ポスター
A. 分子・細胞生物学
A. Neural Excitability, Synapses, and Glia: Cellular Mechanisms |
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| 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-001
セロトニントランスポーター遺伝子活性と高齢者における認知機能・うつ傾向の関連
Gene activity of serotonin transporter associates with cognitive function, depressive tendency and hippocampal volume in older people: the Arao study
*柳田 悠太朗(1)、仲地 ゆたか(1)、吉川 慧(2)、清田 恵美(1)、渡邊 理紗(1)、村田 唯(1)、菅原 裕子(3,4)、石川 智久(5)、橋本 衛(6)、高野 裕治(7)、瀧 靖之(8)、酒井 規雄(2)、岡本 泰晶(9)、竹林 実(5)、文東 美紀(1)、岩本 和也(1)
1. 熊本大学大学院生命科学研究部分子脳科学講座、2. 広島大学大学院医学系研究科神経薬理学、3. 関西労災病院、4. 大阪大学大学院医学系研究科精神医学教室、5. 熊本大学大学院生命科学研究部神経精神医学講座、6. 近畿大学医学部精神神経科学教室、7. 人間環境大学総合心理学部、8. 東北大学加齢医学研究所臨床加齢医学研究分野、9. 広島大学大学院医歯薬保健学研究科精神神経医科学
*Yutaro Yanagida(1), Yutaka Nakachi(1), Satoshi Kikkawa(2), Emi Kiyota(1), Risa Watanabe(1), Yui Murata(1), Hiroko Sugawara(3,4), Tomohisa Ishikawa(5), Mamoru Hashimoto(6), Yuji Takano(7), Yasuyuki Taki(8), Norio Sakai(2), Yasumasa Okamoto(9), Minoru Takebayashi(5), Miki Bundo(1), Kazuya Iwamoto(1)
1. Department of Molecular Brain Science Graduate School of Medical Sciences Kumamoto University, 2. Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical and Health Sciences, Hiroshima University, 3. Department of Psychiatry, Kansai Rosai Hospital, 4. Department of Psychiatry, Graduate School of Medicine, Osaka University, 5. Department of Neuropsychiatry, Faculty of Life Sciences, Kumamoto University, 6. Department of Neuropsychiatry, Kindai University Faculty of Medicine, 7. Department of Psychological Sciences, University of Human Environments, 8. Department of Aging Research and Geriatric Medicine, Institute of Development, Aging and Cancer, Tohoku University, 9. Department of Psychiatry and Neurosciences, Graduate School of Biomedical Sciences, Hiroshima University
Keyword: SLC6A4, 5-HTTLPR, DNA methylation
Serotonin transporter is encoded by SLC6A4, and it regulates serotonin concentration in synaptic cleft by reuptake of serotonin. Inhibition of reuptake is considered to be the main mechanism of action of antidepressants. SLC6A4 has the functional polymorphism called serotonin transporter linked promoter region (5-HTTLPR), which is roughly classified into short and long alleles that have low and high transcriptional activity, respectively. SLC6A4 also has a functional CpG site called CpG3 near the 5-HTTLPR. DNA methylation at this site causes loss of transcriptional activity, and increased DNA methylation has been reported in psychosis. To date, numerous case-control association studies have been conducted between 5-HTTLPR and several psychiatric disorders. However, the role of SLC6A4 on the aging-related changes of brain structure and functions remains poorly understood. In this research, we employed a cohort study which consisted of people older than 65 (n=1,483), and examined the relationship of SLC6A4 activity with cognitive decline and depressive tendency. We used Mini-Mental State Examination, Geriatric Depression Scale-15, brain volumes measured by MRI, 5-HTTLPR genotype, and DNA methylation level of CpG3 measured by pyrosequencing. We found significant correlation between cognitive decline and depressive tendency in the subjects with low activity 5-HTTLPR alleles. Moreover, the subjects with low activity alleles showed cognitive decline-associated hypermethylation at the CpG3. However, the subjects with high activity allele did not show the correlation or cognitive decline-associated hypermethylation. From the results of multiple regression analysis, the high activity allele had significant positive effects on bilateral hippocampus volumes. Consequently, hippocampal volumes were significantly larger in subjects with high activity alleles compared with those with low activity alleles. These results imply that SLC6A4 activity regulated by 5-HTTLPR and DNA methylation was related to cognitive function and depressive tendency in older people by affecting the hippocampal volume. | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-002
TARPγ-8の分子動態にPSD95とのカップリングが及ぼす効果の解析
Analysis of the effect of TARPγ-8-PSD95 coupling on TARPγ-8 dynamics
*小林 新九郎(1)、並木 繁行(2)、浅沼 大祐(2)、廣瀬 謙造(2)
1. 東京大学医学部医学科、2. 東京大学大学院医学系研究科
*Shinkuro Kobayashi(1), Shigeyuki Namiki(2), Daisuke Asanuma(2), Kenzo Hirose(2)
