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）是催化信息分子一氧化氮（Nitric oxide， NO）形成的酶，广泛分布于一般组织细胞中，高浓度分布于小脑颗粒层。小脑颗粒细胞在出生后分化，小脑层也随之发育。因此，为了研究大鼠出生后小脑发育过程中颗粒细胞的分化和成熟过程，我们分析了NOS在发育阶段的活性和亚型。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。NOS活性在发育过程中在分子层和内颗粒层中均有增加，在外颗粒层中也有活性。我们新开发的微免疫印迹显示神经元NOS （nNOS）和内皮NOS （eNOS）存在于分子层和内部颗粒层中，并且nNOS免疫染色强度在出生后发育过程中增加，代表了两层细胞的分化和成熟。神经母细胞外颗粒层免疫印迹染色eNOS和nNOS。此外，利用我们新设计的微免疫印迹法，在生发层中也检测到了nNOS mRNA。腹腔注射N^ g -硝基- l -精氨酸可抑制体内NOS活性，扰乱发育中的小脑层的形成；因此NO似乎支持颗粒细胞的分化和成熟。从出生后第9天分离的小脑颗粒细胞原代培养中，NOS活性和nNOS mRNA在体外成熟过程中增加。从出生后第9天制备的发育中小脑切片的器官型培养显示颗粒细胞向小脑层迁移成熟，但层结构不规则，很难从切片中制备出良好的微样品。

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.