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

一氧化氮合酶（Nitric oxide synthase，NOS）是催化一氧化氮（nitric oxide，NO）形成的信息分子，广泛分布于一般组织细胞中，在小脑颗粒层中含量较高。小脑颗粒细胞在出生后分化，小脑层也相应地发育。因此，为了探讨大鼠小脑颗粒细胞在生后发育过程中的分化成熟过程，本研究对发育层中NOS活性及其亚型进行了分析。我们开发了一种微量测定NOS的方法，使用酶扩增反应NADP循环。在发育过程中，NOS活性在分子层和内颗粒层均呈上升趋势，在外颗粒层也有表达。免疫印迹结果显示，神经元型NOS（nNOS）和内皮型NOS（eNOS）主要分布于分子层和内颗粒层，且nNOS的免疫染色强度随出生后发育而增强，代表两层细胞的分化和成熟。神经母细胞外颗粒层eNOS和nNOS免疫印迹染色。此外，我们设计的微量免疫印迹法还检测到nNOS mRNA在该细胞层中的表达。腹腔注射N^G-硝基-L-精氨酸可抑制体内NOS活性，干扰小脑发育过程中的成层，NO可能支持颗粒细胞的分化和成熟。在原代培养的颗粒细胞中，分离自出生后9天小脑，NOS活性和nNOS mRNA在体外成熟过程中增加。从出生后第9天的小脑制备的发育切片的器官型培养显示颗粒细胞从小脑层迁移和成熟，但层结构不规则，并且从切片制备良好的微量样品几乎是不可能的。

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.