発達脳のシナプス刈り込みにおけるシンギュラリティ現象のイメージング解析

• Project Area: Singularity biology
• Project/Area Number: 19H05415
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Complex systems
• Research Institution: The University of Tokyo
• Principal Investigator: 狩野 方伸 東京大学, 大学院医学系研究科(医学部), 教授 (40185963)
• Project Period (FY): 2019-04-01 – 2021-03-31
• Project Status: Discontinued (Fiscal Year 2020)
• Budget Amount: ¥8,580,000 (Direct Cost: ¥6,600,000、Indirect Cost: ¥1,980,000); Fiscal Year 2020: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2019: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
• Keywords: シナプス刈り込み / 生後発達 / 小脳 / プルキンエ細胞 / 登上繊維 / 神経活動 / マウス / 発達 / 登上線維

Outline of Research at the Start

出生直後のマウス小脳のプルキンエ細胞は5本以上の登上線維からシナプス入力を受けるが、生後7日目までにこのうちの1本だけが選択的に強くなってシナプス領域を拡大し、その他の登上線維は除去されて、生後20日までにほとんどのプルキンエ細胞が1本の強力な登上線維からシナプス入力を受けるようになる。この過程で、1本の「勝者」の登上線維とそれ以外の「敗者」を決定する過程が決定的に重要で、この過程は典型的なシンギュラリティ現象と言える。本研究では、2光子顕微鏡を用いたin vivo imagingによってこの現象を追跡し、この現象に登上線維とプルキンエ細胞の神経活動がどのように関わるかを明らかにする。

Outline of Annual Research Achievements

生後発達期小脳の登上線維－プルキンエ細胞シナプスの刈り込みにおいて、複数の登上線維のうちから生き残る1本の登上線維が選択されるシンギュラリティ現象に神経活動がどのように関わるかを明らかにするために、以下の研究を行った。
令和元年度の研究から新生マウスの下オリーブ核へのウイルス注入の成功率が低いことが判明したため、令和2年度には、Crh-CreマウスとtdToamto-floxマウスを交配して、一部の登上線維がtdTomatoを発現するマウス（Crh-Cre：tdToamto-flox）を得て、Nefl-EGFPマウスと交配した。生後10日から12日において、このマウスの小脳を、イソフルレン麻酔で、2光子顕微鏡を用いて観察した。その結果、EGFP（緑色）、tdTomato（赤色）、EGFP＋tdTomato（黄色）のそれぞれを発現する登上線維が混在する様子を観察した。一方、in vivoのタイムラプス観察を行う前に、固定した小脳を対象に形態学的解析を行って、生後発達の過程で、登上線維が同一のプルキンエ細胞上でどのように競合するかを調べることとした。そのため、生後1－2日において、Nefl-EGFPマウスの一部の下オリーブ核ニューロンにRFP（tdTomato）と抑制性のDREADDを発現させた。このマウスを生後10日に還流固定して小脳切片を作り、共焦点顕微鏡による観察により、一部のプルキンエ細胞がGFPを発現する登上線維とtdTomatoを発現する登上線維によって多重支配されていることを確認した。このマウスに生後4日から11日まで、1日1回のCNOの腹腔内投与によって、RFPを発現する登上線維の活動を抑制する予定であったが、新型コロナウイルスの影響による活動制限のため実施が遅れている間に、本研究は廃止となった。

