ポスター

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ポスター A. 分子・細胞生物学 A. Neural Excitability, Synapses, and Glia: Cellular Mechanisms

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-001 ラットA11領域内でカルビンディン陽性細胞は様々な機能分子を共発現している Calbindin-positive neurons co-express functional markers in a location-dependent manner within the A11 region of the rat brain 山口剛(1)、小澤継史(2)、山口重樹(2)、濱口眞輔(2)、上田秀一(1)、徳田信子(1) 1. 獨協医科大学解剖学講座、2. 獨協医科大学麻酔科学講座 Tsuyoshi YAMAGUCHI(1), Hidechika OZAWA(2), Shigeki YAMAGUCHI(2), Shinsuke HAMAGUCHI(2), Shuichi UEDA(1), Nobuko TOKUDA(1) 1. Dokkyo Med Univ, Dept. of Anat., Tochigi, Japan, 2. Dokkyo Med Univ, Dept. of Anesth. and Pain Med., Tochigi, Japan Keyword: A11 region, Calbindin, calcitonin gene-related peptide, androgen receptor The A11 dopaminergic cell group is the only group among the A8–A16 dopaminergic cell groups that includes neurons innervating the spinal cord. This brain region plays a role in numerous physiological functions, including pain and locomotor activity, and dysfunction of this brain region is thought to contribute to the pathogenesis of restless legs syndrome. The A11 region consists of a variety of neurons including GABAergic, dopaminergic (DA), and calbindin positive (Calb⁺) neurons. However, the neurochemical nature of Calb⁺ neurons and their regulatory role in the A11 region remain largely unknown. In this study, we examined the kind of functional markers co-expressed in the Calb⁺ neurons using brain sections from 8-week-old rats. To examine a marker related to classical neurotransmitters, we performed in situ hybridization for glutamate decarboxylase (GAD) 65 and 67 or vesicular glutamate transporter 2 (vGluT2), in conjunction with Calb immunohistochemistry. We found cellular co-expression of Calb with vGluT2 or GAD65/67 throughout the A11 region. Nearly all Calb⁺/GAD65/67⁺ neurons were found in the rostral-middle aspect of the A11 region. In contrast, Calb⁺/vGluT2⁺ neurons were found predominantly in the middle-caudal aspect of the A11 region. For receptors and neuropeptides, we performed immunohistochemistry for androgen receptor (AR), estrogen receptors (ERα and ERβ), and calcitonin gene-related peptide (CGRP). We found that Calb⁺ neurons co-expressed AR in the rostral aspect of the A11 region in both male and female rats. However, we rarely find cellular co-expression of Calb with ERα or ERβ in this region. For CGRP, we found both Calb⁺ neurons with or without CGRP expression. These results demonstrate that Calb⁺ neurons co-express many functional markers. Calb⁺ neurons have a distinct distribution pattern and may play a variety of regulatory roles, depending on their location within the A11 region.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-002 L-DOPAによるGPR143を介した海馬神経新生の促進 L-DOPA promotes neurogenesis in the Hippocampus via its receptor, GPR143. 笠原由佳(1,2)、増川太輝(2)、小林憲太(3)、山崎美和子(4)、渡辺雅彦(4)、五嶋良郎(2) 1. 九州大学、2. 横浜市立大学、3. 生理学研究所、4. 北海道大学 Yuka Kasahara(1,2), Daiki Masukawa(2), Kenta Kobayashi(3), Miwako Yamasaki(4), Masahiko Watanabe(4), Yoshio Goshima(2) 1. Kyushu University, 2. Yokohama City University, 3. National Institute for Physiological Sciences, 4. Hokkaido University Keyword: L-DOPA, GPR143, neurogenesis, hippocampus Neurogenesis occurs in the hippocampus through life and is implicated in various physiological brain functions such as memory encoding and mood regulation. However, the mechanisms by which neural stem and progenitor populations are maintained and strictly controlled remain vaguely understood. Some neurotransmitters such as dopamine and acetylcholine are known to regulate hippocampal neurogenesis. In this study, we show that l-3,4-dihydroxyphenylalanine (L-DOPA, a precursor amino acid of dopamine) and its receptor, GPR143, the gene product of ocular albinism 1, regulate neurogenesis in the dentate gyrus 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 cognitive decline and exacerbated depression-like behavior were observed in Gpr143^-/y mice. Replenishment of GPR143 in the dentate gyrus recovered impaired neurogenesis and attenuated the depression-like behavior. Antidepressant treatment with imipramine increased neurogenesis and alleviated cognitive decline and depression-like behavior in Gpr143^-/y mice, suggesting different signaling pathways regulated by antidepressant treatment from those in which GPR143 is involved. Our findings suggest that L-DOPA through GPR143 modulates hippocampal neurogenesis, thereby playing a role in mood regulation in the hippocampus.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-003 細胞内ドーパミン濃度によるチロシン水酸化酵素タンパク質量制御機構の解析 Analysis of the regulation mechanism of the tyrosine hydroxylase protein levels by intracellular dopamine concentration 相田莉奈(1)、杉山菜奈子(1)、原怜(1)、一瀬宏(1) 1. 東京工業大学生命理工学院生命理工学系 Rina Aida(1), Nanako Sugiyama(1), Satoshi Hara(1), Hiroshi Ichinose(1) 1. Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan Keyword: Tyrosine Hydroxylase, Dopamine, Biopterin ドーパミンはカテコールアミンに属する神経伝達物質であり、中枢神経系で運動の制御や情動、学習に深く関わっている。現在統合失調症の治療には、主にドーパミンD2受容体の拮抗薬が使われている。これらの抗精神病薬は、ドーパミンによる過剰な神経伝達を抑制するはたらきを持つが、ドーパミンの過剰分泌を抑制する効果はない。そこで私たちは、ドーパミン量を制御するためのターゲットとしてドーパミン生合成経路の律速酵素であるチロシン水酸化酵素(TH)に着目した。THタンパク質量を人為的に調節することでドーパミン量を調節することができれば、ドーパミンによる過剰な神経伝達をD2受容体アンタゴニストよりもマイルドに抑制することができると考えられる。当研究室では、これまでにTHの補酵素であるテトラヒドロビオプテリン(BH4)の欠乏マウスの脳内でTHタンパク質量が減少し、BH4やドーパミン前駆体のドーパの投与により回復することを見出している。そこで、神経系の培養細胞を用いて細胞内BH4量やドーパミン量を変化させることによるTHタンパク質量変化について検討した。ヒトTHを安定発現させたSH-SY5Y細胞に、L-ドーパをドーパミンに変換する芳香族アミノ酸脱炭酸酵素(AADC)の阻害剤であるNSD-1015を添加した。すると、細胞内モノアミン量は投与後すぐに減少したが、投与6時間後に活性化型であるリン酸化THの増加と並行してTHタンパク質量の減少が観察された。THタンパク質は、生成物であるドーパミンが活性中心にある鉄イオンとキレート結合を介して結合することにより、活性は低いけれどもタンパク質としては安定な構造をとること、リン酸化されるとドーパミンが活性中心から外れて活性型となるが不安定となることが知られている。生理的には細胞内ドーパミンが何らかの理由により持続的に減少した状態が継続するとTHタンパク質量が減少すると考えられる。このように代償反応としては矛盾するように考えられるTHタンパク質量変化の意義と、精神疾患治療への応用を目指していく。		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-004 シナプトタグミン７はphasic 活動依存的Somatodendritic ドーパミン放出のカルシウムセンサーとして働く Syt7 is the primary Ca(2+) sensor for phasic somatodendritic DA release 引間卓弥(1)、Witkovsky Paul (1)、Rice Margaret(1,2) 1. ニューヨーク大学医学部神経外科、2. ニューヨーク大学医学部神経生理学 Takuya Hikima(1), Paul Witkovsky(1), Margaret E Rice(1,2) 1. Dept Neurosurgery, NYU School of Medicine, New York, USA, 2. Dept Neurosci and Physiol, NYU School of Medicine, New York, USA Keyword: dopamine release, synaptotagmin, D2 dopamine receptors, autoreceptors Midbrain dopamine (DA) neurons in the substantia nigra pars compacta (SNc) exhibit somatodendritic release of DA. DA acts at somatodendritic D2 autoreceptors to regulate DA neuron firing patterns that govern DA release throughout the brain. Although somatodendritic DA release was discovered decades ago, questions about molecular mechanisms underlying somatodendritic dopamine release remain unresolved. Our previous work showed the Ca²⁺ dependence of somatodendritic and axonal DA release differ, with the persistence of somatodendritic DA release in submillimolar Ca²⁺ concentrations that are insufficient to support axonal release (Chen et al., 2011). In addition, we reported previously that D2 autoreceptor-dependent regulation of a given SNc DA neuron is governed primarily by DA released from that same cell (Hikima et al., 2021). This allows mechanistic studies of the release process through application of selective antibodies or toxins via the recording pipette used to monitor evoked D2 DA autoreceptor-dependent inhibitory currents (D2ICs). Here we tested the hypothesis that the high-affinity Ca²⁺ sensor, synaptotagmin 7 (Syt7), is a key determinant of somatodendritic DA release and its calcium dependence. Somatodendritic DA release from SNc DA neurons was assessed using whole-cell recording in midbrain slices to monitor D2ICs. Single-cell application of an antibody to Syt7 (Syt7 Ab) decreased the amplitude of pulse-train evoked D2ICs, thereby revealing a functional role for Syt7 in phasic DA release. Consistent with our previous findings, D2ICs persisted in low [Ca²⁺]_o that does not support axonal DA release. However, this high sensitivity to Ca²⁺ was lost when anti-Syt7 was applied via the recording pipette and in Syt7 knockout (KO) mice. In millimolar [Ca²⁺]_o, pulse-train evoked D2ICs in Syt7 KOs showed a greater decrease when [Ca²⁺]_o was reduced than seen in wild-type (WT) mice. Single-pulse evoked D2ICs, however, were not altered in Syt7 KOs versus WT. Single-cell application of an antibody to synaptotagmin 1 (Syt1), the Ca²⁺ sensor underlying axonal DA release, had no effect on train-evoked D2ICs in WT SNc DA neurons, implying that Syt1 plays no role in phasic somatodendritic DA release in WT mice. However, Syt1 Ab did cause a decrease in D2ICs in SNc DA neurons from Syt7 KO mice, suggesting that Syt1 can substitute functionally for Syt7 . Together, these data show a key role for Syt7, but not Syt1 in phasic somatodendritic DA release.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-005 58種類のクラスター型プロトカドヘリン（cPcdh）のうち，PcdhγC4はマウスの生存に唯一必須である． Among 58 clustered protocadherin (cPcdh), PcdhγC4 is an essential isoform for mice survival. 樋口流音(1)、梅澤遥香(1)、萩暸(1)、渡辺雅彦(2)、三宝誠(3)、平林真澄(3)、八木健(1) 1. 大阪大学大学院生命機能研究科、2. 北海道大学大学院医学研究院、3. 生理学研究所行動・代謝分子解析センター Ryuon Higuchi(1), Haruka Umezawa(1), Akira Hagi(1), Masahiko Watanabe(2), Makoto Sanbo(3), Masumi Hirabayashi(3), Takeshi Yagi(1) 1. Grad Sch Front. Biosci, Osaka Univ, 2. Grad Sch Med, Hokkaido Univ, 3. Cent for Gen Analy of Behav, Natio Insti for Physi Sci Keyword: clustered protocadherin, neural network diversity, homophilic interaction Cell adhesion molecule-mediated cell-cell recognition is essential for neural circuit formation to obtain advanced brain functions. In mammals, clustered protocadherins (cPcdhs) gene cluster is the candidate proteins bringing the network diversity to the brain. Clustered protocadherin (cPcdh) family has more than 50 isoforms and is classified into three subfamilies (Pcdha, Pcdhb, Pcdhg) based on the amino acid sequence homology. In a previous study, our laboratory showed that only PcdhgC4 among clustered protocadherin is essential for mouse pup survival. However, the concrete molecular and neuronal function of every single isoform except for PcdhaC2 is not known. So, we have been focusing on this molecule to reveal the gene function of PcdhgC4 and the relationship to other clustered protocadherins. PcdhgC4 consists of variable (V) exon and 3 Pcdhg constant (C) exons. The V exon of PcdhgC4 encodes 6 cadherin extracellular domains (EC1-6), a transmembrane domain (TM), membrane-proximal variable cytoplasmic domain (VCP), and 3 C exons encode a Pcdhg common C-terminal cytoplasmic domain (CP). First, we generated the PcdhgC4 specific functional domain deletion mice using CRISPR/Cas9 to two-cell injection of the murine fertilized egg. We have remarked two domains, the homophilic interaction domain (EC2) and the PcdhgC4 specific up/downstream signaling domain (VCP). Both EC2 and VCP domain deletion mice died within one day after birth, surprisingly the body phenotypes and cell-death levels were more severe than PcdhgC4 null mice. Next, we assessed the dynamics of PcdhgC4 specific domain deletion proteins in K562 cells and mouse brains. We found that the possibility of post-translational modification of cPcdh protein via PcdhgC4 VCP domain. Furthermore, we detected the characteristics for heterophilic cis dimerization with a difference between the Pcdha cluster and PcdhgC4 in the EC5-6 domain. In this presentation, we will discuss the functional significance of each PcdhgC4 specific domain both in vitro and in vivo.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-006 巨大神経網様核Gigantocellularにおけるグルタミン酸, GABA性神経伝達物質ダブルポジティブ細胞の特性解析 Characterization of double-positive glutamatergic and GABAergic neurons in the gigantocellular reticular nucleus in the brainstem. 大須賀智輝(1)、和氣弘明(3)、洲崎悦生(4,2)、松本桂彦(2)、八木健(1) 1. 大阪大学生命機能研究科、2. 生命機能科学研究センター、3. 名古屋大学大学院医学研究科、4. 順天堂大学大学院医学研究科 Tomoki Osuka(1), Hiroaki Wake(3), Etsuo Susaki(4,2), Katsuhiko Matsumoto(2), Takeshi Yagi(1) 1. Grad Sch FBS, Univ of Osaka, Osaka, Japan, 2. Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, Osaka, Japan, 3. Grad Sch Med, Univ of Nagoya,Nagoya, Japan, 4. Grad Sch Med, Univ of Juntendo, Tokyo, Japan Keyword: GABA/glutamate, Gigantocellular, Reticular formation, Brainstem The nervous system of organism is regulated by a delicate balance between glutamate excitatory neurotransmission and GABA inhibitory neurotransmission. Previously, it has been thought that these two neurotransmissions were released from independent populations of neurons. However, recent studies have shown that some neurons have ability to co-release both glutamate and GABA from single neurons. We characterize double-positive glutamatergic and GABAergic neurons in Nucleus reticular gigantocellularis (NRG) which name derives from giant neuronal cells. NRG neurons locate in brainstem as in reticular activating system supporting consciousness and have both ascending and descending projections. Furthermore, neurons in the brainstem are known to develop early, and among them the NRG neurons are one of the earliest differentiations in the brain. Here, we have prepared the transgenic mouse capable of visualizing both glutamatergic and GABAergic neurons by crossing transgenic mice expressing FLP recombinase specifically for glutamate expression (Vglut2) and Cre recombinase specifically for GABA (Gad67) expression with mice of the Ai65 (RCFL-tdT) line expressing tdTomato specifically for both Cre and FLP positive cells and clarified the localization distribution of double-positive NRG cells. To observe the distribution of these cells in the whole brain and the detailed connections of nerve fibers, we have performed a 3D analysis of the whole brain by using the whole-brain transparency imaging method with CUBIC transparency technology. As a result, we have found concentrated groups of neurons that double- positive Gad67 and VGlut2, within subdivisions of the ventral tegmental area (VTA), supramammillary nuclei (SUM), Habenula, NRG and able to observe ascending and descending projections from double-positive NRG. Next, we examined more detailed neural connections in a double-positive NRG specific by using that mouse and trans-synaptic virus generated. Specifically, we visualized pre-cells using rabies virus, which is transmitted retrogradely through synapses. As a result, we have found pre-cells in thalamus, hypothalamus, sensory cortex, and spinal cord. In future, we would like to establish cellular and optogenetic manipulating for double-positive NRG neurons in the brainstem on supporting consciousness.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-007 代謝型グルタミン酸6型受容体の細胞内輸送に関与するC末端特異的配列の探索 Exploration of C-terminal specific sequences involved in the intracellular trafficking of mGluR6 下畑充志(1)、赤木巧(1)、荻原郁夫(1)、金田誠(1) 1. 日本医科大学 Atsushi Shimohata(1), Takumi Akagi(1), Ikuo Ogiwara(1), Makoto Kaneda(1) 1. Dept Physiol, Nippon Med Sch, Tokyo, Japan Keyword: Metabotropic glutamate receptor 6, surface localization, ER retention motif The metabotropic glutamate receptor 6, mGluR6, is predominantly expressed in the retinal bipolar cells, and plays critical roles for the signal processing in the retina. The intracellular C-terminal domain (CTD) of mGluR6 interacts with scaffold proteins and Gβγ subunits, and is involved in mGluR6 cell surface localization and intracellular signaling. We recently proposed the endoplasmic reticulum (ER) retention motif-like domains within mGluR6 CTD may participate in the regulation of receptor surface localization (Rai et al. J. Neurochem. 2021). We herein determined amino acids within the mGluR6 CTD required for surface localization in further details. We constructed rat mGluR6 CTD deletion mutants, and tested their subcellular localization on HEK293T cells using immunocytochemistry. While deletions of up to 14-amino acids (Δ858-871) from the C terminus unaffected mGluR6 surface localization, 15- and 16-amino acid deletions (Δ857-871 and Δ856-871) significantly attenuated mGluR6 surface localization, as we recently showed. However, we here found that 17- and 18-amino acid deletions (Δ855-871 and Δ854-871) did not affect mGluR6 surface localization. mGluR6 surface localization was again attenuated by 19- and 20-amino acids deletions (Δ853-871 and Δ852-871), but was reverted to the full-length level by a 21-amino acid deletion (Δ851-871). Using ER and Golgi markers, we found that the surface-deficient deletion mutants were localized to ER. On the basis of these observations, we hypothesized that two separately located ER retention motif-like sequences (848-arginine-lysine-arginine-serine-851 and 853-lysine-lysine-854) within rat mGluR6 CTD might negatively regulate receptor surface localization. We furthermore found that the position 854 was alanine for human while 848-arginine-lysine-arginine-serine-851 was conserved among mammalians. We then introduced alanine substitutions in the ER retention motif-like sequences in the surface-deficient deletion constructs, and observed that these alanine substituted mutants were localized on cell surface. Taken altogether, we suggest that the highly conserved, canonical ER retention motif (848-arginine-lysine-arginine-serine-851) is very likely involved in mGluR6 intracellular trafficking.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-008 AMPA受容体クラスターとGABAA受容体クラスターに対する細胞外タウの影響 Effect of Extracellular Tau on AMPAR and GABA_AR clusters 町田彩乃(1)、櫻木繁雄(1)、添田義行(2)、高島明彦(2)、坂内博子(1) 1. 早稲田大学大学院先進理工学研究科、2. 学習院大学理工学部 Ayano Machida(1), Shigeo Sakuragi(1), Yoshiyuki Soeda(2), Akihiko Takashima(2), Hiroko Bannai(1) 1. Grad Sch of Advanced Science and Engineering, Univ of Waseda, Tokyo, Japan, 2. Faculty of Science, Univ of Gakushuin, Tokyo, Japan Keyword: Tau Neurofibrillary tangles (NFT), which is one of the neuropathological lesions of Alzheimer's disease (AD), are composed of aggregated form of a microtubule-associated protein “Tau”. In addition to intracellular NFT, Tau protein also forms toxic oligomers in extracellular space. Importantly, long-term potentiation (LTP) in excitatory synapses is inhibited in the presence of extracellular tau oligomers, indicating that extracellular Tau oligomer affect the synaptic activity. Considering that the synapse is the basis of memory and learning, it is essential to understand the mechanism how extracellular Tau oligomers affect the synapse. However, how the structure of excitatory synapses is affected by extrasynaptic Tau oligomer still remains unclear. In addition, it is possible that extracellular Tau may also influence the behavior of GABA_ARs, but the effect of Tau on GABAergic synapses has not been examined. 　Based on the above, in this study, we aimed to clarify the effects of extracellular Tau on receptor clusters at AMPAergic synapses and GABA_Aergic synapses. In this study, we found that the presence of extracellular Tau altered the size of AMPAR and GABA_AR clusters at the postsynaptic terminal. Primary neurons from the rat hippocampus were exposed to extracelluar Tau (53 nM or 0.36 nM) for 1 hour or 1 day, and the cluster size of AMPARs and GABA_ARs were quantified by Immunocytochemistry (ICC). Incubation with 53 nM extracellular Tau for 1 hour or 1 day significantly increased the size of GABA_AR clusters, while the same treatment did not affect the cluster size of GluA1. In addition, when 0.36 nM Tau was extracellularly administered for 1 hour, the synaptic GluA1 cluster sizes remained unchanged, but the synaptic GluA2 cluster sizes increased. 　These results indicate that the presence of extracellular Tau alters the number of receptors that accumulate at AMPAergic and GABA_Aergic synapses, suggesting that extracellular tau administration may affect both excitatory and inhibitory neurotransmission.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-009 小脳プルキンエ細胞特異的なmGluR5の発現はmGluR1の運動協調およびシナプス刈り込みの機能を代替できる mGluR5 is substitutable for mGluR1 in cerebellar Purkinje cells for motor coordination and developmental synapse pruning ハーベスまりあ(1)、中尾晴美(1)、渡邉貴樹(2)、岸本泰司(3)、狩野方伸(2)、饗場篤(1) 1. 東京大学大学院医学系研究科疾患生命工学センター動物資源学部門、2. 東京大学大学院医学系研究科神経生理学分野、3. 帝京大学薬学部 Maria Harbers(1), Harumi Nakao(1), Takaki Watanabe(2), Yasushi Kishimoto(3), Masanobu Kano(2), Atsu Aiba(1) 1. Lab Animal Resources, CDBIM, Grad Sch Med, Univ of Tokyo, Tokyo, Japan, 2. Department of Neurophysiology, Grad Sch Med, Univ of Tokyo, Tokyo, Japan, 3. Faculty of Pharma-science, Teikyo University, Tokyo, Japan Keyword: mGluR5, Purkinje cells, cerebellum Two subtypes of group I metabotropic glutamate receptor, mGluR1 and mGluR5, activate phospholipase C-β via Gq proteins to induce phosphoinositide hydrolysis, while they exhibit nearly complementary distributions spatially or temporally in the brain. For example, mGluR1 is expressed in the thalamus, hippocampal dentate gyrus and cerebellum, while mGluR5 is expressed in the cerebral cortex, striatum and hippocampal CA1-CA3. In cerebellar Purkinje cells (PCs), mGluR5 expression is seen until postnatal day 12 (P12) and decreases afterward. In contrast, the mGluR1 protein level increases substantially from P12 to P16 and remains high thereafter throughout life. Many studies have shown that mGluR1 and mGluR5 are involved in synaptic plasticity and implicated in various diseases. For example, dysregulated expression of mGluR1 and mGluR5 is known in spinocerebellar ataxia type 1 (SCA 1) model mice, where reduced mGluR1 and increased mGluR5 expression in PCs are observed. However, significance of the complementary expression of mGluR1 and mGluR5, and relevance of their reciprocal expression during disease development remain unknown. To tackle this issue, we generated “mGluR5-rescue” mice in which mGluR5 is specifically expressed in PCs in global mGluR1-knock-out (KO) mice. First, we generated L7-mGluR5 transgenic (Tg) mice in which mGluR5 is expressed under the control of the PC-specific L7 promoter. Next, we obtained mGluR5-rescue mice by crossing mGluR1^+/- mice with L7-mGluR5 Tg mice. The immunohistochemical analysis confirmed that mGluR5 expression was restricted to the cerebellar molecular layer in mGluR5-rescue mice. We found that mGluR5-rescue mice exhibited normal motor coordination and developmental elimination of redundant climbing fiber (CF)-PC synapses both of which were severely impaired in mGluR1-KO mice. In the immunoprecipitation study of mGluR5-rescue and L7-mGluR5 Tg mice using mGluR5-antibody, we found that mGluR5 interacted with Homer in PCs and that the amount of Homer in mGluR5 complexes depended on the presence of endogenous mGluR1. We also found that mGluR5 formed a complex with mGluR1 when mGluR1 and mGluR5 coexisted in vivo. To clarify whether mGluR5 and mGluR1 are functionally redundant in mGluR1-related functions other than motor coordination and CF synapse elimination, we plan to examine the delay eyeblink conditioning in mGluR5-rescue mice.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-010 培養アストロサイトにおける平衡型ヌクレオシド輸送体の発現と機能調節 Expression and functional regulation of equilibrative nucleoside transporters in cultured astrocytes 田中康一(1,2,3)、井澤琢人(1)、児玉優理(1)、鈴木航(1)、富田和男(2,1)、五十嵐健人(2,1)、北中順惠(3)、北中純一(1,3)、佐藤友昭(2)、西山信好(1) 1. 兵庫医科大学薬学部、2. 鹿児島大学大学院医歯学総合研究科、3. 兵庫医科大学医学部 Koh-ichi Tanaka(1,2,3), Takuto Izawa(1), Yuri Kodama(1), Wataru Suzuki(1), Kazuo Tomita(2,1), Kento Igarashi(2,1), Nobue Kitanaka(3), Junichi Kitanaka(1,3), Tomoaki Sato(2), NobuyoshiNishiyama Nishiyama(1) 1. Dept Pharmacy, Hyogo Col Med, Hyogo, Japan, 2. Grad Sch Med & Dent Sci, Kagoshima Univ, Kagoshima, Japan, 3. Dept. Med, Hyogo Col Med, Hyogo, Japan Keyword: ASTROCYTE, HYDROGE PEROXIDE, NUCLEOSIDE TRANSPORTER, THYMIDINE INCORPORATION We have found that cultured differentiated astrocytes pretreated with N⁶, 2^'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP), a permeable analogue of cAMP, incorporate thymidine, but not uridine, via nucleoside transporters including ENTs into TCA insoluble fraction for repair on DNA injury in the presence of hydrogen peroxide (H₂O₂) at an early time, and these phenomena are specific in differentiated astrocytes, but not undifferentiated astrocytes and neurons. We studied expression of equilibrative nucleoside transporters in cultured astrocytes by RT-PCR, western blot analysis and immunocytochemistry. We could confirm ENT1, that is hypersensitive nucleoside transporter, and ENT2, that is low-sensitive nucleoside transporter in cultured astrocytes by RT-PCR and western blot analysis. This time, astrocyte is stained by anti-GFAP antibody and anti-ENT3 antibody, followed by treated by Texas Red and FITC-conjugated secondary antibody. We could confirm ENT3, that is assumed to be presented in lysosome, in cultured astrocytes co-stained by GFAP. H₂O₂-induced thymidine incorporation into cultured astrocytes decreased by S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), dilazep and dipyridamole, equilibrative nucleoside transporter inhibitors, at nanomolar concentrations but not micromolar concentrations, so thymidine was incorporated via ENT2, but not ENT1.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-011 G蛋白質共役型受容体の１次繊毛への局在を制御する機構の解析 Analysis of the mechanism regulating localization of G protein-coupled receptors to primary cilia 三好耕(1,2)、銀生卓瑪(1)、秦圓圓(1)、吉村武(1)、片山泰一(1) 1. 大阪大学大学院　連合小児発達学研究科　分子生物遺伝学、2. 大阪大学大学院　連合小児発達学研究科　子どものこころの分子統御機構研究センター KO MIYOSHI(1,2), YINSHENGZHUOMA WANG(1), YUANYUAN QIN(1), TAKESHI YOSHIMURA(1), TAIICHI KATAYAMA(1) 1. Dept of Child Develop and Molecular Bra Sci, United Grad Sch of Child Develop, Osaka Univ, Suita, Japan, 2. Molecular Res Center for Child Mental Develop, United Grad Sch of Child Develop, Osaka Univ, Suita, Japan Keyword: PRIMARY CILIA, GPCR Almost all vertebrate cells have an immotile primary cilium that singly extends like an antenna into the environment surrounding the cell and transduces sensory stimuli to the cell body. In the rodent brain each neuron has a solitary primary cilium for nearly all regions. The neuronal primary cilium is thought to be a non-synaptic neurotransmission device, while its exact nature is poorly understood. In humans, functional disruption of primary cilia results in a spectrum of pleiotropic disorders, referred to as ciliopathies, such as Bardet-Biedl syndrome and Joubert syndrome. Ciliopathies share some clinical features including developmental abnormalities in the nervous system. Recent studies have suggested that hedgehog signaling mediated by the primary cilia of progenitor cells regulates the development of the hippocampal dentate gyrus, cerebral cortex and cerebellum. Adenylyl cyclase 3 and several G protein-coupled receptors (GPCRs), including serotonin receptor type 6, somatostatin receptor type 3 and melanin-concentrating hormone receptor type 1, have been found to be localized in primary cilia of neuronal cells in rodents, and it might then be possible that a G protein/cAMP signaling cascade in neuronal primary cilia transduces the extracellular stimuli to the neuronal cell body. In this study, we analyzed ciliary localization of several GPCRs as well as their variants in cultured hTERT-RPE1 cells to determine the possible domain that targets GPCRs to the ciliary compartment. We also examined ciliary localization of several GPCRs in hTERT-RPE1 cells carrying biallelic genomic mutations that disrupt genes known to be responsible for Bardet-Biedl syndrome. The present study provides some insight into the possible mechanism by which several GPCRs can be selectively localized to primary cilia and the association of the ciliary localization of GPCRs with ciliopathies.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-012 マウス脳におけるNMDA受容体サブユニット蛋白質の動態解析 Dynamics of NMDA receptor subunit proteins in mouse brain 﨑村建司(1)、鈴木康浩(1)、中本千尋(1)、渡辺雅彦(2)、笹岡俊邦(1)、阿部学(1) 1. 新潟大学脳研究所、2. 