurons had lower input resistance and firing frequency than 5-HT neurons. As revealed by c-Fos immunohistochemistry, neurons in the DRL, particularly 5-HT/GAD67 neurons, showed higher responsiveness to exposure to an open field arena than those in the DRD and DRV. By contrast, exposure to contextual fear conditioning stress showed no such regional differences. These findings indicate that 5-HT/GAD67 neurons constitute a unique neuronal population with distinctive neurochemical and electrophysiological properties and high responsiveness to innocuous stressor.

Lack of Molecular-Anatomical Evidence for GABAergic Influence on Axon Initial Segment of Cerebellar Purkinje Cells by the Pinceau Formation
Atsushi Iwakura, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 32, 27, 9438, 9448, SOC NEUROSCIENCE, 2012年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The axon initial segment (AIS) of cerebellar Purkinje cells (PCs) is embraced by ramified axons of GABAergic basket cells (BCs) called the pinceau formation. This unique structure has been assumed to be a device for the modulation of PC outputs through electrical and/or GABAergic inhibition. Electrical inhibition is supported by enriched potassium channels, absence of sodium channels, and developed septate-like junctions between BC axons. The neurochemical basis for GABAergic inhibition, however, has not been well investigated. Here we addressed this issue using C56BL/6 mice. First, we confirmed previous observations that typical synaptic contacts were rare and confined to proximal axonal portions, with the remaining portions being mostly covered by astrocytic processes. Then we examined the expression of molecules involved in GABAergic signaling, including GABA synthetic enzyme glutamic acid decarboxylase (GAD), vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT), cytomatrix active zone protein bassoon, GABA receptor GABA(A)R alpha 1, and cell adhesion molecule neuroligin-2. These molecules were recruited to form a functional assembly at perisomatic BC-PC synapses and along the AIS of hippocampal and neocortical pyramidal cells. GAD and VIAAT immunogold labeling was five times lower in the pinceau formation compared with perisomatic BC terminals and showed no accumulation toward the AIS. Moreover, bassoon, neuroligin-2, and GABA(A)R alpha 1 formed no detectable clusters along the ankyrin-G-positive AIS proper. These findings indicate that GABAergic signaling machinery is organized loosely and even incompletely in the pinceau formation. Together, BCs do not appear to exert GABAergic synaptic inhibition on the AIS, although the mode of action of the pinceau formation remains to be explored.

Rewiring of Afferent Fibers in the Somatosensory Thalamus of Mice Caused by Peripheral Sensory Nerve Transection
Yuichi Takeuchi, Miwako Yamasaki, Yasuyuki Nagumo, Keiji Imoto, Masahiko Watanabe, Mariko Miyata
JOURNAL OF NEUROSCIENCE, 32, 20, 6917, 6930, SOC NEUROSCIENCE, 2012年05月, ［査読有り］
英語, 研究論文（学術雑誌）, The remodeling of neural circuitry and changes in synaptic efficacy after peripheral sensory nerve injury are considered the basis for functional reorganization in the brain, including changes in receptive fields. However, when or how the remodeling occurs is largely unknown. Here we show the rapid rewiring of afferent fibers in the mature ventral posteromedial thalamic nucleus of mice after transection of the peripheral whisker sensory nerve, using the whole-cell voltage-clamp technique. Transection induced the recruitment of afferent fibers to a thalamic relay neuron within 5-6 d of injury. The rewiring was pathway specific, but not sensory experience dependent or peripheral nerve activity dependent. The newly recruited fibers mediated small EPSCs, and postsynaptic GluA2-containing AMPA receptors were selectively upregulated at the new synapses. This rapid and pathway-specific remodeling of thalamic circuitry may be an initial step in the massive axonal reorganization at supraspinal levels, which occurs months or years after peripheral sensory nerve injury.

Ca(v)2.1 in Cerebellar Purkinje Cells Regulates Competitive Excitatory Synaptic Wiring, Cell Survival, and Cerebellar Biochemical Compartmentalization
Taisuke Miyazaki, Miwako Yamasaki, Kouichi Hashimoto, Maya Yamazaki, Manabu Abe, Hiroshi Usui, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 32, 4, 1311, 1328, SOC NEUROSCIENCE, 2012年01月, ［査読有り］
英語, 研究論文（学術雑誌）, In the adult cerebellum, each Purkinje cell (PC) is innervated by a single climbing fiber (CF) in proximal dendrites and 10(5)-10(6) parallel fibers (PFs) in distal dendrites. This organized wiring is established postnatally through heterosynaptic competition between PFs and CFs and homosynaptic competition among multiple CFs. Using PC-specific Ca(v)2.1 knock-out mice (PC-Ca(v)2.1 KO mice), we have demonstrated recently that postsynaptic Ca(v)2.1 plays a key role in the homosynaptic competition by promoting functional strengthening and dendritic translocation of single "winner" CFs. Here, we report that Ca(v)2.1 in PCs, but not in granule cells, is also essential for the heterosynaptic competition. In PC-Ca(v)2.1 KO mice, the extent of CF territory was limited to the soma and basal dendrites, whereas PF territory was expanded reciprocally. Consequently, the proximal somatodendritic domain of PCs displayed hyperspiny transformation and fell into chaotic innervation by multiple CFs and numerous PFs. PC-Ca(v)2.1 KO mice also displayed patterned degeneration of PCs, which occurred preferentially in aldolase C/zebrin II-negative cerebellar compartments. Furthermore, the mutually complementary expression of phospholipase C beta 3 (PLC beta 3) and PLC beta 4 was altered such that their normally sharp boundary was blurred in the PCs of PC-Cav2.1 KO mice. This blurring was caused by an impaired posttranscriptional downregulation of PLC beta 3 in PLC beta 4-dominant PCs during the early postnatal period. A similar alteration was noted in the banded expression of the glutamate transporter EAAT4 in PC-Cav2.1 KO mice. Therefore, Cav2.1 in PCs is essential for competitive synaptic wiring, cell survival, and the establishment of precise boundaries and reciprocity of biochemical compartments in PCs.

Developmental Switching of Perisomatic Innervation from Climbing Fibers to Basket Cell Fibers in Cerebellar Purkinje Cells
Ryoichi Ichikawa, Miwako Yamasaki, Taisuke Miyazaki, Kohtarou Konno, Kouichi Hashimoto, Haruyuki Tatsumi, Yoshiro Inoue, Masanobu Kano, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 31, 47, 16916, 16927, SOC NEUROSCIENCE, 2011年11月, ［査読有り］
英語, 研究論文（学術雑誌）, In early postnatal development, perisomatic innervation of cerebellar Purkinje cells (PCs) switches from glutamatergic climbing fibers (CFs) to GABAergic basket cell fibers (BFs). Here we examined the switching process in C57BL/6 mice. At postnatal day 7 (P7), most perisomatic synapses were formed by CFs on to somatic spines. The density of CF-spine synapses peaked at P9, when pericellular nest around PCs by CFs was most developed, and CF-spine synapses constituted 88% of the total perisomatic synapses. Thereafter, CF-spine synapses dropped to 63% at P12, 6% at P15, and <1% at P20, whereas BF synapses increased reciprocally. During the switching period, a substantial number of BF synapses existed as BF-spine synapses (37% of the total perisomatic synapses at P15), and free spines surrounded by BFs or Bergmann glia also emerged. By P20, BF-spine synapses and free spines virtually disappeared, and BF-soma synapses became predominant (88%), thus attaining the adult pattern of perisomatic innervation. Parallel with the presynaptic switching, postsynaptic receptor phenotype also switched from glutamatergic to GABAergic. In the active switching period, particularly at P12, fragmental clusters of AMPA-type glutamate receptor were juxtaposed with those of GABA(A) receptor. When examined with serial ultrathin sections, immunogold labeling for glutamate and GABA(A) receptors was often clustered beneath single BF terminals. These results suggest that a considerable fraction of somatic spines is succeeded from CFs to BFs and Bergmann glia in the early postnatal period, and that the switching of postsynaptic receptor phenotypes mainly proceeds under the coverage of BF terminals.