1. Fac Med, Univ of Tokyo, Japan, 2. Grad Sch Med, Univ of Tokyo, Japan
Keyword: AMPA RECEPTOR, POSTSYNAPTIC DENSITY, TRANSMEMBRANE AMPAR REGULATORY PROTEIN, SINGLE-PARTICLE TRACKING
TARPγ-8, an auxiliary subunit of AMPA receptor (AMPAR), is essential for AMPAR-mediated transmission at hippocampal excitatory synapses. Since TARPγ-8 binds synaptic scaffolding proteins such as PSD95, it is considered to mediate the trapping of AMPARs by postsynaptic density (PSD). However, it is not unclear how PSD proteins regulate localization and dynamics of TARPγ-8. Here we present analyses of TARPγ-8 dynamics and its alteration by PSD95. First, immunostaining of TARPγ-8 with PSD 95 in cultured hippocampal neurons revealed that TARPγ-8 is accumulated at the excitatory synapses. We next attempted to recapitulate the TARPγ-8 coclustering with PSD95 in heterologous expression in cells. When dually expressed in COS7 cells, TARPγ-8 and PSD95 formed molecular clusters with a typical size of ~1 μm on the plasma membrane. In contrast, cells solely expressing TARPγ-8 showed a diffuse pattern. For analysis of TARPγ-8 motion within the cluster at single molecule level, we performed a long-term single-particle tracking (SPT) technique that affords a sufficient number of tracks by employing a newly developed fluorescence labeling technique called DeQODE technology. In DeQODE technology, a QODE probe, an organic dye conjugated to a quencher moiety, is converted from a non-fluorescent to a fluorescent state upon binding to DeQODE tag which is a single-chain antibody against the quencher moiety. To achieve sparse fluorescence labeling of TARPγ-8 suitable for SPT experiments, we expressed DeQODE tag-fused TARPγ-8 in COS7 cells and then applied a low concentration (100 pM) of QODE probe to the cells. The procedure enabled SPT of TARPγ-8 molecules for over 10 minutes. In cells solely expressing TARPγ-8, TARPγ-8 molecules were found to diffuse on the plasma membrane with a diffusion coefficient (D) of ~0.05 μm2/s. In cells expressing TARPγ-8 and PSD95, TARPγ-8 molecules within TARPγ-8/PSD95 clusters diffused slowly (D < 0.01 μm2/s) and the rate of diffusion was slower than that outside the clusters, indicating that they are physically “trapped” by PSD95. To our knowledge, these data are the first to show that PSD95 traps TARPγ-8 in its PSD-like clusters. Considering that TARPγ-8 is a dominant AMPAR binder, our results suggest that PSD95-TARPγ-8 coupling plays a functional role in recruiting AMPAR to PSD. | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-003
近位依存性標識Split-TurboID法による神経回路特異的分子の網羅的探索
A landscape of synaptic molecules identified by synapse-specific proximity labeling with Split-TurboID in the cerebellum
*湯本 蒼也(1)、髙野 哲也(1,2)、山崎 世和(1)、田村 朋則(3)、浜地 格(3)、柚崎 通介(1)
1. 慶應義塾大学医学部生理学教室、2. 科学技術振興機構、さきがけ、3. 京都大学大学院工学研究科、合成・生物化学専攻、浜地研究室
*Soya Yumoto(1), Tetsuya Takano(1,2), Tokiwa Yamasaki(1), Tomonari Tamura(3), Itaru Hamachi(3), Michisuke Yuzaki(1)
1. Department of Neurophysiology, Keio University School of Medicine, 2. PRESTO, Japan Science and Technology Agency, 3. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University
Keyword: Split-TurboID, Proximity labeling, synapse-specific
In the mouse central nervous system, each neuron is thought to be connected to up to 10,000 other neurons through excitatory and inhibitory synapses. Depending on the type of pre- and postsynaptic neurons, synapses are formed at specific subcellular locations and contain different sets of neurotransmitter receptors, enabling synapse-specific functions. However, it remains largely unknown how such synapse-specific characteristics are regulated mainly because the information about molecules constituting each type of synapse is scarce. Here, we aimed to obtain a landscape of synaptic molecules by synapse-specific proximity labeling with Split-TurboID focusing on two types of cerebellar synapses in mice: excitatory synapses between parallel fibers (PF) and Purkinje cells (PC) and inhibitory synapses between molecular layer interneurons (MLI) and PCs. Immunoblot analyses revealed biotinylated proteins in the cerebellum when N-terminal and C-terminal fragments of Split-TurboID were expressed in