2002 Fiscal Year Final Research Report Summary
Study on the function of intracellular signaling by visualization of enzyme activities
| Project/Area Number |
12680803
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
神経・脳内生理学
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| Research Institution | Okazaki National Research Institutes |
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Principal Investigator |
TSUBOKAWA Hiroshi Okazaki National Research Institutes National Institute for Physiological Sciences Associate Professor, 生理学研究所, 助教授 (30227467)
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| Project Period (FY) |
2000 – 2002
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| Keywords | PKC / Intracellular signaling / Imaging / Hippocampus / Calcium / Neuronal excitability / Neurophysiology / Brain science |
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| Research Abstract |
In neurons of the central nervous system, protein kinase C (PKC) modulates functional properties of ion channels which include kinetics of voltage-gate d Na+ and K+ channels. However, little is known ahout spatial and temporal profiles of the PKC activation in relation to neuronal activities. In this study, we tried to detect translocation of the PKC, which is followed by binding to the substrates in single neuron. A combination of intracellular recording and fluorescence imaging was applied to CA1 pyramidal neurons of mouse hippocampal slices. Cells were loaded with 50 μM fim-1, PKC-binding fluorescent dye, through patch-pipette. Bath application of 100 nM phorbol 12-myristate 13-acetate, an activator of the PKC, reversibly changed the spatial pattern of the fim-1 fluorescence. The fim-1 fluorescence measured in the soma transiently decreased, then a bright area appeared around the cell membrane regions within 15 min. During this period, the amplitude of excitatory postsynaptic currents (EPSCs) induced by stimulation of Shaffer collateral / commissural fibers increased, and the paired pulse facilitation of the EPSCs was reduced. Train of action potentials induced similar transient decreases in the fim-1 fluorescence with increases in [Ca^<2+>]_i in the soma. In the presence of PKC inhibitors, pharmacological or electrical stimulation did not induce significant changes in the spatial patterns of the fim-1 fluorescence without affecting changes in [Ca^<2+>]_i. These results suggest that optical imaging of fim-1 fluorescence is useful for detecting activation of the PKC in specified neurons in the brain slice preparation.
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