回路シフトの制御・可視化・構造解析の効率化に向けた基盤技術の開発

• Project Area: Mechanisms underlying the functional shift of brain neural circuitry for behavioral adaptation
• Project/Area Number: 15H01430
• 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: Kyoto University
• Principal Investigator: 日置 寛之 京都大学, 医学研究科, 助教 (00402850)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥6,630,000 (Direct Cost: ¥5,100,000、Indirect Cost: ¥1,530,000); Fiscal Year 2016: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2015: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
• Keywords: 神経科学 / ウイルスベクター / 可視化 / 透明化技術 / ハイスループット形態解析 / 透明化

Outline of Annual Research Achievements

中枢神経系は認知・思考・記憶・感情といった高次機能を実現しているが、その作動原理は未だ謎のままである。高次機能を実現する素子・構成単位として、神経細胞を想定することは妥当であろう。しかし神経細胞一つ当たりの情報処理速度は高々1KHz程度が限界であり、神経細胞が構成するネットワークにこそ、高次機能を生み出す原理があると考えられる。
平成２８年度は以下の課題に取り組んだ。
(1) 細胞種特異的かつ高発現型ウイルスベクターの開発：レンチウイルスベクターにTet-Offシステムを利用することで、神経細胞特異的に、目的遺伝子の発現量を数十倍程度まで増幅することに成功し、またin vivoウイルス二重感染法の基礎を築いた（Hioki et al., 2007, 2009）。本研究課題では、アデノ随伴ウイルス（AAV）で各種配列の最適化を行い、新たなレポーター遺伝子発現増幅AAVベクター（AAV SynTetOff）を開発した。目的遺伝子の発現量を40倍程度まで増幅することに成功している（Sohn et al., 2017）。新規蛍光プローブや各種移行シグナルの検討を進めているところである。
(2) 回路シフトの可視化：連合学習による神経活動遷移（回路シフト）を可視化する技術開発に取り組んだ。神経活動依存的なプロモーター下で、新規蛍光プローブを発現するAAVを4種作製した。in vitroで検討を進めたが、神経活動遷移を効率的に可視化するためには、更なる改良が必要であり、引き続き検討を進めている。
(3) ハイスループット形態解析法：脳透明化技術を有効に利用すれば、三次元情報が容易に取得できるようになる。時間や労力の大幅な削減（量的変化）が、従来見えなかったものを見るという質的変化をもたらすと期待される。透明化ステップの簡略化に成功している。観察・解析システムの最適化に努めたい。

Research Progress Status

28年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

28年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] Loyola University/New York University Medical Center(米国)
• [Int'l Joint Research] Georg-August-University(Germany)
• [Int'l Joint Research] University of Oxford(英国)
• [Int'l Joint Research] University of Debrecen(Hungary)
• [Int'l Joint Research] IBioBA(Argentina)
• [Int'l Joint Research]
• [Journal Article] A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System. (2017)
  • Author(s): Sohn J, Takahashi M, Okamoto S, Ishida Y, Furuta T, Hioki H.
  • Journal Title: PLoS ONE Volume: 12 Issue: 1 Pages: e0169611-e0169611
  • DOI: 10.1371/journal.pone.0169611
  • NAID: 120005954461
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Structural Basis for Cholinergic Regulation of Neural Circuits in the Mouse Olfactory Bulb. (2017)
  • Author(s): Hamamoto H, Kiyokage E, Sohn J, Hioki H, Harada T, Toida K.
  • Journal Title: J Comp Neurol Volume: 525 Issue: 3 Pages: 574-591
  • DOI: 10.1002/cne.24088
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Individual Mediodorsal Thalamic Neurons Project to Multiple Areas of the Rat Prefrontal Cortex: A Single Neuron-Tracing Study Using Virus Vectors. (2017)
  • Author(s): Kuramoto E, Pan SX, Furuta T, Tanaka YR, Iwai H, Yamanaka A, Ohno S, Kaneko T, Goto T, Hioki H.
