Visualization and Quantitative Analysis of Neuropeptide Secretion from Cultured Individual Neurons
| Project/Area Number |
01570070
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurophysiology and muscle physiology
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| Research Institution | Yokohama City University School of Medicine |
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Principal Investigator |
ARITA J Yokohama City University School of Medicine, Department of Physiology, Associate Professor, 医学部・生理学第2講座, 助教授 (80128587)
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| Co-Investigator(Kenkyū-buntansha) |
FUNABASHI T Yokohama City University School of Medicine Department of Physiology, Instructor, 医学部・生理学第2講座, 助手 (70229102)
ENDO Y Yokohama City University School of Medicine, Department of Physiology, Instructo, 医学部・生理学第2講座, 助手 (90194050)
HASHIMOTO R Yokohama City University School of Medicine Department of Physiology, Assistant, 医学部・生理学第2講座, 講師 (70189512)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1989: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Neuron / Secretion / Neuropeptide / Hypothalamus / Visualization / Assay |
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| Research Abstract |
1 (1) Dispersed cells from 18-day-old fetal rat hypothalamus were maintained in monolayer culture.
(2) The optimum conditions of dispersion and culture, and the optimum culture period were determined by measurement of somatostatin that was secreted into medium from cells cultured on Petri dishes.
(3) The best coverslip and attachment factor were selected since the cell immunoblot assay performed using hypothalamic cells cultured on coverslips. Furthermore, the most sensitive immunostaining kit was selected.
2 The antibody against thyroglobulin-conjugated somatostatin was produced since it has been indicated in a previous study that high background in protein transfer membrane is due to the antibody used in immunostaining that was produced against albumin-conjugated somatostatin. By using this antibody the high background in membrane was markedly decreased.
3 Basal somatostatin secretion from single cultured cells was under detectable levels in the immunoblot assay. Furthermore, the somatostatin secretion induced by depolarization stimuli such as 50 mM KCI and 50 uM veratridine was also undetectable.
4 The neuropeptide substance P, which is localized in the hypothalamus as much as somatostatin is, was neither detected by an immunoblot assay using substance P antibody. However, when the immunoblot assay for substance P was applied to anterior pituitary cells in culture, immunoreactive cell blots were found. These results suggest that failure to detect and visualize somatostatin secretion from hypothalamic cells is due to an extremely low level of somatostatin secretion.
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Report
(3 results)
Research Products
(3 results)