Orexin Neurons Receive Glycinergic Innervations
Mari Hondo, Naoki Furutani, Miwako Yamasaki, Masahiko Watanabe, Takeshi Sakurai
PLOS ONE, 6, 9, e25076, PUBLIC LIBRARY SCIENCE, 2011年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation.

Distinct functions of kainate receptors in the brain are determined by the auxiliary subunit Neto1
Christoph Straub, David L. Hunt, Miwako Yamasaki, Kwang S. Kim, Masahiko Watanabe, Pablo E. Castillo, Susumu Tomita
NATURE NEUROSCIENCE, 14, 7, 866, U83, NATURE PUBLISHING GROUP, 2011年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Ionotropic glutamate receptors principally mediate fast excitatory transmission in the brain. Among the three classes of ionotropic glutamate receptors, kainate receptors (KARs) have a unique brain distribution, which has been historically defined by (3)H-radiolabeled kainate binding. Compared with recombinant KARs expressed in heterologous cells, synaptic KARs exhibit characteristically slow rise-time and decay kinetics. However, the mechanisms responsible for these distinct KAR properties remain unclear. We found that both the high-affinity binding pattern in the mouse brain and the channel properties of native KARs are determined by the KAR auxiliary subunit Neto1. Through modulation of agonist binding affinity and off-kinetics of KARs, but not trafficking of KARs, Neto1 determined both the KAR high-affinity binding pattern and the distinctively slow kinetics of postsynaptic KARs. By regulating KAR excitatory postsynaptic current kinetics, Neto1 can control synaptic temporal summation, spike generation and fidelity.

Molecular and Morphological Configuration for 2-Arachidonoylglycerol-Mediated Retrograde Signaling at Mossy Cell-Granule Cell Synapses in the Dentate Gyrus
Motokazu Uchigashima, Maya Yamazaki, Miwako Yamasaki, Asami Tanimura, Kenji Sakimura, Masanobu Kano, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 31, 21, 7700, 7714, SOC NEUROSCIENCE, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of neurotransmission in the brain. In the present study, we investigated the 2-AG signaling system at mossy cell (MC)-granule cell (GC) synapses in the mouse dentate gyrus, an excitatory recurrent circuit where endocannabinoids are thought to suppress epileptogenesis. First, we showed by electrophysiology that 2-AG produced by diacylglycerol lipase alpha (DGL alpha) mediated both depolarization-induced suppression of excitation and its enhancement by group I metabotropic glutamate receptor activation at MC-GC synapses, as they were abolished in DGL alpha-knock-out mice. Immunohistochemistry revealed that DGL alpha was enriched in the neck portion of GC spines forming synapses with MC terminals, whereas cannabinoid CB(1) receptors accumulated in the terminal portion of MC axons. On the other hand, the major 2-AG-degrading enzyme, monoacylglycerol lipase (MGL), was absent at MC-GC synapses but was expressed in astrocytes and some inhibitory terminals. Serial electron microscopy clarified that a given GC spine was innervated by a single MC terminal and also contacted nonsynaptically by other MC terminals making synapses with other GC spines in the neighborhood. MGL-expressing elements, however, poorly covered GC spines, amounting to 17% of the total surface of GC spines by astrocytes and 4% by inhibitory terminals. Our findings provide a basis for 2-AG-mediated retrograde suppression of MC-GC synaptic transmission and also suggest that 2-AG released from activated GC spines is readily accessible to nearby MC-GC synapses by escaping from enzymatic degradation. This molecular-anatomical configuration will contribute to adjust network activity in the dentate gyrus after enhanced excitation.

Glutamate receptor δ2 is essential for input pathway-dependent regulation of synaptic AMPAR contents in cerebellar Purkinje cells.
Yamasaki M, Miyazaki T, Azechi H, Abe M, Natsume R, Hagiwara T, Aiba A, Mishina M, Sakimura K, Watanabe M
The Journal of neuroscience : the official journal of the Society for Neuroscience, 31, 9, 3362, 3374, 9, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）, The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor delta 2 (GluR delta 2 or G1uD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRS2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluR delta 2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluR delta 2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluR52. These results suggest that GluR delta 2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.

Unique inhibitory synapse with particularly rich endocannabinoid signaling machinery on pyramidal neurons in basal amygdaloid nucleus
Takayuki Yoshida, Motokazu Uchigashima, Miwako Yamasaki, Istvan Katona, Maya Yamazaki, Kenji Sakimura, Masanobu Kano, Mitsuhiro Yoshioka, Masahiko Watanabe
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 108, 7, 3059, 3064, NATL ACAD SCIENCES, 2011年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of synaptic transmission in the brain. 2-AG is synthesized in activated postsynaptic neurons by sn-1-specific diacylglycerol lipase (DGL), binds to presynaptic cannabinoid CB(1) receptors, suppresses neurotransmitter release, and is degraded mainly by monoacylglycerol lipase (MGL). In the basolateral amygdala complex, it has been demonstrated that CB(1) is particularly enriched in axon terminals of cholecystokinin (CCK)-positive GABAergic interneurons, induces short-and long-term depression at inhibitory synapses, and is involved in extinction of fear memory. Here, we clarified a unique molecular convergence of DGL alpha, CB(1), and MGL at specific inhibitory synapses in the basal nucleus (BA), but not lateral nucleus, of the basolateral amygdala. The synapses, termed invaginating synapses, consisted of conventional symmetrical contact and unique perisynaptic invagination of nerve terminals into perikarya. At invaginating synapses, DGL alpha was preferentially recruited to concave somatic membrane of postsynaptic pyramidal neurons, whereas invaginating presynaptic terminals highly expressed CB(1), MGL, and CCK. No such molecular convergence was seen for flat perisomatic synapses made by parvalbumin-positive interneurons. On the other hand, DGL alpha and CB(1) were expressed weakly at axospinous excitatory synapses. Consistent with these morphological data, thresholds for DGL alpha-mediated depolarization-induced retrograde suppression were much lower for inhibitory synapses than for excitatory synapses in BA pyramidal neurons. Moreover, depolarization-induced suppression was readily saturated for inhibition, but never for excitation. These findings suggest that perisomatic inhibition by invaginating synapses is a key target of 2-AG-mediated control of the excitability of BA pyramidal neurons.

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］

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］

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］

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

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

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

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

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］

Cellular expression and subcellular localization of secretogranin II in the mouse hippocampus and cerebellum
Taisuke Miyazaki, Miwako Yamasaki, Motokazu Uchigashima, Ayano Matsushima, Masahiko Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 33, 1, 82, 94, WILEY-BLACKWELL PUBLISHING, INC, 2011年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Secretogranin II (SgII), or chromogranin C, is thought to participate in the sorting and packaging of peptide hormones and neuropeptides into secretory granules and large dense-core vesicle (LDCVs), and also functions as a precursor of neuropeptide secretoneurin. Although SgII is widely distributed in the brain and is predominantly localized at terminals of mossy fibers in the hippocampus and cerebellum and climbing fibers in the cerebellum, its cellular expression and ultrastructural localization remain largely unknown. In the present study, we addressed this issue in the adult mouse brain by multiple-labeling fluorescence in situ hybridization and immunofluorescence and by preembedding and postembedding immunoelectron microscopies. SgII was expressed in various neurons, distributed as either tiny puncta or coarse aggregates in the neuropil, and intensely accumulated in perikarya of particular neurons, such as parvalbumin-positive interneurons and mossy cells in the hippocampus and Purkinje cells in the cerebellum. Coarse aggregates were typical of terminals of mossy fibers and climbing fibers. In these terminals, numerous immunogold particles were clustered on individual LDCVs, and one or two particles also fell within small synaptic vesicle-accumulating portions. SgII was further detected as tiny puncta in neural elements lacking LDCVs, such as parallel fibers of cerebellar granule cells, somatodendritic elements of various neurons and Bergmann glia. Thus, SgII is present in LDCV and non-LDCV compartments of various neural cells. The wide subcellular localization of SgII may reflect diverse release sites of neuropeptides and secretorneurin, or suggests its role in the sorting and packaging of molecules other than neuropeptides in non-LDCV compartments.