北海道大学大学院医学研究科 Kenji Sakimura(1), Yasuhiro Suzuki(1), Chihiro Nakamoto(1), Masahiko Watanabe(2), Toshikuni Sasaoka(1), Manabu Abe(1) 1. Brain Res Inst, Niigata Univ, Niigata, Japan, 2. Hokkaido Univ Grad Sch Med, Sapporo, Japan Keyword: NMDA RECEPTOR, QUANTIFICATION, WESTERN BLOT, DEVELOPMENT NMDA-type glutamate receptors (NMDARs) are a group of molecules involved in brain functions such as synaptogenesis and synaptic plasticity in the central nervous system. NMDARs are mainly composed of two molecules of a GluN1 subunit, which is encoded by a single gene and diversified by splicing, and two molecules from four kinds of GluN2 subunits, leading to various combinations of subunits and channel specificities. However, there is no comprehensive quantitative analysis of GluN1 and GluN2 subunit proteins for relative comparison, and their compositional ratios at various regions and developmental stages have not been clarified. Thus we prepared six chimeric subunit molecules, by fusing an N-terminal side of the GluA1 subunit with C-termini of two types of GluN1 and four types of GluN2 in order to measure their antibody titers using the titer of anti-GluA1 antibody as a standard, and quantified protein levels of each subunit by Western blotting. We determined protein amounts of NMDAR component subunits in crude, P2, microsomal and synaptosome enriched fractions each from cortex, hippocampus and cerebellum. We also examined amount changes in the whole brain during developmental stages. Their amounts, when the GluA1 amount in the cortical crude fraction was set as a standard, were almost close to those of mRNA expression, except for some subunits. Interestingly, GluN2D protein was expressed even in the adult stage, although it had been thought to be high only in the developmental stage. In the crude fractions, GluN1 subunit was larger than GluN2 in quantity, but GluN2 subunit increased in the synaptosome enriched fraction, where functional components were enriched, except in cerebellum. These data will help better understand the basic function of NMDARs.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-013 培養ヒトiPS由来神経系細胞において，グルタミン酸トランスポーターEAAT1 とEAAT2は，細胞外グルタミン酸を取り込み，NMDA受容体を介した細胞毒性保護を行っている. Human induced pluripotent stem cell-derived neural cells were protected from excitotoxicity induced by high concentration of L-glutamate through the functions of EAAT1 and 2 高橋華奈子(1)、中條かおり(1)、鈴木郁郎(2)、佐藤薫(1) 1. 国立医薬品食品衛生研究所　薬理部、2. 東北工業大学大学院工学研究科　電子工学 kanako TAKAHASHI(1), Kaori Chujyo(1), Ikuro Suzuki(2), Kaoru Sato(1) 1. Lab. Neuropharmacol., Div. Pharm., Nat. Inst. Hlth. Sci., Kanagawa, Japan, 2. Dept. Electronics, Grad. Sch. Engineering, Tohoku Inst. Tech., Miyagi, Japan Keyword: excitatory amino acids transporter EAAT1 and 2, human induced pluripotent stem cell-derived neural cells, excitotoxicity, safety pharmacology In safety pharmacology, in vitro use of neurons differentiated from human induced pluripotent stem cell (hiPSC-neurons) is expected to improve the prediction accuracy in humans. There is a nervous system-specific toxicity mechanism known as excitotoxicity, which is caused by sustained activation of NMDAR due to an excessive increase in the extracellular concentration of L-glutamate ([L-Glu]_out). In general, it is difficult to reproduce excitotoxicity in hiPSC-neurons by the treatment protocol similar to that of cultured rodent neurons. In the central nervous system (CNS), excitatory amino acids transporters (EAATs), especially astrocytic types (EAAT1 and 2), maintain [L-Glu]_out at a low level. In this study, we examined whether astrocytes were functionally differentiated and whether EAAT1/2 were related to the neuronal sensitivity to [L-Glu]_out in commercially available hiPSC-neurons. hiPSC neurons (XCell Co.) were seeded at 3.0×10⁵ cells/cm² and cultured for 2 months. The expression of astrocyte markers, GFAP and S100b, were detected at 14 DIV and the expression levels greatly increased as the culture period got longer. The expression of EAAT1 and 2 were detected at 14 DIV and these protein expression level greatly increased as the culture period got longer. At 63 DIV, EAAT1 was localized in Nestin+GFAP+ radial glias and GFAP+S100b+ astrocytes, while EAAT2 was localized in HuC/D+MAP2+ neurons and GFAP+S100b+ astrocytes. When we exogenously applied 100 µM L-Glu at 14 DIV and 63 DIV, [L-Glu]_out was decreased to almost zero after 180 min and 60 min, respectively. These decrease in [L-Glu]_out were significantly inhibited by EAAT1 selective blocker UCPH-101 or EAAT2 selective blocker DHK. 100 µM L-Glu alone had little effects on cell viability, however, co-application of L-Glu and UCPH-101 or DHK caused significant decreases in cell viability. We confirmed that NMDAR antagonist AP5 rescued these cell damages. These results suggest that EAAT1 and 2 uptake the excessive L-Glu sufficiently, thereby protecting hiPSC-neurons from excitotoxicity.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-014 末梢神経損傷はミクログリアに依存したGABA作動性の持続性抑制の働きを介して視床皮質投射ニューロンのバースト発火を増強する Peripheral nerve injury facilitates burst firing of thalamocortical neurons via GABAergic tonic inhibition dependent on microglial activity 植田禎史(1)、宮田麻理子(1) 1. 東京女子医科大学 Yoshifumi Ueta(1), Mariko Miyata(1) 1. Tokyo Women's Med Univ Keyword: Peripheral nerve injury, Thalamus, GABAergic tonic inhibition, microglia Thalamic functions crucially involve in various chronic pain conditions. Consistent with this, we have previously shown in mice that peripheral nerve injury-induced ectopic pain associates with structural and functional remodeling of neural circuits in the somatosensory thalamus (Takeuchi et al., 2017), either dependent on enhanced tonic inhibition via extrasynaptic GABAA receptors in thalamocortical neurons or activation of microglia, known as brain-resident immune cells (Nagumo et al., 2020; Ueta and Miyata, 2021). Thalamocortical neurons often show enhanced burst activity under chronic pain. Our in vivo studies report enhanced burst ratio of spontaneous activity of thalamocortical neurons due to reduced tonic firing in unanesthetized mice with whisker deafferentation (Nagumo et al., 2020). However, mechanisms underlying deafferentation-induced alteration of thalamocortical neuronal firing still remain to be characterized. Tonic inhibition can switch thalamocortical neuronal firing from tonic to burst (Nagumo et al., 2020). Thus, tonic inhibition might facilitate burst activity of thalamocortical neurons after whisker deafferentation. To test this, we performed whole-cell patch-clamp recording from thalamocortical neurons after deafferentation using acute slices. Deafferentation shortens interspike intervals and reduces the slope of frequency-current relationships. These changes are occluded by agonist enhancement of tonic inhibition. Additionally, genetic removal of alpha 4 subunits of extrasynaptic GABAA receptors prevents deafferentation-induced changes in firing properties, suggesting the necessity of tonic inhibition for facilitating burst activity after deafferentation. To further examine the mechanism involving in deafferentation-induced enhancement of tonic inhibition, we focus on the role of deafferentation-induced microglial activation. We found that deafferentation-induced enhancement of tonic inhibition is abolished by brain-wide depletion of microglia using oral administration of PLX3397, which inhibits survival and proliferation of microglia. Furthermore, microglial depletion prevents deafferentation-induced facilitation of burst activity of thalamocortical neurons. These findings suggest that increased GABAergic tone by tonic inhibition dependent on microglial activity might be crucial for the development of ectopic pain after peripheral nerve injury by altering thalamocortical information processing through changing neuronal firing pattern together with remodeling thalamic circuits.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-015 生後発達および加齢における線条体コリン作動性介在ニューロンの活動電位特性の変化 Changes in action potential properties of striatal cholinergic interneurons during development and aging. 鈴木江津子(1)、籾山俊彦(1) 1. 東京慈恵会医科大学　薬理学講座 Etsuko Suzuki(1), Toshihiko Momiyama(1) 1. Jikei University, Sch. of Med. Dept. Pharmacology Keyword: striatum, cholinergic interneuron, action potential, ageing Striatal cholinergic interneurons are known to fire spontaneously both in vivo and in vitro. It has been reported that frequency of action potential firing increases during postnatal development. On the other hand, changes in spontaneous frequency and firing properties during aging have not been investigated. In this study, a whole-cell patch-clamp study was carried out to investigate changes in firing properties of striatal cholinergic neurons during development and aging. Brain slices were prepared from 2–3-week-old, 5–6-week-old, 2–3-month-old, 6–7-month-old, 11–12-month-old and 16–18-month-old mice of either sex. The present results have shown that frequency of spontaneous firing was low at 2-3 weeks of age (0.28 ± 0.04 Hz, n = 4) and increased significantly during postnatal development (11–12-month-old: 7.68 ± 1.64 Hz, n = 8, p = 0.035, 16-18-month-old: 8.06 ± 1.91 Hz, n = 9, p = 0.019). The threshold of action potential was significantly increased at 16–18 months of age (-34.8 ± 2.13 mV, n = 5, p = 0.022 compared with 6-7-month-old: -42.9 ± 1.56 mV, n = 8). The rise time of action potential at 16–18 months of age (0.39 ± 0.05 ms) was significantly longer than that of at 2–3-month-old of age (0.26 ± 0.02 ms, p = 0.034). On the other hand, there were no differences in the amplitude of afterhyperpolarization or the sag ratio during postnatal development and aging. These findings suggest prominent changes in firing properties of striatal cholinergic interneurons during development and aging.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-016 Examining the in vivo neural network activity of a Scn2a loss-of-function mouse model Melody Li(1), Bernd Kuhn(1) 1. Okinawa Institute of Science and Technology Keyword:* In vivo imaging, Scn2a Scn2a encodes the alpha subunit of the voltage gated sodium channel Nav1.2 in the brain and plays a critical role in action potential initiation and propagation. Scn2a loss-of-function (LoF) is implicated in a range of neurological disorders such as epilepsy, autism, and intellectual disability. While the consequences of Scn2a LoF on membrane excitability was investigated at single neuron level, effect on in vivo network activity has yet to be elucidated. Here, we will generate a Scn2a LoF mouse model using antisense oligonucleotide (ASO) technology which will achieve over 80 % reduction in Scn2a mRNA expression. To examine in vivo network activity, calcium imaging will be performed with two-photon microscopy in awake mice. The spontaneous activities of layer 5 pyramidal neurons in the somatosensory cortex (S1) and their dendritic activity in layer 1 will be recorded. Electrocorticography (ECoG) will be monitored simultaneously to determine if there are ECoG abnormalities in this Scn2a LoF mouse model, as well as to examine the potential correlation between calcium activity and brain oscillations. Outcomes from this study will be the first to report in vivo neuronal activities of Scn2a LoF mice. This will contribute to the understanding of how Scn2a LoF drives neurological disorders.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-017 細胞体に存在するKv2チャネルは背側蝸牛神経核カートホイール細胞の高頻度発火に重要である Essential Role of Somatic Kv2 Channels in High-Frequency Firing in Cartwheel Cells of the Dorsal Cochlear Nucleus 入江智彦(1) 1. 国立医薬品食品衛生研究所 Tomohiko Irie(1) 1. National Institute of Health Sciences Keyword: Kv2.1, Kv2.2, neuronal excitability, auditory Among all voltage-gated potassium (Kv) channels, Kv2 channels are the most widely expressed in the mammalian brain. However, studying Kv2 in neurons has been challenging because of a lack of high-selective blockers. Recently, a peptide toxin, guangxitoxin-1E (GxTX), has been identified as a specific inhibitor of Kv2, thus facilitating the study of Kv2 in neurons. The mammalian dorsal cochlear nucleus integrates auditory and somatosensory information. In the dorsal cochlear nucleus, cartwheel inhibitory interneurons receive excitatory synaptic inputs from parallel fibers conveying somatosensory information. The activation of parallel fibers drives action potentials in the cartwheel cells up to 130 Hz in vivo, and the excitation of cartwheel cells leads to the strong inhibition of principal cells. Therefore, cartwheel cells play crucial roles in monaural sound localization and cancelling detection of self-generated sounds. However, how Kv2 controls the high-frequency firing in cartwheel cells is unknown. In this study, we performed immunofluorescence labeling with anti-Kv2.1 and anti-Kv2.2 antibodies using fixed mouse brainstem slice preparations. The results revealed that Kv2.1 and Kv2.2 were largely present on the cartwheel cell body membrane but not on the axon initial segment (AIS) nor the proximal dendrite. Whole-cell patch-clamp recordings using mouse brainstem slice preparation and GxTX demonstrated that blockade of Kv2 induced failure of parallel fiber-induced action potentials when parallel fibers were stimulated at high frequencies (30–100 Hz). Thus, somatic Kv2 in cartwheel cells regulates the action potentials in a frequency-dependent manner and may play important roles in the dorsal cochlear nucleus function.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-018 TRPV2の酸化修飾は化学応答・熱応答を感作するが、機械刺激応答には影響しない Oxidation sensitizes TRPV2 to chemical and heat stimuli, but not mechanical stimulation 柴崎貢志(1)、花村衣咲(1) 1. 長崎県立大学大学院　人間健康科学研究科　細胞生化学講座 Koji Shibasaki(1), Isaki Hanamura(1) 1. Laboratory of Neurochemistry, Graduate School of Human Health Science, University of Nagasaki Keyword: TRPV2, oxidization, heat, mechanical stimulation The transient receptor potential vanilloid 2 (TRPV2) ion channel is activated by a chemical ligand (2-aminoethoxydiphenyl borate; 2-APB), noxious heat and mechanical stimulation. In a heterologous mammalian cell expression system, the oxidant chloramine T sensitizes TRPV2 activation in response to 2-APB and heat by oxidation of methionine residues at positions 528 and 607 in rat TRPV2. Here, we used a Xenopus oocyte expression system to determine whether chloramine T -mediated oxidation can also sensitize TRPV2 to mechanical stimulation. In this system, we confirmed that chloramine T sensitized TRPV2 activation in response to 2-APB and heat, but we detected no sensitization to mechanical stimulation. This result suggests that the activation mechanism of TRPV2 by a chemical ligand and heat differs from that for mechanical stimulation.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-019 Nanoscale computational modeling explains why evolutionary shift happened Martyushev Alexey(1)、De Schutter Erik(1) Alexey Martyushev(1), Erik De Schutter(1) 1. Okinawa Institute of Science and Technology Graduate University (OIST) Keyword: calcium-activated ion channels, evolutionary shift, Purkinje cells, nanoscale computational modeling Darwin’s theory of evolution explains how physiological changes of organisms through generations occur to obtain competitive advantages providing better survival and offspring creation – natural selection [1]. Furthermore, on a large timescale of thousands and millions of years, the changes accumulated by organisms can be so major that new species appear. In accordance with the theory, the contemporary version of the phylogenetic tree of life [2] consists of such species while other, non-competitive ones, are known as fossil, including the precursors of Homo sapiens. The evolutionary changes may occur not only at the macro-physiological level, but also at the micro-level including changes in cell morphology, homeostasis, and plasma membrane properties. This research is devoted to the changes of cell membrane proteins, in particular, the calcium-activated ion channels. This class of the channels was previously found on various cells, including the neurons of organisms of different complexities. However, the properties of the channels changed dramatically once the complexity of species increased. The presence of the channels on the neurons of very different species highlights their vital role for neuron functioning, but the observed changes of their properties remained unexplained. Potentially, the micro-level changes may be directly associated with macro-level features such as information processing by the nervous system. Application of nanoscale modeling of stochastic ion diffusion and channel activation in 3D Purkinje cell morphology [3] provided evidence on the differences in the spiking properties of neuron models caused by different evolutionary versions of the channel. The computational experiments demonstrated that the competition for intracellular ions between the channels may cause the evolutionary shift. Furthermore, as shown, in mammals, including Homo sapiens, the shift achieved its apogee providing the most precise timing of spikes. References: 1. On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life. Br Foreign Med Chir Rev, 1860. 25(50): p.367-404. 2. Hinchliff, C.E., et al., Synthesis of phylogeny and taxonomy into a comprehensive tree of life. Proc Natl Acad Sci U S A, 2015. 112(41): p.12764-9. 3. Hepburn, I., et al., STEPS: efficient simulation of stochastic reaction-diffusion models in realistic morphologies. BMC Syst Biol, 2012. 6: p.36.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-020 マウス小脳神経細胞膜におけるホスホイノシチド分布の定量的局在解析 Quantitative analysis of nanoscale phosphoinositide distribution on neuronal cell membranes of mouse cerebellum 江口工学(1)、重本隆一(1) Kohgaku Eguchi(1), Ryuichi Shigemoto(1) 1. IST Austria, Klosterneuburg, Austria Keyword: Phosphoinositide, SDS-FRL, Electron microscope, Synapse Phosphoinositides (PIs) are minor phospholipid components on the cytoplasmic leaflet of eukaryotic cell membranes, but they play essential roles in many aspects of cellular functions, including synaptic transmission in neurons. Although it is important to know how PIs distribute on neuronal cell membranes to understand their roles in synaptic transmission, it is not yet examined at the electron microscopic level. This study aimed to observe nano-scale distribution of PIs on neuronal cell membranes and address their potential roles in neuronal activities including synaptic transmission. To investigate the nanoscale distribution of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) on neuronal cell membranes in brain tissues, we established a labeling method using the SDS-digested freeze-fracture replica labeling (SDS-FRL) with a recombinant GST-tagged pleckstrin homology (PH) domain of phospholipase Cδ1 (PLCδ1) as a specific probe of PI(4,5)P₂. Using replicas of mouse cerebellar tissues, we found that gold particles for PI(4,5)P₂ make clusters broadly distributed on the cytoplasmic side of plasma membranes of Purkinje cells (PCs) and parallel fiber (PF) boutons. At PF-PC synapses, PI(4,5)P₂ was concentrated at active zones (AZs) and co-localized with P/Q-type Ca²⁺ channels (Ca_V2.1) in presynaptic boutons, indicating that PI(4,5)P₂ may contribute to the regulation of synaptic vesicle release. At the postsynaptic sides, PI(4,5)P₂ was co-localized with a metabotropic glutamate receptor mGluR1α and a G protein-gated inward-rectifying potassium channel subunit GIRK3 outside of the postsynaptic density area. The co-localization of PI(4,5)P₂ with mGluR1α may be necessary for effective generation of inositol triphosphate (IP₃) and diacylglycerol (DAG) upon the receptor activation by glutamate and subsequent activation of phospholipase C. The synaptic plasticity induced by the mGluR1α activation may thus be consequently affected by the co-localization with PI(4,5)P₂. The PI(4,5)P₂ distribution close to GIRK3 also suggests its potential role in the regulation of PC excitability since it has been reported that PIP₂ induces conformational changes of GIRK and activates the channels. Manipulation of the co-localization between PI(4,5)P₂ and those key molecules would be useful to elucidate the potential implications of our present findings.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-021 電位依存性ナトリウムチャネルNav1.1と線維芽細胞増殖因子相同因子の相互作用 Insights into the interaction of voltage-gated sodium channel Nav1.1 with fibroblast growth factor homologous factors 荻原郁夫(1)、尹成珠(1)、下畑充志(1)、雁木美衣(1)、金田誠(1) 1. 日本医科大学 Ikuo Ogiwara(1), Chengzhu Yin(1), Atsushi Shimohata(1), Mie Gangi(1), Matoko Kaneda(1) 1. Nippon Medical School Keyword: voltage gated sodium channel, fibroblast growth factor homologous factor Voltage-gated sodium channel alpha subunit type I, Nav1.1, encoded by the SCN1A gene is predominantly expressed in parvalbumin-expressing cells in brain, and plays critical roles in maintaining sustained fast-spiking from these inhibitory neurons. Loss of Nav1.1 function in GABAergic inhibitory neurons is a main cause of Dravet syndrome characterized by early onset of intractable epilepsy with developmental delay. Fibroblast growth factor homologous factors (FHF1-4; also known as FGF11-14) are intracellular, non-secretory forms of FGF. A recurrent mutation of FHF1 is also associated with early onset of intractable epilepsy with developmental delay. It has been reported that the intracellular C-terminal domain of voltage-gated sodium channels contains the domain for binding with the FHFs, and that the FHFs modulate the electrophysiological properties of the sodium channels. However, it has been also reported that Nav1.1 does not bind to FHFs despite its containing the putative FHF-binding domain in the C-terminal domain. Given our recent proteomic data of mouse brain with anti-Nav1.1 antibodies showing a possible interaction between Nav1.1 and FHFs, we here decided to examine whether Nav1.1 interacts to FHFs. We first showed that Nav1.1 is co-precipitated with FHF in mouse brain lysate, and vice versa. We next showed that FHF does not bind to the C-terminal domain of Nav1.1, as reported previously. We however found that FHF interacts with Nav1.1 through the intracellular loop between the transmembrane domains of the channel. We are currently studying whether FHF1, including wild-type construct and a disease-associated mutant, affect the electrophysiological properties of Nav1.1. Our studies will contribute to the understanding of Nav1.1 and FHF1 in the pathophysiology of early-onset intractable epilepsy with developmental delay, and also provide insights into such neurological disorders.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-022 電位依存性ナトリウムチャネルNav1.2結合タンパク質の解析 Analysis of interacting proteins of the voltage-gated sodium channel Nav1.2 宮﨑晴子(1,2)、貫名信行(2) 1. 岡山大学、2. 同志社大学 Haruko Miyazaki(1,2), Nobuyuki Nukina(2) 1. Okayama University, 2. Doshisha University Keyword: Sodium channel, Unmyelinated fiber, Nav1.2, LC-MS/MS Voltage-gated sodium channels play essential roles in the generation and propagation of action potentials in neurons. These channels consist of a pore-forming alpha subunit and one or more auxiliary beta subunits. Nav1.2 is an alpha subunit widely expressed in the central nervous system (CNS). It has been known that Nav1.2 is preferentially expressed in unmyelinated axons of the CNS, such as parallel fibers of cerebellum, mossy fibers of hippocampus and striatonigral fibers. Nav1.2 is diffusely distributed along these axons. This singular Nav1.2 distribution in the unmyelinated axons gives rise to a speculation that Nav1.2 could have another role in the unmyelinated axons. To investigate about this, we examined Nav1.2 interacting proteins in the myelinated axons and unmyelinated axons. First, we generated polyclonal anti-Nav1.2 antibody, which can be used for immunoprecipitation (IP). Next, we fractionated striatonigral fiber enriched mouse brain homogenate to myelin basic protein (MBP) (+) and MBP (-) membrane fraction, containing myelinated and unmyelinated axon properties, respectively. To separate these two fractions, we performed sucrose gradient centrifugation (0.32M/0.85M) using membrane fraction of striatonigral fiber enriched mouse brain homogenate. To identify Nav1.2 interacting proteins, we performed IP of MBP (+) and MBP (-) membrane fractions using anti-Nav1.2 antibody and then IP-samples were analyzed by LC-MS/MS. Comparison of Nav1.2 interacting proteins between MBP (+) and MBP (-) membrane fractions showed many highly enriched proteins in each fraction.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-024 高頻度で持続的なシナプス伝達を支えるシナプス小胞ナノスケール動態 Actin filaments restrict synaptic vesicle movement for rapid tethering of vesicles at a fast central synapse 三木崇史(1)、豊福理央(2)、坂場武史(1) 1. 同志社大学大学院脳科学研究科、2. 同志社大学生命医科学部 Takafumi Miki(1), Rio Toyofuku(2), Takeshi Sakaba(1) 1. Grad Sch Brain Science, Doshisha University, Kyotanabe, Japan, 2. Faculty of Life and Medical Science, Doshisha Universitu Keyword: Synapse, synaptic vesicle, cerebellum A high rate of synaptic vesicle release is required at cerebellar mossy fiber (cMF) terminals for rapid information processing. As the number of release sites is limited, fast vesicle reloading is necessary for achieving sustained release. Our recent study revealed that rapid tethering of vesicles upon stimulation occurred with a similar time course to exocytosis (Miki et al., 2020). However, the mechanism underlying the rapid reloading remains unknown. To study this, we observed synaptic vesicle movement in dissociated cMF terminals using total internal reflection and oblique illumination fluorescence microscopy, and simultaneously performed direct electrophysiological recording from the terminals. As a result, we found that actin disruption by latrunculin A abolished the rapid tethering, and decreased the sustained release. By contrast, induction of actin polymerization and stabilization did not affect the time courses of the vesicle tethering and release, suggesting that the existence of actin filaments was required for the rapid tethering. To investigate the role of the actin filaments in synaptic vesicle dynamics, we performed single-particle tracking experiments of quantum dot-labeled vesicles in the presynaptic terminals. Analysis of synaptic vesicle trajectories indicated that actin disruption made vesicles diffuse faster. Inferring vesicle diffusion states from the trajectories by Hidden Markov modeling revealed that synaptic vesicles under normal conditions had two diffusion states: free-diffusing and trapped. After actin disruption, vesicles tended to have only the free-diffusion state. Based on these results, we suggest that actin filaments limit synaptic vesicle movement to achieve the rapid tethering which is essential for the sustained activity at the cMF terminals.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-025 ゼブラフィッシュの神経筋シナプスにおいて自発的にリサイクリングされるシナプス小胞は即時放出可能な小胞を構成する Spontaneously recycling synaptic vesicles constitute readily releasable vesicles in zebrafish neuromuscular synapses 江頭良明(1)、熊手彩音(2)、王子田彰夫(2)、小野富三人(1) 1. 大阪医科薬科大学大学院医学研究科、2. 九州大学大学院薬学研究院 Yoshihiro Egashira(1), Ayane Kumade(2), Akio Ojida(2), Fumihito Ono(1) 1. Grad Sch Med, Osaka Med Pharm Univ, Takatsuki, Japan, 2. Grad Sch Pharm, Kyushu Univ, Fukuoka Japan Keyword: synaptic vesicles, spontaneous transmission, synaptic vesicle pools, zebrafish Emerging evidence shows that spontaneous synaptic transmission plays crucial roles on neuronal functions through presynaptic molecular mechanisms distinct from that of action potential (AP)-evoked transmission. However, whether the synaptic vesicle (SV) population undergoing the two forms of transmission is segregated remains controversial due in part to the conflicting results observed in cultured neurons. Here we address this issue in intact neuromuscular synapses using transgenic zebrafish larvae expressing two different indicators targeted in the SVs: a pH-sensitive fluorescent protein, pHluorin, and a tag protein, HaloTag. By establishing a quantitative measure of recycled SV fractions, we found that approximately 85% of SVs were mobilized by high-frequency AP firing. In contrast, spontaneously recycling SVs were mobilized only from <8% of SVs with a time constant of 45 min at 25°C, although prolonged AP inhibition mobilized an additional population with a delayed onset. The mobilization of the early-onset population was less temperature-sensitive and resistant to tetanus toxin (TeNT), whereas that of the late-onset population was more sensitive to temperature and was inhibited by TeNT, indicating that prolonged AP inhibition activated a distinct molecular machinery for spontaneous SV fusion. Therefore, the early-onset population limited to <8% was likely the only source of spontaneous release that occurred physiologically. We further showed that this limited population was independent of those reluctant to fuse during AP firing and was employed in both the hypertonic stimulation and the immediate phase of AP-evoked releases, thereby matching the characteristics of the readily releasable pool (RRP). Our study revealed that the origin of spontaneous SV fusion is restricted to the RRP among the SV pools involved in AP-evoked fusion.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-026 大脳皮質錐体ニューロン解析のための生体内二光子顕微鏡と膨張顕微鏡を用いた相関顕微鏡手法の開発 Development of the correlative technique for analyzing cortical pyramidal neurons with in vivo two-photon and expansion microscopy 柏木有太郎(1)、遠藤雅瑛(1)、寺嶋宙(1)、岡部繁男(1) 1. 