感覚入力特異的なスクラップ&ビルドによる大脳皮質内の多細胞ネットワーク形成機構

• Project Area: Dynamic regulation of brain function by Scrap & Build system
• Project/Area Number: 16H06460
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: National Institute for Physiological Sciences
• Principal Investigator: 吉村 由美子 生理学研究所, 基盤神経科学研究領域, 教授 (10291907)
• Co-Investigator(Kenkyū-buntansha): 山本 亘彦 大阪大学, 生命機能研究科, 教授 (00191429)
• Project Period (FY): 2016-06-30 – 2021-03-31
• Project Status: Granted (Fiscal Year 2019)
• Budget Amount: ¥157,430,000 (Direct Cost: ¥121,100,000、Indirect Cost: ¥36,330,000); Fiscal Year 2019: ¥28,470,000 (Direct Cost: ¥21,900,000、Indirect Cost: ¥6,570,000); Fiscal Year 2018: ¥28,080,000 (Direct Cost: ¥21,600,000、Indirect Cost: ¥6,480,000); Fiscal Year 2017: ¥29,640,000 (Direct Cost: ¥22,800,000、Indirect Cost: ¥6,840,000); Fiscal Year 2016: ¥43,290,000 (Direct Cost: ¥33,300,000、Indirect Cost: ¥9,990,000)
• Keywords: 大脳皮質視覚野 / 視覚反応選択性 / 神経回路再編 / RNAシークエンス / 大脳皮質 / 方位選択性 / 経験依存的発達 / 神経活動依存的マーカー / 視覚野 / 神経回路 / 遺伝子発現 / 発達

Outline of Annual Research Achievements

本年度は、単一細胞RNA-seq解析の技術を確立させると共に、その手法により縦縞刺激に反応したVenus陽性細胞と反応しなかったVenus陰性細胞（錐体型細胞）をそれぞれ１０個以上、視覚野の2/3層からパッチピペットにより採取し、RNA抽出、ｃDNA合成・増幅、RNA-seq解析を行った。その結果、採取した細胞の半数以上で単一細胞レベルで十分な遺伝子発現解析が達成され、Venus陽性細胞と陰性細胞に分けて、一方に強く発現する遺伝子が複数個見出された。統計的な解析から、これらの遺伝子の発現がVenus陽性細胞で有意に増大していることも示され、特定の方位選択性を持つ細胞に特有の遺伝子発現があることが示唆された。
また、マウス大脳皮質視覚野において、経験依存的に調節される新たな機能を見出す目的で、生後発達の様々な時期に両目への視覚入力を1週間遮断し、その影響をin vivo2光子励起カルシウムイメージングにより解析した。正常な視覚環境下で飼育したマウスは、発達に伴い視覚野神経細胞が高空間周波数の視覚刺激に強く反応するようになるが、視覚遮断されたマウスの視覚野では低空間周波数に強く反応する細胞が多く、幼若型の反応が維持されていた。また、この過程で、視覚経験に依存して発達する細胞群と非依存的な発達を示す細胞群を見出した。今後は、両群に発現する分子の違いを見出すために、各細胞から採取を行い、RNA-seq解析を行う予定である。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

今年度、特定の方位選択性を有する視覚野細胞の遺伝子発現を単一細胞レベルで解析することに成功し、かつその手法によって特有の遺伝子発現プロファイルを得ることができた。また視覚野において、新たな経験依存的に発達する視覚反応特性を見出した。これらのことから、今後の進展が期待できる。

Strategy for Future Research Activity

これまでは、Venus陽性細胞と陰性細胞との間で遺伝子発現解析を行ったが、次は異なる方位選択性（縦縞と横縞）を有する細胞間で比較し、その遺伝子発現パターンを解析するとともに、変化のあった遺伝子の特徴、機能的な解析を進める。また、個々の神経細胞の視覚反応特性をより詳細に同定した細胞からサンプリングを行うために、in vivo2光子励起カルシウムイメージング下で、パッチピペットにより採取を行うことを試みる。これらの解析により、視覚体験依存的・非依存的な発達の分子機構を明らかにする。