PCs and granule cells, respectively. Immunohistochemical analyses showed biotinylated proteins were localized at PF-PC synapses. Similarly, immunoblot analyses showed reconstituted biotinylation activities in the cerebellum when N-terminal and C-terminal fragments of Split-TurboID were expressed in PCs and MLIs, respectively. Biotinylated proteins were also localized at MLI-PC synapses. We are currently performing mass spectrometric analyses of proteins labeled by synapse-specific expression of Split-TurboID. By comparing molecules identified at PF-PC and MLI-PC synapses, we expect that we could obtain a better understanding of how individual synapses are endowed with synapse-specific characteristics. | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-004
新たなシナプスをつなぐ：シナプス形成分子の改変による新規シナプスコネクターの設計
Bridging synaptic gaps: development of new synthetic synapse connectors based on endogenous synapse organizers
*大和田 響(1)、松田 恵子(1)、柚﨑 通介(1)
1. 慶應義塾大学医学部
*Hibiki Owada(1), Keiko Matsuda(1), Michisuke Yuzaki(1)
1. Keio University School of Medicine, Tokyo, Japan
Keyword: synapse, glutamate receptor, C1q
Synapse organizers, which precisely determine when, where and which types of synapses are formed, play essential roles in higher brain functions by regulating neuronal circuits. It has become increasingly clear that many neuropsychiatric and neurodevelopmental disorders are related to the dysfunction of synapses and neuronal connectivity. To better understand pathological conditions and develop new therapeutics, it is important to clarify molecular mechanisms by which synapses are regulated. Synapse organizers are classified into secreted types, such as Wnt and FGF, and cell adhesion types, such as neurexins and neuroligins. Interestingly, some secreted synapse organizers, which we referred to as extracellular scaffold proteins (ESPs), are retained at the synaptic cleft and rapidly regulate the formation or function of synapses. For example, Cbln1 is secreted from neurons and immediately binds to its pre- and postsynaptic receptors, neurexin and the ionotropic glutamate receptor GluD2. By exploiting the modular architecture and structural information of ESPs, we recently developed a synthetic synapse connector CPTX, which consisted of the pentraxin (PTX) domain of neuronal pentraxin and the cysteine-rich domain (CRR) of Cbln1. CPTX induced synapse formation by simultaneously binding to AMPA receptors and neurexin through the PTX and CRR domains, respectively. These results indicate that new synthetic synapse organizers could be developed to connect desired pre- and postsynaptic sites using structural elements of various ESPs. In the present study, we would like to present our ongoing work on such endeavors and discuss their potential use in modifying neuronal circuits. | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-005
海馬神経細胞におけるクラスター型プロトカドヘリンのホモフィリック相互作用の可視化
Visualization of Homophilic Interaction of Clustered Protocadherin in Cultured Hippocampal Neurons
*高杉 朋実(1)、星野 七海(1)、足澤 悦子(1)、八木 健(1)
1. 大阪大学大学院生命機能研究科
*Tomomi Takasugi(1), Natsumi Hoshino(1), Etsuko Tarusawa(1), Takeshi Yagi(1)
1. Graduate School of Frontier Biosciences, Osaka University , Osaka, Japan
Keyword: synapse formation, clustered protocadherin, Neuroligin, FRET
Self-recognition and non-self-discrimination among neurons via cell adhesion molecules are believed to play a fundamental role in the formation of functional neural assembly in the brain. However, the relationship between cell recognition molecules and a process of synaptic formations remains unresolved. Clustered protocadherin (cPcdh) is one of the cell adhesion molecules belonging to the cadherin superfamily and consists of 58 isoforms divided into three gene clusters, α, β, and γ in mice. cPcdh is widely expressed in the CNS and approximately 15 isoforms out of them are expressed stochastically in individual neurons. In addition, cPcdh proteins homophilically interact with extracellular cadherin domain EC1-EC4 between different cell membrane. These characteristics suggest that a various combination of cPcdh isoforms expressed in each neuron determines a specific neuronal recognition to form synapses. Previous studies have shown that when Pcdhγ interacts with neuloligin-1(Nlg1) or neuloligin-2 (Nlg2), a synaptic adhesion molecule localized at the postsynapse, it inhibits binding to presynaptic partner of Nlg, neurexin, and negatively regulates synapse maturation. However, our immunoelectron microscopic analysis revealed that cPcdhγ is expressed presynaptic and postsynaptic membrane in CA1 pyramidal cells, indicating that cPcdhγ might regulate a selection of synaptic partner via their homophilic interaction. In this study, we examined whether cPcdhγ which is colocalized with Nlg1 or Nlg2 show homophilic interaction of Pcdhγ or not in cultured hippocampal neurons. The homophilic interaction of cPcdhγ was visualized by FRET (Förster resonance energy transfer). FRET is a process that an excited energy of a donor molecule is nonradiatively transferred to a juxtaposed acceptor molecule via dipole-dipole resonance interaction. We produced FRET probes which CFP is inserted into EC1 region of cPcdhγB2 isoform and YFP is inserted into EC5 region. When the cPcdhγB2-FRET probe interacts homophilically, energy transfer from CFP to YFP would occur. This makes it possible to confirm the homophilic interaction of cPcdhγB2-FRET probes. Hippocampal neurons transfected with Nlg1-mRuby3 or Nlg2-mRuby3 and cPcdhγB2-FRET probes were cultured. We for the first time succeeded to observe cPcdhγB2 homophilic interaction in hippocampal neurons. The relationship between cPcdhγB2 homophilic interaction and the localization of Nlg1 or Nlg2 will be discussed. | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-006
floxマウスへのAAVベクター静脈内投与を用いたアストロサイト特異的ノックアウト法の開発
A novel method for astrocyte-targeted knockout of a gene in flox mice by intravenous infusion of AAV vectors
*川畑 勇人(1)、今野 歩(1,2)、佐藤 百美佳(1)、河内 美香(1)、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
*Hayato Kawabata(1), Ayumu Konno(1,2), Yumika Sato(1), Mika Kawachi(1), Hirokazu Hirai(1,2)
1. Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan, 2. Viral Vector Core, Gunma University Initiative for Advanced Research (GIAR), Maebashi, Gunma 371-8511, Japan
Keyword: Autism model transgenic mouse, Viral vector, Cerebral cortex, Rett syndrome (RTT)
Astrocyte plays crucial roles in both health and disease of the brain. Adeno-associated virus (AAV)-PHP.eB, a blood brain barrier-penetrating capsid variant, with the astrocyte-specific GFAP promoter enables astrocyte-specific transgene expression in mouse brain just by a single intravenous infusion. However, upon intravenous injection of AAV-PHP.eB expressing Cre recombinase by the GFAP promoter to floxed Ai14 mice which expressed tdTomato in the presence of Cre, we found only 11% of transduced astrocytes and 51% of transduced neurons among total transduced cells. We supposed that, PHP.eB capsid is more tropic to neurons rather than astrocytes, and some leaky expression of Cre was induced and accumulated also in neurons. Thus, decreasing Cre expression in neurons may suppress untargeted recombination in neurons. To verify this, we used two different AAV-PHP.eB vectors with the GFAP promoter, one expressing FlpO recombinase and another one carrying inverted destabilized Cre (dCre) sequence flanked by flp-dependent double floxed inverted open reading frame (fDIO). These two AAV-PHP.eB vectors were mixed and intravenously infused to Ai14 mice. Although GFAP promoter is used, tiny amount of FlpO may be expressed in neurons, but recombination by FlpO is less efficient than that by Cre. Moreover, retention of Cre can be circumvented by use of dCre. Therefore, we predicted more selective astrocyte transduction. As expected, we found significant increase in the ratio of astrocyte transduction to total transduced cells in the cerebral cortex by 50%. To further increase the ratio, we changed the viral capsid from PHP.eB to AAV-F, since AAV-F was shown to be more tropic to astrocyte than PHP.eB. Intravenous injection of the two AAV-F mixture results in significantly higher transduction ratio of astrocytes to total transduced cells in the cerebral cortex by more than 90%. These results suggest that AAV-PHP.eB-mediated Cre expression by the astrocyte-specific GFAP promoter in flox mice does not cause astrocyte-specific recombination, and that combination of less efficient FlpO and easily degradable dCre with astrocyte-tropic AAV-F can effectively inhibit leaky neuron transduction. In addition, we will present the results of using this method for Methyl-CpG Binding Protein 2 (MeCP2)-flox mice. | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-007
