| Project/Area Number |
12670056
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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 |
Environmental physiology (including Physical medicine and Nutritional physiology)
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| Research Institution | Hamamatsu University School of Medicine |
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Principal Investigator |
SAMEJIMA Michikazu Department of Physiology, Hamamatsu Univ Sch of Med, Associate Professor, 医学部, 助教授 (80135251)
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| Co-Investigator(Kenkyū-buntansha) |
OKABA Akihito Department of Physiology, Hamamatsu Univ Sch of Med, Research Associate, 医学部, 助手 (10313941)
UCHIDA Katsuhisa Department of Physiology, Hamamatsu Univ Sch of Med, Research Associate, 医学部, 助手 (10168693)
FUKUDA Atsuo Department of Physiology, Hamamatsu Univ Sch of Med, Professor, 医学部, 教授 (50254272)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | INTRACELLULAR Cl^- CONCENTRATION / SUPRACHIASMATIC NUCLEUS / INHIBITORY SYNAPTIC TRANSMISSION / Cl^-IMAGIN / MEQ / GABA |
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| Research Abstract |
In this project, change of intracellular Cl^- concentration was directly measured by imaging method. Cl^- sensitive fluorescent dye, MEQ (6-methoxy-N-ethylquinolinium iodide), was loaded to the rat brain slices. The neurons in rat suprachiasmatic nucleus showed blue fluorescence by excitation light of 355 nm. The fluorescence intensity was changed by the bath application of 100 um GABA. In some cases, the fluorescence change of MEQ indicated the increase of [Cl^-]_i, and in other cases, the decrease of [Cl^-]_i. To obtain the final result, it is necessary to measure many neurons. Second, we evaluated if the change in MEQ fluorescence actually indicated the change in [Cl^-]_i, because there was a question that MEQ fluorescence would also changed according to the cellular volume change accompanied with Cl^- or other ion influx and/or eflux. To answer this question, we loaded calcein to the slices so that we could measure the cell volume change simultaneously to [Cl^-]_i change. Calcein is insensitive to any intracellular ionic concentration change or pH change. It is sensitive only to cell volume change. The results indicates that pseudo-ratiometric measurement of [Cl^-]_i is feasible by taking the ratio of MEQ fluorescence change to calcein fluorescence change (F_<MEQ>/F_<calcein>). Finally, we improved the method to obtain the absolute intracellular Cl^- concentration by using MEQ fluorescence. In this case, extracellular Cl^- should be substituted by other anions to take the calibration curve to culculate [Cl^-]_i. For this purpose, the anion which least quenches the intracellular MEQ should be used. Metylsulfric acid potassium salt was found to be the best anion for this purpose.
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