東京大学大学院医学系研究科 Yutaro Kashiwagi(1), Masaaki Endo(1), Hiroshi Terashima(1), Shige Okabe(1) 1. Grad Sch Med, Univ of Tokyo, Tokyo, Japan Keyword: Synapse, In vivo imaging, Superresolution imaging Visualizing the morphology of synapses and neuronal connectivity is crucial to understanding the neural circuit properties in the brain. Most excitatory presynaptic boutons are attached to dendritic spines in neocortical and hippocampal pyramidal neurons. Spines undergo continual formation and elimination, together with their dynamic structural changes in response to neural circuit activity. Previous correlative methods of combining in vivo two-photon spine imaging with electron microscopy (EM) have provided information about the relationship between in vivo spine dynamics and synapse ultrastructure. However, EM-based post hoc analysis of dendritic spines after in vivo two-photon imaging is technically demanding and time-consuming. Therefore, few comprehensive studies of the spine population using EM-based correlative analysis have been carried out. 　We developed a correlative imaging pipeline using the expansion microscopy technique applied to neocortical samples after in vivo two-photon imaging. We first established conditions for reproducibly expanding tissue specimens while maintaining sufficient fluorescence brightness for high-resolution 3D imaging. For precise image correlation, we next established a protocol based on the pattern of blood vessels, which effectively navigates the identification of fluorescent objects in the sample previously imaged by in vivo two-photon microscopy. Correlative imaging of spines by both two-photon and expansion microscopy will be applicable to analyzing a large set of spines, with both their morphological features and molecular distributions. This technique will help understand how thousands of spine synapses formed onto a single pyramidal neuron are collectively regulated in morphology and efficacy.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-027 ドレブリン阻害剤BTP2を投与した初代培養ニューロンの解析 Analysis of primary cultured neurons treated with drebrin inhibitor BTP2 藤瀬なぎさ(1)、関野裕子(2)、白尾智明(3)、児島伸彦(4) 1. 東洋大学生命科学部生命科学科、2. 東京大学大学院薬学系研究科、3. アルメッド（株）、4. 東洋大学生命科学部生命科学科 Nagisa Fujise(1), Yuko Sekino(2), Tomoaki Shirao(3), Nobuhiko Kojima(4) 1. Grad Sch Life science, Univ of Toyo, Gunma, Japan, 2. Grad Sch Pharmaceutical Sciences, Univ of Tokyo, Tokyo, Japan, 3. AlzMed, Inc. Tokyo, Japan, 4. Grad Sch Life science, Univ of Toyo, Gunma, Japan Keyword: DREBRIN, PRIMARY NEURONAL CLUTURE, SYNAPSE, CELL IMAGE ANALYZER Synaptic plasticity is an important neuronal characteristic for learning and memory formation. The efficiency of synaptic transmission can be altered by increasing or decreasing amount of transmitters and the number of receptors at the postsynaptic site. However, much remains to be learned about the molecular mechanisms of synaptic plasticity. Synaptic plasticity involves dynamic changes in the actin cytoskeleton of dendritic spines, postsynaptic structures of excitatory synapses. One of actin-binding proteins drebrin is involved in synaptic plasticity and is associated with cognitive decline in patients of Alzheimer's disease as well as older adults. Activation of NMDA receptor induce drebrin exodus (Mizui et al, 2014), which may be a crucial step for LTP induction. Although drebrin knockout mice can be used to elucidate the role of drebrin, lack of drebrin from early development may lead to developmental abnormalities or compensation of the defects, thereby it may mask the physiological phenotypes in adult. We recently found that the intraventricular injection of BTP2, a compound known as an inhibitor of drebrin-F-actin-binding, impaired learning of novel object recognition test and object location test. To better understand the molecular basis of the learning deficit by a transient inhibition of drebrin, we examined the effect of BTP2 on spine morphology of cultured hippocampal neurons. Rat hippocampal neurons (SKY neurons, Alzmed, Inc.) were placed in a 96-well plate at the density of 10,000 cells/well. The cells were grown in B27-containing Neurobasal medium at 37℃, 5% CO₂ humidified incubator for 21 days in vitro. Twenty-four hours or 30 minutes before fixation various doses of BTP2 (0 to 30 µM) were administered to cultured neurons, then fixed and stained with anti-MAP2 and anti-drebrin antibodies and DAPI. Fluorescent images were acquired by a cell image analyzer, and analyzed the number of cells, the length of dendrites and the number of drebrin-immunopositive clusters. We found an exposure to higher doses (>3 µM) of BTP2 for 24 hours, but not for 30 minutes, resulted in a significant decrease in the numbers of cells and dendrite length, as well as drebrin clusters in dendrites, as compared to non-treated controls. These results indicate a prolonged exposure of BTP2 exhibits cytotoxic effect. On the other hand, lower doses (<3 µM) of BTP2 showed no apparent effects on these measures. We now examined the effects of BTP2 on localization of drebrin in dendritic spines as well as morphological changes of dendritic spines after chemical LTP and LTD stimulations. We think the study is important for better understanding the function of drebrin during synaptic changes at the cellular level, and useful for improving cognitive function and developing diagnostic agents for Alzheimer's disease.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-029 バクロフェンはラット体性感覚野におけるカフェイン依存性神経オシレーションを弱化する Baclofen attenuates activities of caffeine-induced neural oscillations in the somatosensory cortex of rat 金山宏幸(1,2)、福田嵩子(1)、冨永貴志(3)、冨永洋子(3)、加藤伸郎(4)、吉村弘(1) 1. 徳島大学大学院・医歯薬学研究部・口腔分子生理学分野、2. 国立病院機構大阪医療センター・口腔外科、3. 徳島文理大学・神経科学研究所、4. 金沢医科大学・医学部・生理学 Hiroyuki Kanayama(1,2), Takako Fukuda(1), Takashi Tominaga(3), Yoko Tominaga(3), Nobuo Kato(4), Hiroshi Yoshimura(1) 1. Department of Molecular Oral Physiology, Institute of Biomedical Sciences, Tokushima University Graduate School, 2. National Hospital Organization Osaka National Hospital, Oral and Maxillofacial Surgery , 3. Institute of Neuroscience, Tokushima Bunri University, 4. Department of Physiology, Kanazawa Medical University Keyword: NMDA receptor, GABA receptor, Baclofen, oscillation One of important properties of neocortical networks is generation of membrane potential oscillations. The neural oscillations based on synaptic activities may be an important strategy for information processing. Activities of GABA receptors modulate synaptic function in the central nervous systems. However, modulation of the oscillatory neural activities by activation of GABA_B receptors have not been well understood. We previously found that, in the presence of caffeine, electrical stimulation generates membrane potential oscillations at about 10 Hz in the somatosensory cortex slice of rat. In the present study, we used the same experimental design, and performed field potential recordings from the somatosensory cortex. After caffeine-induced oscillations were generated, application of baclofen, an agonist of GABA_B receptor, reversibly attenuated the oscillatory activities. Interestingly, later oscillatory phase disappeared, but not early phases. We previously reported that the early phase of the oscillation is non-NMDA receptor-dependent, whereas the late phase is NMDA receptor-dependent. Thus, baclofen depressed NMDA receptor-dependent phase, but not non-NMDA receptor-dependent phase. Recent studies revealed that GABA_B receptors express at both presynaptic and postsynaptic synapses in almost all regions of the brain, and when postsynaptic GABA_B receptor is activated, function of NMDA receptor is reduced via inhibition of adenylyl cyclase (AC). Together with these previous findings, the present results suggest that the site of active GABA_B receptor in the presence of caffeine may be post-synapse, and activation of postsynaptic GABA_B receptor downregulates NMDA receptor activity, which causes attenuation of oscillatory neural activities. Furthermore, we investigated spatio-temporal behavior of the caffeine-induced oscillation using optical recording method with voltage-sensitive dye. The somatosensory cortex was equipped with one localized oscillator at the upper layer, and the oscillator delivered excitatory signals repeatedly. In conclusion, activation of postsynaptic GABA_B receptor may affect local oscillator in the somatosensory cortex by way of reduction of NMDA receptor, resulting in attenuation of repeated signal deliveries.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-030 行動の柔軟性を制御する視床皮質シナプスの分子基盤：前頭前野におけるCbln1-GluD1複合体の機能解明 Function of GluD1-Cbln1 complex in thalamocortical synapse which implements flexible behavior 塩崎茉莉(1)、石川理子(1)、大塚信太朗 (1)、石田綾(1)、柚﨑通介(1) 1. 慶應義塾大学大学院医学部医学研究科 Mari Shiozaki(1), Ayako Wendy Ishikawa(1), Shintaro Otsuka(1), Aya Ito-Ishida(1), Michisuke Yuzaki(1) 1. Grad Sch Med, Univ of Keio, Tokyo, Japan Keyword: SYNAPSE, PREFRONTAL CORTEX, FLEXIBILITY, THALAMO-CORTICAL Neuropsychiatric disorders are often caused by the alteration in genes encoding synaptic proteins and are considered as “synaptopathy.” In humans, mutations of the gene encoding glutamate receptor delta1 (GluD1) are associated with schizophrenia, major depressive disorders, and autism spectrum disorders. Behavioral analyses of the GluD1-null mice also display a variety of phenotypes, such as decreased sociability, enhanced depressive-like behavior, and increased aggression. Consistently, GluD1 is abundantly expressed in the frontal cortex, however, how it functions remains unclear. Although previous findings from our laboratory demonstrated that GluD1 binds to Cbln family proteins in vitro to mediate synapse formation, whether and how the Cbln-GluD1 complex is necessary for the cortical function in vivo remained unknown. Here, we studied the functional significance of the Cbln1-GluD1 complex in the excitatory synapses of the medial prefrontal cortex (mPFC), which is crucial for higher cognitive functions. First, we conducted immunohistochemical analysis combined with viral tracing to clarify the identity of synapses where Cbln1-GluD1 exists. We found that the Cbln1-GluD1 complex was preferentially localized at excitatory synapses in layer 1 of mPFC, which was positive for the thalamic presynaptic marker, vesicular glutamate transporter2 (vGluT2). By conducting anterograde and retrograde tracing, we confirmed that Cbln1 in the mPFC was produced and secreted from the neurons in the anteromedial (AM) nucleus, one of the thalamic nuclei implied in epilepsy. Next, we examined the impact of deleting Cbln1-GluD1 in the thalamocortical synapses using the Cre-lox system. The localization of Cbln1 and GluD1 were mutually dependent and both Cbln1 and GluD1 were necessary for the maintenance of layer 1-vGluT2-expressing terminals in mPFC. Finally, to address whether Cbln1-GluD1 is necessary for the higher cognitive function, we used “Operant House,” a recently developed operant conditioning device that automatically examines a variety of behaviors in a home cage. Interestingly, selective deletion of GluD1 in mPFC pyramidal neurons led to impaired flexibility and elevated impulsivity. In contrast, mice whose Cbln1 was deleted in AM showed reduced impulsivity and no changes in flexibility. The discrepant behavioral phenotypes suggest that the Cbln1-GluD1 complex may be involved non only in AM-mPFC synapses, but also in other synapses, such as those made with the ventromedial (VM) thalamic nucleus. These findings indicate that the Cbln1-GluD1 complex mediates thalamocortical synapses which regulate behavioral flexibility. Because Cbln1 is a potent soluble synapse inducer that can modify the cerebellar function in vivo, our findings suggest that Cbln1 may be used as a therapeutic tool to modify higher cognitive dysfunctions caused by altered thalamocortical connectivity.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-031 Arcナノボディの開発と応用：Arcの細胞内動態・機能解析のための新規プローブ Development and validation of Arc nanobodies: new tools for probing Arc dynamics and function 石塚佑太(1,5)、Tadiwos Mergiya(1,2)、Rodolfo Baldinotti(1,2)、Ju Xu(1)、Erik Hallin(1)、Sigurbjörn Markússon(1)、Petri Kursula(1,3,4)、Clive Bramham(1,2) 1. Dept Biomed, Univ of Bergen, Bergen, Norway、2. Mohn Ctr for Res on the Brain, Univ of Bergen, Bergen, Norway、3. Fac Biochem & Mol Med, Univ of Oulu, Oulu, Finland、4. Biocenter Oulu, Univ of Oulu, Oulu, Finland、5. 川崎医科大学病態代謝学 Yuta Ishizuka(1,5), Tadiwos F. Mergiya(1,2), Rodolfo Baldinotti(1,2), Ju Xu(1), Erik I. Hallin(1), Sigurbjörn Markússon(1), Petri Kursula(1,3,4), Clive R. Bramham(1,2) 1. Dept Biomed, Univ of Bergen, Bergen, Norway, 2. Mohn Ctr for Res on the Brain, Univ of Bergen, Bergen, Norway, 3. Fac Biochem & Mol Med, Univ of Oulu, Oulu, Finland, 4. Biocenter Oulu, Univ of Oulu, Oulu, Finland, 5. Dept Pathophysiol & Metab, Kawasaki Med Sch, Kurashiki, Okayama Keyword: Arc, nanobody, intrabody, chromobody Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc and analysis of capsid domain specific functions.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-032 成熟後小脳においてC1ql1-Bai3シグナルは登上線維分枝のシナプス結合を制御する C1ql1-Bai3 signaling regulates synaptic connectivity of climbing fiber branches in adult cerebellum 会見昂大(1)、柚﨑通介(1) 1. 慶應義塾大学大学院医学研究科 Takahiro Aimi(1), Michisuke Yuzaki(1) 1. Keio University Graduate School of Medicine, Tokyo, Japan Keyword: synapse, cerebellum, climbing fiber, purkinje cell Synapses receive continuous modifications throughout life by neuronal activities, resulting in adaptation to new environments. However, the molecular mechanisms by which synapses are modified have remained largely unknown. Climbing fibers (CF) -Purkinje cell (PC) synapses in the cerebellum have served as a model synapse that undergoes activity-dependent modification. PCs are initially innervated by multiple CFs, but most CF synapses are eliminated during development, leaving a single winner CFs. Interestingly, CFs retain highly motile branches that run in the transverse direction without forming functional synapses even in adulthood. Whether and how such transverse branches make synapses under certain conditions are unknown. Here, we addressed this issue focusing on the role of C1q-like protein 1 (C1ql1) and brain-specific angiogenesis inhibitor 3 (Bai3) signaling. We have previously shown that C1ql1, released from CFs, regulates the establishment of the single CF innervation pattern during development by binding Bai3 expressed on PC dendrites. Immunohistochemical analyses of the cerebellum, in which C1ql1 was overexpressed by adeno-associated virus, revealed that transverse CF branches became positive for synaptic markers. Electrophysiological recordings showed that PCs became innervated by multiple CFs, which had slow excitatory postsynaptic current kinetics. When C1ql1 was overexpressed in CFs in Bai3 knockout mice, these changes were not observed, indicating that overexpression of C1ql1 in CFs induced innervation of PCs by multiple CFs, including the transverse CF branches, by activating Bai3 signaling. When Kir2.1 (inward-rectifier potassium ion channel) was overexpressed in PCs to inhibit PCs firing, activating Bai3 signaling did not cause innervations of PCs by multiple CFs in the adult cerebellum. Similarly, when Kir2.1 was expressed in CFs, C1ql1 was less accumulated at CF-PC synapses. These results indicate that neuronal activity regulates C1ql1-Bai3 signaling in the adult cerebellum. Since family proteins related to C1ql1 and Bai3 are widely expressed in various brain regions, mechanisms similar to those mediated by C1ql1-Bai3 signaling in the cerebellum may contribute to activity-dependent changes in synaptic connectivity.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-033 学習を制御する運動野の機能的回路 Functional organization of motor cortical circuitry for learning 大塚岳(1)、川口泰雄(2) 1. 生理学研究所　時系列細胞現象解析室、2. 玉川大学　脳科学研究所 Takeshi Otsuka(1), Yasuo Kawaguchi(2) 1. Section of Cellular Electrophysiology, National Institute for Physiological Sciences, 2. Brain Science Institute, Tamagawa University Keyword: Motor learning, Motor cortex, pyramidal cell The cortex outputs the information to several brain areas through different sets of pyramidal cells (PC). We have previously shown that cortical PCs form intra- and inter-laminar subnetworks, depending on pyramidal projection subtypes. However, little is known how individual PC subtypes concern in cortical information processing in the local circuits and between cortical areas. In the present study, we investigated effects of activity manipulation of PC subtypes on learning in the motor cortex. Pattern learning task was examined using forced wheel running system, which animals learn irregularly-patterned intervals between step bars to drink water from the supply port. To evaluate motor learning, we used the falling rate from step bars of a rotating wheel. Channel rhodopsin (ChR2) or archaerhodopsin (eArch) was selectively expressed in L2/3 PCs or L5 PC subtypes of rat motor cortical areas by in utero electroporation. Time courses of pattern learning formation were affected by optogenetic manipulations of specific PC subtypes in primary motor cortex (M1) during learning task. To examine cortical network activity during a task, in vivo recordings were obtained in M1. We found that oscillatory activities in local field potentials during learning were generated and enhanced by light stimulations to specific PC subtype. We also examined involvement of secondary motor cortex (M2) in learning. Activity of PC subtypes in M2 was selectively suppressed by optogenetic manipulation during task. Depending on PC subtypes, time courses of pattern learning formation were affected. Our results suggest that oscillatory activity induced by specific L5 PC subtype in M1 and synaptic inputs from M2 to M1 play important roles in motor learning.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-034 シナプス小胞膜融合のカンナビノイドによる調節の軸索終末パッチクランプ記録による生物物理学的解析 Biophysical analysis of cannabinoids-mediated modulation of synaptic vesicle exocytosis by direct patch-clamp recordings 井下拓真(1)、川口真也(1) 1. 京都大学 Takuma Inoshita(1), Shin-ya Kawaguchi(1) 1. Kyoto University Keyword: exocytosis, presynapse, cannabinoid, Purkinje cell Exocytosis of synaptic vesicles are triggered by Ca²⁺ influx upon action potentials depending on elaborate interactions of multiple presynaptic proteins. Particularly, the mechanisms to determine the readily releasable pool (RRP) of vesicles and to define their release competency have been extensively studied. It is well known that the exocytic transmitter release is negatively regulated by endocannabinoids at various synapses in the central nerves system, such as excitatory and inhibitory synapses on a cerebellar Purkinje cell (PC). To date, two types of endocannabinoid receptors (CBRs), CB1R and CB2R have attracted attention and have been shown by axonal Ca²⁺ imaging experiments to suppress presynaptic exocytosis mainly through reduction of Ca²⁺ influx into a presynaptic bouton. However, it remains unclear whether cannabinoid receptors affect the function of release machinery working downstream of Ca²⁺ influx. To test this possibility, we exogenously transfected a type of CBRs in cultured PCs with an AAV vector together with EGFP, allowing us to perform direct patch-clamp recordings from an axonal varicosity. Membrane capacitance measurements failed to detect any effect of CB2R activation on the amount of RRP vesicles, supporting the idea that CBRs negatively regulate synaptic transmission mainly through down-regulation of Ca²⁺ channels. On the other hand, another type of CBR, specifically named as G-protein coupled receptor 55 (GPR55), was suggested to affect the size of RRP vesicles without affecting presynaptic Ca²⁺ influx in a PC bouton. Thus, our results altogether suggest distinct mechanisms for negative control of presynaptic transmitter release by cannabinoid receptors in a subtype-specific manner acting on Ca²⁺ channels and their downstream release machinery.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-035 シナプス小胞放出サイトの形成に関わるMunc13-1とアクティブゾーンタンパク質の相互作用の同定 Identification of interactions between Munc13-1 and other active zone proteins vital for the formation of synaptic vesicle release sites 小島佑介(1)、坂本寛和(1)、並木繁行(1)、廣瀬謙造(1) 1. 東京大学大学院医学系研究科 Yusuke Kojima(1), Hirokazu Sakamoto(1), Shigeyuki Namiki(1), Kenzo Hirose(1) 1. Grad Sch Med, Univ of Tokyo, Tokyo, Japan Keyword: Synaptic vesicle exocytosis, Presynaptic terminal, Neurotransmission Neurotransmitters are released at presynaptic active zones, where synaptic vesicles are fused with plasma membrane. The active zone consists of several multi-domain proteins, including Munc13-1, RIM, RIM-BP and CAST, which redundantly interact with each other. We have previously shown that Munc13-1 forms supramolecular nanoassemblies that function as the synaptic vesicle release sites. Although the interactions between Munc13-1 and other active zone proteins are thought to be important for the formation of Munc13-1 supramolecular nanoassemblies, its detailed mechanism remains to be fully elucidated. In this study, we aimed to determine which interactions are essential for the formation of Munc13-1 supramolecular nanoassemblies. For this purpose, we obtained a series of isolated domains of RIM, CAST, and RIM-BP, which directly bind to Munc13-1, and expressed them in cultured hippocampal neurons. By quantitative immunocytochemistry we found that the expression of Zn²⁺ finger domain of RIM (RIMZF), which interacts with C₂A domain of Munc13-1, significantly reduced the level of Munc13-1 at presynaptic terminals. Furthermore, stochastic optical reconstruction microscopy (STORM) revealed that the number of Munc13-1 molecules per supramolecular nanoassembly was selectively reduced in RIMZF expressed neurons without changing the number of the supramolecular nanoassemblies per active zone. Finally, we found that the expression of RIMZF caused a significant decrease in neurotransmitter release by glutamate imaging, even though the previous research showed that RIMZF partially restores the impaired neurotransmitter release in RIM knock-out neurons. Taken together, these results suggest that the interaction of RIMZF with Munc13-1 C₂A domain itself is necessary but not sufficient for normal release site formation and neurotransmission, and that coupling between Munc13-1 and the other domains of RIM via RIMZF is important for maintaining the appropriate amount of Munc13-1 at the release site.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-036 乳頭体上核―歯状回顆粒細胞シナプスにおける脱分極誘導性LTP Depolarization-induced long-term potentiation at supramammillary-dentate granule cell synapses 橋本谷祐輝(1)、田淵詠梨(1)、坂場武史(1) 1. 同志社大学大学院脳科学研究科 Yuki Hashimotodani(1), Eri Tabuchi(1), Takeshi Sakaba(1) 1. Graduate School of Brain Science, Doshisha University Keyword: co-release, supramammillary nucleus, dentate gyrus, LTP The supramammillary nucleus (SuM) of the hypothalamus has strong connections with the hippocampal formation and sends its afferents exclusively to the dentate gyrus and the CA2 region. We recently reported that SuM neurons make monosynaptic connections to granule cells (GCs) and GABAergic interneurons in the dentate gyrus and co-release glutamate and GABA at these synapses. Although SuM-dentate gyrus circuits have been implicated in spatial learning, contextual novelty, and sleep and arousal, it remains unknown how the SuM projections to the dentate gyrus contribute to brain functions at the cellular, synaptic and circuit levels. Here, we investigated whether SuM-GC synapses can undergo any form of synaptic plasticity. To this end, we performed whole-cell patch-clamp recordings from GCs in acute hippocampal slices. To optogenetically activate SuM afferents, we injected AAV carrying channelrhodopsin-2(H134R) into the SuM of VGluT2-Cre mice. By light illumination, SuM-GC EPSCs were monitored in the presence of GABA_A receptor antagonist picrotoxin. We found that repetitive postsynaptic depolarizations induced long-term potentiation (LTP) of SuM-GC EPSCs. This depolarization-induced LTP (depol-LTP) required a postsynaptic increase in calcium ions through L-type voltage dependent calcium channels. We also found that depol-LTP required postsynaptic CaMKII activity, and exocytosis of AMPARs, and is expressed postsynaptically. We further found that depol-LTP is exclusively induced at SuM-GC synapses but not at perforant-path-GC, interneuron-GC or SuM-CA2 pyramidal neuron synapses. Importantly, GABAergic co-transmission at SuM-GC synapses did not show depol-LTP, suggesting that depol-LTP changes glutamatergic/GABAergic co-transmission balance at SuM-GC synapses. By monitoring GC firing, we found that SuM inputs triggered GC firing after induction of depol-LTP. Our results demonstrate that glutamatergic/GABAergic co-transmission balance is rapidly changed in an activity-dependent manner, and such dynamic change may modulate dentate gyrus network activity.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-037 Fingolimodの慢性投与がマウス海馬におけるParvalbumin陽性ニューロンとWFA陽性perineuronal Netsへ与える影響 Effects of chronic administration of fingolimod hydrochloride (FTY720) on parvalbumin immunoreactivities in the mouse hippocampus 上野浩司(1)、髙橋優(2)、岡本基(3)、石原武士(2) 1. 川崎医療福祉大学医療技術学部臨床検査学科、2. 川崎医科大学精神科学教室、3. 岡山大学保健学研究科 Hiroshi Ueno(1), Yu Takahashi(2), Motoi Okamoto(3), Takeshi Ishihara(2) 1. Kawasaki Univ of Med Welf, Okayama, Japan, 2. Kawasaki Medical School, Okayama, Japan, 3. Okayama University, Okayama, Japan Keyword: fingolimod, mouse, parvalbumin, perineuronal nets In recent years, the likelihood of resuming the critical period for the central nervous system of mature animals has been demonstrated. It has long been reported that some central nervous system-targeted agents can increase neural plasticity. In this study, we focused on fingolimod hydrochloride (FTY720). Clinically, fingolimod is used for the treatment of multiple sclerosis. The effects of chronic fingolimod administration on parvalbumin-positive interneurons and Wisteria floribunda Lectin (WFA)-positive perineuronal nets in the mouse hippocampus were investigated. This study showed an increase in parvalbumin-positive interneurons in mice treated with fingolimod in the hippocampal CA1 region. Chronic administration of fingolimod also activated glial fibrillary acidic protein (GFAP)-positive astrocytes in the hippocampal CA1 region. The results of this study indicate that chronic administration of fingolimod affects hippocampal neurons and astrocytes.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-038 Stochastic spatial modeling of vesicle-mediated trafficking of AMPA receptors for understanding hippocampal synaptic plasticity mechanisms Sarah Yukie Nagasawa(1), Iain Hepburn(1), Andrew R. Gallimore(1), Erik De Schutter(1) 1. Okinawa Institute of Science and Technology Graduate University Keyword:* Synaptic Plasticity, Stochastic spatial modeling The regulation of AMPA-type glutamate receptor (AMPARs) in the postsynaptic membrane is central for maintaining the normal functioning of hippocampal synapses, and sustaining long-term storage of memories. Dysregulation of AMPARs contributes to cognitive deficits in neurodegenerative diseases, such as Alzheimer's disease. The trafficking of vesicles is hypothesized to function in the fine-tuning of AMPARs in the synapse and is believed to promote different forms of synaptic plasticity. Improvements in biochemical and imaging methods have led to advances in studies of membrane vesicle trafficking. However, it remains challenging to directly observe these processes in intact neurons during synaptic plasticity. Here we use computational approaches to study the relationship between changes in vesicle trafficking and the population of AMPARs maintained in the synapse. Specifically, we focus on the spatial aspects of vesicle trafficking, such as positioning of vesicle processes within a dendritic spine. In this project, we use a stochastic reaction-diffusion simulator, STEPS, to build models that simulate at the molecular level interactions that influence vesicle trafficking processes.¹ Models are based on quantitative data derived from structural, biochemical, and modeling studies. Importantly, a high level of spatiality is preserved in the model by implementing vesicle objects that interact within a multi-compartmental 3D morphology of a dendritic spine. We aim to use this model to identify the important factors of vesicle trafficking that contribute to the regulation of AMPARs during hippocampal synaptic plasticity. Bibliography 1 Hepburn, I., Chen, W., Wils, S. & De Schutter, E. STEPS: efficient simulation of stochastic reaction–diffusion models in realistic morphologies. BMC Systems Biology (2012).

