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Development of a total fluorescence collecting-two photon laser scanning microscope and analysis of intracellular Ca^<2+> dynamics

Research Project

Project/Area Number 10357001
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section展開研究
Research Field General physiology
Research InstitutionNagoya University

Principal Investigator

KUBA Kenji  School of Medicine, Physiology, Nagoya University, Professor, 医学部, 教授 (60080561)

Co-Investigator(Kenkyū-buntansha) TAKABE Kouji  Niken Company, Researcher, 研究員
NAKAYAMA Shinsuke  School of Medicine, Physiology, Nagoya University, Associate Professor, 医学部, 助教授 (30192230)
武部 絋二  株式会社ニコン, 技術工房, 研究員
Project Period (FY) 1998 – 2000
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥32,500,000 (Direct Cost: ¥32,500,000)
Fiscal Year 2000: ¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1999: ¥12,800,000 (Direct Cost: ¥12,800,000)
Fiscal Year 1998: ¥12,900,000 (Direct Cost: ¥12,900,000)
KeywordsTotal fluorescence collecting-two photon laser scanning microscope / Ca^<2+> -dependent K^+ channel / Frng motor nerve terminal / Ryanodine receptor / Intracellular Ca^<2+> release / Exocytosis / Short-term plasticity / Bullfrog sympathetic panglion cell / Ca^<2+>依存性K^+チャンネル / ライアノジン受容体 / カゴメ化合物活性化 / ウシガエル交換神経節細胞
Research Abstract

A total fluorescence collecting-two photon laser scanning microscope that records the sliced images of a cell at a higher signal to noise ratio (S/N) was developed. An optical system with an additional objective, a monochrometer and a photomultiplier replaced the original condenser of an inverted microscope and functioned as a condenser, a monocular viewing optics or a photometric device for visual wavelength fluorescence. The fluorescence images recorded through the original and additional objectives showed similar resolutions in horizontal and axial planes, yielding an image of a better S/N by their summation.
Ca^<2+> entry into frog motor nerve terminals by long-lasting repetitive tetanic stimulation primed ryanodine receptors via activation of Ca^<2+>/calmodulin-dependent kinase II and production of cyclic ADP-ribose, subsequently activated Ca^<2+>-induced Ca^<2+> release (CICR) through ryanodine receptors and finally inactivated it. The inactivation of CICR, however, was inhibited … More by the activation of protein kinase C especially during the initial phase of stimulation. Repetitive Ca^<2+> entry into the preganglionic terminals of bullfrog sympathetic ganglion primed CICR via IP_3 receptors at the terminals and produced the protein synthesis-dependent, pre- and postsynaptic long-term potentiation.
In bullfrog sympathetic ganglion cells, N-type voltage-gated Ca^<2+> channels were found to functionally couple with ryanodine receptors and BK-type Ca^<2+> -dependent K^+ channels, forming a functional triad, and loosely with SK-type Ca^<2+>-dependent K^+ channels. The activation of CICR and subsequent opening of BK and SK channels in these couplings shapes the spike repolarization and the slow afterhyperpolarization, respectively. The mechanism of spike repolarization wanes due to the inactivation of CICR during repetitive action potentials, while the slow afterhyperpolarization increases for the increase in Ca^<2+> entry due to the prolongation of the spike duration. The network of Ca^<2+> stores endowed with ryanodine receptors in the submembrane regions spread into the nuclear membrane envelopes and caused Ca^<2+> release in the nucleus. Less

Report

(4 results)
  • 2001 Final Research Report Summary
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • Research Products

    (47 results)

All Other

All Publications (47 results)