Research Progress Status

令和2年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和2年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Phospholipase C β3 is required for climbing fiber synapse elimination in Aldolase C-positive compartments of the developing mouse cerebellum. (2021)
  • Author(s): Rai Y, Watanabe T, Matsuyama K, Sakimura K, Uesaka N, Kano M
  • Journal Title: Neuroscience Volume: in press
  • Peer Reviewed
• [Journal Article] Tonic GABAergic inhibition is essential for nerve injury-induced afferent remodeling in the somatosensory thalamus and associated ectopic sensations (2020)
  • Author(s): Nagumo Y, Ueta Y, Nakayama H, Osaki H, Takeuchi Y, Uesaka N, Kano M, Miyata M
  • Journal Title: Cell Rep Volume: 31 Issue: 12 Pages: 107797-107797
  • DOI: 10.1016/j.celrep.2020.107797
  • Peer Reviewed / Open Access
• [Journal Article] Sparse activity of hippocampal adult-born neurons during REM sleep is necessary for memory consolidation (2020)
  • Author(s): Kumar D, Koyanagi I, Carrier-Ruiz A, Vergara P, Srinivasan S, Sugaya Y, Kasuya M, Yu TS, Vogt K, Muratani M, Ohnishi T, Singh S, Teixeira CM, Cherasse Y, Naoi T, Wang Y, Nondhalee P, Osman BAH, Kaneko N, Sawamoto K, Kernie SG, Sakurai T, McHugh TJ, Kano M, Yanagisawa M, Sakaguchi M
  • Journal Title: Neuron Volume: 107 Issue: 3 Pages: 1-14
  • DOI: 10.1016/j.neuron.2020.05.008
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Traceable stimulus-dependent rapid molecular changes in dendritic spines in the brain. (2020)
  • Author(s): Kuboyama K, Inoue T, Hashimotodani Y, Itoh T, Suzuki T, Tetsuzawa A, Ohtsuka Y, Kinoshita R, Takara R, Miyazawa T, Gusain P, Kano M, Yamada MK
  • Journal Title: Scientific Reports Volume: 10 Pages: 15266-15266
  • Peer Reviewed / Open Access
• [Journal Article] Improved hyperacuity estimation of spike timing from calcium imaging (2020)
  • Author(s): Hoang Huu、Sato Masa-aki、Shinomoto Shigeru、Tsutsumi Shinichiro、Hashizume Miki、Ishikawa Tomoe、Kano Masanobu、Ikegaya Yuji、Kitamura Kazuo、Kawato Mitsuo、Toyama Keisuke
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 17844-17844
  • DOI: 10.1038/s41598-020-74672-y
  • Peer Reviewed / Open Access
• [Journal Article] AUTS2 Governs Cerebellar Development, Purkinje Cell Maturation, Motor Function and Social Communication (2020)
  • Author(s): Yamashiro Kunihiko、Hori Kei、Lai Esther S.K.、Aoki Ryo、Shimaoka Kazumi、Arimura Nariko、Egusa Saki F.、Sakamoto Asami、Abe Manabu、Sakimura Kenji、Watanabe Takaki、Uesaka Naofumi、Kano Masanobu、Hoshino Mikio
  • Journal Title: iScience Volume: 23 Issue: 12 Pages: 101820-101820
  • DOI: 10.1016/j.isci.2020.101820
  • NAID: 120006979860
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] G Protein-Coupled Glutamate and GABA Receptors Form Complexes and Mutually Modulate Their Signals (2020)
  • Author(s): Sakairi Hakushun、Kamikubo Yuji、Abe Masayoshi、Ikeda Keisuke、Ichiki Arata、Tabata Toshihide、Kano Masanobu、Sakurai Takashi
  • Journal Title: ACS Chemical Neuroscience Volume: 11 Issue: 4 Pages: 567-578
  • DOI: 10.1021/acschemneuro.9b00599
  • Peer Reviewed / Open Access
• [Journal Article] Comprehensive analysis of a novel mouse model of the 22q11.2 deletion syndrome: a model with the most common 3.0-Mb deletion at the human 22q11.2 locus (2020)
  • Author(s): Saito Ryo、Koebis Michinori、Nagai Taku、Shimizu Kimiko、Liao Jingzhu、Wulaer Bolati、Sugaya Yuki、Nagahama Kenichiro、Uesaka Naofumi、Kushima Itaru、Mori Daisuke、Maruyama Kazuaki、Nakao Kazuki、Kurihara Hiroki、Yamada Kiyofumi、Kano Masanobu、Fukada Yoshitaka、Ozaki Norio、Aiba Atsu
  • Journal Title: Translational Psychiatry Volume: 10 Issue: 1 Pages: 35-35
  • DOI: 10.1038/s41398-020-0723-z
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A Glial-Neuronal Circuit in the Median Eminence Regulates Thyrotropin-Releasing Hormone-Release via the Endocannabinoid System (2020)