Inositol 1,4,5-trisphosphate signaling maintains the activity of glutamate uptake in Bergmann glia
Masato Mashimo, Yohei Okubo, Toshiko Yamazawa, Miwako Yamasaki, Masahiko Watanabe, Toshihiko Murayama, Masamitsu Iino
EUROPEAN JOURNAL OF NEUROSCIENCE, 32, 10, 1668, 1677, WILEY-BLACKWELL PUBLISHING, INC, 2010年11月, ［査読有り］
英語, 研究論文（学術雑誌）, The maintenance of synaptic functions is essential for neuronal information processing in the adult brain. Astrocytes express glutamate transporters that rapidly remove glutamate from the extracellular space and they play a critical role in the precise operation of glutamatergic transmission. However, how the glutamate clearance function of astrocytes is maintained remains elusive. Here, we describe a maintenance mechanism for the glutamate uptake capacity of Bergmann glial cells (BGs) in the cerebellum. When inositol 1,4,5-trisphosphate (IP(3)) signaling was chronically and selectively inhibited in BGs in vivo, the retention time of glutamate around parallel fiber-Purkinje cell synapses was increased. Under these conditions, a decrease in the level of the glutamate/aspartate transporter (GLAST) in BGs was observed. The same effects were observed after chronic in vivo inhibition of purinergic P2 receptors in the cerebellar cortex. These results suggest that the IP(3) signaling cascade is involved in regulating GLAST levels in BGs to maintain glutamate clearance in the mature cerebellum.

Ablation of glutamate receptor GluRδ2 in adult Purkinje cells causes multiple innervation of climbing fibers by inducing aberrant invasion to parallel fiber innervation territory.
Miyazaki T, Yamasaki M, Takeuchi T, Sakimura K, Mishina M, Watanabe M
The Journal of neuroscience : the official journal of the Society for Neuroscience, 30, 45, 15196, 15209, 45, 2010年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Glutamate receptor GluR delta 2 is exclusively expressed in Purkinje cells (PCs) from early development and plays key roles in parallel fiber (PF) synapse formation, elimination of surplus climbing fibers (CFs), long-term depression, motor coordination, and motor learning. To address its role in adulthood, we previously developed a mouse model of drug-induced GluR delta 2 ablation in adult PCs (Takeuchi et al., 2005). In that study, we demonstrated an essential role to maintain the connectivity of PF-PC synapses, based on the observation that both mismatching of presynaptic and postsynaptic specializations and disconnection of PF-PC synapses are progressively increased after GluR delta 2 ablation. Here, we pursued its role for CF wiring in adult cerebellum. In parallel with the disconnection of PF-PC synapses, ascending CF branches exhibited distal extension to innervate distal dendrites of the target and neighboring PCs. Furthermore, transverse CF branches, a short motile collateral rarely forming synapses in wild-type animals, displayed aberrant mediolateral extension to innervate distal dendrites of neighboring and remote PCs. Consequently, many PCs were wired by single main CF and other surplus CFs innervating a small part of distal dendrites. Electrophysiological recording further revealed that surplus CF-EPSCs characterized with slow rise time and small amplitude emerged after GluR delta 2 ablation, and increased progressively both in number and amplitude. Therefore, GluR delta 2 is essential for maintaining CF monoinnervation in adult cerebellum by suppressing aberrant invasion of CF branches to the territory of PF innervation. Thus, GluR delta 2 fuels heterosynaptic competition and gives PFs the competitive advantages over CFs throughout the animal's life.

Cytochemical and cytological properties of perineuronal oligodendrocytes in the mouse cortex.
Takasaki C, Yamasaki M, Uchigashima M, Konno K, Yanagawa Y, Watanabe M
The European journal of neuroscience, 32, 8, 1326, 1336, 8, 2010年10月, ［査読有り］
英語, Neuronal cell bodies are associated with glial cells collectively referred to as perineuronal satellite cells. One such satellite cell is the perineuronal oligodendrocyte, which is unmyelinating oligodendrocytes attaching to large neurons in various neural regions. However, little is known about their cellular characteristics and function. In this study, we identified perineuronal oligodendrocytes as 2',3'-cyclic nucleotide 3'-phosphodiesterase(CNP)-positive cells attaching to neuronal perikarya immunostained for microtubule-associated protein 2, and examined their cytochemical and cytological properties in the mouse cerebral cortex. CNP-positive perineuronal oligodendrocytes were immunonegative to representative glial markers for astrocytes (brain-type lipid binding protein and glial fibrillary acidic protein), microglia (Iba-1) and NG2+ glia(NG2). Instead, almost all perineuronal oligodendrocytes expressed glia-specific or glia-enriched metabolic enzymes, i.e. the creatine synthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase and L-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase. As to molecules participating in the glutamate-glutamine cycle, none of the perineuronal oligodendrocytes expressed the plasmalemmal glutamate transporters GLAST and GLT-1, although nearly half of the perineuronal oligodendrocytes were immunopositive for glutamine synthetase. Cytologically, perineuronal oligodendrocytes were mainly distributed in deep cortical layers (layers IV-VI), and attached directly and tightly to neuronal cell bodies, making a long concave impression to the contacting neurons. Interestingly, they attached more to glutamatergic principal neurons than to GABAergic interneurons, and this became evident at postnatal day 14, when the cerebral cortex develops and maturates. These cytochemical and cytological properties suggest that perineuronal oligodendrocytes are so differentiated as to fulfill metabolic support to the associating principal cortical neurons, rather than to regulate their synaptic transmission.

TARPs gamma-2 and gamma-7 are essential for AMPA receptor expression in the cerebellum
Maya Yamazaki, Masahiro Fukaya, Kouichi Hashimoto, Miwako Yamasaki, Mika Tsujita, Makoto Itakura, Manabu Abe, Rie Natsume, Masami Takahashi, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 31, 12, 2204, 2220, WILEY-BLACKWELL, 2010年06月, ［査読有り］
英語, 研究論文（学術雑誌）, The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, gamma-2 and gamma-7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, gamma-2 and gamma-7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in gamma-2-knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in gamma-7-KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in gamma-2/gamma-7 double-KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in gamma-2-KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber-Purkinje cell synapses, and AMPA receptor-mediated currents at climbing fiber-Purkinje cell synapses were all reduced to approximately 10% of the wild-type levels in DKO mice. On the other hand, the reduction in GluA4 in gamma-7-KO granular layer reflected its loss at mossy fiber-granule cell synapses, whereas that of GluA1 and GluA4 in gamma-7-KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, gamma-2 and gamma-7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression.