Research Products

• [Journal Article] Visual experience regulates the development of long-term synaptic modifications induced by low-frequency stimulation in mouse visual cortex (2017)
  • Author(s): Taketoshi Sugimura, Mariko Yamamoto, Kazumasa Yamada, Yukio Komatsu, and Yumiko Yoshimura
  • Journal Title: Neurosci Res Volume: 印刷中 Pages: 36-44
  • DOI: 10.1016/j.neures.2017.02.006
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Nucleocytoplasmic Shuttling of Histone Deacetylase 9 Controls Activity-Dependent Thalamocortical Axon Branching (2017)
  • Author(s): Ricardo Alchini, Haruka Sato, Naoyuki Matsumoto, Tomomi Shimogori, Noriyuki Sugo, Nobuhiko Yamamoto
  • Journal Title: Sci Rep Volume: 7 Pages: 6024-6024
  • DOI: 10.1038/s41598-017-06243-7
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures (2017)
  • Author(s): Hiroto Tsunematsu, Akiko Uyeda, Nobuhiko Yamamoto, Noriyuki Sugo
  • Journal Title: BMC neurosci Volume: 18 Pages: 55-55
  • DOI: 10.1186/s12868-017-0377-9
  • Peer Reviewed / Open Access
• [Journal Article] Genome Stability by DNA polymerase β in Neural Progenitors Contributes to Neuronal Differentiation in Cortical Development (2017)
  • Author(s): Kohei Onishi, Akiko Uyeda, Mistuhiro Shida, Teruyoshi Hirayama, Takeshi Yagi, Nobuhiko Yamamoto, Noriyuki Sugo
  • Journal Title: J Neurosci Volume: 37 Pages: 8444-8458
  • DOI: 10.1523/jneurosci.0665-17.2017
  • Peer Reviewed
• [Journal Article] Activity-dependent dynamics of the transcription factor CREB in cortical neurons revealed by single-molecule imaging (2017)
  • Author(s): Kitagawa H, Sugo N, Morimatsu M, Arai Y, Yanagida T, Yamamoto N
  • Journal Title: J Neurosci Volume: 37 Pages: 1-10
  • DOI: 10.1523/jneurosci.0943-16.2016
  • Peer Reviewed
• [Journal Article] Establishment of high reciprocal connectivity between clonal cortical neurons is regulated by the Dnmt3b DNA methyltransferase and clustered protocadherins (2016)
  • Author(s): Tarusawa E., Sanbo M., Okayama A., Miyashita T., Kitsukawa T., Hirayama T., Hirabayashi T., Hasegawa S., Kaneko R., Toyoda S., Kobayashi T., Kato-Itoh M., Nakauchi H., Hirabayashi M., Yagi T., Yoshimura Y.
  • Journal Title: BMC Biol Volume: 14 Pages: 103-103
  • DOI: 10.1186/s12915-016-0326-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Visualization of HDAC9 Spatiotemporal Subcellular Localization in Primary Neuron Cultures (2016)
  • Author(s): Sugo N, Yamamoto N
  • Journal Title: Methods Mol Biol Volume: 1436 Pages: 119-127
  • DOI: 10.1007/978-1-4939-3667-0_9
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] 二次視覚野の発達における視覚経験の役割 (2018)
  • Author(s): 吉村由美子
  • Organizer: 第９５回日本生理学会大会
  • Invited
• [Presentation] The roles of visual experience in the maturation of neural circuits and functions in the visual cortex (2017)
  • Author(s): Yoshimura Y
  • Organizer: McGill University-NIPS Symposium
  • Invited
• [Presentation] Novel dependence of neural responses on the contrast of visual stimuli used for an orientation discrimination task in rat primary visual cortex (2017)
  • Author(s): Kimura R, Yoshimura Y
  • Organizer: The 40th Annual Meeting of the Japan Neuroscience Society
• [Presentation] Activity-Dependent Mechanisms for Thalamocortical Circuit Formation (2017)
  • Author(s): Yamamoto, N
  • Organizer: The 48th NIPS International Symposium
  • Invited
• [Presentation] 神経活動依存的な視床皮質軸索の分岐制御機構 (2017)
  • Author(s): 山本 亘彦
  • Organizer: 第39回神経組織培養研究会
  • Invited
• [Presentation] The role of neuronal activity and co-factors in CREB dynamics in cortical neurons: A single-molecule imaging study (2017)
  • Author(s): Kitagawa, H., Sugo, N. & Yamamoto, N.
  • Organizer: Cortical development Conference
  • Int'l Joint Research
• [Presentation] High reciprocal connectivity between clonal cortical neurons is established under the guidance of epigenetic regulation (2016)
  • Author(s): Tarusawa E, Sanbo M, Okayama A, Miyashita T, Kitsukawa T, Hirayama T, Hirabayashi T, Hasegawa S, Hirabayashi M, Yagi T, Yoshimura Y
  • Organizer: Society of Neuroscience 2016
  • Int'l Joint Research
• [Presentation] Experience-dependent maturation of neural circuits and functions in the secondary visual cortex of rats (2016)
  • Author(s): Yoshimura Y
  • Organizer: The 39th Annual Meeting of the Japan Neuroscience Society
  • Invited
• [Presentation] Experience-dependent development of fine-scale networks and feature-selective synchronization in the visual cortex (2016)
  • Author(s): Yoshimura Y
  • Organizer: International Symposium 2016 ‘Circuit Construction in the Mammalian Brain’
  • Int'l Joint Research / Invited
• [Presentation] Visual experience-dependent and independent development of visually-evoked synchronized firing in rat visual cortex (2016)
  • Author(s): Ishikawa AW, Komatsu Y, Yoshimura Y
  • Organizer: The 39th Annual Meeting of the Japan Neuroscience Society
• [Presentation] Synaptic connections are preferentially formed between cortical pyramidal neurons with similar orientation preference in both layers 2/3 and 5. (2016)
  • Author(s): Karaki T, Kim R, Bito H, Yoshimura Y
  • Organizer: The 39th Annual Meeting of the Japan Neuroscience Society
• [Presentation] Positive and negative regulation of activity-dependent axon branching in the developing cortex (2016)
  • Author(s): Yamamoto N
  • Organizer: The 39th Annual Meeting of the Japan Neuroscience society
  • Invited
• [Presentation] Positive and negative regulators for activity-dependent thalamocortical axon branching (2016)
  • Author(s): Yamamoto N
  • Organizer: International Symposium 2016 ‘Circuit Construction in the Mammalian Brain’
  • Int'l Joint Research / Invited
• [Presentation] Positive and negative mechanisms of activity-dependent circuit formation in the developing cortex (2016)
  • Author(s): Yamamoto N
  • Organizer: Molecular Neurobiology Program Seminar in New York University
  • Int'l Joint Research / Invited
• [Presentation] DNA polymerase β function in neural progenitors is required for postmitotic neuronal survival and differentiation in the developing cortex (2016)
  • Author(s): Ohnishi, K., Sugo, N., Toyoda, S., Hirayama, T., Yagi, T., Yamamoto, N.
  • Organizer: The 39th Annual Meeting of the Japan Neuroscience Society