  • Journal Title: J Comp Neurol Volume: 525 Issue: 1 Pages: 166-185
  • DOI: 10.1002/cne.24054
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Application of tissue-clearing methods for dissecting brain structure (2017)
  • Author(s): 日置寛之、濱裕、孫在隣、黄晶媛、並木香奈、星田哲志、黒川裕、宮脇敦史
  • Journal Title: Folia Pharmacologica Japonica Volume: 149 Issue: 4 Pages: 173-179
  • DOI: 10.1254/fpj.149.173
  • NAID: 130005548847
  • ISSN: 0015-5691, 1347-8397
  • Peer Reviewed
• [Journal Article] 組織の透明化技術 (2017)
  • Author(s): 濱裕、日置寛之、並木香奈、星田哲志、黒川裕、宮脇敦史
  • Journal Title: 生体の科学 Volume: 68 Pages: 85-93
• [Journal Article] Differential Inputs to the Perisomatic and Distal-Dendritic Compartments of VIP-Positive Neurons in Layer 2/3 of the Mouse Barrel Cortex. (2016)
  • Author(s): Sohn J, Okamoto S, Kataoka N, Kaneko T, Nakamura K, Hioki H.
  • Journal Title: Front Neuroanat Volume: 10 Pages: 124-124
  • DOI: 10.3389/fnana.2016.00124
  • NAID: 120005997790
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Deep Imaging of Cleared Brain by Confocal Laser-Scanning Microscopy. (2016)
  • Author(s): Hama H, Hioki H, Namiki K, Hoshida T, Kurokawa H, *Miyawaki A.
  • Journal Title: Protocol Exchange Volume: inpress
  • DOI: 10.1038/protex.2016.019
  • Open Access
• [Journal Article] ScaleS: an optical clearing palette for biological imaging. (2016)
  • Author(s): Hama H, Hioki H, Namiki K, Hoshida T, Kurokawa H, Ishidate F, Kaneko T, Akagi T, Saito T, Saido T, *Miyawaki A.
  • Journal Title: Nature Neuroscience Volume: 18 Issue: 10 Pages: 1518-1529
  • DOI: 10.1038/nn.4107
  • Peer Reviewed / Open Access
• [Journal Article] Morphological analysis of the early development of telencephalic and diencephalic gonadotropin-releasing hormone neuronal systems in the enhanced green fluorescent protein-expressing transgenic medaka lines. (2016)
  • Author(s): Takahashi A, Islam MS, Abe H, Okubo K, Akazome Y, Kaneko T, Hioki H, *Oka Y.
  • Journal Title: J Comp Neurol Volume: 524 Issue: 4 Pages: 896-913
  • DOI: 10.1002/cne.23883
  • Peer Reviewed / Open Access
• [Journal Article] Dorsal Horn Circuits for Persistent Mechanical Pain. (2015)
  • Author(s): Peirs C, Williams SP, Zhao X, Walsh CE, Gedeon JY, Cagle NE, Goldring AC, Hioki H, Liu Z, Marell PS, *Seal RP.
  • Journal Title: Neuron Volume: 87 Issue: 4 Pages: 797-812
  • DOI: 10.1016/j.neuron.2015.07.029
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Convergence of Lemniscal and Local Excitatory Inputs on Large GABAergic Tectothalamic Neurons. (2015)
  • Author(s): Ito T, Hioki H, Sohn J, Okamoto S, Kaneko T, Iino S, Oliver DL.
  • Journal Title: J Comp Neurol Volume: In Press Issue: 15 Pages: 2277-96
  • DOI: 10.1002/cne.23789
  • Peer Reviewed / Open Access
• [Journal Article] Sequence of molecular events during maturation of developing mouse prefrontal cortex. (2015)
  • Author(s): Ueda S, Niwa M, Hioki H, Sohn J, Kaneko T, Sawa A, *Sakurai T.
  • Journal Title: Mol Neuropsychiatry Volume: 1 Issue: 2 Pages: 94-104
  • DOI: 10.1159/000430095
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Different Cortical Projections from Three Subdivisions of the Rat Lateral Posterior Thalamic Nucleus: A Single Neuron Tracing Study with Viral Vectors. (2015)
  • Author(s): Nakamura H, Hioki H, Furuta T, Kaneko T.
  • Journal Title: Eur J Neurosci Volume: In Press Issue: 10 Pages: 1-7
  • DOI: 10.1111/ejn.12882
  • NAID: 120005674600
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Structural basis for serotonergic regulation of neural circuits in the mouse olfactory bulb. (2015)
  • Author(s): Suzuki Y, Kiyokage E, Sohn J, Hioki H, Toida K.