  • [Publications] S.Suzuki: "Ca^<2+> dependent Ca^<2+> clearance via mitochondrial uptake and plasmalemmal extrusion in frog motor nerve terminals"J. Neurophysiol.. (in press). (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] K.Narita: "Functional coupling of Ca^<2+> channels to ryanodine receptors at presynaptic terminals : amplification of exocytosis and plasticity"J. Gen. Physiol.. 115. 519-532 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S.Suzuki: "Ca^<2+> dynamics at the frog motor nerve terminal"Pflugers Archiv. 440. 351-365 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] F.-M.Lu: "Synchronized Ca^<2+> signals mediated by Ca^<2+> action potentials in the hippocampal neuron network in vitro"Cell Calcium. 29. 379-394 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T.Akita: "Functional Triads Consisting of Ryanodine Receptors, Ca^<2+> channels and Ca^<2+>-activated K^+ channels in Bullfrog Sympathetic Neurons"J. Gen. Physiol.. 116. 697-720 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] M.Nohmi: "Ryanodine-and thapsigargin-insensitive Ca^<2+> induced Ca^<2+> release is primed by lowering external Ca^<2+> in rabbit autonomic neurous"Pflugers Achiv. 440. 588-599 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] F.-M.Lu: "Synchronous and asynchronous transmitter exocytosis induced by subthreshold high K^+ at Cs^+-loaded terminals of rat hippocampal neurons"J. Neurophysiol.. (in press). (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S.-Y.Hua: "Modes of propagation of Ca^<2+>-induced Ca^<2+> release in bullfrog sympathetic ganglion cells"Cell Calcium. 27. 195-204 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] C.Liu: "Activity-dependent enhancement of miniature excitatory postsynaptic current amplitude and its modulation by protein kinase C in cultured rat sympathetic neurons"Neurosc. Lett.. 280. 57-60 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] K.Kuba: "Two-photon laser-scanning microscopy : Tests of objective lenses and Ca^<2+> probes"Neurosci. Res.. 32. 281-294 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] K.Narita: "A Ca^<2+>-induced Ca^<2+> release mechanism involved in asynchronous exocytosis at frog motor nerve terminals"J. Gen. Physiol.. 112. 593-1998 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S.-M.Huang: "Long-term use-dependent enhancement of impulse-induced exocytosis by adrenaline at frog motor nerve terminals"Neurosci. Res.. 33. 239-244 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] K.Kuba 他: "Slow Synaptic Responses and Modulation pp173-181, 274-296, 215-234, 163-172"Springer Verlag 社. 451 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Suzuki, S., Osanai, M., Mitsumoto, N., Akita, K, Narita, K., Kijima, H. and Kuba, K.: "Ca^<2+>-dependent Ca^<2+> clearance via mitochondrial uptake and plasmalemmal extrusion in frog motor nerve terminals"J. Neurophysiol.. (In press). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Lu, F.-M. and Kuba, K.: "Synchronous and asynchronous transmitter exocytosis induced by subthreshold high K^+ at Cs^+ -loaded terminals of rat hippocampal neurons"J. Neurophysiol. (In press). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Lu, F.-M. and Kuba, K.: "Synchronized Ca^<2+> signals mediated by Ca^<2+> action potentials in the hippocampal neuron network in vitro"Cell Calcium. 29. 379-394 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Akita, T. and Kuba, K.: "Functional Triads Consisting of Ryanodine Receptors, Ca^<2+> Channels and Ca^<2+>-activated K^+ Channels in Bullfrdg Sympathetic Neurons Plastic modulation of action potential"J. Gen. Physiol.. 116. 697-720 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Nohmi, M., Hua, S.-Y., Liu, C. and Kuba, K.: "Ryanodine- and thapsigargin-insensitive Ca^<2+>- induced Ca^<2+> release is primed by lowering external Ca^<2+> in rabbit autonomic neurons jahfeit-autonomicjieuraa"Pflugers Archiv.. 440. 588-599 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Narita, K., Akita, T., Hachisuka, J. Huang, S.-M., Ochi, K. and Kuba, K.: "Functional coupling of Ca^<2+> channels to ryanodine receptors at presynaptic terminals : amplification of exocytosis and plasticity"J. Gen. Physiol.. 115. 519-532 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Hua, S.-Y. Liu, C. Lu, F.-M., Nohmi, M. and Kuba, K.: "Modes of propagation of Ca^<2+>-induced Ca2+ release in bullfrog sympathetic ganglion cells"Cell Calcium. 27. 195-204 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Suzuki, S., Osanai, M., Murase, M., Suzuki, N., Ito, K., Shirasaki, T., Narita, K., Ohnuma, K., Kuba, K. and Kijima, H.: "Ca^<2+> dynamics at the frog motor nerve terminal"Pflugers Archiv. 440. 351-365 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Liu, C., Shirasaki, T., Tanaka, S. & Kuba, K.: "Activity-dependent enhancement of miniature excitatory postsynaptic current amplitude and its modulation by protein kinase C in cultured rat sympathetic neurons"Neurosc. Lett.. 280. 57-60 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Huang, S. M., Akita,T., Kitamura, A., Nakayama, S., Tokuno, H. and Kuba, K.: "Long-term use-dependent enhancement of impulse-induced exocytosis by adrenaline at frog motor nerve terminals"Neurosci. Res.. 33. 