ヒト特異的な遺伝的変異を持つマウス作製と行動表現型の解析
Behavioral analysis of a transgenic mouse expressing human-specific alpha-1 adrenergic receptor (ADRA1A) isoforms on the astrocytes
*森本 梨々花(1)、中谷 一真(3,4)、小林 千紘(2)、末永 雄介(3)、毛内 拡(1,2)
1. お茶の水女子大学大学院人間文化創成科学研究科、2. お茶の水女子大学理学部生物学科、3. 千葉県がんセンター発がん制御研究部、4. 千葉大学大学院医学薬学府
*Ririka Morimoto(1), Kazuma Nakatani(3,4), Kobayashi Chihiro(2), Yusuke Suenaga(3), Hiromu Monai(1,2)
1. Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan, 2. Department of Biology, Faculty of Science, Ochanomizu University, Tokyo, Japan, 3. Division of Molecular Carcinogenesis, Chiba Cancer Center Research Institute, Chiba, Japan, 4. Graduate School of Medical and Pharmaceutical Sciences, Chiba University, Chiba, Japan
Keyword: glia, synaptic plasticity, neuromodulator, noradrenaline
Astrocytes abundantly express receptors of neuromodulators such as noradrenaline, and their interaction with neural networks plays a role in the neuromodulation system. Besides, some studies, including ours, have shown that astrocytic Ca2+ elevation mediated by alpha-1 adrenergic receptor (ADRA1A) plays an essential role in the induction of synaptic plasticity. Moreover, we found human-specific ADRA1A exhibits remarkable genetic variants on the astrocytes, and only humans expressed three isoforms 5-7 simultaneously by genome-wide analysis of the astrocytic membrane protein. One of them, ADRA1A isoform 7, has been identified as being expressed in the cerebral cortex, but the neurophysiological function of these proteins remains poorly understood. To determine the role that ADRA1A isoform 5-7 plays on synaptic plasticity, we generated a new BAC transgenic mouse line that expresses all three isoforms on astrocytes. The line was generated from background strain C57BL/6N by insertion of a transgenic construct expressing ADRA1A isoforms 5-7 under the control of the GfaABCD1D promoter. To understand behavioral phenotypes in this line of Tg mice, we first tested their pain-associated behavior, which was shown to be related to ADRA1A function. A hot plate test and a tail-flick test did not reveal any significant differences between Tg and wild-type mice. In addition, a marble-burying test and cliff avoidance test, which indicate anxiety, showed no noticeable changes. In contrast, we found the bodyweight of Tg mice tends to increase. We also noted a significant increase in daytime activities by measuring automated 24-hour tracking for seven days. We will now examine their intake and sleep/wake cycle in order to discover how their body weight increases. Furthermore, we will investigate an anatomical characteristic of ADRA1A especially in the cerebral cortex in parallel. | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-008
Clec7aは神経細胞死後のマイクログリアの増殖を制御する
Clec7a mediates microglial proliferation after neuronal death
*川名 哲(1)、池谷 裕二(1,2,3)、小山 隆太(1,2)
1. 東京大学大学院薬学系研究科、2. 東京大学Beyond AI、3. 脳情報通信融合研究センター
*Tetsu Kawana(1), Yuji Ikegaya(1,2,3), Ryuta Koyama(1,2)
1. Grad Sch of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan, 2. Institute for AI and Beyond, The University of Tokyo, Tokyo, Japan, 3. Center for Information and Neural Networks, National Institute of Information and Communications Technology, Suita City, Osaka, Japan
Keyword: Microglia , Clec7a, epilepsy, gliosis
Microglia are tissue resident immune cells that maintain brain homeostasis. Robust microgliosis are found in the hippocampal CA3 after neuronal loss in individuals with mesial temporal lobe epilepsy and their animal models. Mesial temporal lobe epilepsy is one of most common type of intractable epilepsy in adults, approximately 30% of patient exhibit resistance to antiepileptic drugs that target neuronal hyperactivity. Thus, it is of interest to examine possible roles of glial cells in epilepsy. It has been suggested that microgliosis is induced by the proliferation of microglia after neuronal death, but the underlying mechanisms remain unclear.