Imaging extrasynaptic glutamate dynamics in the brain
Yohei Okubo, Hiroshi Sekiya, Shigeyuki Namiki, Hirokazu Sakamoto, Sho Iinuma, Miwako Yamasaki, Masahiko Watanabe, Kenzo Hirose, Masamitsu Iino
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 107, 14, 6526, 6531, NATL ACAD SCIENCES, 2010年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Glutamate is the major neurotransmitter in the brain, mediating point-to-point transmission across the synaptic cleft in excitatory synapses. Using a glutamate imaging method with fluorescent indicators, we show that synaptic activity generates extrasynaptic glutamate dynamics in the vicinity of active synapses. These glutamate dynamics had magnitudes and durations sufficient to activate extrasynaptic glutamate receptors in brain slices. We also observed crosstalk between synapses-i.e., summation of glutamate released from neighboring synapses. Furthermore, we successfully observed that sensory input from the extremities induced extrasynaptic glutamate dynamics within the appropriate sensory area of the cerebral cortex in vivo. Thus, the present study clarifies the spatiotemporal features of extrasynaptic glutamate dynamics, and opens up an avenue to directly visualizing synaptic activity in live animals.

Preferential Localization of Muscarinic M-1 Receptor on Dendritic Shaft and Spine of Cortical Pyramidal Cells and Its Anatomical Evidence for Volume Transmission
Miwako Yamasaki, Minoru Matsui, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 30, 12, 4408, 4418, SOC NEUROSCIENCE, 2010年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Acetylcholine (ACh) plays important roles for higher brain functions, including arousal, attention, and cognition. These effects are mediated largely by muscarinic acetylcholine receptors (mAChRs). However, it remains inconclusive whether the mode of ACh-mAChR signaling is synaptic, so-called "wired," transmission mediated by ACh released into the synaptic cleft, or nonsynaptic, so-called " volume," transmission by ambient ACh. To address this issue, we examined cellular and subcellular distribution of M-1, the most predominant mAChR subtype in the cerebral cortex and hippocampus, and pursued its anatomical relationship with cholinergic varicosities in these regions of adult mice. M-1 was highly expressed in glutamatergic pyramidal neurons, whereas it was low or undetectable in various GABAergic interneuron subtypes. M-1 was preferentially distributed on the extrasynaptic membrane of pyramidal cell dendrites and spines. Cholinergic varicosities often made direct contact to pyramidal cell dendrites and synapses. At such contact sites, however, synapse-like specialization was infrequent, and no particular accumulation was found at around contact sites for both M-1 and presynpatic active zone protein Bassoon. These features contrasted with those of the glutamatergic system, in which AMPA receptor GluA2 and metabotropic receptor mGluR5 were recruited to the synaptic or perisynaptic membrane, respectively, and Bassoon was highly accumulated in the presynaptic terminals. These results suggest that M-1 is so positioned to sense ambient ACh released from cholinergic varicosities at variable distances, and to enhance the synaptic efficacy and excitability of pyramidal cells. These molecular-anatomical arrangements will provide the evidence for volume transmission, at least in M-1-mediated cortical cholinergic signaling.

Spatiotemporal dynamics of glutamate spillover
Okubo Yohei, Sekiya Hiroshi, Namiki Shigeyuki, Sakamoto Hirokazu, Iinuma Sho, Yamasaki Miwako, Watanabe Masahiko, Hirose Kenzo, Iino Masamitsu
JOURNAL OF PHARMACOLOGICAL SCIENCES, 112, 110P, 2010年, ［査読有り］

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］

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

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

Lentiviral vector-mediated rescue of motor behavior in spontaneously occurring hereditary ataxic mice
Akira Iizuka, Kiyohiko Takayama, Takashi Torashima, Miwako Yamasaki, Chiho Koyama, Kazuhiro Mitsumura, Masahiko Watanabe, Hirozaku Hirai
NEUROBIOLOGY OF DISEASE, 35, 3, 457, 465, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2009年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Hotfoot5J mice are spontaneously occurring ataxic mice that lack delta 2 glutamate receptor (GluR82) protein in cerebellar Purkinje cells. Here we aimed to rescue the ataxic phenotype of hotfoot5J mice by lentiviral vector-mediated expression of recombinant GIuR82 in Purkinje cells. Lentiviral vectors expressing GluR delta 2 were injected into the cerebellar cortex of hotfoot5J mice 6 or 7 days after birth, and the effects were studied on postnatal day 30. The motor behavior of hotfoot5J mice treated with vectors expressing GluR82 was markedly rescued, whereas the ataxia of hotfoot5J mice treated with vectors expressing GFP was comparable to that of non-injected hotfoot5J littermates. Furthermore, the impaired release probability of glutamate from parallel fiber terminals and the failure of developmental elimination of surplus climbing fibers from Purkinje cells in hotfoot5J mice were completely rescued by GIuR82 expression. These results indicate the therapeutic potential of viral vector-based gene therapy for hereditary cerebellar ataxia and other neuronal disorders. (C) 2009 Elsevier Inc. All rights reserved.

NMDA Receptor GluN2B (GluR epsilon 2/NR2B) Subunit Is Crucial for Channel Function, Postsynaptic Macromolecular Organization, and Actin Cytoskeleton at Hippocampal CA3 Synapses
Kaori Akashi, Toshikazu Kakizaki, Haruyuki Kamiya, Masahiro Fukaya, Miwako Yamasaki, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura
JOURNAL OF NEUROSCIENCE, 29, 35, 10869, 10882, SOC NEUROSCIENCE, 2009年09月, ［査読有り］
英語, 研究論文（学術雑誌）, GluN2B (GluR epsilon 2/NR2B) subunit is involved in synapse development, synaptic plasticity, and cognitive function. However, its roles in synaptic expression and function of NMDA receptors (NMDARs) in the brain remain mostly unknown because of the neonatal lethality of global knock-out mice. To address this, we generated conditional knock-out mice, in which GluN2B was ablated exclusively in hippocampal CA3 pyramidal cells. By immunohistochemistry, GluN2B disappeared and GluN1 (GluR zeta 1/NR1) was moderately reduced, whereas GluN2A (GluR epsilon 1/NR2A) and postsynaptic density-95 (PSD-95) were unaltered in the mutant CA3. This was consistent with protein contents in the CA3 crude fraction: 9.6% of control level for GluN2B, 47.7% for GluN1, 90.6% for GluN2A, and 98.0% for PSD-95. Despite the remaining NMDARs, NMDAR-mediated currents and long-term potentiation were virtually lost at various CA3 synapses. Then, we compared synaptic NMDARs by postembedding immunogold electron microscopy and immunoblot using the PSD fraction. In the mutant CA3, GluN1 was severely reduced in both immunogold (20.6-23.6%) and immunoblot (24.6%), whereas GluN2A and PSD-95 were unchanged in immunogold but markedly reduced in the PSD fraction (51.4 and 36.5%, respectively), indicating increased detergent solubility of PSD molecules. No such increased solubility was observed for GluN2B in the CA3 of GluN2A-knock-out mice. Furthermore, significant decreases were found in the ratio of filamentous to globular actin (49.5%) and in the density of dendritic spines (76.2%). These findings suggest that GluN2B is critically involved in NMDAR channel function, organization of postsynaptic macromolecular complexes, formation or maintenance of dendritic spines, and regulation of the actin cytoskeleton.