  • Journal Title: The Journal of Comparative Neurology Volume: 523 Issue: 2 Pages: 262-80
  • DOI: 10.1002/cne.23680
  • Peer Reviewed / Open Access
• [Journal Article] Efficient and graded gene expression in glia and neurons of primary cerebellar cultures transduced by lentiviral vectors. (2015)
  • Author(s): Kumar S, Zimmermann K, Hioki H, Pfeifer A, Baader SL.
  • Journal Title: Histochem Cell Biol Volume: 143(1) Issue: 1 Pages: 109-121
  • DOI: 10.1007/s00418-014-1260-8
  • Peer Reviewed / Open Access
• [Journal Article] Ventral medial nucleus neurons send thalamocortical afferents more widely and more preferentially to layer 1 than neurons of the ventral anterior-ventral lateral nuclear complex in the rat. (2015)
  • Author(s): Kuramoto E, Ohno S, Furuta T, Unzai T, Tanaka YR, Hioki H, Kaneko T.
  • Journal Title: Cereb Cortex Volume: 25 Issue: 1 Pages: 221-235
  • DOI: 10.1093/cercor/bht216
  • Peer Reviewed / Open Access
• [Presentation] A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System. (2017)
  • Author(s): Hioki H, Sohn J, Takahashi M, Okamoto S, Ishida Y, Furuta T.
  • Organizer: The 7th International Neural Microcircuit Conference
  • Place of Presentation: Okazaki
  • Year and Date: 2017-12-08
  • Int'l Joint Research
• [Presentation] Differential Inputs to the Prismatic and Distal-Dendritic Compartments of VIP-Positive Neurons in Layer 2/3 of the Mouse Barrel Cortex. (2017)
  • Author(s): Sohn J, Okamoto S, Kataoka N, Kaneko T, Nakamura K, Hioki H.
  • Organizer: The 7th International Neural Microcircuit Conference
  • Place of Presentation: Okazaki
  • Year and Date: 2017-12-08
  • Int'l Joint Research
• [Presentation] Dorsal and ventral parts of the rat thalamic submedial nucleus provide two independent inputs to different areas of orbital cortex. (2017)
  • Author(s): Kuramoto E, Iwai H, Yamanaka A, Seki H, Ohno S, Sendo R, Koyanagi K, Toyodome S, Furuta T, Hioki H, Goto T.
  • Organizer: 第122回日本解剖学会総会・全国学術集会
  • Place of Presentation: 長崎
  • Year and Date: 2017-03-28
• [Presentation] A Single Vector Platform for High-Level Gene Transduction of Central Neurons: AAV Vector Equipped with the Tet-Off System (2017)
  • Author(s): 日置寛之、孫在隣、高橋慧、岡本慎一郎、石田葉子、古田貴寛
  • Organizer: 第122回日本解剖学会総会・全国学術集会
  • Place of Presentation: 長崎
  • Year and Date: 2017-03-28
• [Presentation] Efficient Gene Delivery into Neuronal Cells using Adeno-Associated Virus Vector Equipped with the Tet-Off System. (2017)
  • Author(s): Hioki H.
  • Organizer: The 1st ABiS Symposium -Towards the Future of Advanced Bioimaging for Life Sciences-
  • Place of Presentation: Okazaki
  • Year and Date: 2017-02-19
  • Int'l Joint Research
• [Presentation] 大脳新皮質GABA細胞が構成する特異的ネットワーク構造 (2016)
  • Author(s): 日置寛之
  • Organizer: 第57回日本組織細胞化学会総会・学術集会
  • Place of Presentation: 三鷹
  • Year and Date: 2016-09-03
  • Invited
• [Presentation] Cholecystokinin- and/or vasoactive intestinal polypeptide-positive neurons preferentially innervate the somatic compartment of parvalbumin-expressing neurons in the mouse primary somatosensory cortex. (2016)
  • Author(s): 日置寛之、孫在隣、中村悠、岡本 慎一郎、髙橋慧、亀田浩司
  • Organizer: 第39回日本神経科学学会
  • Place of Presentation: 横浜
  • Year and Date: 2016-07-20
• [Presentation] Two types of orbitofrontal cortical projection from the rat submedial thalamic nucleus: Possible function in descending pain modulation. (2016)
  • Author(s): 倉本恵梨子、岩井治樹、山中淳之、千堂良造、大野幸、古田貴寛、日置寛之、後藤哲哉
  • Organizer: 第39回日本神経科学学会
  • Place of Presentation: 横浜
  • Year and Date: 2016-07-20
• [Presentation] Neocortical GABAergic malfunction underlying ASD-like social deficits. (2016)
  • Author(s): Kazuki Okamoto, Chiaki Kobayashi, Natsuko Hitora-Imamura, Hiroyuki Hioki, Yuji Ikegaya
  • Organizer: The 10th FENS FORUM OF NEUROSCIENCE
  • Place of Presentation: Copenhagen
  • Year and Date: 2016-07-02
  • Int'l Joint Research
• [Presentation] Basic architecture of neocortical circuits (2016)
  • Author(s): Hioki H.