239-244 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kuba, K. & Nakayama, S.: "Two-photon laser-scanning microscopy : Tests of objective lenses and Ca^<2+> probes ca*T orooohp"Neurosci. Res.. 32. 281-294 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Narita, K., Akita, T., Osanai, M., Shirasaki, T., Kijima, H. & Kuba, K.: "A Ca^<2+>-induced Ca^<2+> release mechanism involved in asynchronous exocytosis at frog motor nerve terminals"J. Gen. Physiol. 112. 593-609 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kuba, K., Narita, K., and Akita, T.: "Ca^<2+>-Induced Ca^<2+> Release in Presynaptic Terminals and Exocytosis. In Slow Synaptic Responses and Modulation edited by K. Kuba, H. Higashida, D. A. Brown and T. Yoshioka"Springer Verlag, Tokyo. 173-181 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Yoshioka, T. and Kuba, K.: "Synaptic plasticity and modulation : Introductory review. In slow Synaptic Responses and Modulation, Eds. Kuba, K., Higashida, H., Brown, D. A. & Yoshioka, T."Springer Verlag, Tokyo. 274-296 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Mochida, S. and Kuba, K.: "Exocytosis and modulation : Introductory review. In Slow Synaptic Responses and Modulation, eds. Kuba, K., Higashida, H., Brown, D. A. & Yoshioka, T."Springer Verlag, Tokyo. 215-234 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kuba, K.: "Ca^<2+> dynamics and modulation : Introductory review. In Slow synaptic Responses and Modulation, eds. Kuba, K., Higashida, H., Brown, D. A. & Yoshioka, T."Springer Verlag, Tokyo. 163-172 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] C.Liu: "Activity-dependent enhancement of miniatune excitatony postsymapth cunnent amplitude and its modulation by protein kinase C in cultured rat sympathetic neurons"Neurosci,Letters. 280. 57-60 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] K.Narita: "Functional coupling of Ca^<2+> channels to ryanodine receptors at presynaptic terminals"J.Ger.Physiol. 115. 519-532 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] S.Suzuki: "Ca^<2+> dynamics at the frog motor nerve terminal"Pflugers Archiv. 440. 351-365 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] S.Y.Hua: "Modes of Propagation of Ca^<2+>-induced Ca^<2+> release in bullfrog sympathetic ganglion cells."Cell Calcium. 27. 195-204 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] T.Akita: "Functional triads consisting of ryanodine receptors, Ca^<2+> channels and Ca^<2+> activated K^+ channels in bullfrog sympathetic neurons"J.Gm.Physiol. 116. 697-720 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] M.Nohmi: "Ryanodine- and thapsigargin-insensitive Ca^<2+>-induced Ca^<1+> release is primed by lowering external Ca^<2+> in rabbit autonomic neurons"Pflugers Archiv. 440. 588-599 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] K.Kuba: "Slow Synaptic Responses and Modulation"Springer.Tokyo. 451 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] S.M.Huang: "Long-term use-dependent enhancement of impulse-induced exocytosis by adrenaline at frog motor nerve terminals."Neuroscience Research. 33. 239-244 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] C.Liu: "Activity-dependent enhancement of miniature excitatory post synaptic current amplitude and its modulation by protein kinase C in cultured rat sympathetic neurons."Neuroscience Letters. 280. 57-60 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] S.Suzuki: "Ca^<2+>dynamics at the frog motor nerve terminals."Pflugers Archiv. (印刷中). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] S.Y.Hua: "Modes of propagation of Ca^<2+>-induced Ca^<2+>release in bullfrog sympathetic ganglion cells."Cell Calcium. (印刷中). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] K.Narita: "Functional coupling of Ca^<+2> channels to ryanodine receptors at presynaptic terminals: Amplification of exocytosis and plasticity"J.Gen.Physiol.. (印刷中). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] 久場健司: "シナプス前終末の細胞内^<2+>遊離とシナプスの可塑性"蛋白質・核酸・酵素. 45・3. 498-503 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] K.KUBA: "Ca^<2+>dynamics and modulation. In Slow synaptic responses and modulation"Springer. 10 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] K.KUBA: "Exocytosis and modulation"Springer. 20 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] K.Narita, K.Kuba et al.: "A Ca^<2+>-induced Ca^<2+> release mechanisin involved in asynchronous exocytosis at frog motor norve terminals" Journal of General Physiology. 112. 593-609 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Kuba, S.Nakayama: "Two-photon laser-scanning microscopy:tests of objective lenses and Ca^<2+> probes" Neuroscience Research. 32. 281-294 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] S.H.Huang, K.Kuba et al.: "Long-term use-dependent enhancement of impulse-induced exocytosis by adrenaline at frog motor norve terminals" Neuroscience Research. (In Press). (1999)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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