Rescue of abnormal phenotypes in delta 2 glutamate receptor-deficient mice by the extracellular N-terminal and intracellular C-terminal domains of the delta 2 glutamate receptor
Takashi Torashima, Akira Iizuka, Hajime Horiuchi, Kazuhiro Mitsumura, Miwako Yamasaki, Chiho Koyama, Kiyohiko Takayama, Masae Iino, Masahiko Watanabe, Hirozaku Hirai
EUROPEAN JOURNAL OF NEUROSCIENCE, 30, 3, 355, 365, WILEY-BLACKWELL PUBLISHING, INC, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The delta 2 glutamate receptor (GluR delta 2) is expressed predominantly in cerebellar Purkinje cells. GluR delta 2 knock-out mice show impaired synaptogenesis and loss of long-term depression (LTD) at parallel fiber/Purkinje cell synapses, and persistent multiple climbing fiber (CF) innervation of Purkinje cells, resulting in severe ataxia. To identify domains critical for GluR delta 2 function, we produced various GluR delta 2 deletion constructs. Using lentiviral vectors, those constructs were expressed in Purkinje cells of GluR delta 2-deficient mice at postnatal day (P) 6 or 7, and rescue of abnormal phenotypes was examined beyond P30. Most constructs failed to rescue the defects of GluR delta 2-deficient mice, mainly because they were not efficiently transferred to the postsynaptic sites. However, a construct carrying only the extracellular N-terminal domain (NTD) and the intracellular C-terminal domain (CTD) linked with the fourth transmembrane domain of GluR delta 2 (NTD-TM4-CTD) caused incomplete, but significant rescue of ataxia, consistent with relatively better transport of the construct to the synapses. Notably, the expression of NTD-TM4-CTD in GluR delta 2-deficient Purkinje cells restored abrogated LTD, and aberrant CF territory in the molecular layer. Although the expression of NTD-TM4-CTD failed to rescue persistent multiple CF innervation of GluR delta 2-deficient Purkinje cells, a similar construct in which only TM4 was replaced with a transmembrane domain of CD4 successfully rescued the multiple CF innervation, probably due to more efficient transport of the protein to postsynaptic sites. These results suggest that NTD and CTD are critical domains of GluR delta 2, which functions substantially without conventional ligand binding and ion channel structures.

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］

Imaging extrasynaptic glutamate dynamics in the brain
Okubo Yohei, Sekiya Hiroshi, Namiki Shigeyuki, Sakamoto Hirokazu, Iinuma Sho, Yamasaki Miwako, Watanabe Masahiko, Hirose Kenzo, Iino Masamitsu
NEUROSCIENCE RESEARCH, 65, S142, S143, 2009年, ［査読有り］

GluR epsilon 2 subunit is crucial for NMDA receptor activity and regulation of postsynaptic macromolecular organization
Akashi Kaori, Kakizaki Toshikazu, Kamiya Haruyuki, Fukaya Masahiro, Yamasaki Miwako, Abe Manabu, Watanabe Masahiko, Sakimura Kenji
NEUROSCIENCE RESEARCH, 65, S139, S140, 2009年, ［査読有り］

PREFERENTIAL EXPRESSION OF M1 mAChR IN DENDRITES AND SPINES OF CORTICAL PRINCIPAL CELLS AND ITS ANATOMICAL EVIDENCE FOR VOLUME TRANSMISSION
Miwako Yamasaki, Masahiko Watanabe
JOURNAL OF PHYSIOLOGICAL SCIENCES, 59, 142, 142, SPRINGER TOKYO, 2009年, ［査読有り］
英語

Use-dependent amplification of presynaptic Ca2+ signaling by axonal ryanodine receptors at the hippocampal mossy fiber synapse
Hidemi Shimizu, Masahiro Fukaya, Miwako Yamasaki, Masahiko Watanabe, Toshiya Manabe, Haruyuki Kamiya
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105, 33, 11998, 12003, NATL ACAD SCIENCES, 2008年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Presynaptic Ca2+ stores have been suggested to regulate Ca2+ dynamics within the nerve terminals at certain types of the synapse. However, little is known about their mode of activation, molecular identity, and detailed subcellular localization. Here, we show that the ryanodine-sensitive stores exist in axons and amplify presynaptic Ca2+ accumulation at the hippocampal mossy fiber synapses, which display robust presynaptic forms of plasticity. Caffeine, a potent drug inducing Ca2+ release from ryanodine-sensitive stores, causes elevation of presynaptic Ca2+ levels and enhancement of transmitter release from the mossy fiber terminals. The blockers of ryanodine receptors, TMB-8 or ryanodine, reduce presynaptic Ca2+ transients elicited by repetitive stimuli of mossy fibers but do not affect those evoked by single shocks, suggesting that ryanodine receptors amplify presynaptic Ca2+ dynamics in an activity dependent manner. Furthermore, we generated the specific antibody against the type 2 ryanodine receptor (RyR2; originally referred to as the cardiac type) and examined the cellular and subcellular localization using immunohistochemistry. RyR2 is highly expressed in the stratum lucidum of the CA3 region and mostly colocalizes with axonal marker NF160 but not with terminal marker VGLUT1. Immunoelectron microscopy revealed that RyR2 is distributed around smooth ER within the mossy fibers but is almost excluded from their terminal portions. These results suggest that axonal localization of RyR2 at sites distant from the active zones enables use dependent Ca2+ release from intracellular stores within the mossy fibers and thereby facilitates robust presynaptic forms of plasticity at the mossy fiber-CA3 synapse.

Glutamate transporters regulate lesion-induced plasticity in the developing somatosensory cortex
Chihiro Takasaki, Rieko Okada, Akira Mitani, Masahiro Fukaya, Miwako Yamasaki, Yuri Fujihara, Tetsuo Shirakawa, Kohichi Tanaka, Masahiko Watanabe
JOURNAL OF NEUROSCIENCE, 28, 19, 4995, 5006, SOC NEUROSCIENCE, 2008年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Glutamate transporters are involved in neural differentiation, neuronal survival, and synaptic transmission. In the present study, we examined glutamate transporter 1 (GLT1) expression in the neonatal somatosensory cortex of C57BL/6 mice, and pursued its role in somatosensory development by comparing barrel development between GLT1 knock-out and control mice. During the first few neonatal days, a critical period for barrels, GLT1 expression is strikingly upregulated in cortical astrocytes, whereas it was downregulated in neuronal elements to below the detection threshold. GLT1 knock-out neonates developed normally in terms of body growth, cortical histoarchitecture, barrel formation, and critical period termination. However, when row C whiskers were lesioned during the critical period, reduction of lesioned row C barrels and reciprocal expansion of intact row B/D barrels were both milder in GLT1 knock-out mice than in control littermates. Accordingly, the map plasticity index, calculated as (B + D)/2C, was significantly lowered in GLT1 knock-out mice. We also found that extracellular glutamate levels in the neonatal somatosensory cortex were significantly elevated in GLT1 knockout mice. Diminished lesion-induced plasticity was further found in mutant mice lacking glutamate-aspartate transporter (GLAST), an astrocyte-specific glutamate transporter throughout development. Therefore, glutamate transporters regulate critical period plasticity by enhancing expansion of active barrels and shrinkage of inactive barrels. Because cortical contents of glutamate receptors and GLAST were unaltered in GLT1 knock-out mice, this action appears to be mediated, at least partly, by keeping the ambient glutamate level low. Considering an essential role of glutamate receptors in the formation of whisker-related thalamocortical synapse patterning, glutamate transporters thus facilitate their activity-dependent remodeling.