  • Organizer: LEICA MEETS SCIENCE
  • Place of Presentation: Mannheim
  • Year and Date: 2016-06-14
  • Int'l Joint Research / Invited
• [Presentation] かたちの意味 -形態解析から迫る神経機構の原理- (2016)
  • Author(s): 日置寛之
  • Organizer: 第121回日本解剖学会・全国学術集会
  • Place of Presentation: ビックパレットふくしま
  • Year and Date: 2016-03-30
  • Invited
• [Presentation] 自閉症の社会性低下は前帯状皮質の過剰興奮により生じる (2016)
  • Author(s): 岡本和樹、人羅（今村）菜津子、日置寛之、池谷裕二
  • Organizer: 第89回 日本薬理学会年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2016-03-09
• [Presentation] Dual innervation modes of inhibitory inputs to vasoactive intestinal polypeptide-positive neurons in mouse primary somatosensory cortex. (2016)
  • Author(s): Jaerin Sohn and Hiroyuki Hioki
  • Organizer: 新学術領域「適応回路シフト」国際シンポジウム
  • Place of Presentation: 同志社大学室町キャンパス寒梅館ハーディーホール
  • Year and Date: 2016-03-03
  • Int'l Joint Research
• [Presentation] Dual innervation modes of inhibitory inputs to vasoactive intestinal polypeptide-positive neurons in mouse primary somatosensory cortex. (2016)
  • Author(s): Hiroyuki Hioki
  • Organizer: 新学術領域「大脳新皮質構築」第三回国際シンポジウム
  • Place of Presentation: 東京大学小柴ホール
  • Year and Date: 2016-02-12
  • Int'l Joint Research / Invited
• [Presentation] 大脳新皮質のグランドデザイン解読を目指して (2016)
  • Author(s): 日置寛之
  • Organizer: 次世代脳プロジェクト冬のシンポジウム
  • Place of Presentation: 東京
  • Invited
• [Presentation] 大脳新皮質のグランドデザインを読み解く～解析例と技術開発～ (2016)
  • Author(s): 日置寛之
  • Organizer: 第39回日本分子生物学会（イブニングフォーラム）
  • Place of Presentation: 横浜
  • Invited
• [Presentation] Strong and specific gene expression in neurons by adeno-associated virus with Tet-Off system. (2015)
  • Author(s): 日置寛之、孫在隣、孫在隣、岡本慎一郎、高橋慧
  • Organizer: 包括脳ネットワーク冬のシンポジウム
  • Place of Presentation: 一橋大学一橋講堂
  • Year and Date: 2015-12-18
• [Presentation] ヒゲ感覚システムにおいて新しく発見した視床覚醒α振動発火と皮質視床連関. (2015)
  • Author(s): 平井大地、柴田憲一、中村公一、田中琢真、日置寛之、金子武嗣、古田貴寛
  • Organizer: 包括脳ネットワーク冬のシンポジウム
  • Place of Presentation: 一橋大学一橋講堂
  • Year and Date: 2015-12-18
• [Presentation] An examination of sex differences in neocortical excitatory synapse number in the aged rat (2015)
  • Author(s): *Borkowski V, Tsai SY, Martin JL, Hioki H, Marinopoulos AE, Hsu KS, Papadapoulos CM, Kartje GL.
