|Budget Amount *help
¥16,300,000 (Direct Cost: ¥16,300,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1989: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1988: ¥13,100,000 (Direct Cost: ¥13,100,000)
Recent studies have demonstrated that the intracellular Ca^<2+> plays an important role in various neuronal functions and neuronal cell death in the mammalian central nervous system. Therefore, it is significant to understand how intracellular Ca^<2+> concentration changes under various conditions ([Ca^<2+>]i) not only in the field of basic neuroscience but also clinically to elucidate mechanisms of neuronal impairments and their prevention. In this research project, the primary purpose was to establish a method for measuring [Ca^<2+>]i on the single cell basis. First, either the hippocampus or striatum was removed from matured fetal rats and dissociated cell cultures were done. After about 14 days in vitro, cells were used for measurement of [Ca^<2+>]i. A Ca^<2+> indicator, fura-2, was loaded into neurons. Cells were mounted on a fluorescence inverted microscope. A xenon lamp as a light source and interference filters for 340 nm and 360 nm were used. Microscope images were obtained th
rough a silicon-intensified target video camera. An optic fiber was vertically fixed on the cell image, and the other end of the fiber was connected to a photodiode, the output of which was amplified, integrated and recorded with a pen recorder. In addition, an automatic image analyzer, ARGUS II, was combined and changes of [Ca^<2+>]i were detected as image.
At the next step, we applied the method to neuropharmacological studies, that is, changes of [Ca^<2+>]i in response to various neurotransmitters and abnormalities of these responses after hypoxic exposure of cells were detected. Somatostatin has been considered to be an inhibitory neuropeptide throughout the nervous and endocrine systems. Nevertheless, we obtained the data that somatostatin induced an increase of [Ca^<2+>]i in cultured rat hippocampal neurons. The Ca^<2+> source for the effect of somatostatin was an influx from extracellular fluids. From experiments using - conooxin GVIA, it was indicated that somatostatin receptors couple with N-type Ca^<2+> channels in the rat hippocampus. In addition, it was demonstrated that cholecystokinin increased [Ca^<2+>]i in cultured rat striatum in the same mechanism.
This method is useful for observing changes of [Ca^<2+>]i in neurons and understanding the cellular functions, since regulation of intracellar [Ca^<2+>]i is one of the most important parts of signal transduction. Less