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-039 海馬CA1におけるアセチルコリンによるシナプス可塑性誘導ダイナミクスの解明 Dynamic mechanisms of cholinergically modulated synaptic plasticity in the hippocampal CA1 area 杉崎えり子(1)、福島康弘(2)、相原威(1) 1. 玉川大学脳科学研究所、2. 川崎医療福祉大学 Eriko Sugisaki(1), Yasuhiro Fukushima(2), Takeshi Aihara(1) 1. Tamagawa University, Tokyo, Japan, 2. Kawasaki University of Medical Welfare, Okayama, Japan Keyword: Hippocampus, Spike timing-dependent plasticity (STDP), Acetylcholine It is known that the hippocampus is a crucial function for memory encoding and retrieval, so that the spike timing-dependent plasticity (STDP), a basic phenomenon of learning and memory, is observed in the CA1 region of hippocampus. When we pay an attention to an episode, acetylcholine (ACh) is released form cholinergic neurons originated in the media septum, and this release is also seen in the CA1. Therefore, STDP can be modulated by the activation of ACh receptors (AChR) on pyramidal neurons and interneurons if ACh is effective in the network. However, what kind of synaptic changes are occurred and how the direction and the magnitude of STDP are decided during the STDP inducing stimuli in the presence of ACh are barely known. In order to clarify the mechanisms of synaptic changes during the stimuli, characteristics of membrane potentials and action potentials modulated by ACh were investigated by making patch clamp recordings to the soma of pyramidal neurons in the CA1 using rat hippocampal slices. Baseline amplitude, defined as a difference between the lowest value of response induced by paired stimuli at the positive timing of STDP-inducing stimulus in the Schaffer collaterals and the membrane potential before the stimuli application, was evaluated to see the ACh contribution to the synaptic changes. Also, half widths of action potentials were quantitated. As the results, baseline amplitudes were elevated if ACh was effective in the GABA_A receptor (GABA_AR) blocked network, especially muscarinic ACh receptors (mAChR) contributed more on the elevation. Moreover, STDP was enhanced along with the magnitude of baseline amplitude. Contrary in the GABA_AR activated network, baseline amplitudes were balanced regardless of ACh concentration. This may be due to an up regulation related to ACh was highly down regulated by cholinergically modulated interneuron activities. Although in a nicotinic ACh receptor (nAChR) activated condition, STDP was enhanced even baseline amplitude was low. Therefore, the half widths of action potential were next focused on and it was clarified that the STDP enhancement was related to a broadened width of action potential when nAChR were activated. These results suggest that the synaptic changes during memory encoding along with the activation of mAChRs and nAChRs, contributors for baseline amplitude and action potential respectively, are crucial function for memory enhancement.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-040 1型代謝型グルタミン酸受容体に対するB型GABA受容体の相互作用効率：漸進性アクセプタ・フォトブリーチングによる解析 Gradual acceptor photobleaching reveals the interactability of B-type GABA receptor with type-1 metabotropic glutamate receptor 桐木賢吾(1)、森拓哉(2)、高橋泰斗(2)、上窪裕二(3)、坂入伯駿(3)、櫻井隆(3)、田端俊英(4) 1. 富山大学大学院理工学教育部、2. 富山大学大学院医薬理工学環、3. 順天堂大学医学部、4. 富山大学学術研究部工学系 Kengo Kirinoki(1), Takuya Mori(2), Taito Takahashi(2), Yuji Kamikubo(3), Hakushun Sakairi(3), Takashi Sakurai(3), Toshihide Tabata(4) 1. Grad Sch Sci Eng, Univ of Toyama, Toyama, Japan, 2. Grad Sch Pharma-Med Sus, Univ of Toyama, Toyama, Japan, 3. Juntendo Univ Fac Med, Tokyo, Japan, 4. Fac Eng, Univ of Toyama, Toyama, Japan Keyword: SYNAPTIC PLASTICITY, CEREBELLUM, GPCR, FRET We have previously shown that in cerebellar Purkinje cells, activation of B-type gamma-amino butyric acid receptor (GABAbR) leads to facilitation of type-1 metabotropic glutamate receptor (mGluR1)-mediated long-term depression of postsynaptic glutamate responsiveness, a cellular basis for cerebellar motor learning. Our previous co-immunoprecipitation and double-fluorescence confocal microscopy indicated that GABAbR and mGluR1 intimately interact with each other. However, the fraction of the receptors that participates in inter-receptor interaction was unknown. Here we estimated the fraction using a gradual acceptor photobleaching technique. We created HEK293 cells co-expressing GABAbR subunit 1 with a Halo-tagged N-terminus, GABAbR subunit 2, and mGluR1 subunit with a SNAP-tagged N-terminus. The corresponding subunits were labeled with membrane-impermeant donor (HaloTag Alexa Fluor 488 Ligand) and acceptor (SNAP-Surface 594) fluorescent substrates. For each fluorescence punctum, the intensity of the donor’s fluorescence excited with a 488 nm laser (power, 10%) (Id) was measured once every 10 repetitions of photobleaching of the acceptor with a 638 nm laser (100%) using a Leica TCS SP8 confocal microscope. Along with the progress of acceptor photobleaching, Id first increased due to a decrease of donor-acceptor pairs in which Förster resonance energy transfer (FRET) took place and later decayed due to photobleaching of the donor itself. The overall trajectory of Id could be described by a function Id(i) = Id(0)•[1 – E(0)•exp(–10i/tau_a)]•exp(–i/tau_d) + C, where Id(i), E(0), tau_d, tau_a, and C were Id at the i-th excitation, initial FRET efficiency, time constants of donor and acceptor photobleaching in i-scale, and offset, respectively. The value of tau_d or tau_a was obtained from a calibration experiment in which the bare donor or acceptor placed on a coverslip was photobleached with the above protocol. In normal HBSS, 32.0%, 67.4%, and 0.6% of 181 puncta fell into low (0-0.05), middle (0.05-0.9), and high (0.9-1) ranges of E(0), respectively. In HBSS containing 3 µM GABA, 32.5%, 61.4%, and 6.0% of 83 puncta fell into the low, middle, and high ranges, respectively. In HBSS containing 10 µM L-glutamate, 40.2%, 56.9%, and 2.9% of 102 puncta fell into the low, middle, and high ranges, respectively. These results suggest that a considerable fraction of GABAbR population can interact with mGluR1 on the cell surface.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-041 GluA2/3 AMPA受容体に対する光不活性化技術 Optical inactivation technology for GluA2/3 AMPA receptor. 實木亨(1,2)、高橋琢哉(2)、竹本研(1) 1. 三重大学大学院医学系研究科、2. 横浜市立大学大学院医学研究科 Susumu Jitsuki(1,2), Takuya Takahashi(2), Kiwamu Takemoto(1) 1. Grad Sch Med, Mie University, 2. Grad Sch Med, Yokohama City University Keyword: AMPA receptors, synaptic plasticity, CALI method, hippocampal learning Ionotropic neurotransmitter receptors commonly form protein complex by various combinations with subunits and have distinct functional properties. For example, AMPA type glutamate receptors (AMPA-Rs), which are well known to be important glutamate receptors for learning, are composed of variable combinations of four subunits, GluA1-4. The combinations of GluA1 homomer, GluA1/2 and GluA2/3 were known to be expressed in adult brain. AMPA-Rs with GluA1 subunits require plasticity-inducing stimuli and NMDA-Rs activation to be driven into synapses and serve to enhance neurotransmission. In contrast, GluA2/3 complex continuously replace synaptic receptors in a manner that maintains transmission (Shi, S-H. et al. Cell 2001, Takahashi, T. et al. Science 2003　etc.).　Since subunit combinations affect the functions of ion channel, complexes lacking GluA2 subunit shows calcium permeability and high single channel conductance (Dingledine R et al. Pharmacol Rev 1999, Coombs ID et al. J. Neurosci. 2012). These observations support the idea that AMPA-Rs complexes should have different physiological functions in vivo. To elucidate their complex-specific functions in vivo, we have developed an optical technology for acute inactivation of synaptic GluA1 homomeric AMPA-Rs in vivo by chromophore assisted light inactivation (Takemoto et al. Nat. Biotechnol. 2017). This technology enabled us to elucidate the function of GluA1 homomer in the acquisition of contextual fear memory in hippocampus. Based on this study, we are currently developing a CALI method for GluA2/3, an AMPA receptor that does not contain GluA1.　To achieve inactivation of GluA2/3, We screened GluA3 antibody by using flow cytometry and electrophysiology. We obtained antibodies that showed high CALI efficiency of GluA2/3. Using this antibody could be clarify GluA2/3-specific physiological functions in vivo.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-042 Heterosynaptic spine structural plasticity across dendritic branches Thomas Chater(1), Yukiko Goda(1,2) 1. RIKEN, Japan, 2. OIST, JAPAN Keyword:* Heterosynaptic plasticity, Long term potentiation, Synaptic plasticity Neurons receive thousands of synaptic inputs of varying strengths. These strengths are determined by the activity history of a particular synapse (e.g. Hebbian plasticity), but are also modulated by other mechanisms that monitor local and global neuronal activity levels such as those represented by heterosynaptic and homeostatic plasticity. It is not well understood how these different forms of synaptic plasticity subserving specific demands of the circuit to which individual synapses are embedded in, are coordinated across the dendritic arbour. By measuring the size and distribution of postsynaptic dendritic spines on GFP-expressing excitatory pyramidal neurons, coupled with targeted glutamate uncaging to trigger synaptic plasticity at defined sets of inputs, we find that the dynamics of both Hebbian and heterosynaptic plasticity is modulated by the number of stimulated spines, and that heterosynaptic effects are confined to within a few micrometres of the stimulated region. Calcium imaging during the uncaging stimulus demonstrates that calcium rises are locally confined to spines and the dendrite close to the homosynaptic stimulated spines, suggesting that distant heterosynaptic changes are likely triggered by diffusible factors downstream of homosynaptic activity. Our results can be explained by a local dynamic distribution of synaptic resources, in which stimulated synapses signal for the production and transport of critical synaptic components, and neighbouring weakly active synapses take advantage of this and recruit relevant plasticity-related proteins. We are now working to determine the molecules that control this behaviour.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-043 生体内における樹状突起スパインでの分子可塑性 Molecular plasticity in dendritic spines in vivo 山田麻紀(1,2)、久保山和哉(1,4)、窪田剛志(1,2)、吉岡美紅(1)、中谷音葉(1)、藤島利江(3)、足達俊吾(5)、夏目徹(5) 1. 徳島文理大・香川薬学部・薬理、2. 徳島文理大・神経研・神経薬理、3. 徳島文理大・香川薬学部・薬化学、4. 名古屋市大・医学系・神経研・神経発達再生医学、5. 産業技術総合研・細胞分子工学 Maki K. Yamada(1,2), Kazuya Kuboyama(1,4), Takeshi Kubota(1,2), Miku Yoshioka(1), Otoha Nakatani(1), Toshie Fujishima(3), Shungo Adachi(5), Tohru Natsume(5) 1. Neuropharmacol, Kagawa Pharma Sci, Tokushima Bunri Univ, Kagawa, Japan, 2. Neuropharmacol, Inst Neurosci, Tokushima Bunri Univ , 3. Med Chem, Kagawa Pharma Sci, Tokushima Bunri Univ , 4. Dev Regen Neurobio, Inst Brain Sci, Grad Sch Med, Nagoya City Univ, 5. Cell Mol Biotech Res Inst, AIST Keyword: MS proteomics, Protein Profiling, Phencyclidine, Schizophrenia Long-term changes in synapses as a basis for brain plasticity, such as memory, have been studied mainly in vitro, and it is still unclear whether the same things take place in vivo. To investigate what changes occur at synaptic sites in response to stimuli in the living brain, the authors generated a transgenic mouse line expressing CapZ protein, with HA and EGFP tags by an Arc7k-promoter. CapZ is a reduced 2D-DIGE spot protein in one side of the hippocampus, where memory is thought to be impaired by unilateral fornix-transection. This CapZ signal in immunostaining was increased in layers that underwent DG-LTP in vivo (Genes Cells 2010 https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2443.2010.01411.x ). In the AiCE-Tg, we confirmed EGFP-CapZ fluorescence changes in the postsynaptic spines and published the results in a recent paper (SciRep2020 https://www.nature.com/articles/s41598-020-72248-4 ). Specifically, twenty minutes after visual stimulation with treatment of one eye closure and fifteen minutes after sensory stimulation with Rotarod after blocking the sciatic nerve of one leg, there was a relative increase in the stimulated side of the population in EGFP-CapZ fluorescence at the postsynaptic spine in the input area (layer IV) of the responsible area of the cortex. (In the used AiCE-Tg line, about a hundred copies of the transgene are thought to have been inserted, and bright fluorescence is maintained even after more than ten years of passaging.) In this presentation, we will report on some unexpected molecules that have come up as the activity-dependent CapZ-binding proteins. In order to investigate the differences in the spines having high EGFP-HA-CapZ fluorescence from a molecular science point of view, we performed pull-down and elution of molecules using magnetic beads with HA-tagged antibodies, and performed comprehensive molecular analyses by mass spectrometry, which revealed a large number of binding molecules. Among them, we focused on a group of molecules that is thought to be important for plasticity regulation, but whose details have not yet been clarified. The possibility of stimulus-dependent co-localization was considered in immunostaining experiments, and is now under analysis.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-044 ケタミンの持続的な抗うつ作用の機構 Mechanism of the long-lasting antidepressant action of ketamine 岡田大助(1,2)、天野史稔(1)、萬代研二(1,2) 1. 北里大学大学院医療系研究科、2. 北里大学 Daisuke Okada(1,2), Fuminari Amano(1), Kenji Mandai(1,2) 1. Kitasato University, Grad Sch Med Sciences, Sagamihara, Japan, 2. Kitasato Univ, Sagamihara, Japan Keyword: ketamine, hydroxynorketamine, cytochromeP450, late phase of LTP Ketamine and its major metabolite, (2R,6R)-hydroxynorketamine (R-HNK), are fast-acting and long-lasting antidepressants. These compounds reactivate depressed neural circuits by facilitating functional connections between distinct brain regions. This activation is associated with persistent enhancement of glutamatergic synaptic transmission by protein synthesis-dependent synaptic plasticity, such as late-phase of long-term potentiation (L-LTP). R-HNK does not have aversive effects seen in ketamine such as analgesic, anesthetic, and psychotomimetic effects, and thus could be a more specific antidepressant. However, the intravenous administration of ketamine or R-HNK would evoke heterogenous responses from multiple brain regions, because it acts on the whole brain through blood circulation, which is inconsistent with the specific action of R-HNK. To understand mechanisms underlying the circuit-specific antidepressant actions, we hypothesized that R-HNK is synthesized from ketamine in specific cell populations in the brain, and exerts circuit-specific actions by locally modifying protein synthesis involved in L-LTP. We found the following actions of ketamine and R-HNK on the L-LTP processes by using primarily cultured rat hippocampal cells: (1) Ketamine and HNK enantiospecifically suppressed synaptic tagging, a mechanism required for synaptic localization of the soma-derived newly synthesized proteins in L-LTP. (2) Ketamine did not suppress synaptic tagging in the presence of cytochromeP450 (CYP) inhibitors. (3) RT-PCR demonstrated that some of the ketamine-metabolizing CYPs are expressed mainly in neurons. (4) HNK synthesis from ketamine was detected in cultured hippocampal cells. (5) Both ketamine and R-HNK enhanced the expression of GluA1 in the culture. (6) GluA1 increase by R-HNK, but not ketamine, was rapamycin-sensitive, in other words, R-HNK activated local protein synthesis, another mechanism required for rapid protein synthesis near synapses in L-LTP. These results suggested that ketamine and R-HNK affect the plasticity differently. The cell-dependent synthesis of R-HNK contributes to its specific antidepressant action, because R-HNK synthesized in neurons can act differently from liver-derived HNK due to different accesses to their targets.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-045 Methamphetamine-induced remodelling of hippocampal neurons is orchestrated via cdc42 pathway Terceiro Ana(1,2)、Lobo Andrea(1)、Carvalhais Lia(3,4)、Aroso Miguel (5)、Sousa Mafalda(6)、Magalhães Ana(1)、Aguiar Paulo(5)、Relvas João (7,8)、Summavielle Teresa(1,9) Ana Filipa Terceiro(1,2), Andrea Lobo(1), Lia Carvalhais(3,4), Miguel Aroso(5), Mafalda Sousa(6), Ana Magalhães(1), Paulo Aguiar(5), João Relvas(7,8), Teresa Summavielle(1,9) 1. Addiction Biology Group, i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal, 2. Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal, 3. Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, 4. Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal, 5. Neuroengineering and Computational Neuroscience, i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal, 6. i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal, 7. Glial Cell Biology, i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal, 8. Faculdade de Medicina da Universidade do Porto (FMUP), Porto, Portugal, 9. ESS.PP, Escola Superior de Saúde do Politécnico do Porto, Porto, Portugal Keyword: Methamphetamine, Hippocampus, Cdc42, Intersectin1 The development of addiction is strongly influenced by synaptic and morphological adaptations in the brain provoked by drugs of abuse such as methamphetamine (Meth), a powerful psychostimulant. The hippocampus is one the regions more affected, contributing to the formation of drug-context associations and relapse. However, particularly in this region, the mechanisms regulating this complex process are not clear. We observed that in primary hippocampal neuronal cultures, exposure to Meth (24h) increased neurite outgrowth, dendritic spine density and impacted the expression of synaptic proteins. In vitro electrophysiology assays using multi-electrode arrays, we have also demonstrated that Meth decreases spontaneous neuronal activity. RhoGTPases, as key regulators of the actin cytoskeleton, have been linked to drug-induced maladaptive neuronal restructuring. Here, we demonstrate, using FRET assays, that cdc42 activity is increased at dendritic spines 5 min after drug exposure. Concurrently, the downstream pathway (N-Wasp, Arp3) is activated in synaptoneurosomes of hippocampal neuronal cultures, 15 min following Meth exposure. Inhibition of Intersectin1(Itsn1, a cdc42 specific activator)/cdc42 interaction, as well as Itsn1 knockdown prevented Meth effects on neuronal remodelling. Importantly, in WT mice exposed to a Meth binge regimen (5 mg/kg x4, 2h intervals), cdc42 activity was increased in hippocampal synaptoneurosomes 15 min after the last drug administration. Meth also increased neurite length, dendritic spines density and decreased PSD95 expression 24h following the last administration of Meth. Currently, we are assessing whether silencing Itsn1 in vivo can prevent Meth-elicited neuronal remodelling. Collectively, our data demonstrates cdc42 as an important mediator of Meth-induced remodelling in the hippocampus.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-046 海馬顆粒細胞における入力の連合応答 Associative responces for inputs to hippocampal granule cell 亀井和久(1)、門傅忠叡(1)、杉崎えり子(2)、中島直樹(1)、小池亮太朗(1)、相原威(2) 1. 玉川大学大学院工学研究科、2. 玉川大学工学部 kazuhisa kamei(1), Tadayosi Monden(1), Eriko Sugisaki(2), Naoki nakajima(1), Ryoutarou Koike(1), Takesi Aihara(2) 1. Tamagawa University Graduate School of Engineering, 2. Tamagawa University Keyword: hippocampus, dentate gyrus, granule cell, information integration The dentate gyrus granule cells (GCs) at an entrance of the hippocampus have two-types input though the lateral perforant path (LPP) and the medial perforant path (MPP) form the entorhinal cortex (EC). The LPP sends non-spatial information, such as odor, to the medial dendrite (DD). On the other hand, the MPP sends spatial information, relating the place, to the distal dendrite (MD) of GCs. However, the scheme of information integration between the two inputs is not clear yet. In this study, we investigated the input-timing dependence of association responses for inputs to DD and MD and discussed the integration of input information in GCs. “Arithmetic waveforms” of the field excitatory-postsynaptic membrane-potential (fEPSP) were calculated using two fEPSP obtained by a single electrical stimulation to LPP or MPP in GCs, respectively. Each input strength was limited under the firing threshold. “Measured waveforms” of fEPSP were obtained by using paired stimulation with the different timing from -40 msec to 40 msec to LPP and MPP. To compared the difference between those two fEPSP, the ratio in the peak amplitude of fEPSP for the following stimulus in pairing stimuli, which was superposed on the fEPSP for the preceding stimulus, and observed as the second peak amplitude or single peak (0msec) in the “measured fEPSP” for phase-contrast stimuli. Furthermore, to investigate the effect of inhibitory cells on the association response, an inhibitor of GABA(A) receptors (picrotoxin) was applied. In addition, to investigate the influence of NMDA receptors on the association response, an inhibitor of NMDA receptors (D-AP5) was applied. As the result, a linear response was seen only when two inputs were simultaneously applied to LD and MD (⊿t=0ms) in the control state (naive). Results for other timings showed the nonlinear responses. Next, when we blocked the GABA(A) inhibitory connection, results were closer to the linear response as in the naive state, though the nonlinearity remained at the negative timing. When NMDA receptors were inhibited by D-AP5, the nonlinearity was larger than that in the naive state. Simultaneous blocking of NMDA receptors and GABA(A) receptor, the response were similar to that in the naive state. These results suggest that GCs have properties as a coincidence detection of inputs to MD and DD, and both NMDA and GABA(A) receptors play a crucial role in the information association for the two inputs to DGs.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-047 海馬神経細胞における樹状突起内での興奮性及び抑制性シナプス入力の独自修飾 Distinct modulation of excitatory and inhibitory synaptic inputs in hippocampal neuronal dendrites 森田雅登(1)、川口真也(1) 1. 京都大学大学院理学研究科生物物理学教室 Masato Morita(1), Shin-ya Kawaguchi(1) 1. Dept Biophys, Grad Sch Sci, Kyoto Univ, Kyoto, Japan Keyword: neuron, membrane potential imaging Neurons form many synapses and receive a lot of different inputs there. Many studies have shown how synaptic potentials travel in dendrites mainly by somatic patch-clamp techniques and mathematical models. However, this issue remains elusive because of the difficulty in simultaneous measuring membrane potentials at several subcellular compartments. To study this issue, we used a genetically encoded voltage indicator together with a spot uncaging of glutamate or GABA. We examined how excitatory and inhibitory postsynaptic potentials spread in dendrites. A fluorescent voltage-sensitive probe, ASAP detects membrane potential changes as fluorescently intensity changes. An improved version of ASAP was transfected in hippocampal cultured neurons. EPSP was caused by locally activating MNI-caged-glutamate around a spine by illuminating 405 nm lazar spot (2 µm diameter). In line with the cable theory, fluorescent detected EPSP attenuated during the propagation toward the soma. In contrast, surprisingly, EPSP was amplified during the propagation toward the distal region of a dendritic branch. Thus, our results suggest that excitatory synaptic inputs are oppositely modulated in a dendrite depending on the direction of propagation. Then, IPSP was caused by activating DPNI-caged-GABA in a manner similar to the EPSP experiments. The IPSPs showed a different pattern of modulation during dendritic propagation from that of excitatory inputs. In order to address the mechanisms how EPSPs and IPSPs exhibit distinct modulation in dendrites, we tested the involvement of various types of ion channels by pharmacological approaches on voltage imaging together with direct patch-clamp recordings from a thin dendritic branch. In the poster, we are going to demonstrate the comprehensive view of the dynamic modulation mechanism of excitatory and inhibitory synaptic potentials in an elaborate dendritic arborizations.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-048 樹状突起形成に関与するSez6スプライスバリアントの脳内分布 The expression pattern of alternative splicing variant of Sez6 in mouse brain 日高千晴(1)、三井真一(2)、大迫洋治(1)、高橋弘(1)、田中健二朗(1)、由利和也(1) 1. 高知大学医学部解剖学講座、2. 群馬大学大学院保健学研究科リハビリテーション学講座 Chiharu Hidaka(1), Shinichi Mitsui(2), Yoji Osako(1), Kou Takahashi(1), Kenjiro Tanaka(1), Kazunari Yuri(1) 1. Department of Neurobiology and Anatomy, Kochi Medical School, Kochi University, Kochi, Japan, 2. Department of Rehabilitation Science, Gunma University Graduate School of Health Science, Gunma, Japan Keyword: Sez6, dendrite branching, neurite Seizure-related gene 6 (Sez6) is a transmembrane protein specifically localized on neuronal dendrites and is responsible for its dendritic branching and synapse formation. Alternative splicing produces three isoforms of Sez6 mRNAs, and the dominant isoform encodes a transmembrane-type protein, whereas the other two recessive isoforms encode transmembrane and secrete-type proteins. In the present study, to clarify the differential functions of these isoforms, the expression pattern of Sez6 splicing was investigated in the adult mouse brain. The whole brains were sliced on coronal sections by each 1 mm in thickness and punched out brain area from a coronal brain section by φ2.5 mm. The mRNA levels of each Sez6 isoform in the prefrontal cortex, cingulate cortex, striatum, hippocampus, and amygdala, where Sez6 expression has been reported previously, were analyzed using a qPCR technique. The result showed that the splicing pattern of Sez6 was modulated in a brain area-specific manner. In particular, the striatum showed characteristic splicing pattern of recessive isoforms. Moreover, neuronal activation by convulsant drug stimulation increased recessive isoforms in cultured cortical neurons at 5 or 10 days in vitro. In conclusion, alternative splicing of Sez6 may be modulated in brain area-specific and neural activity-dependent manners. In the future, we will intend to investigate whether the overexpression of each Sez6 isoform affects the formation of dendritic branching and synapse.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-049 タウタンパク質をモデルとした軸索輸送の分子メカニズムについて A study of molecular mechanism for the axonal transport employing the Tau protein as a model cargo 中田里奈穂(1)、御園生裕明(1) 1. 同志社大学　脳科学研究科 Rinaho Nakata(1), Hiroaki Misonou(1) 1. Graduate School of Brain Science, Doshisha University Keyword: Tau, Axonal transport The tau protein is known to be transported by the slow axonal transport and localized to the axons of neurons. However, in Alzheimer disease, it accumulates abnormally in the cell bodies and dendrites as hyper-phosphorylated forms. In this study, we aim to understand the mechanism of this abnormal localization of tau through understanding the molecular mechanism of tau transport and disruption. We have previously developed an inducible expression system, with which human tau is properly localized to the axon in cultured rat hippocampal neurons. Using this, we employed human tau tagged with a photoconvertible fluorescent protein, Dendra2, to visualize its axonal transport. To achieve this, we performed pulse-chase imaging of Dendra2-tau in immature neurons, in which tau is actively localized to the axon, and analyzed how dendritic tau migrates into the axon. We were able to detect preferential transport of tau to the axon at a comparable speed to that of the slow axonal transport and verify that the transport was energy (ATP)-dependent. With the tool to investigate the slow axonal transport of tau, we plan to identify the structural determinants of tau for its transport. We have previously identified the proline-rich region 2 (PRR2) as a critical domain for the axonal localization of tau. We will analyze Dendra2-tau lacking PRR2 and show whether preferential transport to axons can be observed. Also, we will analyze the mutant lacking the microtube binding domain. We have obtained a surprising finding with this mutant in that it did not bind to microtubules in neurons yet localized properly to the axon. By comparing this mutant with free Dendra2, we would like to discriminate diffusion, microtubule-binding, and transport behavior of tau in living cells. Lastly, we will also investigate how phosphorylation affects tau transport. We have generated a phosphorylation-mimetic mutant, in which eight phosphorylation sites in PRR2 were replaced by glutamate (Tau-8E). Immunostaining results showed no axon specific localization of Tau-8E, suggesting transport disruption (Iwata et al., 2019). By analyzing this mutant in the pulse-chase imaging, we hope to determine how tau transport is regulated by phosphorylation. In the future, in order to identify the molecular mechanism of tau transport, we will examine whether tau transport may be controlled by the motor protein kinesin or other transport mechanisms, using knockdown, pharmacological, and biochemical analysis.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-050 TC10は小胞上からの特異的なシグナル伝達経路による微小管の安定化を介して軸索伸長を促進する Active TC10 on vesicles promotes axon outgrowth by stabilizing microtubules through the specific phosphorylation pathway 鯉沼真吾(1)、宮地美沙(1)、保科光(1,2)、前澤創(2)、和田直之(2)、竹村裕(2)、郡山恵樹(3)、桐生寿美子(4)、木山博資(4)、五十嵐道弘(5)、中村岳史(1) 1. 東京理科大学生命医科学研究所、2. 東京理科大学理工学部、3. 鈴鹿医療科学大学薬学部、4. 名古屋大学大学院医学系研究科、5. 新潟大学大学院医歯学総合研究科 Shingo Koinuma(1), Misa Miyaji(1), Akira Hoshina(1,2), So Maezawa(2), Naoyuki Wada(2), Hiroshi Takemura(2), Yoshiki Koriyama(3), Sumiko Kiryu-Seo(4), Hiroshi Kiyama(4), Michihiro Igarashi(5), Takeshi Nakamura(1) 1. RIBS, Tokyo Univ of Sci, Chiba, Japan, 2. Faculty of Sci and Tech, Tokyo Univ of Sci, Chiba, Japan, 3. Faculty of Pharm Sci, Suzuka Univ of Med Sci, Mie, Japan, 4. Grad Sch of Med, Nagoya Univ, Aichi, Japan, 5. Grad Sch of Med and Dental Sci, Niigata Univ, Niigata, Japan Keyword: Rho family, axon regeneration, microtubule Mammalian CNS neurons lose axon regeneration ability as they mature. To find more efficient strategies for repairing injured CNS, the elucidation of the intracellular signaling pathways that promote axon outgrowth has been increasingly important. TC10, a member of Rho-family G proteins, is a signaling molecule that acts on neurite/axon outgrowth through membrane trafficking. We recently showed that TC10 ablation significantly reduced axon regeneration after injury in hypoglossal and optic nerves. Here we aimed to find another TC10’s downstream pathway which elucidates axon outgrowth. We obtained the following results using primary hippocampal neurons. (1) Time-lapse imaging for 10 hr revealed that the frequency of axon retraction in TC10 KO neurons was four times higher than that in WT neurons. (2) In distal axons, the level of acetylated tubulin, a marker of stable microtubules, was significantly reduced by TC10 ablation. (3) The increase in retraction frequency and the decrease in tubulin acetylation in TC10 KO neurons were rescued by expression of wild-type TC10, but not by a TC10 mutant (TC10-K-RasCT) which was localized to the plasma membrane only. This result suggests that microtubule stabilization is mediated by active TC10 on vesicles and is consistent with our previous observation that TC10 activity on vesicles was higher than that on the plasma membrane. (4) We checked the phosphorylation levels of regulatory factors involved in microtubule stabilization and found that the phosphorylation levels of SCG10 and MAP1B were lower in the E18 brains of TC10 mice than that in control. The phospho-stathmin1 level was not changed. Immunostaining analysis of TC10 KO neurons also showed that the SCG10 and MAP1B phosphorylation levels were significantly decreased in distal axons compared to the control. These results suggest that active TC10 on vesicles is implicated in microtubule stabilization through phosphorylation of SCG10 and MAP1B. Based on these results and our previous studies, we propose that TC10 promotes axon elongation by regulating microtubule stabilization and membrane trafficking, thereby contributing to axon regeneration.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-051 神経細胞局所における微小管安定性の解析 Analysis of microtubule stability in neurons 今井貴之(1)、宮坂知宏(1)、御園生裕明(2) 1. 同志社大学大学院生命医科学研究科、2. 同志社大学大学院脳科学研究科 Takayuki Imai(1), Tomohiro Miyasaka(1), Hiroaki Misonou(2) 1. Department of Neuropathology, Doshisha University, Kyoto, Japan, 2. Graduate School of Brain Science, Doshisha University, Kyoto, Japan Keyword: Microtubules Microtubules (MTs) are a major cytoskeleton in eukaryotes and have roles in a spectrum of cell functions. They are cylindrical and polar tubule structures consisting of parallel protofilaments built from head-to-tail assembly of α/β-tubulin heterodimer subunits. The plus-end where the β-tubulin subunit is located actively repeats its depolymerization and polymerization. This is called dynamic instability of MTs, and its frequency is thought to vary depending on the tissue and cell. Especially in neurons which have highly differentiated cell morphology, regulation of microtubule stability is essential for development and maintenance of the neuronal function. MTs in neurons are consisted with distinct pools of tubulin, that appeared to be free tubulin dimers and MTs with different stability signatures, which may correspond to stable and labile MTs. It is plausible that these are appropriately maintained through nerve cell differentiation to maturation. However, populations of these three pools of tubulin in neurons remains unclear. Here, we analyzed the MT stability in neurons using fluorescence recovery after photobleaching (FRAP) analyses. GFP-labeled α-tubulin was expressed in primary cultured rat hippocampal neurons using a lentiviral expression system. FRAP analysis demonstrated that there were three components of tubulin: that recovered rapidly, that recovered slowly, and that did not recover even after 1800 seconds. These are considered to be free tubulin, labile MTs, and stable MTs, respectively. In the axons at DIV14, it was estimated that 6.6 ± 2.5% of tubulin was free tubulin, 12.2 ± 4.9% was labile MTs, and 81.2 ± 4.7% was stable MTs. There were no differences with those in dendrites. However, MTs in neuron were extremely stable than that in COS7. Furthermore, we found that the MT stability was increased from DIV3 to DIV14, indicating that, during maturation, neuronal MTs acquired high stability than that in dividing cells.