Here we examined the possible involvement of Clec7a (C-type lectin domain family 7member A), a pattern recognition receptor expressed in the surface of various myeloid cells, in the proliferation of microglia. Clec7a recognizes β-glucans and elicits antifungal proinflammatory immune responses and recent genomics have revealed that microglia that surround neurodegenerative site express Clec7a in response to neuronal death. Using mouse hippocampal slice cultures in which neuronal death in CA3 was induced by kainic acid, we found that Clec7a expression in microglia is increased. Further, Clec7a stimulation with its agonist Curdlan induced ERK signaling in microglia. Finally, we found that microglia which are immunopositive for Ki67, a cellular maker for proliferation, expressed significantly higher levels of Clec7a than negative microglia. These finding suggest that Clec7a mediates microglial proliferation in response to neuronal cell death. | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-009
分化過程にあるオリゴデンドロサイトのカルシウム活動を記録する
Recording calcium activity of oligodendrocytes in the process of differentiation
*横山 貴一(1)、平岡 優一(2)、田中 謙二(1)
1. 慶應義塾大学医学部 先端医科学研究所 脳科学研究部門、2. 東京医科歯科大学 難治疾患研究所 先端分子医学研究部門 分子神経科学
*Kiichi Yokoyama(1), Yuichi Hiraoka(2), Kenji Tanaka(1)
1. Division of Brain Science, Institute for Advanced Medical Research, Sch Med, Keio Univ, Tokyo, Japan, 2. Molecular Neuroscience, Division of Advanced Molecular Medicine, Medical Research Institute, Tokyo Medical and Dental Univ, Tokyo, Japan
Keyword: OLIGODENDROCYTE, MYELIN, NON-CODING RNA, CALSIUM
中枢神経系で髄鞘を形成する細胞であるオリゴデンドロサイトは、その前駆細胞であるOPCから成熟オリゴデンドロサイトに至るまで、突起数の増加、突起の伸張、突起の巻き付きなど動的な形態変化を示す。一方で、近年のシングルセル解析によって、オリゴデンドロサイトは分化途中で時空間特異的な遺伝子発現パターンを示すことが報告された。形態学的、遺伝学的なオリゴデンドロサイトの詳細な特徴が個別に明らかになりつつある中で、両者の定義の間にどのような対応関係が存在するのか報告したものは少ない。また、遺伝学的に新たに定義されたオリゴデンドロサイトのサブポピュレーションが、生体内でどのような活動性を示すのかはわかっていない。
そこで我々は、オリゴデンドロサイトの特定の分化段階においてのみ発現が特異的に上昇する遺伝子のプロモーター配列の活性を利用して、分化段階の早い（若い）オリゴデンドロサイトの一部のみを蛍光タンパクで可視化することができるマウスを作成し、可視化された細胞の形態と遺伝子発現を同一切片上で観察した。これにより、若いオリゴデンドロサイトの形態学的な定義と遺伝学的な定義の間の対応を明らかにした。また、それらのオリゴデンドロサイトにおいて、IP3受容体2型の発現が上昇していることに着目し、カルシウムを介した細胞内シグナル伝達の変化を記録することで、特定の若いオリゴデンドロサイトの活動性を生体内で計測できる系を樹立することを試みた。
結果として、今回細胞の可視化に利用したシステムでは、実際に遺伝子プロモーターが活性を持ってから蛍光分子が発現するまでの間に、オリゴデンドロサイトの分化が進み、プロモーターが活性を持つ段階よりも後の段階にあたる細胞群で、蛍光が観察されることがわかった。一方で、このシステムで直接誘導されるカルシウムインディケーターの発現量は非常に低く、感度の高い方法でカルシウム活動を計測する必要があると予想された。
今後はカルシウムインディケーターの発現量が高い脳領域を選択し、ウイルス注入を検討することによって、可視化された細胞群におけるカルシウム活動を明らかにして行きたい。 | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-010
C. elegansの感情に伴う全脳活動の理解に向けて
Toward understanding whole brain activity associated with emotion in C. elegans
*鈴木 涼月(1)、遠藤 雄人(2)、Tee Ling Fei(1)、Wen Chentao(1)、木村 幸太郎(1,2)
1. 名古屋市立大学総合生命理学部、2. 大阪大学大学院理学研究科
*Ryoga Suzuki(1), Yuto Endo(2), Ling Fei Tee(1), Chentao Wen(1), Kotaro Kimura(1,2)
1. Sch Biol Integr Sci, Nagoya City Univ, Nagoya, Japan, 2. Grad Sch Sci, Osaka Univ, Osaka, Japan
Keyword: whole brain activity, calcium imaging, 4D physiology
An animal's brain receives environmental information, processes it, and outputs the results as behavior. How brain functions are emerged from its neural circuit structure is one of the most central questions in modern neuroscience. In the nematode C. elegans, all the anatomical connections between neurons (i.e. connectome) have been revealed, and whole brain neural activity in the transparent body can be measured using calcium imaging at cellular resolution. Moreover, Yemini et al. developed the NeuroPAL technology, which allows researchers to identify all the neurons with uniquely assigned four-color fluorescence in individual worms subjected to calcium imaging (Yemini et al., Cell 2021). Recently, we reported a deep learning-based software for efficient whole brain calcium imaging, 3DeeCellTracker, that automatically tracks nearly all the neurons in a semi-constrained worm's brain (as well as ~100 heart cells in a zebrafish heart and ~1,000 cells in a tumor spheroid) with ≥95% efficiency in most of the cases (Wen et al., eLife 2021). We have also established a new microscope system that integrates spinning disk confocal sub-system for whole brain calcium imaging plus four-color imaging sub-system for cell identification with NeuroPAL. By using the above-mentioned system, we are now monitoring whole brain neural activity caused by repellent odorant 2-nonanone stimuli (Kimura et al., J Neurosci 2010) and revealing the relationship between the neural activities and the circuit structure. We are also trying to reveal whole brain activity caused by electric shock, which causes persistent behavioral response associated with fear-like emotion (Tee et al., bioRxiv 2021). | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-011
