| Project/Area Number |
11680786
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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 |
Neuroscience in general
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| Research Institution | Nagoya University |
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Principal Investigator |
TATSUMI Hitoshi School of medicine, Nagoya University, Asociate Prof., 医学部, 助教授 (20171720)
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| Co-Investigator(Kenkyū-buntansha) |
SOKABE Masahiro School of medicine, Nagoya University, Prof., 医学部, 教授 (10093428)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Near Field / Growth cone / GFP / TIR / Imaging / Synapse / Vesicle / Synaptotagumin / 成長円錐 / シナプス / カルシウム / 開口放出 |
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| Research Abstract |
Stable transfection of GFP conjugated synaptotagmin-I (Syt-I-GFP) gene was established to visualize Syt-I-GFP containing vesicles in the living PC12. Depolarizations of the voltage clamped PC12 cells decreased the fluorescence intensity of Syt-I-GFP.The properties of the changes in Syt-I-GFP fluorescence was examined to reveal the mechanism of Syt-I-GFP fluorescence decrease in relation to exocytosis process. The fluorescence decrease was voltage dependent started from -20 mV and reached a maximum decrease at +0 mV.The intensity of Syt-I-GFP fluorescence also decreased when a high potassium solution (60 mM) or calcium ionophore (ionomycin) was applied to the PC12 cells. The fluorescence decrease started from 100 nM of intracellular calcium ion concentration ([Ca^<2+>]_i) and reached a maximum level at 1μM.Syt-I-GFP fluorescence intensity changes underneath the cell membrane (100 nm from the substrate) was examined with a total internal reflection fluorescence microscope (TIRFM). Stimulation of live PC12 cells which increases [Ca^<2+>]_i led to dark punctuate loss of Syt-I-GFP fluorescence spots. Staining the cells with FM4-64 a membrane marker of secretory granules, the fluorescence intensity changes from both Syt-I-GFP amd FM4-64 were examined simultaneously. FM4-64 and Syt-I-GFP fluorescence spots colocalized and both were decreased when high K stimulation was applied. The time lapse analysis showed that Syt-I-GFP decreased 0.4 sec faster than the FM4-64. These results suggest that Ca^<2+> influx through voltage dependent Ca^<2+> channels cause the Syt-I-GFP fluorescence decrease and this process is followed by the exocytosis of vesicles in PC12 cells.
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