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-052 シナプス構築部位におけるテニューリン2はタンパク質集積のための標しである Teneurin-2 at Synapse construction site is a signpost for protein accumulation 一ノ瀬聡太郎(1)、岩﨑広英(1) 1. 群馬大学大学院医学系研究科 Sotaro Ichinose(1), Hirohide Iwasaki(1) 1. Gunma University Graduate School of Medicine Keyword: Microtubules, Synapse In contrast to excitatory postsynapses, which are formed and matured on a characteristic structure called a spine, inhibitory postsynapses are formed directly on the dendritic shaft. Therefore, it is currently presumed that only the accumulation of inhibitory synapse-specific proteins distinguishes the inhibitory postsynaptic membrane from the non-synaptic membrane. Motor proteins are responsible for the accumulation of inhibitory synaptic components. We hypothesized that there should be signs for efficient transport regulation at the synapse construction site. We found that Teneurin-2 (TEN2), an adhesion molecule that is required for initial contact between axons and dendrites and subsequently promotes synaptic maturation, recruits dynamic microtubules (MTs) to synaptic sites via binding to EB1, facilitating the release of cargoes at synaptic sites, and accumulating molecules required for inhibitory synapse maturation.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-053 DNA損傷による小胞体膜局在転写因子OASISの活性化を介したp53非依存的なアストロサイトの増殖制御 p53-independent regulation of astrocyte proliferation mediated by ER-resident transcription factor OASIS activated by DNA damage 齋藤敦(1)、上川泰直(1)、今泉和則(1) 1. 広島大学大学院医系科学研究科分子細胞情報学 Atsushi Saito(1), Yasunao Kamikawa(1), Kazunori Imaizumi(1) 1. Department of Biochemistry, Institute of Biomedical & Health Sciences, Hiroshima University Keyword: astrocyte, cell cycle, OASIS, p53 Cells determine to progress or stop cell cycle and the proliferation via the surveillance of the internal conditions including DNA damage and incomplete replication to avoid the loss of DNA integrity due to the aberrant proliferation. ER-resident transmembrane transcription factor OASIS, specifically expressed in astrocytes in central nervous system, is cleaved by regulated intramembrane proteolysis (RIP) in response to various cellular stresses. The cleavage by RIP generates the N-terminal fragments containing a basic leucine zipper-type DNA binding domain, which act as a transcription factor. OASIS full length and the N-terminus were gradually increased in the late stage of primary astrocytes that undergo cell cycle arrest due to the loss of DNA integrity by the telomere shortening. The N-terminal fragments were also increased by DNA damage prompted by the insults from the external stimuli such as anti-cancer drugs. The activated OASIS by DNA damage led to the transcriptional induction of p21. We found that the deficiency of OASIS caused the accelerated proliferation of long-term-cultured astrocytes. Flow cytometric analysis showed that the number of Oasis +/+, but not -/- astrocytes in G2/M phase was increased by the long-term-culture. The introduction of OASIS or p21 into the late stage of Oasis -/- astrocytes decreased in the number of BrdU-positive cells. The increased population in G2/M phase was observed in these cells, suggesting that OASIS-p21 axis stops cell cycle at G2/M phase in long-term-cultured astrocytes. Previous studies have shown that p53 is a main regulator for the induction of p21. The levels of p53 and phosphorylated p53 (the active form) were not changed in Oasis -/- astrocytes. The activation of OASIS was not also affected by the p53-knockdown, indicating that there is not any crosstalk between OASIS and p53 pathways for the induction of p21. The number of astrocytes in G2/M phase was not decreased in p53-knockdown cells. The overexpression of p53 into Oasis -/- astrocytes triggered the promotion of apoptosis. However, the number of BrdU-positive cells was not decreased, indicating that OASIS has priority over p53 to stop cell cycle of astrocytes. Our findings suggest the critical role of OASIS in astrocyte proliferation as a main regulator alternative to p53.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-054 Calcineurin欠損腸管グリア細胞における増殖能低下と分泌物質量変化：小腸の機能異常及び炎症との関連 Reduced proliferative capacity and altered levels of secreted substances in calcineurin-deficient enteric glial cells in relation to the dysfunction and inflammation of the small intestine 田中正彦(1)、寺本光(1)、平嶋尚英(1) 1. 名古屋市立大学大学院・薬学研究科・生体超分子システム解析学 Masahiko Tanaka(1), Hikaru Teramoto(1), Naohide Hirashima(1) 1. Dept Cell Biophys, Grad Sch Pharmaceut Sci, Nagoya City Univ, Nagoya, Japan Keyword: calcineurin, enteric glial cell, small intestine, inflammation Background & objective: Calcineurin (CN), a Ca²⁺/calmodulin-dependent protein phosphatase composed of A (catalytic) and B (regulatory) subunits, is involved in a number of intracellular signaling processes. Although CN is involved in various aspects of neuronal development and function, its roles in glial cells remains to be elucidated. To investigate the roles of CN in glial cells, we previously generated conditional knockout (CKO) mice lacking CNBα in glial cells by crossing floxed CNBα mice with glial fibrillary acidic protein (GFAP)-Cre mice. Because the CKO mice showed the dysfunction and inflammation in the small intestine in addition to growth retardation and postweaning death (Fujita et al., 2018; Okura et al., 2019), we have focused on enteric glial cells (EGCs) in the small intestine. EGCs have been recognized as an important cell type constituting the enteric nervous system, which controls intestinal function and homeostasis by interacting with enteric neurons and epithelial cells via secreted substances. In this study, we examined the effects of CN deficiency on proliferative capacity of EGCs and the expression and secretion of EGC-derived substances in culture to reveal the mechanisms of the dysfunction and inflammation of the small intestine in the CKO mice. Results: In primary cultures of the myenteric plexus of the small intestine, EGCs from the CKO mice showed reduced numbers and proliferative capacity compared to EGCs from control mice. In purified EGC cultures from the CKO mice, Western blot analysis showed increased expression of S100β, NF-κB p65 and GDNF in addition to GFAP. Because S100β activates the NF-κB pathway, the increased expression of S100β, as well as NF-κB p65, may result in increased production of inflammatory cytokines downstream of the NF-κB pathway. In the supernatant of purified EGC cultures from the CKO mice, ELISA analysis showed reduced secretion of TGF-β1, which has a protective role in the gut. In contrast, GDNF secretion was normal in purified EGC cultures from the CKO mice. Conclusion: CN deficiency leads to reduced proliferative capacity of EGCs and abnormal expression and secretion of EGC-derived substances, which may contribute to the dysfunction and inflammation of the small intestine.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-055 グリア細胞におけるシンタキシン１B欠損はGABA放出の異常を引き起こす Impaired GABAergic synaptic transmission was caused by loss of syntaxin1B in glial cells 小藤剛史(1)、三嶋竜弥(2)、齋藤綾子(2)、藤原智徳(2,3) 1. 杏林大学医学部RI部門、2. 杏林大学医学部病態生理学、3. 埼玉医科大学保健医療学部臨床検査学科 Takefumi Kofuji(1), Tatsuya Mishima(2), Ayako Saito(2), Tomonori Fujiwara(2,3) 1. RI Lab, Kyorin Univ Sch Med, 2. Dept Med Physiol, Kyorin Univ Sch Med, 3. Sch Med Tech, Fac Health Med Care, Saitama Med Univ Keyword: syntaxin1B, glial cells, GABAergic transmission Syntaxin1B (STX1B) is abundantly expressed in neuronal plasma membrane as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, and regulates the exocytosis of synaptic vesicles. We previously reported that STX1B null mutant mice died within 2 weeks after birth, and exhibited structural abnormality in CNS partly caused by low cell viability of neurons. Both of glutamatergic and GABAergic fast synaptic transmission was impaired in STX1B null mutant mice. Furthermore, STX1B null mutant mice exhibited motor function impairment, and STX1B heterozygote mutant mice tended to be seizure phenotype which was caused by abnormal GABAergic system. We also found that STX1B was predominantly expressed in glial cells compared to another isoforms of STX1, HPC-1/syntaxin1A (STX1A). Therefore, STX1 is associated with vesicle exocytosis in both neurons and glial cells, however, little is known about roles of STX1 in glial cells. In this study, we found that GABA uptake through GABA transporter was reduced in STX1B null and heterozygote glial cells, but not in STX1A null glial cells. Expression of GABA transporters, such as GAT-1 and GAT-3, was decreased in STX1B null glial cells compared to that of WT glial cells. On the other hand, glutamate uptake into STX1B null glial cells was normal. Furthermore, we examined if loss of STX1 in glial cells affect on the synaptic transmission or synapse formation using WT hippocampal neurons cultured on wild type (WT) or STX1B null glial cells. At DIV 7 or 14, the frequency of miniature IPSC (mIPSC) in WT neurons was not affected by loss of STX1B in glial cells, and number of main branches or that of synaptophysin puncta on dendrites of WT neurons was not different among each genotype of glial cells. However, the frequency of mIPSC in WT neurons cultured on STX1B null glial cells was reduced compared to that on WT glial cells at DIV 20 or more. Thus, loss of STX1B in glial cells caused impaired GABAergic synaptic transmission, and might be associated with an abnormal GABAergic system caused by an imbalance of excitation and inhibition. STX1B roles in glial cells on the neuronal network will be further discussed.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-056 高速分化技術によりヒトiPS細胞から作成したアストロサイトの神経細胞サポート機能の評価 Functional evaluation of iPSC-derived astrocytes generated by a rapid differentiation method 和田唯奈(1)、矢本梨恵(1)、瀬尾学(2)、細谷俊彦(1) 1. 株式会社リコー RFS バイオメディカル事業センターバイオメディカル研究開発室、2. Elixirgen Scientific, Inc. Yuina Wada(1), Rie Yamoto(1), Manabu Seo(2), Toshihiko Hosoya(1) 1. Biomedical Research and Development Department, Biomedical Business Center, RICOH Futures BU, Ricoh Company, Ltd., Kanagawa, Japan, 2. Elixirgen Scientific, Inc., Baltimore, USA Keyword: iPSC, astrocytes, neurons, in vitro assay Astrocytes provide various supportive functions to neurons and contribute to the regulation of the central nervous system. Recent studies have revealed that astrocytes play important roles in the brain function and nervous system diseases. The neuron-astrocyte co-culture system is an in vitro tool that can evaluate the supportive functions of astrocytes and therefore is an attractive test bed for drug screenings, toxicity assays, and disease research. The Quick-Tissue^TM technology (Elixirgen Scientific, Inc.) is a transcription factor-based method for rapid differentiation of human induced pluripotent stem cells (iPSCs) into desired cell types. The method can generate a pure population of astrocytes (Es astrocytes) within 28 days, which is about three times faster than the conventional methods. In the present study, we examined whether Es astrocytes provide supportive functions that are similar to that of in vivo astrocytes. We co-cultured neurons with Es astrocytes and characterized their responses to various drugs using calcium imaging. The cells were labeled with the calcium indicator Cal-520 and imaged using the microplate reading system FDSS μCell system. The co-cultured neurons responded to antagonists and agonists for receptors of various neurotransmitters as expected. Calcium oscillations of the co-cultured neurons exhibited higher frequency and stronger synchrony compared to that of neuronal cultures without astrocytes, suggesting supportive functions of the astrocytes. The observed supportive functions were robust under a wide range of culture conditions. These results suggest that Es astrocytes provide supportive functions that are similar to those of in vivo astrocytes and that they are useful for pharmacological assays of glial functions.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-057 神経回路形成因子LOTUSはアミロイドβによるミクログリアの活性化状態を制御する A neural circuit formation factor LOTUS regulates amyloid-β protein-induced activation states in microglia. 井上愛海(1)、松林潤平(1)、川口祐生(1)、竹居光太郎(1) 1. 横浜市立大学　生命医科学研究科　生体機能医科学研究室 Ami Inoue(1), Junpei Matsubayashi(1), Yuki Kawaguchi(1), Kohtaro Takei(1) 1. Molecular Medical Bioscience Laboratory, Yokohama City University Graduate School of Medical Life Science, Yokohama, Japan Keyword: microglia, Alzheimer's Disease, LOTUS Microglia is an important component maintaining functions of the central nervous system (CNS). Microglia shows various functions such as synaptic depletion, phagocytosis of dead cells and producing inflammation or anti-inflammation factors in the CNS. Amyloid-beta protein (Aβ) is one of the onset factor for Alzheimer's disease (AD). In AD, microglia plays a central role in regulation of Aβ clearance and degradation. On the other hand, Aβ triggers the overactivation of microglia producing pro-inflammatory cytokines, thereby inducing neuroinflammation and neuronal cell death. Lateral olfactory tract usher substance (LOTUS) contributes to the neural circuit formation and nerve regeneration. However, the functional relationship between LOTUS and microglia function is completely unknown. In this study, we examined whether LOTUS suppresses the Aβ induced-activation of microglia that produces pro-inflammatory cytokines in AD. To address this issue, we first investigated expression of LOTUS in microglia and found no expression of LOTUS in primary cultured microglial cells. Next, we examined the effect of LOTUS on the Aβ binding to microglia and found that soluble-form of LOTUS (sLOTUS) inhibited the binding of Aβ to microglia. The data suggest the possibility of antagonistic action of LOTUS in Aβ-induced activation of microglia. Therefore, we examined if inhibition of Aβ binding to microglia could suppress the production of pro-inflammatory cytokines. We found that increase of pro-inflammatory cytokines increased by Aβ was suppressed by sLOTUS administration. These findings suggest that LOTUS may suppress Aβ-induced activation states inducing neuroinflammation in microglia, and lead to the idea that LOTUS may be involved in inhibition microglia-mediated pathological progression in AD.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-058 生体外微小物質に対する脳内免疫機構 Brain immune mechanisms against exogeneous nanoparticles 大柿安里(1)、池谷裕二(1,2,3)、小山隆太(1,3) 1. 東京大学大学院薬学系研究科、2. 脳情報通信融合研究センター、3. 東京大学 Beyond AI 研究推進機構 Ari Ogaki(1), Yuji Ikegaya(1,2,3), Ryuta Koyama(1,3) 1. Grad Sch Pharmaceut Sci, Univ Tokyo, Tokyo, Japan, 2. Institute for AI and Beyond, Univ Tokyo, Tokyo, Japan, 3. Center for Information and Neural Networks, National Institute of Information and Communications Technology, Osaka, Japan Keyword: Glial cells, Brain immune mechanisms, Exogeneous nanoparticles, Complement マイクログリアは、脳内の主要な免疫細胞であり、生体由来の異物を貪食することで脳内恒常性を維持することが知られてきた。しかし、脳内に侵入した生体外の異物に対して、脳内でどのような免疫機構が働くのかについては未解明であった。本研究では、血管を介して脳内に侵入した生体外異物に対する脳内免疫機構の解明を目的とした。　我々は、脳内に侵入することが報告されている外因性微粒子であるParticulate Matter2.5 (PM_2.5) に着目し、これを生体外異物として皮下投与することで、生体外異物の脳内侵入モデルを作製した。脳内に侵入した生体外微小物質の周囲では、マイクログリアの形態が変化し生体外微小物質はマイクログリアに貪食されていた。マイクログリアによる貪食の分子メカニズムを検討するため、生体外微小物質曝露マウス全脳においてタンパク質発現量解析を行ったところ、ケモカインCC chemokine ligand 17 (CCL17) が増加した。CCL17の受容体CC chemokine Receptor 4 (CCR4) のアンタゴニストC-021を処置すると、マイクログリアによる貪食が抑制された。したがって、マイクログリアによる生体外微小物質の貪食はCCL17-CCR4シグナルを介する可能性が示唆された。　CCL17は血管内皮細胞の刺激によって分泌促進されることが知られている。また、その受容体であるCCR4は血管周囲のアストロサイトに高発現していた。このことから、生体外微小物質の侵入に伴い血管内皮細胞が刺激されることでCCL17が増加し、血管周囲アストロサイトに高発現するCCR4が活性化される可能性が考えられた。そこで、アストロサイトの単離培養系にCCL17を処置したところ、補体Complement 3 (C3)の分泌が増加した。さらに、生体外微小物質曝露マウス脳の免疫染色によって、C3が脳内に侵入した生体外微小物質に付着していることを発見した。C3はアストロサイトで高発現し、マイクログリアによるシナプス貪食において”eat-me”シグナルとして機能することが知られている物質である。従って、アストロサイトにおけるCCR4が活性化することでC3の分泌が促進され、そのC3が生体外微小物質に付着することでマイクログリアに貪食される可能性が示唆された。　以上の結果から、血管を介して脳内に侵入した生体外微小物質は血管-アストロサイト-マイクログリアが協調的に働くことでマイクログリアに貪食されることが明らかとなった。これは、生体外の異物に対する脳内免疫機構の一端であり、現在まで未解明である生体外の異物に対する脳内防御機構の分子的メカニズムを明らかにした点で意義深い。

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-059 Exploring prefrontal cortex astrocytic contributions to neural circuits Bolati Wulaer(1), Jun Nagai(1) 1. RIKEN Center for Brain Science Laboratory for Glia-Neuron Circuit Dynamics Keyword:* Prefrontal cortex, Astrocytes, DREADD, Behavior The prefrontal cortex (PFC) is a central hub circuit that has long been implicated in adaptive behavior and psychiatric disorders. Due to rapid developments of tools, recent reports in the last few years have been shining a light on glial cells, including astrocytes, indicating that they may have much more significant roles in mediating behavior than previously thought. However, the neural circuit and behavioral functions of PFC astrocytes are incompletely understood. Therefore, the present study set out to systematically explore how astrocyte signaling occurs and regulates the PFC microcircuits in ways that are behaviorally consequential. We firstly characterized when and how astrocyte signals can be evoked by various neurotransmitters with 2-photon excitation microscopy and found that norepinephrine triggers astrocyte Ca²⁺ signaling via Gq-coupled a1-adrenoceptors. Second, we mimicked this signaling pathway by deploying chemogenetic activation of Gq-coupled DREADD (hM3Dq) and carefully validated that both acute and repeated ex vivo and in vivo CNO application evoked astrocyte Gq/Ca²⁺ signaling in PFC. Third, to explore the behavioral consequences of this, we then performed a behavioral testing battery. By exploring how astrocytes regulate neural circuits and behavior, the present study will serve as a basis for future studies and aim to uncover the roles of astrocytes in nervous system function and, therefore, and also their potentials as targets to be exploited to correct disease-associated behavioral changes.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-060 ミクログリアにおけるCX3CR1とSIRPαの機能的相互作用 Functional interaction between CX3CR1 and SIRPα in microglia 今井武史(1)、富山飛鳥(1)、水谷瑠依(1)、松下紗世子(1)、森田紋子(1)、浦野江里子(1)、神宮大輝(1)、林由里子(2)、的崎尚(3)、大西浩史(1) 1. 群馬大学大学院保健学研究科、2. 群馬パース大学大学院保健科学研究科、3. 神戸大学大学院医学研究科 Takeshi Imai(1), Asuka Tomiyama(1), Rui Mizutani(1), Sayoko Matsushita(1), Ayako Morita(1), Eriko Urano(1), Daiki Jingu(1), Yuriko Hayashi(2), Takashi Matozaki(3), Hiroshi Ohnishi(1) 1. Gunma University Graduate School of Health Sciences, 2. Gunma Paz University Graduate School of Health Sciences, 3. Kobe University Graduate School of Medicine Keyword: MICROGLIA, SIRPα, CX3CR1 SIRPα (Signal regulatory protein α) is expressed in macrophages and negatively regulates phagocytosis. In the central nervous system, SIRPα is dominantly expressed in neurons and microglia (MG). MG, a macrophage-like cell in the brain, plays an important role in the removal and repair of damaged brain tissue. We have reported that MG was spontaneously activated in the brain of microglia-specific SIRPα conditional knockout (MG-SIRPα cKO) mice, and the mutant mice were resistant to animal model of demyelination. MG-SIRPα cKO mice are also resistant to age-related motor learning disability. Thus, SIRPα-deficient microglia may have a protective effect against demyelination and brain damage due to aging. In these previous studies, we used MG-SIRPα cKO mice generated by crossing Cx3cr1-CreERT2 mice, which had tamoxifen (TAM)-dependent microglial-specific Cre drivers, and SIRPα-flox mice. During the research process, we noticed that the TAM untreated Cx3cr1-CreERT2: SIRPα-flox mice, which had intact SIRPα gene, exhibited impaired motor learning in accelerated rotarod test. On the other hand, motor learning disabilities recovered almost completely when TAM was administered to these mice to induce SIRPα gene deletion in microglia. In the Cx3cr1-CreERT2: SIRPα-flox mice, the CreERT2 construct is knocked in to the endogenous Cx3cr1 gene, thus these are Cx3cr1 heterozygous knockout mice. Motor learning disabilities in TAM-untreated Cx3cr1-Cre: SIRPα-flox mice is likely due to the heterozygous lack of Cx3cr1, and TAM-induced deletion of SIRPα seems to complement the effects. CX3CR1 is expressed in microglia and functions as a receptor for the chemokine CX3CL1. The CX3CR1-CX3CL1 signal regulates microglial survival, motility, and phagocytosis, and has also been reported to participates in synaptic pruning. Although the physiological function of SIRPα in microglia is unclear yet, it may act antagonistically with Cx3CR1-CX3CL1 signal and thereby control the function of microglia.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-061 Delayed Reduction of Excitatory Synapses in CA3 following Systemic Inflammation Tatsuya Manabe(1,2), Ildiko Racz(1,2), Stephanie Schwartz(1,2), Francesco Santarelli(2), Julius V. Emmrich(3), Jonas J. Neher(4,5), Michael T. Heneka(1,2,6) 1. Department of Neurodegenerative Diseases and Geriatric Psychiatry, University of Bonn Medical Center, Bonn, Germany, 2. German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany, 3. Department of Neurology and Experimental Neurology, Charite – Universitatsmedizin Berlin, Berlin, Germany, 4. Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tubingen, Tubingen, Germany, 5. German Center for Neurodegenerative Diseases (DZNE), Tubingen, Germany, 6. Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, USA Keyword:* Microglia, Sepsis, Synaptic pruning, Complement Background Sepsis-associated encephalopathy (SAE) represents acute cerebral dysfunctions caused by sepsis (such as delirium and coma). Notably, elderly individuals who survive sepsis show persisting cerebral disturbances because they are at higher risk of dementia onset for up to 14 years following the recovery (Manabe and Heneka, 2021). As an animal model of systemic inflammation that simulates sepsis, intraperitoneal injection of lipopolysaccharide (LPS) led to glial activation and destruction of neurons and synapses (Weberpals et al. 2009; Tejera et al. 2019; Beyer et al. 2020). However, it remains unexplored whether activated microglia may play a role in cerebral damage that can ultimately drive cognitive impairments. Methods Middle-aged mice (14–16 months old) received two structurally distinct bacterial strains of LPS (i.e. Salmonella enterica and E. coli O55 B5) via intraperitoneal route on two consecutive days. Neuroinflammation, synapse density and synaptic pruning by microglia were histologically assessed using the brains collected at 7 or 63 days post-injection (dpi). To this end, we analysed microglial morphology, synaptic puncta density and density of synaptic puncta inside microglial lysosomes using super-resolution microscopy images. Results Analysis of microglial morphology in the hippocampus demonstrated a reduction of the number and length of microglial processes at 7 dpi. Despite the progressive microglial priming during ageing, morphology was normalised by 63 dpi. Both excitatory pre- and post-synaptic puncta density decreased selectively in the CA3 subfield of the hippocampus at 63 dpi. Likewise, we found a reduction of synaptic complement factor C3 (that is, an important molecule frequently required for microglial synaptic pruning). In contrast, similar changes in synapses and synaptic C3 were not found in CA3 at 7 dpi or in other regions of the hippocampus (CA1 and dentate gyrus). Nevertheless, our analysis of synaptic pruning suggested that the density of excitatory synaptic puncta inside CA3 microglia remained unchanged at 7 dpi and 63 dpi. Conclusion Systemic LPS challenge induced persistent neuroinflammation in the hippocampus for more than seven days in middle-aged mice. The number of excitatory synapses, together with synaptic C3, decreased in CA3 between 7 dpi and 63 dpi. Because this particular region of the hippocampus is known to be vulnerable to age-related, complement-dependent synapse loss (Shi et al., 2015), it seems possible that LPS injection may accelerate the synaptic changes during ageing. Due to the simultaneous reduction of the synaptic C3, microglia may engulf more synaptic puncta between two points of our investigation. In the future, it will be of great importance to study whether the altered abundance of CA3 synapses can worsen the cognitive functions of mice that survive the LPS challenge.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-062 オリゴデンドロサイトへ分化誘導したグリア前駆細胞から放出されるエクソソーム Analysis of exosomes released from a glial progenitor cell-line after induction of oligodendrocyte differentiation 小野健治(1)、伊藤友香(1)、大橋和哉(1)、鈴木弘美(1)、澤田誠(1) 1. 名古屋大学環境医学研究所 Kenji Ono(1), Yuka Ito(1), Kazuya Ohashi(1), Hiromi Suzuki(1), Makoto Sawada(1) 1. Res Inst Env Med, Nagoya Univ, Aichi, Japan Keyword: EXOSOMES, DEMYELINATION, GLIA, MICROGLIA OS3ChR2, a glial progenitor cell-line expressing channelrhodopsin-2 (ChR2), activates signaling pathways such as Akt and ERK1/2 in response to alteration in the intracellular ionic environment by blue light exposure. The phosphorylation of Akt induces the differentiation of OS3ChR2 into oligodendrocytes. When photo-activated OS3ChR2 was injected into a mouse model of demyelination, it was thought that OS3ChR2 not only functions as an oligodendrocyte but also alleviates demyelination symptoms by improving cell-cell interaction around demyelination, but the mechanism is not well understood. In this study, we focused on exosomes, which are responsible for intercellular communication, and investigated the alteration in the properties of exosomes during induction of differentiation by photo-activation. Since exosome release is known to occur in both endosormal sorting complexes required for transport (ESCRT)-dependent and -independent pathways, we examined alteration in exosome release in the presence of inhibitors of each pathway (Manumycin A and GW4869). The increase in exosomes released from photo-activated OS3ChR2 was inhibited in the presence of Manumycin A or GW4869. The increase in phosphorylation of Akt and ERK1/2 was also inhibited in the presence of these inhibitors. Analysis of miRNAs in exosomes showed that the content of miRNAs that exacerbate inflammation was reduced in the photo-activated group. When exosomes from OS3ChR2 were added to primary microglia, there was an increase in the expression of iNOS (M1 marker) and a decrease in the expression of CD206 (M2 marker), but these changes were hardly observed in the photo-activated group. Similar results were obtained when exosomes were injected into demyelinated model mice. These results suggest that glial progenitor cells induced to differentiate into oligodendrocytes change not only the amount of released exosome but also the functional substances contained in exosomes. In addition, glial progenitor cells may communicate with microglia via exosomes at the site of demyelination.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-063 加齢による運動学習能低下が改善するミクログリア特異的SIRPα欠損マウスにおける遺伝子発現解析 Gene expression analysis of microglia-specific SIRPα knockout mice exhibiting improved age-related motor learning disability 水谷瑠依(1)、富山飛鳥(1)、今井武史(1)、松下紗世子(1)、森田紋子(1)、浦野江里子(1)、橋本美穂(2)、林由里子(3)、的崎尚(4)、大西浩史(1) 1. 群馬大学大学院保健学研究科、2. 九州保健福祉大学薬学部、3. 群馬パース大学大学院保健科学研究科、4. 神戸大学大学院医学研究科 Rui Mizutani(1), Asuka Tomiyama(1), Takeshi Imai(1), Sayoko Matsushita(1), Ayako Morita(1), Eriko Urano(1), Miho Sato-Hashimoto(2), Yuriko Hayashi(3), Takashi Matozaki(4), Hiroshi Ohnishi(1) 1. Gunma Univ Grad Sch Health Sci, Gunma, Japan, 2. Sch Pharm Sci, Kyushu Univ Health Welfare, Miyazaki, Japan, 3. Gunma Paz Univ Grad Sch Health Sci, Gunma, Japan, 4. Kobe Univ Grad Sch Med, Kobe, Japan Keyword: microglia, aging, motor leaning SIRPα (Signal regulatory protein α) is a member of immunoglobulin superfamily membrane proteins. SIRPα is phosphorylated on tyrosine residues in its cytoplasmic region and thereby binds and activates protein tyrosine phosphatase Shp2. The extracellular region of SIRPα interacts with its ligand CD47, another membrane protein, and thereby constitutes a cell-cell contact signal. The interaction between SIRPα on phagocytes and CD47 on phagocytic targets negatively regulates phagocytosis. In the central nervous system, microglia (MG), the tissue macrophages of the brain, also express SIRPα. We have found that MG-specific ablation of SIRPα results in spontaneous activation of MG in healthy intact mouse brain. Furthermore, in the MG-specific SIRPα conditional knockout (MG-SIRPα cKO) mice, experimentally-induced demyelination was attenuated compared with normal control mice, suggesting that SIRPα-deficient MG have protective effect on brain injury. More recently, by the use of rotarod test, we found that genetic ablation of microglial SIRPα improved age-related motor learning disability in aged mice. Here we compared gene expression profiles in the brains of aged MG-SIRPα cKO and control mice by microarray analysis and RT-qPCR analysis. We identified gene products, expressions of which were significantly altered in the brain of MG-SIRPα cKO mice compared with age-matched control mice. We also investigated the characteristics of microglia and age-related degeneration in the brains of mutant mice by immunohistochemical analysis. Age-related degeneration of the cerebellum was not significantly different between mutant and control mice, while mutant MG exhibited characteristics specific to protective MG. SIRPα could act as a checkpoint, which suppresses induction of a subgroup of protective MG, and inactivation of microglial SIRPα may be an effective approach to prevent age-related decline of the brain function.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-064 低酸素環境順応下における皮質方向の深さに依存したアストロサイトの形態変化 Morphological changes of astrocytes along the cortical depth by hypoxic adaptation 野田陽太(1)、西野智博(2)、田中草介(1)、正本和人(1)、宮脇陽一(1) 1. 電気通信大学大学院情報理工学研究科、2. 電気通信大学情報理工学部 Yota Noda(1), Tomohiro Nishino(2), Sosuke Tanaka(1), Kazuto Masamoto(1), Yoichi Miyawaki(1) 1. Graduate School of The University of Electro-Communications, Tokyo, Japan, 2. The University of Electro-Communications, Tokyo, Japan Keyword: Astrocyte, DCNN, High Dimensional Image Feature , Cell Recognition Astrocytes change their cellular morphology in various pathological conditions. Deep Convolutional Neural Network (DCNN) is useful to comprehensively describe such changes in morphological features of astrocytes, and the DCNN-defined features can predict whether astrocytes are under hypoxic adaptation more accurately than the human-defined (Nishino et al., 2017). We also found a certain amount of variations in the morphological changes over individual cells, but it remains unclear what major factors cause such variations. In this study, we focused on individual cellular location in the cortical tissues and examined whether it has systematic effects on morphological variations. We analyzed the data sets of astrocyte images acquired from the same mouse taken by a two-photon microscope at days 0, 7, 14, and 21 after the onset of hypoxic adaptation and those from a different mouse at days 0 and 21 after the onset of hyperoxic adaptation. After all the images were normalized and spatially-smoothed, hierarchical image features were extracted using AlexNet (Krizhevsky et al., 2012) for each layer. The morphological changes were represented by differential vectors of the image features between pre and post-adaptation for each cell, and its amount was defined by the Euclidean norm of the differential vectors. The similarity in the morphological changes was quantified by the directional cosine between the differential vectors. Hierarchical clustering was then performed on the similarity, and cellular locations for estimated clusters, particularly the depth from the cortical surface, were investigated. Results showed that the amount of the morphological change defined by the higher-layer features increased as days elapsed and were larger for the hypoxic adaptation than those for the hyperoxic adaptation. Furthermore, the similarity of morphological changes across astrocytes was divided into two clusters, and the mean depth from the cortical surface was significantly different between the clusters. These results confirm that DCNN can extract image features with descriptive power sufficient to predict the hypoxic status of astrocytes and further demonstrate that hypoxic influence on cellular morphology depends on its depth from the cortical surface.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-065 サル大脳皮質におけるグリア細胞の時期および領野特異的な動態変化 Time- and area-specific dynamics of glial cells during postnatal development of the monkey cerebral cortex. 山中智子(1)、田中江美子(1)、高田昌彦(1)、井上謙一(1) 1. 