神経回路基盤から探る慢性疼痛の色光線療法メカニズム
Neuronal circuit-based mechanisms of chromotherapy for chronic pain
*加藤 菫(1)、竹田 育子(1,2)、和氣 弘明(1,2)
1. 名古屋大学大学院医学系研究科分子細胞学、2. 生理学研究所多細胞回路動態研究部門
*Sumire Kato(1), Ikuko Takeda(1,2), Hiroaki Wake(1,2)
1. Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate school of Medicine , 2. Division of Multicellular Circuit Dynamics, National Institute for Physiological Sciences
Keyword: neuronal circuit, neuronal activity, chromotherapy, chronic pain
Chromotherapy is used to improve emotional symptoms and memory impairments by illuminating specific color. Photosensitive retinal ganglion cells (ipRGCs) intrinsically activated by specific colorhave a widespread influence on hypothalamic,preoptic areas, and limbic regions and regulatenonimage-forming visual functions such as circadian clock and mood. Recent studies have shown that green light exposure has antinociceptive and moodeffects. In this experiment, we aim to clarify the therapeutic mechanism of chromotherapy on chronic pain and their neural circuits basis.Partial sciatic nerve ligation mice (PSL mice, a model of neuropathic pain) partially ligated sciatic nerve 2 weeks before chromotherapy, were exposed to red-, green-, or white-light 12 hours daily for five days as chromotherapy and assessed the mechanical threshold using von Frey test. Allodynia in PSL micetended to be restored after green-light exposure, while red-light exposure induced high withdrawal threshold as like hypoalgesia. To extract the locus related to chromotherapy, we analyzed c-fosexpression, which is a marker of neural activity, following 5-day chromotherapy using immunohistochemistry. The anterior cingulate cortexand somatosensory cortex increased c-fos expression after green- and red-light compared with white-light exposure. We will visualize the neuronal activity by two photon microscope and extract functional connectivity of local microcircuits by holographic microscope before and after chromotherapy in the c-fos expressed regions. In this way, we try to reveal the neuronal circuit modulation by chromotherapy. The mechanism of chromotherapy will clarify anovel therapeutic target for pain and psychological symptoms of chronic pain. | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-012
静磁場刺激によるマウス皮質由来初代培養細胞の自発活動に対する抑制効果：Ca(2+)イメージングによる研究
Inhibitory effects of static magnetic fields on spontaneous activations of primary mouse cortical neurons in culture: a Ca(2+) imaging study
*山本 拓都(1)、岡 雄一郎(1,4,6)、佐藤 真(1,2,4,6)、北澤 茂(1,2,3,5)
1. 大阪大学医学部、2. 大阪大学大学院生命機能研究科、3. 大阪大学医学系研究科脳生理学教室、4. 大阪大学医学系研究科神経機能形態学教室、5. 脳情報通信融合研究センター、6. 大阪大学大学院連合小児発達学研究科
*Takuto Yamamoto(1), Yuichiro Oka(1,4,6), Makoto Sato(1,2,4,6), Shigeru Kitazawa(1,2,3,5)
1. Faculty of Medicine, Osaka University, Osaka, Japan, 2. Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan, 3. Department of Brain Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan, 4. Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan, 5. Center of Information and Neural Networks, Osaka University, Osaka, Japan, 6. Molecular Brain Science, Division of Developmental Neuroscience, Department of Child Development, United Graduate School of Child Development, Osaka University, Suita, Osaka, Japan
Keyword: static magnetic field stimulation, calcium imaging