Role of the internal Shank-binding segment of glutamate receptor delta 2 in synaptic localization and cerebellar functions
Misato Yasumura, Takeshi Uemura, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe, Masayoshi Mishina
NEUROSCIENCE LETTERS, 433, 2, 146, 151, ELSEVIER IRELAND LTD, 2008年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Glutamate receptor (GluR) delta 2 selectively expressed in cerebellar Purkinje cells (PCs) plays key roles in cerebellar long-term depression (LTD), motor learning and formation of parallel fiber (PF)-PC synapses. We have recently shown that the PDZ [postsynaptic density (PSD)-95/Discs large/zona occludens-1]-binding domain at the C-terminal, the T site, is essential for LTD induction and the regulation of climbing fiber (CF) territory, but is dispensable for synaptic localization of GluR delta 2, PF-PC synapse formation and CF elimination process. To investigate the functional roles of the S segment, the second PDZ-binding domain in the middle of the C-terminal cytoplasmic region, we generated GluR delta 2 Delta S mice carrying mutant GluR delta 2 lacking this segment. The amount of GluR delta 2 Delta S in mutant mice was reduced compared with that of GluR delta 2 in wild-type mice. However, the extent of decrease was much larger in the PSD fractions than in cerebellar homogenates, suggesting the requirement of the S segment for efficient synaptic localization. Furthermore, mismatched PF synapses and free spines emerged and CF-innervation territory on PC dendrites expanded in GluR delta 2 Delta S mice. On the other hand, the performance in the rotarod test was comparable between wild-type and GluR delta 2 Delta S mice. These results suggest that the S segment and T site, the two PDZ-binding domains in the C-terminal cytoplasmic region, are differentially involved in diverse GluR delta 2 functions. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Roles of metabotropic glutamate receptor subtype-1 in the adult cerebellum
Nakao Harumi, Hashimoto Kouichi, Yamasaki Miwako, Nakao Kazuki, Watanabe Masahiko, Kano Masanobu, Ailba Atsu
NEUROSCIENCE RESEARCH, 61, S147, 2008年

ERK is required for developmental synapse elimination in the cerebellum
Harada Takeshi, Hirai Yoshie, Yamasaki Miwako, Hashimoto Kouichi, Nakao Harumi, Tabata Toshihide, Watanabe Masahiko, Kano Masanobu, Aiba Atsu
Neuroscience Research, 61, S57, 2008年, ［査読有り］
研究論文（国際会議プロシーディングス）

P3-c2O Role of the internal Shank-binding segment of GluR delta 2 in cerebellar functions
Yasumura Misato, Uemura Takeshi, Yamasaki Miwako, Sakimura Kenji, Watanabe Masahiko, Mishina Masayoshi
NEUROSCIENCE RESEARCH, 61, S221, 2008年, ［査読有り］

The C-terminal domain of GluR delta 2 is essential for cerebellar LTD and regulation of climbing fiber territory at parallel fiber synapses in cerebellar Purkinje cells
Uemura Takeshi, Kakizavva Sho, Yamasaki Miwako, Sakimura Kenji, Watanabe Masahiko, Iino Masamitsu, Mishina Masayoshi
JOURNAL OF PHARMACOLOGICAL SCIENCES, 106, 80P, 2008年, ［査読有り］

Hole of the C-terminal PDZ binding domain of glutamate receptor delta 2 in synaptic plasticity and climbing fiber wiring
Uemura Takeshi, Kakizawa Sho, Yamasaki Miwako, Sakimura Kenji, Watanabe Masahiko, Lino Masamitsu, Mishina Masayoshi
NEUROSCIENCE RESEARCH, 61, S219, 2008年, ［査読有り］

Roles of the internal PDZ-binding domain of GluR delta 2 in synaptic localization and cerebellar functions
Yasumura Misato, Uemura Takeshi, Yamasaki Miwako, Sakimura Kenji, Watanabe Masahiko, Mishina Masayoshi
JOURNAL OF PHARMACOLOGICAL SCIENCES, 106, 133P, 2008年, ［査読有り］

Regulation of long-term depression and climbing fiber territory by glutamate receptor delta 2 at parallel fiber synapses through its c-terminal domain in cerebellar Purkinje cells
Takeshi Uemura, Sho Kakizawa, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe, Masamitsu Iino, Masayoshi Mishina
JOURNAL OF NEUROSCIENCE, 27, 44, 12096, 12108, SOC NEUROSCIENCE, 2007年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Glutamate receptor ( GluR) delta 2 selectively expressed in cerebellar Purkinje cells ( PCs) plays key roles in long-term depression ( LTD) induction at parallel fiber ( PF)-PC synapses, motor learning, the matching and connection of PF-PC synapses in developing and adult cerebella, the elimination of multiple climbing fibers ( CFs) during development, and the regulation of CF territory on PCs. However, it remains unsolved how GluR delta 2 regulates cerebellar synaptic plasticity, PF-PC synapse formation, and CF wiring. One possible signaling mechanism through GluR delta 2 is signaling by protein-protein interactions. The C-terminal region of GluR delta 2 contains at least three domains for protein-protein interactions. The PDZ ( postsynaptic density-95/Discs large/zona occludens 1)-binding domain at the C terminal, named as the T site, interacts with several postsynaptic density proteins. Here, we generated GluR delta 2 Delta T mice carrying mutant GluR delta 2 lacking the T site. There were no significant differences in the amount of receptor proteins at synapses, histological features, and the fine structures of PF-PC synapses between wild-type and GluR delta 2 Delta T mice. However, LTD induction at PF-PC synapses and improvement in the accelerating rotarod test were impaired in GluR delta 2 Delta T mice. Furthermore, CF territory expanded distally and ectopic innervation of CFs occurred at distal dendrites in GluR delta 2 Delta T mice, but the elimination of surplus CF innervation at proximal dendrites appeared to proceed normally. These results suggest that the C-terminal T site of GluR delta 2 is essential for LTD induction and the regulation of CF territory but is dispensable for PF-PC synapse formation and the elimination of surplus CFs at proximal dendrites during development.

Miniature synaptic events elicited by presynaptic Ca2+ rise are selectively suppressed by cannabinoid receptor activation in cerebellar Purkinje cells
M Yamasaki, K Hashimoto, M Kano
JOURNAL OF NEUROSCIENCE, 26, 1, 86, 95, SOC NEUROSCIENCE, 2006年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Activation of cannabinoid receptors suppresses neurotransmitter release in various brain regions. In cerebellar Purkinje cells (PCs), cannabinoid agonists suppress both EPSC and IPSC evoked by stimulating the corresponding inputs. However, cannabinoid agonists suppress miniature IPSC (mIPSC) but not miniature EPSC (mEPSC) at normal external Ca2+ concentration ([Ca2+](o)). Therefore, cannabinoid agonists are thought to suppress release machinery for IPSCs but not that for EPSCs. Here we investigated the possible cause of this difference and found that cannabinoid agonists selectively suppressed Ca2+-enhanced miniature events. A cannabinoid agonist, WIN55,212-2 (5 mu M), did not affect mEPSC frequency with 2 mM extracellular Ca2+ (Ca2+(o)). However, WIN55,212-2 became effective when mEPSC frequency was enhanced by elevation of presynaptic Ca2+ level by perfusion with 5mMCa(o)(2+) or bath application of A23187, a Ca2+ ionophore. In contrast, WIN55,212-2 suppressed mIPSC frequency with 2mM Ca-o(2+), but it became ineffective when the presynaptic Ca2+ level was lowered by perfusion with a Ca2+-free solution containing BAPTA-AM. Experiments with systematic [Ca2+](o) changes revealed that mIPSC but not mEPSC regularly involved events elicited by presynaptic Ca2+ rise with 2mM Ca-o(2+). Importantly, Ca2+-enhancement of mEPSC and mIPSC was not attributable to activation of voltage-dependent Ca2+ channels. Activation of GABA(B) receptor or group III metabotropic glutamate receptor, which couple to G(i/o)-protein, also preferentially suppressed Ca2+-enhanced miniature events in PCs. These results suggest that the occurrence of Ca2+-enhanced miniature events at normal [Ca2+](o) determines the sensitivity to the presynaptic depression mediated by cannabinoid receptors and other G(i/o)-coupled receptors in PCs.