  • Author(s): Farkas Erzsevet、Varga Edina、Kovacs Balazs、....、Watanabe Masahiko、Kano Masanobu、...、Charli Jean-Louis、Joseph-Bravo Patricia、Fekete Csaba
  • Journal Title: iScience Volume: 23 Issue: 3 Pages: 100921-100921
  • DOI: 10.1016/j.isci.2020.100921
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Rational engineering of XCaMPs, a multicolor GECI suite for in vivo imaging of complex brain circuit dynamics. (2019)
  • Author(s): Inoue M, Takeuchi A, Manita S, Horigane S-i, Sakamoto M, Kawakami R, Yamaguchi K, Otomo K, Yokoyama H, Kim R, Yokoyama T, Takemoto-Kimura S, Abe M, Okamura M, Kondo Y, Quirin S, Ramakrishnan C, Imamura T, Sakimura K, Nemoto T, Kano M, Fujii H, Deisseroth K, Kitamura K, Bito H.
  • Journal Title: Cell Volume: 177 Issue: 5 Pages: 1346-1360
  • DOI: 10.1016/j.cell.2019.04.007
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] mGluR1 in cerebellar Purkinje cells is essential for the formation but not expression of associative eyeblink memory (2019)
  • Author(s): Nakao Harumi、Kishimoto Yasushi、Hashimoto Kouichi、Kitamura Kazuo、Yamasaki Miwako、Nakao Kazuki、Watanabe Masahiko、Kano Masanobu、Kirino Yutaka、Aiba Atsu
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 7353-7353
  • DOI: 10.1038/s41598-019-43744-z
  • Peer Reviewed / Open Access
• [Journal Article] Developmental synapse remodeling in the cerebellum and visual thalamus. (2019)
  • Author(s): Kano M, Watanabe T.
  • Journal Title: F1000Research Volume: 8 Pages: 1191-1191
  • DOI: 10.12688/f1000research.18903.1
  • Peer Reviewed / Open Access
• [Journal Article] Monoacylglycerol lipase blockade impairs fine motor coordination and triggers cerebellar neuroinflammation through cyclooxygenase-2 (2019)
  • Author(s): Martinez-Torres Sara、Cutando Laura、Pastor Antoni、Kato Ako、Sakimura Kenji、de la Torre Rafael、Valjent Emmanuel、Maldonado Rafael、Kano Masanobu、Ozaita Andres
  • Journal Title: Brain, Behavior, and Immunity Volume: 81 Pages: 399-409
  • DOI: 10.1016/j.bbi.2019.06.036
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Modular organization of cerebellar climbing fiber inputs during goal-directed behavior. (2019)
  • Author(s): Tsutsumi S, Hidaka N, Isomura Y, Matsuzaki M, Sakimura K, Kano M, Kitamura K.
  • Journal Title: Elife. Volume: 8
  • DOI: 10.7554/elife.47021
  • Peer Reviewed / Open Access
• [Journal Article] Endocannabinoid signaling from 2-arachidonoylglycerol to CB1 cannabinoid receptor facilitates reward-based learning of motor sequence. (2019)
  • Author(s): Tanigami H, Yoneda M, Tabata Y, Echigo R, Kikuchi Y, Yamazaki M, Kishimoto Y, Sakimura K, Kano M, Ohno-Shosaku T
  • Journal Title: Neuroscience Volume: 421 Pages: 1-16
  • DOI: 10.1016/j.neuroscience.2019.09.040
  • Peer Reviewed
• [Presentation] Shaping mature neural circuits through synapse pruning (2020)
  • Author(s): Masanobu Kano
  • Organizer: 第43回日本神経科学大会
  • Int'l Joint Research / Invited
• [Presentation] The transcription factor ZFP64 facilitates climbing-fiber synapse elimination along signaling pathway mediated by P/Q-type voltage-dependent Ca2+ channel in the developing cerebellum (2020)
  • Author(s): Jianling Zhang, Takaki Watanabe, Naofumi Uesaka, Masanobu Kano
  • Organizer: 第43回日本神経科学大会
  • Int'l Joint Research
• [Presentation] Protocadherin 10 delays developmental climbing fiber synapse elimination in a subset of aldolase C-positive Purkinje cells in the cerebellum (2020)
  • Author(s): Takaki Watanabe, Kazuto Sakoori, Honoka Suzuki, Tsubasa Akamatsu, Kyoko Matsuyama, Shutaro Inoue, Manabu Abe, Kenji Sakimura, Naofumi Uesaka, Masanobu Kano
  • Organizer: 第43回日本神経科学大会
  • Int'l Joint Research
• [Presentation] Visualizing Network Level Endocannabinoid Signaling in the Hippocampus (2020)
  • Author(s): Henry Paul Denny, Ao Dong, Kaikai He, Yulong Li, Yuki Sugaya, Masanobu Kano
  • Organizer: 第43回日本神経科学大会
  • Int'l Joint Research
• [Presentation] Protocadherin 10 delays developmental climbing fiber synapse elimination in a subset of aldolase C-positive Purkinje cells in the cerebellum (2020)