京都大学霊長類研究所 Tomoko Yamanaka(1), Emiko Tanaka(1), Masahiko Takada(1), Kenichi Inoue(1) 1. Primate research institute, Kyoto university, Aichi, Japan Keyword: Postnatal Development, Primate , Cerebral Cortex, Glia The areal diversity of cortical growth in the primate brain is essential for age-appropriate acquisition of various abilities in sensory, motor, and cognitive functions during postnatal development. Ample evidence from rodent models suggests that glial cells, such as astrocytes, microglia, oligodendrocytes, and oligodendrocyte precursor cells (OPCs), may play critical roles in organizing the functional architecture of the cerebral cortex. However, the possible involvement of glial cells in the cortical development in primates remain to be understood. In the present study, we first examined the expression patterns of glial fibrillary acidic protein (GFAP), which has been reported to be differentially expressed in the primate cortex depending on the age and subtype of astrocytes, in different cortical areas of macaque monkeys from neonate to adult. In our immunohistochemical analysis, we found that global level of GFAP expression in all areas tested was rather weak until puberty, and was gradually increased toward adult. Interestingly, clusters of protoplasmic astrocytes displaying strong GFAP immunoreactivity emerged in late infancy and reached a peak in puberty. These clusters were prominently located in the middle cortical layers of anterior cingulate cortex (ACC; area 24). Given that no such event was observed in the developing mouse brain, the time- and area-specific occurrence of protoplasmic astrocytes during cortical development seemed unique to the primate brain. We further identified that the protoplasmic astrocytes seen in the ACC was composed of the reactive phenotype, similar to that detected in the damaged brain, and appeared to surround activated microglia and elongated oligodendrocytes/OPCs, which also emerged in puberty. In summary, we defined that astrocytes, microglia, and oligodendrocytes/OPCs were coordinately activated in the monkey ACC during postnatal development. This suggests that such glial cell dynamics may contribute to the maturation of the ACC, one of the executive centers of cognitive and emotional control in the brain, leading to the age-appropriate acquisition of these functional roles.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-066 ミクログリアの炎症性サイトカインの誘導におけるNFkBの関与 Involvement of NFκB in the induction of inflammatory cytokines in microglia 中嶋一行(1)、石嶋貴志(1) 1. 創価大学 Kazuyuki Nakajima(1), Takashi Ishijima(1) 1. Soka University Keyword: Inflammatory cytokine, microglia, NFkB As reported previously, we indicated that microglia are a major glial cell type inducing inflammatory cytokines including tumor necrosis factor alpha (TNFα), interleukin 1beta (IL-1β) and interleukin-6 (IL-6). Some signaling molecules have been speculated to contribute to the induction of the inflammatory cytokines. However, a requirement of NFκB in these inductions was not clear and remains to be solved. In this study, we examined a relation of NFκB to the induction of the inflammatory cytokines in lipopolysaccharide (LPS)-stimulated rat microglia in vitro. At first, we confirmed that the three inflammatory cytokines were induced in microglia by the stimulus of LPS. In this reaction, NFκB (p65) was phosphorylated and IκB was degraded, indicating that NFκB was activated. We next examined that a NFκB inhibitor ammonium pyrrolidine dithiocarbamate (APDC) can suppress LPS-dependent NFκB activation in microglia. Pretreatment of microglia with APDC prior to LPS stimulation significantly decreased the phosphorylation of NFκB (p65) and the degradation of IκB, indicating that APDC is an effective NFκB inhibitor. We thus investigated whether or not APDC inhibits the induction of inflammatory cytokines in LPS-stimulated microglia. As the result, LPS-dependent IL-1β induction was significantly suppressed by pretreatment with APDC, but those of TNFα and IL-6 were not affected by the NFκB inhibitor. Taken together, these results strongly suggest that NFκB is associated with the induction of IL-1β, but not TNFα and IL-6, in LPS-stimulated microglia.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-067 急性鼻腔炎症モデルマウスにおける末梢免疫細胞の嗅球への浸潤 Infiltration of peripheral immune cells into the olfactory bulb in a mouse model of acute nasal inflammation 浅野妃南(1)、島田厚良(2)、石井さなえ(2) 1. 杏林大学大学院保健学研究科、2. 杏林大学保健学部 Hinami Asano(1), Atuyohsi Shimada(2), Sanae Ishii(2) 1. Grad Sch Health Sci, Kyorin Univ, Tokyo, Japan, 2. Fac Health Sci, Kyorin Univ, Tokyo, Japan Keyword: NASAL INFLAMMATION, MONOCYTES, OLFACTORY BULB, MICROGLIA 環境中に含まれる細菌やウイルス、花粉やカビなどの有害物質は、呼吸により容易に鼻腔に入り、鼻腔炎症を引き起こす。ヒトでは、慢性的な鼻腔炎症は副鼻腔炎の原因となり、うつ病や不安障害といった精神疾患のリスクを高める。先行研究では、マウスに慢性鼻腔炎症を起こすと、脳の最吻側に位置する嗅球に変化が起きた。3週間以内に嗅球におけるグリア細胞の活性化、介在ニューロンの活性減少、嗅球投射ニューロンのシナプス減少が顕著に表れ、10週間以内に嗅球が有意に萎縮した。これは、鼻腔炎症が単に末梢の炎症というだけでなく、脳にも影響を及ぼすことを示している。しかしながら、嗅上皮で起こる炎症が嗅球へと伝播する様式は、まだ未解明である。そこで、本研究では、鼻腔炎症がどのようにして嗅球に影響を及ぼすのか、その最初期の現象を明らかにすることを目的とし、鼻腔炎症の急性期における嗅上皮と嗅球の変化を、組織学的及び遺伝子発現的に解析した。8週齢の成体雄マウスに、鼻腔炎症モデルとして細菌の内毒素であるリポ多糖（LPS、1μg/μL）を、対照として生理食塩水を、それぞれ両側鼻腔に10 μLずつ投与した。組織学的解析として、生理食塩水投与48時間後、LPS投与12, 24, 48, 72時間、2週間後に4％PFAにて灌流固定し、凍結切片を作製し、各種免疫染色を行った。また、遺伝子発現解析として、生理食塩水もしくはLPSを両側鼻腔に投与し、12, 24, 48時間後に新鮮脳組織を採取した後、qRT-PCRを行った。LPS投与12から48時間後にかけて、24時間後をピークに、単球・好中球、リンパ球などの血球系細胞が嗅球実質内に浸潤した。血球系細胞の浸潤は局所的であり、主に嗅球の外側2層である嗅神経層、糸球体層に多く浸潤した。単球は嗅神経層の神経線維に沿った部分と、糸球体層の血管周囲に多く分布した。好中球やリンパ球は、嗅神経層にはあまり見られなかったが、糸球体層の血管周囲に多く分布した。血球系細胞の浸潤に先立ち、血管内皮細胞はLPS投与12時間後に細胞接着分子の発現を増加した。また、血管内皮細胞や浸潤した血球系細胞は、単球や好中球を誘引するケモカイン及び炎症性サイトカインを発現した。嗅球実質のミクログリアは、LPS投与48時間後に最も活性化し、分裂・増加しながら、抗炎症性サイトカインの発現を増加した。嗅球におけるこれらの反応は2週間後には収束した。以上の結果から、鼻腔炎症の急性期に血球系細胞が一過性に嗅球に浸潤し、炎症性サイトカインを産生するが、その後、活性化したミクログリアが抗炎症性サイトカインを発現することで、嗅球の炎症収束に寄与すると考えられた。また、血球系細胞の嗅球への浸潤経路として、嗅上皮から嗅神経を介する経路と、嗅球の血管を介する経路の2つが考えられた。これら一連の反応が鼻腔炎症の最初期に起こる嗅球の反応であり、このような反応が繰り返し起こると嗅球は萎縮し、精神疾患のリスクが高まると考えられた。		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-068 光遺伝学とカルシウムイメージングによるホヤ幼生のグリア上衣細胞の生理機能の解析 Analysis of neuron-glia interactions by optogenetics and calcium imaging in ascidian larvae 大川奈菜子(1)、保田一貴(2)、谷口倫菜(2)、本村晴佳(1)、岡畑美咲(1,2)、久原篤(1,2)、日下部岳広(1,2) 1. 甲南大学自然科学研究科、2. 甲南大学　理工学部　統合ニューロバイオロジー研究所 Nanako Okawa(1), Kazuki Yasuda(2), Michina Taniguchi(2), Haruka Motomura(1), Misaki Okahata(1,2), Atsushi Kuhara(1,2), Takehiro G. Kusakabe(1,2) 1. Graduate School of Natural Science, Konan university, 2. Inst. for Inte. Neurobiol., Dept. of Biol., Konan Univ., Kobe, Japan Keyword: optogenetics, Calcium imaging, Ciona, glia The human brain contains about 100 billion neurons. In addition, there are as many glial cells as there are neurons in the brain, and the glial cells occupy a large portion of the brain volume. There are four types of glial cells: astrocytes, oligodendrocytes, microglia, and ependymal cells. Glial cells were once thought to be "glue-like" cells that filled the gaps between neurons, but in recent years, a growing number of studies suggest that glial cells actively exchange information with neurons and play important roles in higher brain functions. However, it is still unclear that how glial cells are involved in higher brain functions. In this study, we adopt the ascidian Ciona intestinalis type A, the closest invertebrate relative of vertebrates, as a simple model to study neuron-glia interactions in the brain function. The Ciona larva has a central nervous system homologous to that of vertebrates. Its nerve cord, the spinal cord homolog, is mainly composed of glial ependymal cells. We previously reported that active Ca²⁺transients in the nerve cord ependymal cells were associated with swimming behavior and suggested that the glial cells are involved in the control of neurons and muscle activity (Okawa et al. Sci. Rep. 10, 18590, 2020). In this study, we analyzed the causal mechanism of glial cell activation using optogenetics in combination with calcium imaging. To analyze the relationship between neuronal activation and glial cell activity, ChrimsonR, a light-gated ion channel, was expressed in cholinergic neurons, excitatory neurons involved in tail movement, and calcium imaging of the glial cells in the nerve cord was performed while cholinergic neurons were activated by light irradiation. When the cholinergic neurons were activated, the Ca²⁺concentration in glial cells increased in a few seconds. We also examined whether acetylcholine receptors were expressed in the glial cells of the nerve cord by single-cell transcriptome analysis. We found that the glial cells in the nerve cord express genes encoding nicotinic acetylcholine receptors. These results suggest that glial ependymal cells receive information from cholinergic neurons, which in turn triggers Ca²⁺ transients in the glial cells. The Ciona larva has morphologically only one type of glial cells, the ependymal cells, but they may exhibit functional properties of both oligodendrocytes and astrocytes in addition to those of ependymal cells.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-069 ミクログリアにおけるスフィンゴ脂質代謝と炎症反応に対するスフィンゴシンキナーゼ１阻害の効果 Effect of sphingosine kinase 1 inhibition on sphingolipid metabolic pathway and inflammatory responses in microglia 南畑朋輝(1)、高野桂(1)、森山光章(1) 1. 大阪府立大学 Tomoki Minamihata(1), Katsura Takano(1), Mitsuaki Moriyama(1) 1. Osaka Prefecture University Keyword: microglia, sphingosine kinase, sphingosine-1-phosphate, Alzheimer's disease Previous studies demonstrated that neuroinflammation by microglia contribute to the pathogenesis and exacerbation of Alzheimer’s disease (AD). Alteration in sphingolipid metabolism have been linked to AD. For example, postmortem brain of AD patients showed the reduction in expression of sphingosine kinase 1 (SK1), a rate-limiting enzyme in sphingosine-1-phosphate (S1P) production, followed by decrease in the amount of S1P. Furthermore, accumulating evidence have suggested that the balance in sphingolipid metabolism including S1P synthesis can regulate neuroinflammation such as neuronal cell death and glial cell activation. Thus, controlling sphingolipid, especially S1P, metabolic balance is considered helpful to regulate neurodegeneration. However, the effect of the change in S1P metabolic balance on microglial inflammatory responses remain unclear. Therefore, it is worthwhile to determine the effect of the decrease in SK1 expression and S1P production on whole sphingolipid metabolic pathway and inflammatory responses in microglia for elucidating AD pathology. In the present study, we explored the influence of SK1 inhibition on sphingolipid metabolic pathway and inflammatory responses by using mouse microglial cell line, BV-2 and primary microglia. Treatment with lipopolysaccharide (LPS) increased SK1 mRNA expression in BV-2 cells. Treatment with the SK1 inhibitor (PF-543) decreased SK1 mRNA expression both in non-treated and LPS-treated BV-2 cells. SK1 inhibition also decreased LPS-induced mRNA expression of ceramidase which degrade ceramide into sphingosine, following the decrease in SK1 expression. Moreover, SK1 inhibition suppressed the LPS-induced inflammatory responses such as nitric oxide synthesis, generation of reactive oxygen species and phagocytosis both in BV-2 and primary microglia. S1P treatment exacerbated these inflammatory responses in LPS-treated BV-2 microglia. However, SK1 inhibition decreased the uptake of amyloid-beta protein in LPS-treated primary microglia. These results suggest that the inhibition of SK1 and sequentially decrease in S1P cause the imbalance in sphingolipid metabolism such as ceramide degradation and have an anti-inflammatory effect on microglia. In contrast, decrease in SK1 expression may exacerbate AD pathology through increase in amyloid-beta deposits due to low microglial uptake of amyloid-beta.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-070 PKCによりリン酸化された L-MPZ は末梢神経ミエリン形成期に有意に増加する Significant increase of PKC-phosphorylated L-MPZ in peripheral myelin formation 山口宜秀(1)、岡本紘佳(1)、杉本拓真(1) 1. 東京薬科大学薬学部 Yoshihide Yamaguchi(1), Hiroka Okamoto(1), Takuma Sugimoto(1) 1. Sch of Pharm, Tokyo Univ of Pharm and Life Sci, Hachioji, Japan Keyword: MYELIN PROTEIN ZERO, TLANSLATIONAL READTHROUGH, PROTEIN KINASE C, PHOSPHOLYLATION Large myelin protein zero (L-MPZ) is a translational readthrough isoform of peripheral myelin protein zero (MPZ/P0), that contains an additional domain at the cytoplasmic C terminus. L-MPZ is synthesized from the P0 mRNA by translational readthrough of the canonical stop codon. In P0 protein, there is a protein kinase C (PKC)-dependent phosphorylation site in the cytoplasmic C-terminal domain, which is related to the cell adhesion property. L-MPZ has another PKC-dependent phosphorylation site in the extra L-MPZ specific domain as well as the same P0 PKC- phosphorylation site. Previously, we revealed that L-MPZ is phosphorylated and has two phosphorylation states. While synthesis of L-MPZ increases with that of P0 protein during peripheral myelin formation, the physiological function of L-MPZ is still unclear. To elucidate the role of L-MPZ in myelin formation, we investigated the changes of PKC-mediated phosphorylated L-MPZ in mouse sciatic nerve development. For Western blot analysis, an anti-PKC-phosphorylated L-MPZ-specific domain antibody was generated. Using this antibody, Western blot analyses of sciatic nerve homogenates from postnatal day 3 (P3), P5, P7, P10, P14, P21, P28, and adult mice were performed. Significant increase of phosphorylated L-MPZ was observed during a period of active myelin formation in the sciatic nerve (peaks at P14 and P21). Especially, increase of two-site phosphorylated L-MPZ form was demonstrated by Western blotting using a trapping reagent of phosphorylated protein (Phos-tag). These results suggest that PKC-phosphorylated (particularly, two-site phosphorylated) form of L-MPZ may be significant in peripheral myelin formation.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-071 Toll様受容体7を介した全身性エリテマトーデスモデルマウスにおけるグリア細胞特異的な炎症機序について Glial cell-type specific inflammation in Toll-like receptor 7 agonist-induced mouse model for systemic lupus erythematosus 下川梨津子(1)、祖父江顕(1,2)、小峯起(1)、伊藤綾香(3,4)、菅波孝祥(3)、山中宏二(1) 1. 名古屋大学環境医学研究所病態神経科学分野、2. 名古屋大学環境医学研究所附属MIRAIC-未来の医学研究センター、3. 名古屋大学環境医学研究所分子代謝医学分野、4. 名古屋大学高等研究院 Ritsuko Shimogawa(1), Akira Sobue(1,2), Okiru Komine(1), Ayaka Ito(3,4), Takayoshi Suganami(3), Koji Yamanaka(1) 1. Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, 2. Medical Interactive Research and Academia Industry Collaboration Center, Research Institute of Environmental Medicine, Nagoya University, 3. Department of Molecular Medicine and Metabolism, Research Institute of Environmental Medicine, Nagoya University, 4. Institute for Advanced Research, Nagoya University Keyword: Astrocyte, Microglia, Toll-like receptor 7, neuroinflammation Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting multiple organs as well as peripheral and central nervous system (CNS) involvement. Previous studies have reported a higher risk of developing dementia in the patients with SLE. However, the mechanisms of systemic and central immune system crosstalk in SLE are not fully understood.In this study, we examined the neuroinflammation in the brains of Toll-like receptor 7 (TLR7) agonist-induced SLE model mice, which are established by the chronic skin administration of imiquimod (IMQ) on the ear of wild-type C57BL/6 female mice at 6 weeks of age. We used Vaseline-treated mice as control. We confirmed a significant increase in splenic weight and an elevated level of anti-nuclear antibody in serum of IMQ-treated mice as a hallmark of SLE. In addition to validation of SLE model, we measured the mRNA expressions in inflammatory cytokines and activated glial genes using the cerebral cortices of IMQ-induced model by quantitative RT-PCR. The mRNA levels of inflammatory cytokines such as Ccl5 and Cxcl10 were significantly increased in the cerebral cortices of IMQ-induced model comapered to the ones in Vaseline-trearted mice. To identify the cell types that were affected by IMQ treatment, we isolated microglia and astrocytes from the cerebral cortices of IMQ-induced model by magnetic activated cell sorting (MACS). IMQ-induced SLE mice also showed increases in Cxcl10 mRNA both in isolated microglia and astrocytes. In addition, activated (A1) astrocyte markers such as H-2d and Fkbp5 were upregulated in the isolated astrocytes from IMQ-induced SLE mice.These results suggest that SLE model may upregulate the expression levels of chemokines in CNS through peripheral TLR7-induced systemic inflammation and activated glial cells. We plan to investigate neuroinflammation and cognitive function in a TLR7 agonist-induced model and the genetic SLE model, B6^lpr/lpr mice.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-072 Role of astrocyte APLPs on astrocyte/synapse interaction and synaptic plasticity Margaux Saint-Martin(1), Yukiko Goda(1) 1. RIKEN, CBS, Wako, Japan Keyword:* Astrocytes, APLPs, Synaptic plasticity Synaptic adhesion proteins such as neurexins, neuroligins or ephrins, are involved in synapse formation in developing neurons, and regulate synaptic transmission and synapse remodeling in mature neurons. Astrocytes, a type of glial cells, are also known to contribute to the formation, function and plasticity of synapses. Their processes surround synapses, respond to neurotransmitters and actively modulate synaptic transmission, to constitute the tripartite synapse (Pre-synapse, Post-synapse and Perisynaptic astrocyte process). Recently, neuroligins expressed in astrocytes were shown to play a key role in morphological elaboration of astrocytes and synaptogenesis highlighting a novel mechanism by which astrocytes regulate synaptic formation though cell adhesion proteins that are expressed also in astrocytes. These results beg the need to re-consider the synapse adhesion code, traditionally focused on the interactions of pre/post synaptic elements. We recently carried out single cell RNA-seq (patch-Seq) analysis from hippocampal astrocytes and found the expression of the cell adhesion proteins APLP1 and APLP2 (Amyloid precursor like proteins 1 and 2). These proteins are also expressed in neurons, in the pre- and post-synaptic compartment and have been previously reported to induce synaptic differentiation in vitro. Their function in astrocytes have not yet been described, however. Using astrocytes grown on top of HEK cells overexpressing APLPs we showed that APLPs as a substrate are able to modify astrocyte morphology by increasing their complexity. Then, using shRNAs to knockdown (KD) APLPs in astrocytes co-cultured with neurons, we showed that KD of APLP1 induced a decrease of astrocyte branching. Along with this decrease, we observed a slight effect of APLP1 KD on excitatory synapses. Together these results suggest a role of APLPs in astrocyte morphology and potentially regulating synaptic plasticity. Further experiments will determine if this function relies on heterophilic and/or homophilic interactions between astrocyte APLPs and neuronal APLPs.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-073 ラット脳出血後の前肢伸長運動は小脳オリゴデンドロサイト新生を促し、運動機能回復を促進する Forelimb reaching exercise after intracerebral hemorrhage causes better motor function recovery with adaptive cerebellar oligodendrogenesis in rats 清水健史(1)、佐藤碧惟(1)、國料美月(1)、上野新也(1)、小林憲太(2,3)、飛田秀樹(1) 1. 名古屋市立大学医学研究科脳神経生理学、2. 生理学研究所・ウイルスベクター開発室、3. 総合研究大学院大学　生理科学 Takeshi Shimizu(1), Aoi Sato(1), Mitsuki Kokuryoh(1), Shinya Ueno(1), Kenta Kobayashi(2,3), Hideki Hida(1) 1. Department of Neurophysiology and Brain Science, Graduate School of Medical Sciences, Nagoya City University, 2. Section of Viral Vector Development, National Institute for Physiological Sciences, 3. Graduate University of Advanced Studies (SOKENDAI) Keyword: oligodendrocyte, cerebellum, rehabilitation, stroke We previously reported that rehabilitative training of forced limb use after internal capsule hemorrhage (ICH), a situation of neural activity increase, is due to a causal relationship between the cortico-rubral tract and the functional recovery. However, it is still unclear whether dynamic change of the cerebellum in the motor regulatory system is also induced by rehabilitation after ICH. On the other hand, growing number of studies demonstrates that oligodendrocyte (OL) remodeling in adult mice is involved in motor learning, in which neural activity increase modulates myelination. Therefore, we focus on the adaptive change of motor regulatory system in the cerebellum from the aspect of OL remodeling: we assessed whether rehabilitative exercise of forelimb reaching itself influences cerebellar oligodendrogenesis. ICH model was made by the injection of Type IV collagenase (15 units/ml, 1.4µl) into the internal capsule of male rats. Reaching exercise (forelimb reaching for taking 30 pellets) was given for 8 days from 1 day after the lesion, followed by the assessment of skilled reaching test. We observed that the reaching exercise significantly induced better functional recovery of skilled forelimb reaching in ICH model. We also revealed the increase in the number of newly-born OLs by 3.5 times and CC1+ mature OLs in the cerebellar nucleus of the exercise group. These data suggest that OL remodeling under reconstruction of injured brain circuits by intervention therapy plays roles in functional recovery. In addition, molecular mechanisms underlying the rehabilitative effects after ICH and the influence of oligodendrogenesis inhibition in the cerebellum on functional recovery will be presented.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-074 生体ミクログリアの機能応答における時空間的多様性 Spatiotemporal divergence of microglial Ca(2+) activity 堀内浩(1)、石田順子(1)、鍋倉淳一(1) 1. 生理学研究所 Hiroshi Horiuchi(1), Junko Ishida(1), Junichi Nabekura(1) 1. National Institute for Physiological Sciences Keyword: Microglia, Calcium imaging, Neuronal activity, 2 photon imaging Microglia are the sole resident immune cells in the central nervous system. Microglial Ca²⁺ activity is critical to their surrounding environment according to various spatiotemporal scales. However, region-of-interest-based analyses on microglial activity used in previous reports have remained limited in their scope. Here, we accurately characterize the spatiotemporal properties of individual microglial Ca²⁺ events in an awake state according to onset location using an event-based approach. Almost all events originate at their processes, and had non-propagative features. Their spatiotemporal dynamics significantly depended on the origin and propagative features. Microglial Ca²⁺ propagative features of events generated at processes are unrelated to Ca²⁺ amplitude. Surprisingly, their propagation and direction are precisely regulated at their process branches. Furthermore, the Ca²⁺ activity is clearly suppressed by both the inhibition of neuronal activity and purinergic P2 receptor signaling. These results indicate a certain degree of spatiotemporal diversity in microglial Ca²⁺ activity.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-075 アストロサイトにおけるグルタミン合成酵素の発現はHippo-YAPシグナル伝達経路によって制御される Glutamine synthetase expression in astrocytes is regulated by Hippo-YAP signaling pathway 那須優介(1)、今井真美子(2)、横山望実(2)、照沼美穂(1) 1. 新潟大学大学院医歯学総合研究科、2. 新潟大学歯学部 Yusuke Nasu(1), Mamiko Imai(2), Nozomi Yokoyama(2), Miho Terunuma(1) 1. Grad Sch Med Dent Sci, Niigata Univ, Niigata, Japan, 2. Facl Dent, Niigata Univ, Niigata, Japan Keyword: Glutamine synthetase, Astrocyte, Hippo-YAP signaling pathway, Epilepsy Astrocytes, one of the major glial cells in the central nervous system, are the only cell type that express glutamine synthetase (GS) to protect neurons from the neurotoxicity induced by glutamate and ammonia. In epileptic brain, excessive glutamate and ammonia have been observed, and their metabolism are important for preventing from the seizure aggravation. In our previous research, we found that co-application of glutamate and NH₄Cl, the sources of glutamine, to the rat primary cultured astrocytes reduced GS expression at both protein and mRNA levels, consistent with findings in previous reports studying the epileptic brain. In this study, we planned to determine the regulator of GS expression in astrocytes, which could be the target for the treatment of epilepsy. We found that Yes-associated protein (YAP), which has been reported to regulate GS in the liver, is also a regulator of GS in astrocytes. We confirmed it by the overexpression of YAP as well as by the pharmacological inhibition of YAP nuclear localization. In addition, when astrocytes were exposed with glutamate and NH₄Cl, the expression level of YAP decreased in a time-dependent manner, similar to that of GS. YAP nuclear translocation was also decreased. To examine if decreased expression of GS observed by the glutamate and NH₄Cl co-treatment is due to the reduced YAP in the nucleus, we tested XMU-MP-1, an inhibitor of the Hippo pathway kinases MST1/2, an upstream regulator of YAP. We found that XMU-MP-1 promotes the nuclear translocation of YAP and increases the expression of GS even under glutamate and NH₄Cl condition. We are currently investigating the pharmacological efficacy of XMU-MP-1 in the animal model of epilepsy.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-076 擬似ウイルス感染モデルラットでの脳内炎症は倦怠感の惹起に関わる Peripheral pseudoviral infection-induced regional neuroinflammation responsible for fatigue symptoms in rats: A [(18)F]DPA-714 PET study 越智祐太(1)、新垣和貴子(2)、胡迪(1)、李丹渓(1)、重田美香(1)、林中恵美(3)、和田康弘(3)、土居久志(2)、渡辺恭良(3)、崔翼龍(1) 1. 国立研究開発法人理化学研究所生命機能科学研究センター生体機能動態イメージング研究チーム、2. 国立研究開発法人理化学研究所生命機能科学研究センター標識化学研究チーム、3. 国立研究開発法人理化学研究所生命機能科学研究センター健康・病態科学研究チーム Yuta Ochi(1), Wakiko Arakaki(2), Di Hu(1), Danxi Li(1), Mika Shigeta(1), Emi Hayashinaka(3), Yasuhiro Wada(3), Hisashi Doi(2), Yasuyoshi Watanabe(3), Yilong Cui(1) 1. Laboratory for Biofunction Dynamics Imaging, RIKEN Center for Biosystems Dynamics Research, 2. Laboratory for Labeling Chemistry, RIKEN Center for Biosystems Dynamics Research, 3. Laboratory for Pathophysiological and Health Science, RIKEN Center for Biosystems Dynamics Research Keyword: fatigue, neuroinflammation, peripheral infection A range of symptoms including fever, widespread pain, and fatigue, are thought to be accompanied by various infections, such as COVID-19. Although underlying mechanisms of infection induced fever and pain have been well studied, the neuronal/molecular mechanisms of fatigue induced by infection remains to be elucidated. Recently, the results from preclinical and clinical studies proposed that fatigue may be attributed to neuroinflammation, which is thought to be induced by peripheral infection. In order to investigate whether and how the peripheral infection relates to fatigue via regional neuroinflammation, we performed brain-wide quantitative evaluation of neuroinflammation in a peripheral pseudoviral infection-model rats using positron emission tomography (PET) imaging with [¹⁸F]DPA-714, a selective PET probe for activated microglia, and assessed the correlations between the regional neuroinflammation and the symptoms related to fatigue. Peripheral pseudoviral infection-model was generated by intraperitoneal injection of polyriboinosinic: polyribocytidylic acid (poly I:C, 10 mg/kg), a synthetic double-stranded RNA, in male Wistar rats. Consistent with the previous report, poly I:C treatment evoked transient fever for several hours and subsequent suppression of spontaneous activity lasting for a few days. Furthermore, significant elevation of plasma pro-inflammatory cytokines was observed at 2 and 4 h after poly I:C treatment, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. PET image analysis revealed that the brain uptake of [¹⁸F]DPA-714 was significantly increased in several brain regions one day after poly I:C treatment, such as dorsal raphe nucleus (DR) parvicellular part of red nucleus (RPC), A5 and A7 noradrenergic nuclei, and solitary nucleus, as compared with control rats. Immunohistochemical analysis also showed that the brain accumulation of [¹⁸F]DPA-714 was well consistent with the distribution of activated microglia indicated by CD11b-staining, a marker for activated microglia. Besides, the accumulation of [¹⁸F]DPA-714 in the DR, RPC and A5 positively correlated with the fatigue-like behavior. These results suggest that peripheral infection could trigger regional neuroinflammation in these brain regions which may cause fatigue-like symptoms.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-077 Substrate preference of fatty acid oxidation in murine astrocytes in vitro Laarni Grace Corales(1), Hitoshi Inada(1,2), Noriko Osumi(1) 1. Department of Developmental Neuroscience, Graduate School of Medicine, Tohoku University, 2. Laboratory of Health and Sports Sciences, Division of Biomedical Engineering for Health and Welfare, Tohoku University Graduate School of Biomedical Engineering Keyword:* astrocytes, fatty acid oxidation, palmitic acid, fatty acid binding proteins Brain is the most energy demanding organ in the body. The brain utilizes glucose as its main source of energy, but previous studies have shown that the organ can also use fatty acids as an energy source, to a minor extent. Astrocytes, the most abundant glial cells in the brain, are thought to be the cells where oxidation of fatty acids primarily occurs for energy generation. In this study, we determined the beta-oxidation activity of fatty acids in the mitochondria and the preference of fatty acids as an energy source in primary cultured astrocytes from the neonatal mouse brain to better understand the usage of the fatty acids in the astrocytes. We also analyzed the fatty acid oxidation activity using astrocytes derived from fatty acid binding protein (FABP) knockout mice to determine whether FABPs play a role in fatty acid transport to the mitochondria. Oxidation of long chain fatty acids such as palmitic acid, oleic acid, arachidonic acid (ARA), and docosahexaenoic acid (DHA) was measured through changes in oxygen consumption rate (OCR) using the extracellular flux analyzer. Our results showed that the OCR of all the four fatty acids at the maximal respiration was significantly higher compared to the control, indicating that these fatty acids were oxidized in the murine cultured astrocytes. Palmitic acid had the highest OCR value among the four fatty acids tested, being followed by oleic acid. ARA and DHA showed similar OCR values but significantly lower compared to palmitic acid. The astrocytes lacking FABPs did not show significant difference in OCR values compared to those derived from the wild type, suggesting that FABPs may not be the major carrier of these fatty acids for beta-oxidation in the astrocytes. Our study highlights the preferential oxidation of palmitic acid in the murine cultured astrocytes.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-078 Sensory Glia Detect Repulsive Odorants and Drive Olfactory Adaptation Xiaomin Yue(1) 1. Zhejiang University Keyword:* Glia, olfactory transduction, C. elegans Glia are typically considered as supporting cells for neural development and synaptic transmission. Here, we report an active role of a glia in olfactory transduction. As a polymodal sensory neuron in C. elegans, the ASH neuron is previously known to detect multiple aversive odorants. We reveal that the AMsh glia, a sheath for multiple sensory neurons including ASH, cell-autonomously respond to aversive odorants via G-proteincoupled receptors (GPCRs) distinct from those in ASH. Upon activation, the AMsh glia suppress aversive odorant-triggered avoidance and promote olfactory adaptation by inhibiting the ASH neuron via GABA signaling. Thus, we propose a novel two-receptor model where the glia and sensory neuron jointly mediate adaptive olfaction. Our study reveals a non-canonical function of glial cells in olfactory transduction, which may provide new insights into the glia-like supporting cells in mammalian sensory procession.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-079 Astrocytic inward rectifying K+ channels 4.1 subtype modulate glutamate release probability and LTP at individual CA3-CA1 synapses Olga Tiurikova(1,2), Olga Kopach (1), Dmitri Rusakov(1) 1. University College London, 2. Institute of Bioorganic Chemistry, Russia Keyword:* Astrocyte, Kir4.1 channels, potassium signalling Perisynaptic astrocytic processes (PAPs) surround both pre- and postsynaptic neuronal terminals form an ‘astrocytic cradle’ and ensure an effective clearance of extracellular K+ during synaptic activity. Among the mechanisms for spatial redistribution of extracellular K+, K+ uptake through inwardly rectifying K+ channels 4.1 subtype (Kir4.1) is the most abundant. The perturbed function or expression level of Kir4.1 channels in astrocytes has been associated with several neurodegenerative disorders, including hippocampal sclerosis, Huntington’s disease, and epilepsy. Given the slow progress of pharmacological treatment of these diseases manipulating the functional expression of astrocytic Kir4.1 channels by gene therapy might open new directions for potential therapeutic strategies. In this study, we used gene transduction in vivo to upregulate expression level of functional Kir4.1 channels in astrocytes and investigate their role in the modulation of synaptic function. First, taking advantage of acute hippocampal slices we confirmed that over-expression of astrocytic Kir4.1 channels enhances K+ currents in astrocytes. Evoked astrocytic currents were sensitive to both Ba2+ and selective Kir4.1 channel antagonist, assigning predominant contribution to the current enhancement to Kir4.1 channels. Next, using Tornado line-scan technique we monitored single-synapse glutamate releases from neurons expressing glutamate sensor (iGluSnFR). We showed that the glutamate release probability depends on an extracellular K+ concentration ([K+]e) and increase with [K+ ]e elevation. However, when the axon passed through astrocytic PAPs with Kir4.1 channels over-expression, [K+ ]e elevation did not affect either the iGluSnFR fluorescence intensity or the release probability of the recorded responses. These data suggest that over-expression of functional Kir4.1 channels in astrocytes provide more rapid redistribution of extracellular K+ and precludes the [K+]e-induced enhancement of glutamate release. Moreover, recorded glutamate transporter (EAAT)-mediated currents from astrocytes over-expressing Kir4.1 channels demonstrated reduced current amplitude during burst stimulation, suggesting that less amount of glutamate is released. Finally, induced long-term potentiation at CA3-CA1 synapses, trespassing astrocytes with over-expression of Kir4.1 channels was significantly reduced. We have therefore uncovered that astrocytic Kir4.1 channels-mediated mechanism plays an important role in the local regulation of synaptic function at individual synapses. This may represent a new avenue towards the potential treatment of neurodegenerative diseases.