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］

3-Phosphoglycerate dehydrogenase, a key enzyme for L-serine biosynthesis, is preferentially expressed in the radial glia/astrocyte lineage and olfactory ensheathing glia in the mouse brain
Miwako Yamasaki, Keiko Yamada, Shigeki Furuya, Junya Mitoma, Yoshio Hirabayashi, Masahiko Watanabe
Journal of Neuroscience, 21, 19, 7691, 7704, 19, 2001年10月01日, ［査読有り］
研究論文（学術雑誌）, L-Serine is synthesized from glycolytic intermediate 3-phosphoglycerate and is an indispensable precursor for the synthesis of proteins, membrane lipids, nucleotides, and neuroactive amino acids D-serine and glycine. We have recently shown that L-serine and its interconvertible glycine act as Bergmann glia-derived trophic factors for cerebellar Purkinje cells. To investigate whether such a metabolic neuron-glial relationship is fundamental to the developing and adult brain, we examined by in situ hybridization and immunohistochemistry the cellular expression of 3-phosphoglycerate dehydrogenase (3PGDH), the initial step enzyme for de novo L-serine biosynthesis in animal cells. At early stages when the neural wall consists exclusively of the ventricular zone, neuroepithelial stem cells expressed 3PGDH strongly and homogeneously. Thereafter, 3PGDH expression was downregulated and eventually disappeared in neuronal populations, whereas its high expression was transmitted to the radial glia and later to astrocytes in the gray and white matters. In addition, 3PGDH was highly expressed throughout development in the olfactory ensheathing glia, a specialized supporting cell that thoroughly ensheathes olfactory nerves. These results establish a fundamental link of the radial glia/astrocyte lineage and olfactory ensheathing glia to L-serine biosynthesis in the brain. We discuss this finding in the context of the hypothesis that 3PGDH expression in these glia cells contributes to energy metabolism in differentiating and differentiated neurons and other glia cells, which are known to be vulnerable to energy loss.

L-Serine and glycine serve as major astroglia-derived trophic factors for cerebellar Purkinje neurons
Shigeki Furuya, Toshihide Tabata, Junya Mitoma, Keiko Yamada, Miwako Yamasaki, Asami Makino, Toshifumi Yamamoto, Masahiko Watanabe, Masanobu Kano, Yoshio Hirabayashi
Proceedings of the National Academy of Sciences of the United States of America, 97, 21, 11528, 11533, 21, 2000年10月10日, ［査読有り］
研究論文（学術雑誌）, Glial cells support the survival and development of central neurons through the supply of trophic factors. Here we demonstrate that L-serine (L-Ser) and glycine (Gly) also are glia-derived trophic factors. These amino acids are released by astroglial cells and promote the survival, dendritogenesis, and electrophysiological development of cultured cerebellar Purkinje neurons. Although L-Ser and Gly are generally classified as nonessential amino acids, 3-phosphoglycerate dehydrogenase (3PGDH), a key enzyme for their biosynthesis, is not expressed in Purkinje neurons. By contrast, the Bergman glia, a native astroglia in the cerebellar cortex, highly expresses 3PGDH. These data suggest that L-Ser and Gly mediate the trophic actions of glial cells on Purkinje neurons.

Critical period for activity-dependent synapse elimination in developing cerebellum
S Kakizawa, M Yamasaki, M Watanabe, M Kano
JOURNAL OF NEUROSCIENCE, 20, 13, 4954, 4961, SOC NEUROSCIENCE, 2000年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Synapse elimination is considered to be the final step in neural circuit formation, by causing refinement of redundant connections formed at earlier developmental stages. The developmental loss of climbing fiber innervation from cerebellar Purkinje cells is an example of such synapse elimination. It has been suggested that NMDA receptors are involved in the elimination of climbing fiber synapses. In the present study, we probed the NMDA receptor-dependent period of climbing fiber synapse elimination by using daily intraperitoneal injections of the NMDA receptor antagonist MK-801. We found that blockade of NMDA receptors during postnatal day 15 (P15) and P16, but not before or after this period, resulted in a higher incidence of multiple climbing fiber innervation and caused a mild but persistent loss of motor coordination. Neither basic synaptic functions nor cerebellar morphology were affected by this manipulation. Chronic local application of MK-801 to the cerebellum during P15 and P16 also yielded a higher incidence of multiple climbing fiber innervation. During P15-P16, large NMDA receptor-mediated EPSCs were detected at the mossy fiber-granule cell synapse, but not at the parallel fiber-Purkinje cell or climbing fiber-Purkinje cell synapse. It is therefore likely that the NMDA receptors located at the mossy fiber-granule cell synapse mediate signals leading to the elimination of surplus climbing fibers. These results suggest that an NMDA receptor-dependent phase of climbing fiber synapse elimination lasts 2 d at most. During this phase, the final refinement of climbing fiber synapses occurs, and disruption of this process leads to permanent impairment of cerebellar function.

Gq protein alpha subunits G alpha q and G alpha 11 are localized at postsynaptic extra-junctional membrane of cerebellar Purkinje cells and hippocampal pyramidal cells
J Tanaka, S Nakagawa, E Kushiya, M Yamasaki, M Fukaya, T Iwanaga, MI Simon, K Sakimura, M Kano, M Watanabe
EUROPEAN JOURNAL OF NEUROSCIENCE, 12, 3, 781, 792, BLACKWELL SCIENCE LTD, 2000年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Following cell surface receptor activation, the alpha subunit of the Gq subclass of GTP-binding proteins activates the phosphoinositide signalling pathway. Here we examined the expression and localization of Gq protein alpha subunits in the adult mouse brain by in situ hybridization and immunohistochemistry. Of the four members of the Gq protein alpha subunits, G alpha q and G alpha 11 were transcribed predominantly in the brain. The highest transcriptional level of G alpha q was observed in cerebellar Purkinje cells (PCs) and hippocampal pyramidal cells, while that of G alpha 11 was noted in hippocampal pyramidal cells. Antibody against the C-terminal peptide common to G alpha q and G alpha 11 strongly labelled the cerebellar molecular layer and hippocampal neuropil layers. In these regions, immunogold preferentially labelled the cytoplasmic face of postsynaptic cell membrane of PCs and pyramidal cells. Immunoparticles were distributed along the extra-junctional cell membrane of spines, dendrites and somata, but were almost excluded from the junctional membrane. By double immunofluorescence, G alpha q/G alpha 11 was extensively colocalized with metabotropic glutamate receptor mGluR1 alpha in dendritic spines of PCs and with mGluR5 in those of hippocampal pyramidal cells. Together with concentrated localization of mGluR1 alpha and mGluR5 in a peri-junctional annulus on PC and pyramidal cell synapses (Baude et al. 1993, Neuron, 11, 771-787; Lujan et al. 1996, Eur. J. Neurosci., 8, 1488-1500), the present molecular-anatomical findings suggest that peri-junctional stimulation of the group I metabotropic glutamate receptors is mediated by G alpha q and/or G alpha 11, leading to the activation of the intracellular effector, phospholipase C beta.