  • Author(s): Takaki Watanabe, Honoka Suzuki, Kazuto Sakoori, Shutaro Inoue, Tsubasa Akamatsu, Manabu Abe, Kenji Sakimura, Naofumi Uesaka, Masanobu Kano
  • Organizer: 第97回日本生理学会
  • Int'l Joint Research
• [Presentation] The endocannabinoid 2-arachidonoyl glycerol in dentate granule cells suppresses kainate-induced seizures (2020)
  • Author(s): Yuki Sugaya, Kenji Sakimura, Masanobu Kano
  • Organizer: 第97回日本生理学会
  • Int'l Joint Research
• [Presentation] Cerebellar climbing fiber signals represent voluntary forelimb lever-pull trajectories (2019)
  • Author(s): Naoki Hidaka, Koji Ikezoe, Shinichiro Tsutsumi, Yoshikazu Isomura, Masanobu Kano, Kazuo Kitamura
  • Organizer: 第42回日本神経科学大会
  • Int'l Joint Research
• [Presentation] Loss of the psychiatric disorder-related epigenetic factor reduces excitatory synaptic inputs to pyramidal neurons of the medial prefrontal cortex and causes abnormal behavior in mice (2019)
  • Author(s): Kenichiro Nagahama, Kazuto Sakoori Takaki Watanabe, Michinori Koebis, Naofumi Uesaka, Atsu Aiba, Masanobu Kano
  • Organizer: 第42回日本神経科学大会
  • Int'l Joint Research
• [Presentation] Rational engineering of XCaMPs, a multiclolor GECI suite for in vivo imaging of complex brain circuit dynamics (2019)
  • Author(s): Masatoshi Inoue, Atsuya Takeuchi, Satoshi Manita, ..., Masanobu Kano, Hajime Fujii Deisseroth Karl, Kazuo Kitamura, Haruhiko Bito
  • Organizer: 第42回日本神経科学大会
  • Int'l Joint Research
• [Presentation] AiCE mouse: A novel tool to probe LTP-related change in dendritic spines (2019)
  • Author(s): Kazuya Kuboyama, Takafumi Inoue, Yuki Hashimotodani, Takuya Itoh, Tohsuke Suzuki, Yosuke Ohtuska, Ryo Kinoshita, Pooja Gusain, Masanobu Kano, Shigeo Okabe, Maki K Yamada
  • Organizer: 第42回日本神経科学大会
  • Int'l Joint Research
• [Presentation] Assessment of visual evoked potential in a mouse line with 22q11.2 deletion, a putative mouse model of schizophrenia (2019)
  • Author(s): Ryo Saito, Michinori Koebis, Yuki Sugaya, Masanobu Kano, Atsu Aiba
  • Organizer: 第42回日本神経科学大会
  • Int'l Joint Research
• [Presentation] Climbing fiber synapse elimination in aldolase C-positive Purkinje cells is delayed compared to aldolase C-positive Purkinje cells and requires phospholipase C β3 in the developmental cerebellum (2019)
  • Author(s): Takaki Watanabe, Yurie Rai, Honoka Suzuki, Tsubasa Akamatsu, Naofumi Uesaka, Masanobu Kano
  • Organizer: 第42回日本神経科学大会
  • Int'l Joint Research
• [Presentation] Modulation of dentate granule cell activity during fear memory extinction in freely moving mice (2019)
  • Author(s): Alvaro Carrier Ruiz, Yuki Sugaya, Masanobu Kano
  • Organizer: 第42回日本神経科学大会
  • Int'l Joint Research
• [Presentation] Neural mechanisms of synapse remodeling in the developing brain (2019)
  • Author(s): Masanobu Kano
  • Organizer: The 10th IBRO World Congress of Neuroscience
  • Int'l Joint Research / Invited
• [Presentation] Function of adult-born neurons in maturation of fear memory engram during sleep (2019)
  • Author(s): Masanori Sakaguchi, Iyo Koyanagi, Alvaro Carrier-Ruiz, Pable Vergara, ... Masanobu Kano, Masashi Yanagisawa, Deependra Kumar
  • Organizer: The 10th IBRO World Congress of Neuroscience
  • Int'l Joint Research
• [Presentation] Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain (2019)
  • Author(s): Kohtarou Konno, Chihiro Nakamoto, Kenji Sakimura, Masanobu Kano, Masahiko Watanabe
  • Organizer: The 10th IBRO World Congress of Neuroscience
  • Int'l Joint Research
• [Presentation] Synapse remodeling in developing Purkinje cells (2019)
  • Author(s): Kano, Masanobu
  • Organizer: Society for Neuroscience
  • Int'l Joint Research
• [Presentation] Cellular and Circuit Mechanisms of Developmental Synapse Pruning in the Cerebellum (2019)
  • Author(s): Kano, Masanobu
  • Organizer: NUS Neuroscience & Technology Symposium
  • Int'l Joint Research / Invited