FUNCTION OF NITRIC OXIDE AS A TROPHIC FACTOR SUPPORTING DIFFERENTIATION AND MATURATION OF NEURONS ; ANALYSIS USING ULTRAMICRO TECHNIQUES

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Nitric oxide synthase (NOS), which catalyzes the formation of the information molecule nitric oxide (NO), is widely distributed in general tissue cells and its high concentrations are found in the cerebellar granular layr. Cerebellar granule cells differentiate postnatally and the cerebellar layrs are known to develop accordingly. Thus, in order to investigate the differentiation and maturation process of granule cells in the postnatal development of rat cerebellum, NOS activities and isoforms were analyzed in the developing layrs. We developed a microassay method for NOS,using an enzymatic amplification reaction NADP cycling. NOS activities were increased in the molecular and internal granular layrs during development and the activity was detected in the external granular layrs. Our newly developed micro-immunoblot showed neuronal NOS (nNOS) and endothelial NOS (eNOS) in the molecular layr and internal granular layr, and the nNOS immunostaining intensity increased during the postnatal development, representing differentiation and maturation in both layrs. eNOS and nNOS were stained in immunoblot of the neuroblastic external granular layr. Further-more, nNOS mRNA was also detected in this germinal layr, using our newly devised micro-immunoblot. Intraperitoneal administration of N^G-nitro-L-arginine inhibited NOS activity in vivo and disturbed the layr formation in developing cerebellum ; thus NO seems to support the differentiation and maturation of granule cells. In the primary culture of granules cells, isolated from the postnatal day 9 cerebella, NOS activity and nNOS mRNA increased during maturation in vitro. The organotypic culture of the developing slices, prepared from the postnatal day 9 cerebella, showed migration and maturation of granule cells to from the cerebellar layrs, but the layr structure was irregular and the preparation of good microsamples form the slices was hardly possible.