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2012 Fiscal Year Annual Research Report

Oxonol系電位感受性色素の新規応用による新しい細胞膜電位イメージング法の開発

Research Project

Project/Area Number 22300129
Research InstitutionKanto Gakuin University

Principal Investigator

佐藤 容子  関東学院大学, 人間環境学部, 教授 (70251501)

Project Period (FY) 2010-04-01 – 2015-03-31
Keywords膜電位感受性色素 / イメージング / 神経系 / 鶏胚 / 細胞
Research Abstract

ニューロン電位活動の光学的測定法は、膜電位感受性色素で標本を染色し、膜電位変化に応じてその色素が示す吸光や蛍光の変化を光学的に計測するものである。組織標本で活動電位やシナプス後電位などの速い時間経過の電位変化を測定する際には、通常fast-response dyeと呼ばれる膜電位感受性色素が用いられるが、この色素は、シグナルが背景光の10-4~10-3程度と非常に小さく、single cellレベルでの応答を加算なしに検出するのはほとんど不可能であった。本研究は、新しいoxonol系色素を用いた細胞膜電位イメージング法を開発し、ニューロン電位活動の光学的計測法と中枢神経系の機能的システム解析に新機軸をもたらすことを目的とする。
本年度は、以下の手順で解析を行った。
1 色素の最適励起・蛍光波長の同定:蛍光分光光度計を用いた波長スキャンによって、色素の最適励起波長、蛍光波を同定した。得られた励起波長、蛍光波長のピークは以下の通りである。
DiBAC4(3): Ex490 nm, Em520 nm; DiBAC4(5): Ex586 nm, Em610 nm; DiSBAC2(3): Ex534 nm, Em556 nm; DiSBAC2(5): Ex628 nm, Em648 nm。
2 応答シグナルの大きさ:分離培養細胞系を用い、単一細胞の膜電位変化をどれだけの精度で計測できるのかを調べた。灌流液をnormal solution ([K+]0=5.0 mM)からhigh K+ solution ([K+]0=50.0 mM)に変化させ、細胞脱分極に対する蛍光シグナルのfractional change(ΔF/F)を測定した。

Current Status of Research Progress
Current Status of Research Progress

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

Reason

研究は、申請書に記載されたスケジュールに沿って順調に進んでいる。

Strategy for Future Research Activity

当初色素の合成を依頼する予定であった林原生物化学研究所/感光色素研究所が倒産したので、当面市販のDiBAC4(3)とそのアナログを用いてスクリーニングを行う。

  • Research Products

    (11 results)

All 2013 2012

All Journal Article (5 results) (of which Peer Reviewed: 5 results) Presentation (6 results)

  • [Journal Article] Optical imaging of the spontaneous depolarization wave in the mouse embryo: Origins and pharmacological natures.2013

    • Author(s)
      Momose-Sato, Y. and Sato, K.
    • Journal Title

      Annals of the New York Academy of Sciences

      Volume: 1279 Pages: 60-70

    • DOI

      10.1111/j.1749-6632.2012.06806.x

    • Peer Reviewed
  • [Journal Article] Large-scale synchronized activity in the embryonic brainstem and spinal cord.2013

    • Author(s)
      Momose-Sato, Y. and Sato, K.
    • Journal Title

      Frontiers in Cellular Neuroscience

      Volume: 7, Article 36 Pages: 1-15

    • DOI

      10.3389/fncel.2013.00036

    • Peer Reviewed
  • [Journal Article] Spontaneous depolarization wave in the mouse embryo: origin and large-scale propagation over the CNS identified with voltage-sensitive dye imaging.2012

    • Author(s)
      Momose-Sato, Y., Nakamori, T. and Sato, K.
    • Journal Title

      European Journal of Neuroscience

      Volume: 35 Pages: 1230-1241

    • DOI

      10.1111/j.1460-9568.2012.07997.x

    • Peer Reviewed
  • [Journal Article] Pharmacological mechanisms underlying the switching from the large-scale depolarization wave to segregated activity in the mouse CNS.2012

    • Author(s)
      Momose-Sato, Y., Nakamori, T. and Sato, K.
    • Journal Title

      European Journal of Neuroscience

      Volume: 35 Pages: 1242-1252

    • DOI

      10.1111/j.1460-9568.2012.08040.x

    • Peer Reviewed
  • [Journal Article] Optical analysis of developmental changes in synaptic potentiation in the neonatal rat corticostriatal projection.2012

    • Author(s)
      Mullah, S. H.-E.-R., Inaji, M., Nariai, T., Momose-Sato, Y., Sato, K. and Ohno, K.
    • Journal Title

      Neuroscience

      Volume: 201 Pages: 338-348

    • DOI

      10.1016/j.neuroscience.2011.11.023

    • Peer Reviewed
  • [Presentation] Effects of in ovo blockade of the spontaneous depolarization wave on functional synaptogenesis in the embryonic NTS.2013

    • Author(s)
      Momose-Sato, Y., Tashiro, Y., Nakamori, T., Mullah H.-E-R. S. and Sato, K.
    • Organizer
      The 90th Annual Meeting of the Physiological Society of Japan
    • Place of Presentation
      タワーホール船堀(東京)
    • Year and Date
      20130327-20130329
  • [Presentation] Developmental changes in the LTP in the neonatal rat corticostriatal projection: Optical imaging with the voltage-sensitive dye.2013

    • Author(s)
      Sato, K., Mullah H.-E-R. S., Inaji, M., Nariai, T., Momose-Sato, Y. and Ohno, K.
    • Organizer
      The 90th Annual Meeting of the Physiological Society of Japan
    • Place of Presentation
      タワーホール船堀(東京)
    • Year and Date
      20130327-20130329
  • [Presentation] Effects of in ovo blockade of the spontaneous depolarization wave on functional synaptogenesis in the embryonic brainstem nucleus.2012

    • Author(s)
      Momose-Sato, Y., Tashiro, Y., Nakamori, T., Mullah, S. H.-E.-R. and Sato, K.
    • Organizer
      Society for Neuroscience 42nd Annual Meeting
    • Place of Presentation
      Ernest N, Memorial Convention Center New Orleans (USA)
    • Year and Date
      20121013-20121017
  • [Presentation] Developmental changes in the LTP in the neonatal rat corticostriatal projection: Optical imaging with a voltage-sensitive dye.2012

    • Author(s)
      Mullah, S. H.-E.-R. , Inaji, M., Nariai, T., Urmy, J., Momose-Sato, Y., Ohno, K. and Sato, K.
    • Organizer
      Society for Neuroscience 42nd Annual Meeting
    • Place of Presentation
      Ernest N, Memorial Convention Center New Orleans (USA)
    • Year and Date
      20121013-20121017
  • [Presentation] From embryonic correlated activity to the respiratory and locomotor rhythms: pharmacological mechanisms underlying the switching.2012

    • Author(s)
      Momose-Sato, Y., Nakamori, T., Mullah H.-E-R. S. and Sato, K.
    • Organizer
      The 35 Annual Meeting of the Japan Neuroscience Society
    • Place of Presentation
      名古屋国際会議場(名古屋)
    • Year and Date
      20120918-20120921
  • [Presentation] Optical analysis of developmental changes in the LTP in the neonatal rat corticostriatal projection.2012

    • Author(s)
      Sato, K., Mullah H.-E-R. S., Inaji, M., Nariai, T., Momose-Sato, Y. and Ohno, K.
    • Organizer
      The 35 Annual Meeting of the Japan Neuroscience Society
    • Place of Presentation
      名古屋国際会議場(名古屋)
    • Year and Date
      20120918-20120921

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Published: 2014-07-24  

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