Olig2アストロサイトの脳内分布と抑制性シナプスとの関与
辰巳晃子, 石西綾美, 山崎美和子, 河邉良枝, 森田(竹村)晶子, 中原一貴, 奥田洋明, 田中達英, 和中明生, 日本解剖学会総会・全国学術集会講演プログラム・抄録集, 124th, 2019年

Unique neural characteristics of GAD67-containing 5-HTergic neurons in the rat dorsal raphe nucleus
Shikanai Hiroki, Yoshida Takayuki, Konno Kohtarou, Yamasaki Miwako, Izumi Takeshi, Ohmura Yu, Shimamura Keiichi, Watanabe Masahiko, Yoshioka Mitsuhiro, JOURNAL OF PHARMACOLOGICAL SCIENCES, 121, 65P, 2013年, ［査読有り］

離乳期の新奇環境ストレス曝露に対して優先的に活性化する背側縫線核ニューロンの発見とその細胞特性解析
鹿内 浩樹, 吉田 隆行, 今野 幸太郎, 山崎 美和子, 泉 剛, 大村 優, 渡邊 雅彦, 吉岡 充弘, 北海道醫學雜誌 = Acta medica Hokkaidonensia, 87, 6, 288, 288, 2012年11月01日
日本語

小脳プルキンエ細胞の軸索初節はGABA作動性シナプス伝達に必要な分子形態学的基盤を欠如する
岩倉 淳, 内ヶ島 基政, 宮崎 大輔, 山崎 美和子, 渡辺 雅彦, 北海道醫學雜誌 = Acta medica Hokkaidonensia, 87, 6, 273, 273, 2012年11月01日
日本語

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
日本語

扁桃体基底核の特異な抑制性シナプス結合「陥入型シナプス」の発見と機能的役割
吉田 隆行, 内ヶ島 基政, 山崎 美和子, イストバンカトナ, 山崎 真弥, 崎村 建司, 狩野 方伸, 吉岡 充弘, 渡辺 雅彦, 北海道醫學雜誌 = Acta medica Hokkaidonensia, 87, 2, 72, 72, 2012年04月01日
日本語

シナプス外グルタミン酸動態の可視化解析
大久保洋平, 関谷敬, 並木繁行, 坂本寛和, 坂本寛和, 飯沼将, 山崎美和子, 渡辺雅彦, 廣瀬謙造, 飯野正光, 日本薬理学会関東部会プログラム・要旨集, 120th, 2009年

3-Phosphoglycerate dehydrogenase, a key enzyme for l-serine biosynthesis, is preferentially expressed in the radial glia/astrocyte lineage and olfactory ensheathing glia in the mouse brain.
Yamasaki M, Yamada K, Furuya S, Mitoma J, Hirabayashi Y, Watanabe M, The Journal of neuroscience : the official journal of the Society for Neuroscience, 21, 19, 7691, 7704, 2001年10月01日
l-Serine is synthesized from glycolytic intermediate 3-phosphoglycerate and is an indispensable precursor for the synthesis of proteins, membrane lipids, nucleotides, and neuroactive amino acids d-serine and glycine. We have recently shown that l-serine and its interconvertible glycine act as Bergmann glia-derived trophic factors for cerebellar Purkinje cells. To investigate whether such a metabolic neuron-glial relationship is fundamental to the developing and adult brain, we examined by in situ hybridization and immunohistochemistry the cellular expression of 3-phosphoglycerate dehydrogenase (3PGDH), the initial step enzyme for de novo l-serine biosynthesis in animal cells. At early stages when the neural wall consists exclusively of the ventricular zone, neuroepithelial stem cells expressed 3PGDH strongly and homogeneously. Thereafter, 3PGDH expression was downregulated and eventually disappeared in neuronal populations, whereas its high expression was transmitted to the radial glia and later to astrocytes in the gray and white matters. In addition, 3PGDH was highly expressed throughout development in the olfactory ensheathing glia, a specialized supporting cell that thoroughly ensheathes olfactory nerves. These results establish a fundamental link of the radial glia/astrocyte lineage and olfactory ensheathing glia to l-serine biosynthesis in the brain. We discuss this finding in the context of the hypothesis that 3PGDH expression in these glia cells contributes to energy metabolism in differentiating and differentiated neurons and other glia cells, which are known to be vulnerable to energy loss., Society of Neuroscience, 英語

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
科学研究費助成事業
2022年04月01日 - 2025年03月31日
宮崎 太輔, 山崎 美和子
日本学術振興会, 基盤研究(C), 北海道大学, 22K06784

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
科学研究費助成事業 基盤研究(B)
2020年04月01日 - 2024年03月31日
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。
日本学術振興会, 基盤研究(B), 北海道大学, 20H03410

認知・運動における多領野間脳情報動態の光学的計測と制御　　　　　　　　　　　　　　　
新学術領域研究（研究領域提案型）
2017年09月 - 2022年03月
喜多村和郎
文部科学省, 競争的資金

抑制性介在細胞の非シナプス結合におけるNR3A受容体選択的集積メカニズムの解明　　　　　　　　　　　　　　　
国際共同研究加速基金（国際共同研究強化）
2018年01月 - 2021年03月
山崎美和子
文部科学省, 研究代表者, 競争的資金

経路選択的な標識・操作技術を応用したマーモセット大脳皮質―基底核 ネットワークの構造・機能マッピング　　　　　　　　　　　　　　　
革新的技術による脳機能ネットワークの全容解明プロジェクト 技術開発個別課題（新技術創出）
2017年04月 - 2021年03月
小林和人
国立研究開発法人日本医療研究開発機構, 競争的資金

非シナプス性結合に入力経路・細胞選択的に集積するNR3A受容体の機能的意義の解明　　　　　　　　　　　　　　　
基盤研究（Ｃ）
2017年04月 - 2020年03月
山崎美和子
文部科学省, 研究代表者, 競争的資金

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
科学研究費助成事業
2014年04月01日 - 2017年03月31日
宮崎 太輔, 山崎 美和子
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。
日本学術振興会, 基盤研究(C), 北海道大学, 26460250

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
科学研究費助成事業
2012年05月31日 - 2017年03月31日
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。
日本学術振興会, 基盤研究(S), 北海道大学, 24220007

成体マウス脳で非シナプス結合に選択的に発現するNR3A受容体の機能的意義　　　　　　　　　　　　　　　
医学系研究奨励
2016年04月 - 2017年03月
山崎美和子
武田科学振興財団, 研究代表者, 競争的資金

海馬におけるアセチルコリン作動性神経依存的な記憶形成機構の分子解剖学的基盤　　　　　　　　　　　　　　　
内藤記念女性研究者研究助成金
2014年04月 - 2016年03月
山崎美和子
内藤記念科学振興財団, 研究代表者, 競争的資金

バレル神経回路の機能発達におけるニコチン性アセチルコリン受容体の役割　　　　　　　　　　　　　　　
基盤研究（Ｃ）
2014年04月 - 2016年03月
山崎美和子
文部科学省, 研究代表者, 競争的資金

ＮＭＤＡ受容体を介する興奮・抑制バランス依存的なバレル神経回路形成制御機構　　　　　　　　　　　　　　　
若手研究（B)
2012年04月 - 2014年03月
山崎美和子
文部科学省, 研究代表者, 競争的資金

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
科学研究費助成事業
2007年 - 2011年
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。
日本学術振興会, 基盤研究(S), 北海道大学, 19100005

１次抗体直接標識プローブの開発と実用性に関する検討　　　　　　　　　　　　　　　
シーズ発掘試験
2009年04月 - 2010年03月
山崎美和子
国立研究開発法人科学技術振興機構, 研究代表者, 競争的資金

余剰登上線維除去に先行して起こるmGluR1依存的な登上線維シナプス強化過程　　　　　　　　　　　　　　　
若手研究（B)
2007年04月 - 2009年03月
山崎美和子
文部科学省, 研究代表者, 競争的資金

活動依存的に誘導されるGPIアンカー蛋白質の小脳内分布とシナプス機能発達との関連　　　　　　　　　　　　　　　
特別研究員奨励費（DC1)
2003年04月 - 2006年03月
山崎美和子
日本学術振興会, 研究代表者, 競争的資金