  • Organizer: The 45th Society for Neuroscience Annual Meeting (Neuroscience 2015)
  • Place of Presentation: Chicago, IL, USA.
  • Year and Date: 2015-10-21
  • Int'l Joint Research
• [Presentation] Excitatory and inhibitory inputs to vasoactive intestinal polypeptide-expressing neurons in the mouse barrel cortex. (2015)
  • Author(s): Sohn J, Okamoto S, kataoka N, Nakamura K, Kaneko T
  • Organizer: The 45th Society for Neuroscience Annual Meeting (Neuroscience 2015)
  • Place of Presentation: Chicago, IL, USA.
  • Year and Date: 2015-10-20
  • Int'l Joint Research
• [Presentation] A morphological analysis of thalamocortical projections arising from the rat mediodorsal nucleus: A single neuron-tracing study using viral vectors. (2015)
  • Author(s): *Kuramoto E, Pan S, Furuta T, Hioki H, Iwai H, Yamanaka A, Ohno S, Goto T, Kaneko T.
  • Organizer: The 45th Society for Neuroscience Annual Meeting (Neuroscience 2015)
  • Place of Presentation: Chicago, IL, USA.
  • Year and Date: 2015-10-20
  • Int'l Joint Research
• [Presentation] A single vector platform for high-level gene transduction of central neurons with Tet-Off adeno-associated virus. (2015)
  • Author(s): *Hioki H, Sohn J, Takahashi M, Okamoto S, Kaneko T.
  • Organizer: The 45th Society for Neuroscience Annual Meeting (Neuroscience 2015)
  • Place of Presentation: Chicago, IL, USA.
  • Year and Date: 2015-10-18
  • Int'l Joint Research
• [Presentation] Spatiotemporal dynamics of sensory processing in the rat whisker tactile system. (2015)
  • Author(s): *Hirai D, Shibata L, Nakamura KC, Tanaka T, Hioki H, Kaneko T, Furuta T.
  • Organizer: The 45th Society for Neuroscience Annual Meeting (Neuroscience 2015)
  • Place of Presentation: Chicago, IL, USA.
  • Year and Date: 2015-10-17
  • Int'l Joint Research
• [Presentation] ヒゲ感覚システムにおいて新たに発見した視床アルファ・オシレーションと皮質視床連関 (2015)
  • Author(s): 平井大地、柴田憲一、中村公一、田中琢真、日置寛之、金子武嗣、古田貴寛
  • Organizer: 第38回日本神経科学学会
  • Place of Presentation: 神戸国際会議場・展示場
  • Year and Date: 2015-07-30
• [Presentation] マウス第一次体性感覚野VIP陽性抑制性細胞に対する興奮性・抑制性入力の形態学的解析 (2015)
  • Author(s): 孫在隣、岡本慎一郎、金子武嗣、日置寛之
  • Organizer: 第38回日本神経科学学会
  • Place of Presentation: 神戸国際会議場・展示場
  • Year and Date: 2015-07-30
• [Presentation] 近傍に位置する間接路・直接路ニューロンから淡蒼球への投射様式 (2015)
  • Author(s): 岡本慎一郎、日置寛之、孫在隣、藤山文乃、金子武嗣
  • Organizer: 第38回日本神経科学学会
  • Place of Presentation: 神戸国際会議場・展示場
  • Year and Date: 2015-07-30
• [Presentation] Tet-Off搭載アデノ随伴ウイルスベクターによる、神経細胞特異的かつ高発現型シングルベクタープラットフォームの構築 (2015)
  • Author(s): 日置寛之、孫在隣、高橋慧、岡本慎一郎、金子武嗣
  • Organizer: 第38回日本神経科学学会
  • Place of Presentation: 神戸国際会議場・展示場
  • Year and Date: 2015-07-29
• [Book] Methods and Protocols (Neuromethods) (2016)
  • Author(s): Hioki H, Nakamura H, Furuta T.
  • Publisher: Springer
• [Book] Methods and Protocols (Neuromethods) (2016)
  • Author(s): Furuta T, Okamoto-Furuta K, Hioki H.
  • Publisher: Springer
• [Remarks] 研究室webページ
  • URL: http://www.mbs.med.kyoto-u.ac.jp
• [Remarks] 高次脳形態学
  • URL: http://www.mbs.med.kyoto-u.ac.jp/default.html