Inhibition of neural activity by static magnetic field stimulation (SMS) has been reported using in vitro electrophysiological techniques since the 1990s (McLean et al. 1995; Cavopol et al. 1995; Lu et al. 2015). These in vitro studies used cultured neurons of the peripheral nervous system but SMS has inhibitory effects on neurons in the cerebral cortex of the cats and monkeys as well (Aguila, Cudeiro, and Rivadulla 2014). SMS is now further applied clinically to suppress pain transmission (Okano et al. 2012) or abnormal activity in epileptic seizures (Rivadulla et al. 2018). However, physiological mechanisms of this inhibition is not yet clear (Nojima, Oliviero, and Mima 2020). To study mechanisms underlying the inhibitory effects of SMS on the cerebral cortex, we aimed to establish an experimental test bed using cultured neurons of the cerebral cortex. Genetically encoded calcium indicators (GCaMP6f) were expressed in the cortical neurons of the mouse using adeno-associated virus. After incubation for more than 21 days, calcium imaging was repeated three times using a confocal laser microscope, before, during, and after the SMS (5 min for each). The strength of SMS was adjusted to 50-150 mT. Spontaneous firing rates of 342 neurons from four chambers were estimated from calcium imaging signals. The median firing rates of the cultured neurons significantly decreased during the SMS period (median = 1.1 spikes/min) as compared to the pre (1.5) and post (1.9) SMS periods (p < 0.0001, Wilcoxon signed rank test). In conclusion, we have successfully established a calcium imaging system for studying inhibitory effects of SMS on cultured cortical neurons. Spatio-temporal information that can be obtained by the calcium imaging, which cannot be obtained by conventional electrophysiological measurements, would be useful to study mechanisms of inhibition in the future. | | 2022年6月30日 13:00～14:00 沖縄コンベンションセンター 展示棟 ポスター会場1
1P-013
分泌タンパク質リーリンの 神経細胞の膜脂質組成に与える影響の解明
The effect of Reelin on the lipid composition of neuronal plasma membrane
*竹腰 祐斗(1)、安藤 飛悠吾(1)、河野 孝夫(1)、有田 誠(2,3)、服部 光治(1)
1. 名古屋市立大学薬学部薬学科、2. 慶応大薬・代謝生理化学、3. 理研IMS・メタボローム
*Yuto Takekoshi(1), Hugo Ando(1), Takao Kohno(1), Makoto Arita(2,3), Mitsuharu Hattori(1)
1. Nagoya City Univ. Faculty of Pharmaceutical science, Nagoya, Japan, 2. Keio Univ. Faculty of Pharmacy, Tokyo, Japan, 3. RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
Keyword: Reelin, neuron, sphingomyelin
The brain is not only a lipid-rich organ but also has a specific composition of lipid molecules. Because the lipid composition of the cellular membrane modulates the function of various proteins, the characteristic lipid compositions of neurons are likely to contribute to brain functions. However, the precise mechanism by which the lipid composition of the neuronal cell membrane is regulated remains poorly understood. Reelin, a large secreted protein, plays important roles in neuronal migration and function. We previously reported that the Reelin deficient mice, reeler, exhibit aberrant lipid composition in the embryonic brain. Although it is likely that Reelin deficiency alters the lipid composition of neurons, the precise mechanism is still unclear. In this study, we aim to understand the effect of Reelin on the lipid composition of the neuronal plasma membrane and its underlying mechanism. We compared the lipid composition of the postsynaptic density (PSD) fraction between wild-type and reeler mice. The cerebral cortex was taken out from these mice and PSD fractions were obtained by using sequential ultracentrifugation with a discontinuous sucrose gradient. The validity of fractionation was confirmed by western blotting. The lipid molecules were extracted by chloroform/methanol (Bligh and Dyer extraction). We performed non-target lipids analysis of these fractions by LC-MS/MS and found that some lipid species were either increased or decreased in the PSD fraction of reeler mice, and particularly, sphingomyelin (SM) synthesis was decreased. SM is an essential component of the so-called lipid-microdomain that plays important role in neuronal functions. It was previously reported that the activation of Fyn, a member of Src family tyrosine kinases, promotes the SM synthesis by preventing the degradation of SM synthases. Because Reelin can activate Fyn, Reelin may upregulate the SM synthesis in neurons. Thus, Reelin may regulate neuronal functions by increasing SM on the plasma membrane of neurons. We are investigating whether Reelin controls the amount of SM on the plasma membrane of primary cultured neurons. | | | |
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