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-080 Microglia, a key player in hippocampal synaptic plasticity and memory deficits associated with sleep deprivation Katherine Picard(1,2), Janine Kox(6), Eva Šimončičová(2), Robbert Havekes(6), Marie-Eve Tremblay(1,2,3,4,5) 1. Dept Med moléculaire, Univ Laval, Québec, Qc, Canada, 2. Division of Medical Sciences, Univ of Victoria, Victoria, BC, Canada, 3. Dept of Biochemistry and Molecular Biology, Univ of British Columbia, Vancouver, BC, Canada, 4. Dept of Neurology and Neurosurgery, McGill Univ, Montréal, QC, Canada, 5. Centre for Advanced Materials and Related Technology (CAMTEC), Univ of Victoria, Victoria, BC, Canada, 6. Neurobiology expertise group, Groningen Institute for Evolutionary Life Sciences, Univ of Groningen, Groningen, The Netherlands Keyword:* Microglia, Sleep deprivation, Synaptic plasticity, Hippocampus In our society focused on efficiency and performance, the lack of sleep has now become the new normal. However, sleep loss can lead to many detrimental consequences, including cognitive impairments. In the brain, the hippocampus is one of the regions that is most vulnerable to sleep deprivation, often leading to neuronal connectivity changes and a reduction of synaptic density. Strikingly, sleep deprivation leads to subregion-specific changes affecting the Cornu Ammonis (CA)1, but not CA3 at the structural plasticity level. Microglia, the resident immune cells of the brain, are important contributors to synaptic plasticity, and their functions are affected by sleep deprivation. As microglia are a highly heterogenous population and their functions can differ among brain regions, we aim to determine if microglia could be involved in the region-dependent synaptic deficits observed after sleep deprivation. To do so, we will expose male mice to 6 hours of sleep deprivation, which is representative of what can be experienced in the everyday life, through gentle handling. Using an immunostaining for ionized calcium-binding adaptor molecule 1 (IBA1), a marker of myeloid cells, we will characterize by brightfield microscopy possible changes in microglial density, distribution, and morphology in the CA1 and CA3 of the hippocampus. We will then perform a double immunofluorescence staining for IBA1 and TMEM119, a marker specific to microglia, to examine potential peripheral immune infiltration, which is known to occur when brain homeostasis is altered. We will further assess microglial phagocytosis of synaptic elements by confocal microscopy using immunofluorescence for cluster of differentiation 68, a phagolysosomal marker, as well as vesicular glutamate transporter 1 and Homer protein homolog 1, which are respectively pre- and post-synaptic markers. Lastly, we will characterize microglial ultrastructure by scanning electron microscopy to provide a better understanding of their functions and interaction with synapses. Elucidating the role of microglia in the synaptic deficits resulting from sleep deprivation may ultimately contribute to the development of novel therapeutic strategies aimed to combat the negative impact of sleep deprivation on cognitive function and brain function as a whole.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-081 細胞外マトリックスタンパク質fibulin-7はオリゴデンドロサイトの分化と神経軸索への接着を制御する Fibulin-7, an extracellular matrix protein, positively regulates oligodendrocyte differentiation and attachment to neuronal axons 山田桃奈(1)、岩瀬未帆(1)、林千香子(1)、Susana de Vega(2)、鈴木喜晴(1) 1. 東京医科歯科大学大学院医歯学総合研究科遺伝子細胞検査学分野、2. 順天堂大学大学院医学研究科整形外科学講座 Momona Yamada(1), Miho Iwase(1), Chikako Hayashi(1), Susana de Vega(2), Nobuharu Suzuki(1) 1. Dept. of Mol. and Cell. Biol., Grad. Sch. of Med. and Dent. Sci., Tokyo Medical and Dental University, 2. Dept. of Med. for Orthop. and Motor Organ, Juntendo University Grad. Sch. of Med. Keyword: Oligodendrocyte, Extracellular matrix protein, Fibulin-7 Fibulin-7 (Fbln7), an extracellular matrix (ECM) protein, comprises an N-terminal sushi domain, three EGF-like motifs, and a C-terminal fibulin-type module. It is known to bind to integrin b1 and heparan sulfate proteoglycan receptors. We previously reported that Fbln7 is overexpressed in glioblastoma and causes abnormal angiogenesis. Also, the RNA-Seq analysis revealed that Fbln7 is specifically expressed in oligodendrocyte (OL)-lineage cells, compared to other resident cells in the CNS. However, the functions of Fbln7 in normal CNS development have not been yet elucidated. Therefore, we aimed to investigate the expression and function of Fbln7 in the CNS. First, we examined the expression of Fbln7 in the mouse spinal cord at embryonic and postnatal stages. The Fbln7 mRNA was highly expressed at postnatal day (P) 3 and P7. In addition, immunohistochemistry studies in the spinal cord of P3 and P7 mice showed that Fbln7 is localized on myelin and between myelin and axon. In primary cell-culture, Fbln7 is expressed along the protrusion of OLs, in particular, at the branching points and the tips of the protrusions. To analyze the function of Fbln7, we performed the co-culture experiment of OLs and neurons. When OLs and neurons were cultured in the presence of recombinant Fbln7 (rFbln7), the number of GalC-positive OLs increased, compared with the control. Moreover, the number of OLs attaching to neurons also increased. These results of the co-culture experiment suggest that Fbln7 promotes OL differentiation and adhesion of OLs to neurons. In conclusion, Fbln7 is highly expressed at the early postnatal stage, when oligodendrocyte precursor cells differentiate to OLs and start to attach on neuronal axons, and is a novel ECM protein, which regulates OL differentiation and the cellular interaction between OLs and neurons.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-082 小脳プルキンエ細胞軸索の恒常性維持におけるパラノーダルジャンクションの重要性 The importance of the paranodal axoglial junctions in Purkinje axonal homeostasis 石橋智子(1)、志村聡栄(1)、菅原航太(1)、馬場広子(2) 1. 東京薬科大学薬学部機能形態学、2. 新潟医療福祉大学 Tomoko Ishibashi(1), Akitaka Shimura(1), Kota Sugawara(1), Hiroko Baba(2) 1. Dept of Mol Neurobiol, Tokyo Univ of Pharm and Life Sciences, 2. Dept of Occupational Therapy, Faculty of Rehabilitation, Niigata Univ of Health and Welfare Keyword: paranodal junction, myelin, axon Myelin loops attach to the axonal membrane and form paranodal axoglial junctions (PNJs) at paranodes adjacent to nodes of Ranvier. We think that PNJs regulate axonal homeostasis in addition to their role as lateral diffusion barriers that restrict the location of nodal axolemmal proteins. To better understand how PNJs regulate axonal functioning, we studied cerebroside sulfotransferase knockout (CST^-/-) mice that partially lack paranodal junctions. Previously we reported that CST^-/- mice develop Purkinje axonal swellings after myelination and that type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1)-positive focal accumulations were the earliest finding therein. In the IP₃R1-positive swellings, accumulation of neurofilament, mitochondria, and proteins that localize to mitochondria-associated ER membranes gradually increased. Because IP₃R1 is an ER-resident Ca²⁺ channel, it is possible that ER stress is involved in swelling formation. On the other hand, it is elusive how IP₃R1 accumulates in the restricted internodal regions in PNJ disrupted Purkinje axons. In the present study, we investigate whether ER stress is involved in the accumulation of IP₃R1 in CST^-/- Purkinje axons, and whether axonal distribution of IP₃R1 is associated with myelination state. Immunohistochemical analysis showed that GRP78/BiP accumulated in the IP₃R1-positive axonal regions, and that these accumulations already appeared by 18 days of age. The number of GRP78/BiP-positive/IP₃R1-positive swellings gradually decreased with age. The positive signal of C/EBP homologous protein (CHOP) was also observed in the IP₃R1-positive swollen regions as well as in Purkinje nuclei in CST^-/- mice. However, these findings were never observed at the non-swollen axons. We also investigated CST^-/-IP₃R1^+/- mice. These mice showed a marked decrease in the number of GRP78/BiP-accumulated-IP₃R1-positive swellings. Furthermore, we observed a difference in the distribution of IP₃R1 under normal myelination. In in vitro cerebellum, before myelination, IP₃R1 was observed throughout unmyelinated Purkinje axons, while IP₃R1-positive signals gradually concentrated into internodal regions with Caspr clustering in the paranodes. These results suggest that the distribution and/or expression level of IP₃R1 may be dependent on the state of PNJs, and that the focal IP₃R1 accumulation may induce the cellular response to ER stress in the axonal swollen regions.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-083 オリゴデンドロサイト系譜細胞の増殖・形態形成におけるアクチン骨格制御分子Arpc1aの分子機能 The molecular function of the actin cytoskeletal regulator Arpc1a in proliferation and morphogenesis of oligodendrocyte-lineage cells 岩瀬未帆(1)、山田桃奈(1)、林千香子(1)、鈴木喜晴(1) 1. 東京医科歯科大学大学院医歯学総合研究科遺伝子細胞学分野 Miho Iwase(1), Momona Yamada(1), Chikako Hayashi(1), Nobuharu Suzuki(1) 1. Dept. of Mol. and Cell Biol., Grad. Sch. of Med. and Dent. Sci., Tokyo Medical and Dental University Keyword: oligodendrocyte, Arpc1a, actin cytoskeleton In the CNS, oligodendrocytes (OLs) form myelin around axons, which plays important roles in motor and cognitive function and learning. When OLs extend their processes, ensheathe axons, and form compacted spirals of myelin, a balance between assembly and disassembly of actin cytoskeleton is precisely controlled. At the initial myelination stage, the assembly of actin filaments are regulated by Arp2/3 complex, consisting of seven subunits, whose functions are still incompletely understood. One of them, Arpc1a exhibits the highest mRNA expression level among the subunits during OL differentiation and myelination. In this study, we analyzed the expression pattern and function of Arpc1a in the myelin specific morphology during CNS myelination. In immunohistochemistry of mouse spinal cords, at postnatal day (P) 3 and P7, the intense expression signal of Arpc1a was observed surrounding axons, while the signal disappeared at P14. In immunocytochemistry of mouse primary cell culture, Arpc1a was expressed in both oligodendrocyte progenitor cells (OPCs) and OLs, and colocalized with F-actin along cell processes. Knockdown experiments of Arpc1a in primary culture cells and the OPC line CG-4 revealed that the proliferation of Arpc1a-knockdown cells was inhibited and their cellular state was shifted from the proliferation stage to the early differentiation stage. When we examined the cell morphology of the differentiated OLs, aberrantly asymmetric processes and abnormal myelin-like membrane sheets were observed in Arpc1a-knockdown cells. Further, overexpression of Arpc1a increased the number and branches of cell processes to form myelin-like membrane sheets. These results suggested that Arpc1a was required for the proliferation of OPCs and for the formation of proper cell morphology with the processes and myelin membrane sheets in OLs. Arpc1a may be an important player in CNS myelination through the actin cytoskeletal organization in OLs.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-084 高分子医薬品の受容体介在性トランスサイトーシスによる脳移行性評価におけるヒト不死化血液脳関門スフェロイドモデルの有用性 Usability of human immortalized cell-based blood-brain barrier spheroid model for evaluation of macromolecular drug permeability via receptor-mediated transcytosis 森尾花恵(1)、北村啓太(1,2)、岡本彩花(1)、伊藤涼(3)、山浦由之(4)、小森高文(5)、降幡知巳(1) 1. 東京薬科大学　個別化薬物治療学教室、2. 千葉大学大学院薬学研究院　薬物学研究室、3. 小野薬品工業　ニューロロジー研究センター、4. 小野薬品工業　薬物動態研究部、5. エーザイ　　薬物動態研究部 Hanae Morio(1), Keita Kitamura(1,2), Ayaka Okamoto(1), Ryo Ito(3), Yoshiyuki Yamaura(4), Takafumi Komori(5), Tomomi Furihata(1) 1. Lab. Clin. Pharm. & Exp. Therap., Sch. Pharmacy., Tokyo Univ. Pharm. & Life Sci. , 2. Lab. DDS Design & Drug Dispos., Grad. Sch. Pharm. Sci., Chiba Univ., 3. Res. Ctr. Neurol., Ono Pharm. Co., Ltd., 4. Dept. Pharmacokinet. Res., Ono Pharm. Co., Ltd., 5. Dept. Drug Metab. & Pharmacokinet., Eisai Co. Ltd. Keyword: Blood-brain barrier, In vitro BBB model, Receptor-mediated transcytosis, Transferrin receptor [Purpose] For the treatment of central nervous system (CNS) disorders, various macromolecular drugs have been developed as new modality drugs. For these delivery across blood-brain barrier (BBB), which restricts the entry of various substances into the brain, receptor-mediated transcytosis (RMT) has gained attention. To evaluate permeability of macromolecular drugs utilizing RMT (RMT-drugs), in vitro BBB models are expected to serve as a useful experimental tool. Accordingly, we previously developed a multicellular spheroidal BBB model using human immortalized BBB cells (hiMCS-BBB model). In this study, we aimed to show its applicability to permeability studies of RMT-drugs. [Methods] The hiMCS-BBB models were developed by integrating HBMEC/ci18 (brain microvascular endothelial cells) and two other different kinds of human immortalized brain cells based on the previously-described method with slight modifications in their seeding order and numbers. The transferrin receptor (TfR) expression was examined by immunocytochemistry (ICC), and its RMT function was examined by fluorescence-labeled transferrin (Tf) and anti-TfR IgG permeability assays. [Results and Discussion] The results of ICC showed the clear expression of TfR at the surface of the spheroids, indicating the prominent TfR expression in the HBMEC/ci18 monolayer. As a result of TfR permeability assays, higher fluorescent level of TfR was detected in the spheroid core at 37℃, compared to that at 4℃ (1.49 ± 0.12-fold). Moreover, in competition assays, the permeability of TfR was significantly inhibited by excess amount of non-labeled Tf at 37℃ (0.23 ± 0.08-fold), which confirms functional expression of TfR in the hiMCS-BBB models. Finally, we performed anti-TfR IgG permeability assays with isotype-matched normal IgG (control). After the incubation at 37°C, the fluorescence intensity derived from the anti-TfR IgG in the spheroid core was detected at higher levels than that of the control IgG (1.7 ± 0.2-fold). Furthermore, under 4°C incubation, the anti-TfR IgG fluorescence levels were significantly reduced (0.6 ± 0.1-fold). [Conclusions] The hiMCS-BBB models firmly express TfR-mediated RMT function in HBMEC/ci18 cells, which accelerates anti-TfR IgG penetration across the BBB in the models. Therefore, the hiMCS-BBB models can provide a useful tool for permeability evaluation of TfR-targeting RMT-drugs and hopefully will be for other RMT-drugs as well.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-085 脳梗塞後のリンパ上皮細胞の役割 LYMPHATIC ENDOTHELIAL CELLS AFTER ISCHEMIC STROKE 桑田康弘(1)、眞木崇州(1)、安田謙(1)、高山直樹(1)、戸田真太郎(1)、髙橋良輔(1) 1. 京都大学大学院医学研究科脳病態生理学講座 Kuwata Yasuhiro(1), Maki Takakuni(1), Yasuda Ken(1), Takayama Naoki(1), Toda Shintaro(1), Takahashi Ryosuke(1) 1. Department of Neurology, Kyoto University Graduate School of Medicine Keyword: Ischemic stroke, Lymphatic endothelial cells , MCAO, Prox-1 [Objective] Stroke is one of the major causes of neurological disability worldwide. Ischemic stroke accounts for over 80% of all types of stroke. However, there is no beneficial treatment for ischemic stroke except thrombolytic therapy or intravascular thrombectomy. There is an urgent need to find novel therapeutic approach for ischemic stroke. It has been reported that brain to cervical lymph node signaling is involved in the pathological interactions between central brain injury and peripheral immune activation after cerebral ischemia. Lymphatic endothelial cells (LECs) constitute the structure of the lymphatic vessels and the sinuses of lymph nodes, contributing to various aspects of immune modulation. From these points of view, we focused on LECs as key players for damage and repair processes after cerebral ischemia. [Methods] We performed both transient middle cerebral artery occlusion (MCAO) and sham operation on Prox-1 GFP mice and compared the distribution of LECs between two groups in order to assess the behavior of LECs and their interaction with other cell types after ischemic stroke. [Results] The number of Prox-1, Lyve-1 and VEGFR-3-positive LECs was significantly increased and closely localized with immune cells in MCAO group compared with sham operation group (n=5, p<0.05). [Conclusions] Our data suggest that LECs actively participate in the damage and repair processes after cerebral ischemia. Further analysis of neurovascular unit crosstalk with systemic biology via lymphatics would enable us to find novel therapeutic approach for ischemic stroke.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-086 神経細胞における初期応答遺伝子調節機構の定量的解析 The quantitative analysis of immediate early genes regulation in neuron 伊藤夏穂(1)、岡田眞里子(1) 1. 大阪大学蛋白質研究所 Kaho Itoh(1), Mariko Okada(1) 1. Institute for Protein Research, Osaka University Keyword: IMMEDIATE EARLY GENES, OMICS Neurons rapidly change their expression levels of immediate early genes (IEGs) in response to external stimuli. IEGs have important roles in regulation and maintenance of neural activity. However, the quantitative mechanism between epigenetic regulation and IEGs expression remains unclear. Therefore, this study aims to clarify the mechanism regulating stimuli-induced gene expression by bioinformatic and experimental approaches. We first analyzed RNA-seq data obtained from mouse cortical neuron stimulated by KCl(Tyssowski et al., Neuron, 2018) to identify IEGs. We identified 144 IEGs such as Npas4 and Nr4a1 transcription factors involved in neuronal functions. We also found that when cells are stimulated briefly, some IEGs were still highly upregulated as shown in the sustained stimulation. To investigate the quantitative mechanism of the difference of IEGs expression pattern evoked by these two stimuli conditions, we treated PC12 cells by sustained or brief NGF stimulation and quantified time-course IEGs expression levels by qRT-PCR. We found that 5 minutes brief stimulation, was sufficient to induce IEGs upregulation at 50 minutes later, suggesting that cells remember the stimuli was given or not even though the stimulation was very briefly. In this experiment, some IEGs in brief stimulation reached the same expression level as sustained stimuli, while others showed lower levels. The lower expression levels induced by 5 min brief stimulation was recovered by extending the stimulation from 5 min to 30 min, suggesting the existence of a positive feedback regulation in the time zone. These results indicates that each IEG is differently regulated by, for example, feedback loops or epigenetic modifications. Currently, time-course RNA-seq analysis is investigated using PC12 cells to find global transcriptome signatures in response to the two different types of stimuli. In addition, time-course Histone H3 lysine 27 chromatin immunoprecipitation sequencing (H3K27ac ChIP-seq) and Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) are conducted. H3K27ac modification activates transcription through chromatin modeling and ATAC-seq reveals chromatin opened regions. Therefore, the transcription factors responsible for IEGs regulation and that contributes the feedback regulation can be predicted using these data. The hypothesis made by the analysis will be verified using mouse primary culture neurons.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-087 アデノ随伴ウイルスベクターに最適化した抑制性ニューロン特異的プロモーターの開発 Development of adeno-associated viral vector-optimized inhibitory neuron-specific promoters 深井悠貴(1)、今野歩(1,2)、松﨑泰教(1,2)、平井宏和(1,2) 1. 群馬大学大学院医学系研究科脳神経再生医学分野、2. 群馬大学未来先端研究機構・ウイルスベクター開発研究センター Yuuki Fukai(1), Ayumu Konno(1,2), Yasunori Matsuzaki(1,2), Hirokazu Hirai(1,2) 1. Department of Neurophysiology and Neural Repair, Gunma University Graduate School of Medicine, 2. Viral Vector Core, Gunma University Initiative for Advanced Research (GIAR) Keyword: AAV, PHP.eB, inhibitory neuron, GABAergic neuron 【Purpose】 Recently, we identified a genomic region upstream of mouse GAD65 gene, which can be used as an inhibitory neuron-specific promoter (mGAD65 promoter) (Hoshino et al. Mol Brain. 2021). However, the mGAD65 promoter has 2.5 kb in length and occupies over a half of the accommodation space of adeno-associated virus (AAV) vector, which substantially compromises the usefulness of the mGAD65 promoter. Here, we tried to obtain shorter inhibitory neuron-specific promoters from the original mGAD65 promoter and mouse GAD67 promoter (mGAD67 promoter). 【Methods】 We produced deletion constructs with different lengths from the 2.5-kb-mGAD65 promoter and the mGAD67 promoter. AAV-PHP.eB, expressing GFP under the control of one of the shortened promoters, was intravenously administered to VGAT-tdTomato mice, which expressed tdTomato specifically in inhibitory GABAergic neurons (Kaneko et al. J Comp Neurol. 2018). Three weeks after the viral injection, GFP expression in the brain was examined. Whether GFP was expressed in inhibitory neurons was verified by overlap of GFP fluorescence with that of tdTomato. In addition, promoter activities were evaluated by measuring mRNA expression levels in GFP. 【Result and Discussion】 Some of the shortened mGAD65 and mGAD67 promoters showed the same or higher promoter activity than that of the original (2.5kb) mGAD65 promoter without compromising the specificity for GABAergic neurons. Intravenous infusion of AAV vectors with the shortened mGAD65 promoter or new mGAD67 promoter allowed to express a larger transgene specifically in inhibitory neurons. These results suggest that AAV vectors carrying a newly developed compact mGAD65 or mGAD67 promoter are useful for studying pathophysiology of inhibitory GABAergic neurons in various brain regions.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-088 ポストシナプスに局在するMRTFBは神経活動依存的に核移行しSRF標的遺伝子の発現を制御する Neuronal activity-dependent SRF target gene expression via nuclear translocation of the SRF coactivator MRTFB 伊原大輔(1)、田邉広樹(1)、今西詩織(1)、小坂彩(1)、佐野友香里(1)、阪上洋行(2)、田渕明子(1) 1. 富山大学学術研究部薬学・和漢系分子神経生物学、2. 北里大学医学部解剖学 Daisuke Ihara(1), Hiroki Tanabe(1), Shiori Imanishi(1), Aya Kosaka(1), Yukari Sano(1), Hiroyuki Sakagami(2), Akiko Tabuchi(1) 1. Lab Mol Neurobiol, Grad Sch of Med & Pharm Sci, Univ of Toyama, 2. Dept of Anatomy, Kitasato Univ Sch of Med Keyword: myocardin-related transcription factor, nuclear translocation, activity-dependent gene expression Activity-dependent gene expression plays crucial roles in neuronal plasticity. It has been reported that CRTC1, a CREB coactivator, mediates the activity-dependent gene expression through its translocation from synapses into a nucleus. Myocardin-related transcription factors (MRTFs), highly expressed in the brain, have actin-binding motifs and function as transcriptional cofactors of serum response factor. Recently, we reported that MRTFs are localized in postsynapses and involved in synaptic maturation. However, it remains unclear how MRTFs are involved in activity-dependent gene expression in neurons. Therefore, we hypothesized that MRTFs, which exist in postsynapses, act as a transducer of synapse-to-nucleus signaling and analyzed their functions in primary cultured rat cortical neurons. Immunostaining revealed that stimulation with bicuculline/4-aminopyridine (Bic/4AP), which causes synaptic activation, induced the transient nuclear translocation of MRTFB, but which was blocked by the pretreatment with APV/nicardipine and FK506, inhibitors of NMDA receptor/L-type voltage-dependent Ca²⁺ channels and calcineurin, respectively. Furthermore, an inhibitor for actin polymerization, latrunculin B, also blocked Bic/4AP-induced nuclear translocation of MRTFB. Next, reporter assay revealed that, pretreatment with APV/nicardipine, FK506 or latrunculin B inhibited the SRF-mediated transcriptional activation caused by Bic/4AP. We further confirmed that knockdown of MRTFA/B decreased the SRF-mediated transcriptional activation increased by Bic/4AP. Using qPCR analyses, we also found that APV/nicardipine or FK506 decreased the Bic/4AP-induced expression of junB, an SRF target gene. Moreover, using ChIP assay, we preliminarily found that MRTFB accumulation on the enhancer of junB gene increased by Bic/4AP. Taken together, these findings suggest that MRTFB is a novel key player of the synapses-to-nucleus signaling not only in neuronal activity- and actin polymerization-dependent manner.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-089 神経細胞識別コードの可視化：クラスター型プロトカドヘリンの発現解析 Visualization of Protocadherin neuronal self-recognition code 金子涼輔(1)、阿部学(2)、井上由紀子(3)、高鶴裕介(4)、渡辺雅彦(5)、崎村建司(2)、柳川右千夫(6)、八木健(1) 1. 大阪大学大学院生命機能研究科、2. 新潟大学脳研究所、3. 国立精神・神経医療研究センター疾病研究第６部、4. 東洋大学食環境科学部、5. 北海道大学大学院医学研究院、6. 群馬大学大学院医学系研究科 Ryosuke Kaneko(1), Manabu Abe(2), Yukiko Ueno Inoue(3), Yusuke Takatsuru(4), Masahiko Watanabe(5), Kenji Sakimura(2), Yuchio Yanagawa(6), Takeshi Yagi(1) 1. FBS, Osaka Univ, Osaka, Japan, 2. BRI, Niigata Univ, Niigata, Japan, 3. Dep of Biochem and Cellular Biol, National Institute of Neurosci, Tokyo, Japan, 4. Toyo Univ, Dept Nutr Health Sci, Gunma, Japan, 5. Grad Sch Med, Hokkaido Univ, Hokkaido, Japan, 6. Grad Sch Med, Gunma Univ, Gunma, Japan Keyword: Protocadherin, stochastic, reporter mouse, distribution Neuronal self-recognition code could specify neuronal identity and possibly be essential to organize complex neural networks in mammalian brain. It has been demonstrated that clustered protocadherins (Pcdhs), which encode cadherin-related transmembrane proteins as gene clusters in vertebrate genome,provide these kinds of molecular codes. The murine Pcdhs are further classifiedinto three subfamilies: Pcdh-a(14 genes), Pcdh-b (22 genes), and Pcdh-g(22 genes). Their loss of function in mice revealed that the Pcdhsplayimportant roles in dendritic self avoidance, neuronal survival, axonal projection, and synapse formation, but its effects showed cell-type dependency. As revealed by histological examinations and single-cell RT-PCR, the most of Pcdhs showed the monoallelic and stochastic expression in each cerebellar Purkinje cell at adult stage [Kaneko R et al, 2006]. Moreover, cell-type dependency of Pcdh expression pattern has also been reported [Mountoufaris G et al, 2017; Chen WV et al, 2017]. These results strongly suggest that there are correlations between expression pattern and physiological role of Pcdhs in each cell type.In order to divulge the neuronal self-recognition code, several key questions should be untangled. For example, which cell type show the monoallelic and stochastic Pcdh expression? How frequently does each cell type express one Pcdh isoform? When does the stochastic Pcdh expression start during development?In order to solve these questions, we generated knock-in mice that harbor cDNA encoding red or green fluorescent protein, tdTomato or GFP, under the control of endogenous Pcdh-b3 or b19 promoter. The mice showed stochastic and monoallelictdTomato/GFP fluorescence in various cell types, including cerebellar Purkinje cells, cerebellar molecular layer interneurons, cerebellar granule cells, hippocampal CA1 and CA3 pyramidal cells, dentate gyrus granule cells, astrocyte etc. However, none of microglia expressed fluorescent protein. The percentage of those cells expressing fluorescent protein showed cell-type dependent transition during development. Interestingly, spatial distributions of fluorescent protein revealed inter-individual variation. Our data provide direct support for a model in which a neuronal self-recognition code play a wide-spread role throughout mouse brain.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-090 PSD95とFXR1のノックダウンは突起形成を抑制する Knockdown of PSD95 and FXR1 inhibits protrusion formation 清水英雄(1)、北條浩彦(1) 1. 国立精神・神経医療研究センター　神経研究所　神経薬理研究部 Hideo Shimizu(1), Hirohiko Hohjoh(1) 1. Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry Keyword: FMRP, FXR1, PSD95, neurite formation Fragile X syndrome (FXS) is an inherited disorder caused by mutant Fragile X mental retardation 1 (FMR1) genes carrying aberrantly expanded CGG triplet repeats: aberrant CGG repeats inhibit the expression of FMR1. The protein encoded by FMR1 (FMRP) is an RNA binding protein and involved in the stabilization and translation of mRNAs. FMRP is present in dendrites and dendritic spines of neurons and plays an important role in local translation of mRNAs. Some previous studies suggest that FMRP is involved in neuronal development. The decrease in dendritic spines and the reduction of basal dendrite length and branching were observed in Fmr1 knockout mice. Thus, FXS may have some adverse effects on neurite formation, but the mechanism is not yet fully understood. In this study, to examine the effects of Fmr1 (FMRP) on process formation, we generated Fmr1-deficient cell lines with mouse neuroblastoma Neuro2a (N2a) cells: Fmr1 was knocked out by gene editing using CRISPR-Cas9. Western blot data indicated little or no expression of FMRP in Fmr1-deficient N2a cell lines. The Fmr1-deficient cells showed impairment of process formation under differentiation conditions. When expression plasmids encoding a full-length FMRP were introduced into the Fmr1-deficient N2a cells, the cells restored process formation. Interestingly, when mutant plasmids encoding FMRP lacking an RNA binding domain were introduced to the Fmr1-deficient N2a cells, the resultant cells hardly restored process formation. Since FMRP is associated with the PSD95 mRNA and FXR1 protein, we performed gene silencing against Psd95 and Fxr1 by RNAi in naïve N2a cells. As a result, the knockdown cells exhibited impairment of process formation under differentiation conditions. In addition, the Psd95 mRNA could be detected, but the protein was hardly detected in naïve N2a cells (without gene silencing): PSD95 could not be detected by western blotting. we further performed gene silencing against Psd95 and Fxr1 by RNAi in primary mouse hippocampal neuron culture. As a result, the knockdown neurons exhibited impairment of neurite formation as with N2a cells. Therefore, the findings suggest that the FXR1 protein and PSD95 mRNA, which are associated with FMRP, may be involved in process formation.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-091 The m6A reader YTHDF1 modulates APC and controls axon and dendrite development. Loic Broix(1), Rohini Roy(2,1), Shengqun Hou(1,2), Momoe Sukegawa(1,2), Hidenori Ito(3), Koh-ichi Nagata(3), Dan Ohtan Wang(1,2) 1. RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan, 2. Graduate School of Biostudies, Kyoto University, Yoshida hon-machi, Kyoto, Japan, 3. Institute for developmental Research, Aichi Developmental Disability Center, Japan Keyword:* RNA modification, m6a reader, Neuronal development, Axon growth A new mode of gene regulation, epitranscriptomics, has recently emerged as a versatile and powerful post-transcriptional regulatory pathway in the central nervous system. RNA modification N6-methyl-adenosine (m6A) has been found in axonal and synaptically localized mRNAs whose local translation is required for axon growth, synaptogenesis, and synaptic plasticity. However, the molecular mechanisms responsible for m6a-mediated regulation of postmitotic neuronal development are not completely understood. Here, we report that cytoplasmic m6A reader YTHDF1, which binds m6a-modified mRNAs, is widely expressed in the mouse brain and enriched in neurons compared to the other brain cell types such as astrocytes, oligodendrocytes or microglia. Time-sensitive downregulation of YTHDF1 in cultured hippocampal neurons indicates that YTHDF1 is required for the proper development of neurons in culture, from early polarization to spine maturation. In addition, using an inducible knockdown system in vivo, we demonstrate that YTHDF1 is required for in vivo development and positioning of hippocampal neurons to dendrate gyrus. To better understand the molecular mechanisms underlying the neuronal development defects, we focus on the growth cone structure, which is crucial for the guidance and elongation of the growing axon and dendrites, and we show that YTHDF1 localizes at a subset of the plus ends of dynamic microtubules in the growth cone and deregulation of YTHDF1 leads to stalled growth cone dynamics. Moreover, we show that YTHDF1 interacts with the scaffold protein Adenomatous Polyposis Coli (APC), which is a known regulator of the microtubule-actin cytoskeleton in the growth cone, and that reducing YTHDF1 expression results in a decrease in APC immunostaining in the axon and growth cone. Given that APC has been identified as a risk gene for autism spectrum disorder and intellectual disabilities, our findings suggest that deregulation of the YTHDF1/m6a/APC axis pathway could contribute to the physiopathology of these neurological disorders.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-092 A cartographic and morphometric analysis of neuronal spines in m6A reader-deficient mice SHENGQUN HOU(1,2), Momoe Sukegawa(1,2), Dan Ohtan Wang(1,2) 1. RIKEN BDR, Kobe, Japan, 2. Grad Sch Biostudies, Univ of Kyoto, Kyoto, Japan Keyword:* spine morphometric analysis, high through-put, dendrite spine, RNA modification The morphological features of dendritic spines are highly dynamic and correlated to synapse maturation, plasticity, learning and memory. Recently, a new mode of gene regulation, epitranscriptomics, has emerged as a versatile and powerful post-transcriptional regulatory pathway to regulate spines and synapses; the prevalent mRNA modification N6-methyladenosine (m6A) is a critical component in multiple learning and memory paradigms such as motor learning, spatial memory, fear consolidation and extinction, etc. Previously, we have cataloged thousands of m6A-modified RNAs in the synaptic compartments and demonstrated that the loss of the cytoplasmic “reader” proteins YTHDF1 and YTHDF3 cause reduced synaptic transmission. However, the role of YTHDF proteins in neurons over the regulation of spine morphology in vivo is unknown. In the current study, we have performed high-resolution morphometric analysis on a large number of dendritic spines over wide brain regions by combining modified Dil-labeling with high-speed, high-resolution, position-registered imaging followed by computer-aided spine detection and quantification. We applied this technique to analyze dendritic spines in YTHDF1 or YTHDF3-conditional knockout mice (CaMKIIa-Cre: to specifically inactivate YTHDF1 or YTHDF3 in mature forebrain excitatory neurons) and quantified parameters such as spine volume, diameters of the spine head and neck, as well as the length of spine neck, etc. We found massively altered spine morphometry and density at the loss of YTHDFs in different brain regions. Intriguingly, morphometric measurements revealed diverse responses to the loss of YTHDFs in different neurons, and even within the neurons in difference subcellular compartments. Golgi staining supported the analysis with consistent albeit less accurate measurements. Further detailed anatomical analysis will be applied in the future studies to understand the role of m6A signaling in regulating spine dynamics.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-093 SARS-CoV-2 Orf9bとその派生ペプチドは神経細胞のPar-1/MARK活性を抑制する SARS-CoV-2 Orf9b and its derived peptide suppress Par-1/MARK activity in neurons 斎藤太郎(1)、大場俊弥(1)、安藤香奈絵(1) 1. 東京都立大学理学研究科 Taro Saito(1), Toshiya Oba(1), Kanae Ando(1) 1. Grad Sch Sci, Tokyo Met Univ, Tokyo, Japan Keyword: SARS-CoV-2, protein kinase, tau, MARK SARS-CoV-2 affects multiple organ systems, including the central nervous system. However, it is still unclear how gene products from the SARS-CoV-2 genome interrupt molecular processes in neurons. Recent comprehensive interactome analysis of human host proteins and proteins derived from the SARS-CoV-2 genome revealed that one of the accessory proteins of SARS-CoV-2, Orf9b, interacts with the members of Par-1/microtubule affinity-regulating kinases (MARKs). MARKs regulate physiological functions and pathological changes of neurons via phosphorylation of microtubule-binding proteins, including tau, which accumulates in the brain in the pathogenesis of Alzheimer’s disease. In this study, we examined how Orf9b affects Par-1/MARK functions in neurons. Here we report that Orf9b suppresses Par-1/MARK4 activity in neurons. We found that the kinase activity of MARK4 and tau phosphorylation was suppressed when Orf9b was expressed in primary mouse neurons. In transgenic Drosophila expressing human tau in the retina, coexpression of Orf9b suppresses tau phosphorylation at MARK-target sites and ameliorated tau-induced neurodegeneration, indicating that Orf9b suppresses Par-1 activity in vivo. We further revealed the minimal sequences required for interaction with MARK4 by using a series of deletion mutants of Orf9b. Bath application of this fragment of Orf9b conjugated with cell-permeable peptide TAT to primary neurons suppressed phosphorylation of tau at MARK-target sites, indicating that this peptide works as a MARK-inhibitory peptide. Furthermore, feeding this peptide to flies expressing human tau in the retina suppressed tau phosphorylation and ameliorated tau-induced neurodegeneration. These results suggest that the infection of SARS-CoV-2 may modulate kinase activities of MARKs, and Orf9b may be utilized to develop novel MARK inhibitors.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-094 パルブアルブミン陽性神経細胞におけるクラスター型プロトカドヘリンγの欠損が大脳皮質神経回路形成に与える影響 Role of clustered protocadherin gamma for the specific synaptic connections between excitatory and parvalbumin-positive inhibitory neurons in the mouse visual cortex 河村菜々実(1)、足澤悦子(1)、吉村由美子(2)、八木健(1) 1. 大阪大学大学院生命機能、2. 総研大院生理学研究所 Kawamura Nanami(1), Tarusawa Etsuko(1), Yoshimura Yumiko(2), Yagi Takeshi(1) 1. Osaka Univ. FBS, Osaka, 2. Dept. Dev. Physiol., NIPS, Okazaki, Japan Keyword: Visual cortex, Parvalbumin, clustered protocadherin, neuronal circuit In the cerebral cortex, functional neural circuits are formed by specific neural connections between inhibitory and excitatory neurons. However, the molecular mechanisms for the recognition of synaptic partners have remained unclear. We focused on clustered protocadherin (cPcdh) which is a diversified membrane adhesion molecule as a candidate for the recognition molecules between neurons. cPcdh consists of 58 isoforms which are divided into three gene cluster structures (α, β, and γ). In a single neuron, approximately 15 isoforms of cPcdhs are expressed with differential combination, and they have a homophilic adhesion property. It has been reported that the deletion of cPcdh in clonally-related excitatory neurons in the barrel cortex causes a reduction of reciprocal connectivity between them. However, the influence of cPcdh deletion on connectivity between excitatory and inhibitory neurons has not been examined because the deletion of cPcdhγ in inhibitory neurons at embryonic stage is accompanied by their apoptosis. To avoid the inhibitory cell death, we used parvalbumin (PV)-Cre mice in which the Cre-expression starts postnatally. In this study, we investigated the influence of cPcdhγ deletion in PV neurons on local neuronal circuits in the primary visual cortex of PV-Cre; cPcdhγ conditional KO mice using a slice whole-cell patch-clamp recording. The connection probability between a PV neuron and a pyramidal neuron within layer 2/3 was similar between control and KO mice. However, we found two types of PV neuron connectivity: a high reciprocal connectivity to neighboring multiple pyramidal neurons (high-reciprocity) and a low-reciprocity in control mice. Surprisingly, that classification was lost in KO mice, indicating that cPcdhγ might determine the differential connectivity of each PV neuron within layer 2/3. To reveal the influence of cPcdhγ KO in interlaminar connectivity, we examined excitatory inputs to layer 2/3 PV neurons from layers 2-6 by laser-scanning photostimulation with caged glutamate. We found that cPcdhγ-deleted PV neurons receive more inputs from layer 5 and layer 6 pyramidal cells compared to control mice. These results indicate that cPcdhγ proteins in PV neurons are involved in both intralaminar and interlaminar connections in the visual cortex.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-095 中脳水道周囲灰白質腹外側部GABA作動性ニューロンのカタプレキシー表出における役割 GABAergic neurons in the ventrolateral periaqueductal gray are implicated in cataplexy of narcoleptic mice. ジョンソル(1)、内田俊太郎(2)、征矢晋吾(1,2)、櫻井武(1,2) 1. 筑波大学医学医療系、2. 国際統合睡眠医科学研究機構 Sol Jeong(1), Shuntaro Uchida(2), Shingo Soya(1,2), Takeshi Sakurai(1,2) 1. Fac of Med, Univ of Tsukuba, Ibaraki, Japan, 2. WPI-IIIS, Ibaraki, Japan Keyword: cataplexy, narcolepsy, venrtrolateral periaqueductal gray Orexin-producing neurons in the lateral hypothalamus (LH) play an important role in maintenance of wakefulness. The loss of orexin neurons results in narcolepsy, a sleep disorder characterized by excessive day-time sleepiness and cataplexy. Cataplexy is a sudden muscle atonia during wakefulness, often triggered by strong positive emotions. It is considered as a sudden intrusion of rapid eye movement (REM) sleep into wakefulness. The mesopontine junction region, including ventolateral periaqueductal gray (vlPAG) is known to be important in regulating REM sleep (Weber et al., 2018). However, the role of vlPAG in cataplexy is incompletely understood. Recent studies showed that glutamatergic neurons in sublaterodorsal tegmental nucleus (SLD) projecting to ventromedial medulla (VMM; Glu^SLD^→^VMM) was involved in muscle atonia during both cataplexy and REM sleep (Uchida et al., 2021). We identified upstream input neurons of Glu^SLD^→^VMM neurons by cell-type-specific TRIO (cTRIO) method in vGlut2-IRES-Cre mice. We injected Cre-dependent retrograde adeno-associated virus (AAVretro) carrying flippase (FLP) into the VMM, FLP dependent AAV carrying TVA-mCherry and RG in the SLD. Later we injected EnvA-pseudotyped, glycoprotein deleted and GFP-expressing rabies viruses (SAD△G-EGFP (EnvA)) into the SLD. Starter neurons (Glu^SLD^→^VMM neruons) expressing RG (rabies glycoprotein) and TVA-mCherry, allow SAD△G infection and complementation, enabling trans-monosynaptic spread to presynaptic neurons. We found input neurons in the vlPAG, DpMe and several brain region, raising possibility that these neurons might be components of neural circuit of cataplexy and REM atonia. Based on the previous research that activating vlPAG GABAergic neurons suppressed REM sleep, we injected Cre-dependent AAV expressing hM3Dq-mCherry into the vlPAG in vGAT-IRES-Cre;orexin-ataxin3 mice. Chemogenetic activation of vlPAG GABAergic neurons by Clozapine-N-Oxide (CNO) intraperitoneal (i.p.) injection significantly decreased total time of cataplexy-like episodes (CLEs) compared to control group in narcoleptic mice during dark phase. These observations suggest that vlPAG GABAergic neurons suppressed cataplexy through SLD.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-096 マウス前障におけるGABA作動性神経細胞の形態学的解析 Morphological analysis of GABAergic neurons in the mouse claustrum. 髙橋慧(1,2,3)、小林朋世(2,3)、岡本慎一郎(2,3)、山内健太(2,3)、岡本和樹(2,3)、小池正人(2)、伊佐正(1)、日置寛之(2,3) 1. 京都大学大学院医学研究科神経生物学、2. 順天堂大学大学院医学研究科神経機能構造学、3. 順天堂大学大学院医学研究科脳回路形態学 Megumu Takahashi(1,2,3), Kobayashi Tomoyo(2,3), Shinichiro Okamoto(2,3), Kenta Yamauchi(2,3), Kazuki Okamoto(2,3), Masato Koike(2), Tadashi Isa(1), Hiroyuki Hioki(2,3) 1. Dept Neurosci, Kyoto Univ, Grad Sch Med, 2. Dept Cell Biol Neurosci, Juntendo Univ, Grad Sch Med, 3. Dept Neuroanat, Juntendo Univ, Grad Sch Med Keyword: Claustrum, GABAergic neuron, adeno-associated virus, tissue clearing The claustrum (Cla) has been revealed to have abundant excitatory reciprocal connections with the cerebral cortex; corticoclaustral inputs integrate multiple cortical information in Cla (Chia et al., 2020) and claustrocortical projections coordinate cortical activities (Narikiyo et al., 2020). The Cla can be divided into two subregions, defined by parvalbmin (PV)-immunoreactive (ir) core region and vesicular glutamate transporter 2 (VGluT2)-ir shell region (Real et al., 2003, 2006). The core region projects to frontal-midline cortical areas, while the shell region to motor-related and temporal cortices. These distinct features suggest that excitatory neurons in the subregions regulate the activity of each cortical network in parallel (Marriott et al., 2021). However, it remains unclear whether the two subregions are interacted through GABAergic neurons in Cla. To clarify the involvement of GABAergic neurons to the neuronal circuits of each subregion, we examined their distribution, neurochemical expression, and dendritic and axonal arborizations of single neurons within the core and shell regions. First, to distinguish the two subregions, we performed three-dimensional immunohistochemistry (3D-IHC) (AbScale; Hama et al., 2015) against PV, VGluT2, and NeuN. We showed that the PV-ir core region has higher NeuN⁺ cell densities than the VGluT2-ir shell region, and that two subregions can be identified from the distribution of NeuN-ir (cytoarchitecture). We then conducted in situ hybridization histochemistry (ISH) for VGluT1 and GAD67 mRNAs, and uncovered that about 10% of Cla neurons were GABAergic in both the core and shell regions. We also combined ISH for GAD67 mRNA and IHC for PV, SOM, and found main GABAergic neurons in the core and shell regions were PV⁺ and SOM⁺, respectively. We further investigated the dendritic and axonal arborization of PV and SOM neurons in Cla. After injecting adeno-associated virus vectors into the Cla of PV- or SOM-Cre knock-in mice, 1-mm-thick brain slices were prepared and optically cleared with ScaleS. Following acquisition of 3D image stacks, the whole dendrites and axons were reconstructed. We have revealed that the PV and SOM neurons are interneurons confined to Cla, and we are currently investigating the distribution of their neurites within the two subregions. These results will contribute to understanding of the influence of GABAergic neurons on excitatory projection neurons in the core and shell regions.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-097 大脳皮質への反復刺激に対する線条体投射ニューロンのシナプス応答は直接路と間接路で異なる Synaptic responses of striatal projection neurons to cortical repetitive stimulation are difference between direct and indirect pathway. 田村篤史(1)、末岡智己(1)、笹川正人(4)、稲垣良(1)、小林和人(2)、小山内実(1,3,4) 1. 大阪大学大学院医学系研究科、2. 福島県立医科大学生体情報伝達研究所、3. 情報通信研究機構・大阪大学脳情報通信融合研究センター、4. 東北大学大学院医学系研究科 Atsushi Tamura(1), Tomoki Sueoka(1), Masato Sasagawa(4), Ryo Inagaki(1), Kazuto Kobayashi(2), Makoto Osanai(1,3,4) 1. Osaka Univ, Grad Sch Medicine, Suita, Japan, 2. Inst Biomed Sci, Fukushima Med Univ, Sch Med, Fukushima, Japan, 3. CiNet, NICT, Suita, Japan, 4. Grad Sch Med, Tohoku Univ, Sendai, Japan Keyword: stiatum, calcium imaging, dopamine, oscillation The basal ganglia play an important role in the motor control. The striatum is one of the primary input nuclei of the basal ganglia and is composed mainly of the medium spiny neuron (MSN). The MSNs project to output nuclei of the basal ganglia through two major projection systems, direct and indirect pathway. The MSNs of direct pathway express dopamine D1 receptor (D1-MSN) and the MSNs of direct pathway express dopamine D2 receptor D2 (D2-MSN). It is known that D1-MSN and D2-MSN oppositely modulate motor functions by facilitation and inhibition the movements, respectively. The beta-band frequency (~13-30Hz) oscillations in scalp electroencephalographic (EEG) are observed over sensorimotor cortex at the time of planning and/or execution of motion. How these cortical inputs in multiple frequency ranges effect on synaptic property of MSN is unclear. Here, we investigated the response properties of D1- and D2- MSN to the various frequency inputs from the cortex. In addition, we examined the involvement of dopaminergic signaling in D1- and D2-MSN activities. To test the synaptic property of MSNs following the cortical inputs in multiple frequency ranges, we observed the intracellular Ca²⁺ concentration ([Ca²⁺]_i) transient on D1- and D2-MSN to the cortical stimulation in 1-50 Hz frequency range in the striatal slices of transgenic mice which express yellow fluorescent protein (YFP) under D1R or D2R promotor (mDrd1-YFP or Drd2-YFP mice). We found that the [Ca²⁺]_i transients to entire stimulation frequency were significantly more elevated in D1-MSN than D2-MSN. This result suggests that D1- and D2-MSN have different synaptic property to cortical input.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-098 マーモセット海馬体から前海馬台への直接投射 Direct hippocampo-presubicular projections in the common marmoset. 本多祥子(1)、下川哲哉(2)、松田正司(2)、小林靖(3)、守屋敬子(4) 1. 東京女子医大・医・解剖（神経分子形態学）、2. 愛媛大学・医・解剖発生、3. 防衛医大・解剖、4. 都医学研・脳発達神経再生 Yoshiko Honda(1), Tetsuya Shimokawa(2), Seiji Matsuda(2), Yasushi Kobayashi(3), Keiko Moriya-Ito(4) 1. Dept Anat & Neurobiol, Sch Med, Tokyo Women’s Med, Univ, Tokyo, Japan, 2. Div Anat & Embryol, Dept Funct Biomed, Ehime Univ, Toon, Japan, 3. Dept Anat & Neurobiol, National Defense Med Col, Tokorozawa, Japan, 4. Dept Brain Development & Neural Regeneration, Tokyo Metropol Inst Med Sci, Tokyo, Japan Keyword: CA1, SUBICULUM, ENTORHINAL CORTEX The marmoset recently attracts much attention as an experimental animal, however, there are few studies that have comprehensively analyzed the neural connectivity of the limbic system. We have been investigated the connectivity among the hippocampal formation (i.e., DG, CA1-3 and the subiculum) and parahippocampal regions (the presubiculum, parasubiculum, entorhinal cortex and retrosplenial cortex), especially focusing on the presubiculum, using retrograde and anterograde tracers (cholera toxin B subunit and biotin dextran amine). We have reported that the major connections of memory circuit in the rats were mostly conserved in the marmoset brain, for example, direct back projection (for instance, from CA1 to the entorhinal cortex) and indirect back projections (for instance, from the subiculum to the entorhinal cortex via the presubiculum). Moreover, we have demonstrated the presence of a direct projection from the CA1 pyramidal cell layer to the deep layers of the presubiculum in the marmoset, which was previously identified in the rabbit brain but not in the rat. In the present study, we investigated the distribution patterns of the presubicular projection cells in CA1 and the subiculum. The cells of origin were distributed in most of the entire rostrocaudal and proximodistal extents of CA1, and cells in the rostral part of CA1 tended to project to a wide range of the presubiculum along the rostrocaudal axis. We also found that the cells of origin of subiculo-presubicular projections were localized in the middle part along the superficial-to-deep axis of the pyramidal cell layer of the distal subiculum in the marmoset. Our results suggest that,during the evolution of the cerebral cortex, information processing became more complex due to increased information transmitted through CA1; as a result, the CA1-presubicular projections, which are minor in rodents, become more major in primates. The present study also revealed a clear sublaminar pattern in the distribution of cortical projection cells in the marmoset subiculum.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-099 パーキンソン病感受性遺伝子GAKの阻害による神経発達への影響 Effects of GAK inhibition on Neurite Outgrowth and Synapse Formation 江川純(1)、Reza Arta(1)、五十嵐道弘(1)、Vance Lemmon(2)、John Bixby(2)、Melissa Barrero(2)、Yan Shi(2)、染矢俊幸(1) 1. 新潟大学大学院医歯学総合研究科、2. マイアミ大学 Jun Egawa(1), Reza Arta(1), Michihiro Igarashi(1), Vance Lemmon(2), John Bixby(2), Melissa Barrero(2), Yan Shi(2), Toshiyuki Someya(1) 1. Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan, 2. University of Miami, Miami, USA. Keyword: GAK, Parkinson disease, High content screening, Neurodevelopment ackground: Cyclin G-associated kinase (GAK) has been identiﬁed as a potential candidate risk gene for Parkinson's disease (PD) by molecular genetic studies, including genome-wide association studies. Although the α-synuclein-mediated neurodegeneration of the reduced GAK function has been shown, the effects of GAK on neurodevelopment have not been well investigated. Method: The hippocampal neurons of E15 mice were cultured on 96 well plates. For neurite length analysis, after plates were incubated for three hours, we added SGC-GAK-1 solution was transferred to each well in the culture plate in final concentrations ranging from 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 µM and a control. Then the plate was incubated for 3days total for the same condition. For synapse numbers analysis, after plates were incubated for 7days, we added SGC-GAK-1 solution was transferred to each well in the culture plate in final concentrations ranging from 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 µM and a control. Then the plate was incubated for 14days total for the same condition. Neurons were fixed and stained with MAP2 antibody only for neurite length analysis, Synaptophysin1, SHANK2, and MAP2 antibodies for synapse number analysis. The image acquisition was performed using a CellInsight^TM CX5. In the Neurite length analysis, the neurite was detected by MAP2 signal. The total of the calculated neurite lengths in the entire visual field divided by the number of neurons was compared. In the synapse number analysis, a synapse signal was defined as a combination of the SHANK2 signal and the Synaptophysin1 signal by 50% or more around the MAP2 neurite signal. The putative positive hit and control images were also analyzed using a custom algorithm developed using the Neuronal Profiling BioApplication v4.2. All data are expressed as mean ± the standard error of the mean. Control wells and wells with each KI concentration added were compared by Student's t-test or Welch's t-test. An alpha level of p < 0.05 was set for significance, and multiple comparisons were corrected by the Bonferroni method in each analysis. Results: We compared the total neurite length per number of neurons in control wells and wells with eight concentrations (40, 20, 10, 5, 2.5, 1.25, 0.625, and 0.3125μM) SGC-GAK-1. Total neurite length per the number of neurons was significantly reduced with 5, 10, 20, and 40μM SGC-GAK-1 additions compared to controls (p < 0.005/8). The total number of synapses per the number of neurons was significantly reduced with 40, 20, and10μM SGC-GAK-1 additions compared to controls (p < 0.005/8). Conclusion: We treated primary neurons with SGC-GAK-1, a selective inhibitor of GAK, suggesting that decreased GAK function was associated with impaired neurodevelopment. The study may contribute to the elucidation of new molecular pathways involved in the pathophysiology of PD and the development of new therapeutic agents.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-100 軟体動物嗅覚中枢のin vitro同期振動ネットワークを駆動するアセチルコリン作動性シナプス Cholinergic induction of synchronous oscillation in the slug neuronal network in vitro. 小林卓(1,2)、太梨穂(1) 1. 徳島文理大学香川薬学部、2. 徳島文理大学神経科学研究所 Suguru Kobayashi(1,2), Futori Riho(1) 1. Kagawa Schl Pharmaceut Sci, Tokushima Bunri Univ, Sanuki, Japan, 2. Inst Neurosci, Tokushima Bunri Univ, Sanuki, Japan Keyword: SYNCHRONOUS OSCILLATION, OLFACTORY CENTER, SLUG, CULTURED NEURON NETWORK Synchronous oscillatory network is vital for cognitive functions of the brain in both vertebrates and invertebrates. In the central nervous system of the terrestrial slugs, spontaneous periodic oscillation (0.5 - 1.0 Hz) is recorded from the surface of the laminar structure of procerebrum (PC), and its frequency changes are suggested to encode the olfactory information and memory. We recently found oscillatory activity is generated spontaneously in dispersed cell culture of PC neurons. Application of acetylcholinesterase inhibitor or nicotine increased the number of spontaneous activities in cultured PC neurons, and furthermore, induced synchronous oscillation in in vitro network activity. On the other hand, biogenic amines or neuropeptides often changed the number of spontaneous activities without generating synchronous oscillation on PC neurons. To investigate how such synchronous oscillation is generated, we tested cholinergic activation and compared between synchronous and asynchronous networks. Previous results suggest that acetylcholine could be function as a driving force on the synchronous oscillatory activity of the PC neuron network via nicotinic acetylcholine receptors activation in vivo. In present study, differences between synchronous and asynchronous network were examined in cultured PC neuron (7-21 days). First, PC neurons cultured more than 10 days could induce synchronous oscillation in vitro. Synchronous networks included two groups of neurons, (a) acetylcholine-sensitive PC neurons, (b) acetylcholine-insensitive PC neurons that activated or driven by excitatory input from a. Second, we found a lower excitability and a higher sensibility to cholinergic activation of PC neurons in synchronous networks than asynchronous networks. Third, muscarinic receptor agonist, pilocarpine, did not induce synchronous oscillation and did not occlude the effects of physostigmine. It is suggested that: (1) in vitro synchronous oscillation was induced by activation of cholinergic synaptic transmission via nicotinic ACh receptors; (2) "synchronous in vitro networks" were characterized by higher ACh-sensitivity (than "asynchronous networks") together with the lateral inhibition of excessive activity in resting states.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-101 ラット顔面神経軸索切断後のc-Jun/c-FosとCREB/ATF2タンパク質の変動 Changes of c-Jun/c-Fos and CREB/ATF2 proteins after rat facial nerve axotomy 石嶋貴志(1)、中嶋一行(1) 1. 創価大学糖鎖生命システム融合研究所 Takashi Ishijima(1), Kazuyuki Nakajima(1) 1. Glycan & Life Systems Integration Center (GaLSIC), Soka University Keyword: axotomy, c-Jun, p-CREB, functional down regulation In adult rats, transection of the facial nerve leads to the functional downregulation of motoneurons, with decreases in the levels of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAchT) and GABA_A receptor alpha 1 (GABA_AR◊1). Subsequently, resident microglia are activated and proliferate around living motoneurons. These cellular responses are thought to promote the survival, repair and regeneration of motoneurons. However, it is still unclear which kinds of signaling molecules are related to these responses. To analyze the responses in vivo, we cut the right facial nerve of 8-week-old male Wister rats at the stylomastoid foramen, and at 1.5, 3, 6, and 12 h, or 1, 3, 5, 7, and 14 d after axotomy, rats were decapitated. The contralateral and ipsilateral facial nuclei of each rat were cut out from the brainstem, and their tissue extract was prepared for Western blot. Immunohistochemistry was also performed to indicate the localization of the signaling molecules. In this study, we investigated the changes and localizations of several signaling molecules, including immediate early genes (IEGs) such as c-Jun and c-Fos and CREB/ATF family members such as CREB and ATF2. Western blot and immunohistochemical analyses revealed the following. Among the IEGs, c-Jun was increased in injured motoneurons, but c-Fos did not respond to neuronal injury. Among the CREB/ATF family members, phosphorylated CREB (p-CREB) was significantly decreased in injured motoneurons. The levels of CREB and ATF2 were immunohistochemically increased in microglia. These results strongly suggested that c-Jun is involved in the survival, repair and regeneration of motoneurons, but p-CREB/CREB are associated with the downregulation of motoneuron-specific molecules. CREB and ATF2 were also suggested to participate in microglial responses.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-102 小脳プルキンエ細胞軸索における活動電位伝播と伝達物質放出のcAMPによる修飾 Modulation of action potential propagation and synaptic outputs by cAMP in cerebellar Purkinje cell axons 古川慧(1)、川口真也(1) 1. 京都大学理学研究科生物物理学教室 Kei Furukawa(1), Shin-ya Kawaguchi(1) 1. Department of Biophysics, Graduate School of Science, Kyoto University Keyword: action potential, cAMP, axon, Purkinje cell At many central nervous system synapses, cAMP potentiates neurotransmitter release from presynaptic terminals. By using an adeno-associated virus (AAV) vector, we fluorescently visualized a cultured Purkinje cell (PC), an inhibitory cerebellar neuron, and performed a patch clamp recording from a PC's target neuron. We induced increase in intracellular cAMP by bath application of forskolin, a cell membrane-permeable adenylyl cyclase activator. Unexpectedly, forskolin only slightly augmented both spontaneous and evoked transmitter releases from PC axon terminals, and the extent of augmentation was marginal compared to other terminals as shown in many studies, for example, on cerebellar parallel fiber and hippocampal mossy fiber terminals. Additionally, we found by simultaneous recordings from a PC soma and its target neuron that forskolin increased the synaptic delay. Paired recordings from the soma and the axon showed that action potential propagation in an axon was delayed by forskolin in a manner dependent on the axon length, suggesting that cAMP slows the action potential conduction velocity in PC axons. Moreover, forskolin decreased the peak amplitude and increased halfwidth of action potentials in an axon, indicating that cAMP negatively regulates action potentials in the course of propagation from a PC soma to an axon. We also voltage-clamp recorded the axonal currents through voltage-gated Na⁺ channels, voltage-gated K⁺ channels, and hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels before and after forskolin application. The action potential attenuation in an axon by cAMP is expected to reduce the Ca²⁺ influx, leading to reduction of transmitter release. Taken all these results together, it was suggested that cAMP attenuates the action potential propagation in PC axons, which counteracts the well-known facilitative action of cAMP on the release of neurotransmitter.

2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-103 成長円錐内の局所的タンパク合成に伴うリボゾームとアクチンフィラメントとの関係について The relationship between ribosomes and actin filament associated with local protein synthesis in axonal growth cones 星治(1)、武井延之(2) 1. 東京医科歯科大学、2. 新潟大学脳研究所 Osamu Hoshi(1), Nobuyuki Takei(2) 1. Tokyo Medical and Dental University, 2. Brain Research Institute, Niigata University Keyword: local protein synthesis, growth cone, ribosomal protein [Background and objective] A neuron has a single axon and multiple dendrites and which are used to transmit and receive information, respectively. Neurons that require rapid functional changes in response to stimuli at sites distant from the cell body, proteins are synthesized in both the cell body and locally in dendrites and axons, allowing more efficient responses. This "local protein synthesis" has been studied post-synaptically, but the detailed molecular bases of translational regulation in response to stimuli is largely unknown. In this study, we stimulated primary cultures of rat spinal dorsal root ganglion cells with brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) to promote local protein synthesis in the growth cone of the axon tip, and analyzed how the stimulation changed localization of actin filaments and ribosomal protein S6. [Materials and methods] Specimens were prepared by culturing primary rat spinal dorsal root ganglion neurons and treating them with 50 ng/ml BDNF and NGF in culture medium for 30 min. Unstimulated cultures were prepared as controls. Both control and treated neurons were fixed in 4% paraformaldehyde solution, reacted with phalloidin 488, followed by anti-ribosomal protein S6 antibody. Alexa Fluor 555 anti-rabbit IgG was labeled as a secondary antibody and super-resolution microscopy was used to analyze the localization of actin filaments and ribosomal protein S6. [Results] A total of 64% of the anti-ribosomal protein S6 antibody-positive sites and phalloidin-positive sites in the P-domain of growth cones were colocalized in control specimens. In contrast, the colocalization rate decreased to 48% in BDNF- and NGF-treated specimens. In addition, a cluster of anti-ribosomal protein S6 antibody-positive sites occurred in the region distant from the phalloidin-positive sites. [Discussion and Future Prospects] Our previous study found that BDNF promotes local protein synthesis in the growth cone. Thus, in this study, we focused on ribosomal protein S6 and actin filaments to analyze how localization of ribosomes changes with overall protein synthesis, using super-resolution microscopy. Our quantitative analysis suggested that ribosomal protein S6 and actin tend to colocalize in the absence of stimulation, and that ribosomal protein S6 tends to move away from actin when local protein synthesis is stimulated/promoted. We propose that this is due to the ribosome being turned into a polysome by protein factory. This study provides a useful finding for further clarification of the role of cytoskeletal components in local protein synthesis that may see further applications in the study of neurological disorders associated with cytoskeletal disruption.		2022年7月1日　11:00～12:00　宜野湾市民体育館　ポスター会場2 2P-104 Effects of fingolimod on vasculature and glial scar formation after closed-head injury Nitin Shrikant Sawant(1), Natsuki Okazaki(1), Mitsuhiro Morita(1) 1. Graduate School of Science, Kobe University, Kobe, Japan Keyword:* Reactive astrocyte, Brain injury, Fingolimod A sphingosine-1-phosphate (S1P) receptor 1 agonist, fingolimod is known to inhibit the egress of T cells from lymph node, and an FDA-approved immunosuppressant for treating multiple sclerosis. This drug is currently under preclinical, as well as clinical studies for treating brain injuries including stroke and traumatic brain injury (TBI) . Since accumulating evidence indicates that T cell plays crucial roles in activating astrocyte after brain injury, we investigated the influence of fingolimod on the pathological processes, especially reactive astrocytes in an originally developed closed-head injury model, photo-injury mouse. This model is created by an intense light exposure through thinned cranial window, and suitable for studying reactive astrocytes because artificial glial activation by craniotomy, which is commonly used for conventional TBI model, such as fluid percussion is avoided. Unexpectedly, photo-injury mouse subjected to daily fingolimod treatment (10 mg / kg) showed significant red blood cell (RBC) deposition around lesion 7-14 days after injury. And this RBC deposition was likely associated with reduced vascular integrity, which was reflected in the increased leakage of Evans blue, and reduced blood vessel density around lesion. Since it is reported that S1P is essential for angiogenesis during development, as well as tissue recovery after injury, and that continuous fingolimod treatment down-regulates S1P receptor 1, the RBC deposition is most likely attributed to the impairment of angiogenesis by suppressing S1P signaling. Fingolimod also reduced the accumulation of laminin, an extracellular matrix protein consisting of basement membrane, along the border of lesion core (LC). Thus, fingolimod was found to affect not only vasculature, but also glial scar formation. GFAP immunohistochemistry revealed that fingolimod reduced a reactive astrocyte subpopulation in the LC (LC reactive astrocyte) without affecting another subpopulation surrounding the LC. Since our previous report (Matsuda et al NEURO2019) indicates that the LC reactive astrocyte is derived from NG2 glia, it is supposed that fingolimod reduces the LC reactive astrocytes and their scar forming laminin production, presumably by suppressing T cell / NG2 glia interaction. Our results highlight complicated long-term influences of fingolimod on pathological processes after brain injury, and provides significant implications for clinical application of this drug to brain injuries.