GABA-A RECEPTOR EXPRESSION IN CEREBELLAR NEURONS

该提案将研究GABA/A受体亚单位的组成， 在体内和培养的小脑神经元中组装。 GABA/A 受体是一种多亚基配体门控离子通道，介导 主要抑制性神经递质γ-氨基丁酸的作用 在中枢神经系统中。 最近的研究表明， 受体由几个亚基组成，其中大部分由 基因家族 每个亚基基因都表现出不同的 小脑中表达的细胞和时间模式。 而 只有一种特定亚基的同种型在一些小脑中表达， 群体中，多个亚基同种型存在于其他细胞类型中。 这些结果，加上电生理学的发现，提高了 小脑神经元表达多种受体亚型的可能性 具有不同的亚基组成和功能特性。 然而，在这方面， 天然受体的亚基组成以及 受体的组装仍是未知的。 为了阐明GABA/A受体亚单位的组成和组装， 为了确定小脑神经元，将进行一些研究。 首先，受体亚基在体内和体外小脑细胞中的表达， 使用亚单位特异性抗血清检查培养物。 变化 亚基水平在出生后的发展将评估西方 印迹分析 此外，颗粒和浦肯野细胞中的亚基分布 通过免疫组织化学检查神经元。 这些研究将 开始确定鉴定细胞中GABA/A受体亚单位组成 群体和表征受体的发育变化 表情 第二，小脑GABA/A的亚基组成 受体将使用免疫沉淀与受体 亚单位特异性抗血清。 这些研究将阐明哪些亚基 来自不同类别的亚基异构体以及来自单个类别的亚基异构体是 共组装成受体复合物。 最后，GABA/A 培养的小脑颗粒神经元中的受体将使用 代谢标记，然后用亚基特异性免疫沉淀 抗血清 这些研究将确定受体亚基是否表现出 生物合成的可比速率，并将检查亚基的过程 组装成寡聚受体复合物。 这些研究一起 将产生关于GABA/A受体亚基的重要新信息 在鉴定的小脑细胞群中的组成和组装。 由于不同的亚基赋予不同的生理特性， 受体，亚基组成的知识是必要的， 了解GABA/A受体在中枢神经系统中的功能 系统

This proposal will examine GABA/A receptor subunit composition and assembly in cerebellar neurons in vivo and in culture. the GABA/A receptor is a multisubunit, ligand-gated ion channel that mediates the actions of gamma-aminobutyric acid, the major inhibitory neurotransmitter in the central nervous system. Recent studies have shown that the receptor is composed of several subunits, most of which are encoded by families of genes. Each of the subunit genes exhibits a distinct cellular and temporal pattern of expression in the cerebellum. While only one isoform of a specific subunit is expressed in some cerebellar populations, multiple subunit isoforms are present in other cell types. These results, coupled with electrophysiological findings, raise the possibility that cerebellar neurons express multiple receptor subtypes having diverse subunit compositions and functional properties. However, the subunit composition of native receptors and the process by which receptors are assembled remain unknown. To elucidate GABA/A receptor subunit composition and assembly in identified cerebellar neurons, a number of studies will be performed. First, receptor subunit expression in cerebellar cells in vivo and in culture will be examined using subunit-specific antisera. changes in subunit levels during postnatal development will be assessed by Western blot analysis. In addition, subunit distribution in granule and Purkinje neurons will be examined by immunohistochemistry. These studies will begin to define GABA/A receptor subunit composition in identified cell populations and characterize developmental changes in receptor expression. Second, the subunit composition of cerebellar GABA/A receptors will be determined using immunoprecipitation with receptor subunit-specific antisera. These studies will elucidate which subunits from different classes and which subunit isoforms from a single class are coassembled into a receptor complex. Finally, the assembly of GABA/A receptors in cultured cerebellar granule neurons will be examined using metabolic labeling followed by immunoprecipitation with subunit-specific antisera. These studies will determine whether receptor subunits exhibit comparable rates of biosynthesis and will examine the process of subunit assembly into oligomeric receptor complexes. Together, these studies will yield important new information concerning GABA/A receptor subunit composition and assembly in identified cerebellar cell populations. Since the different subunits confer distinct physiological properties to the receptor, knowledge of subunit composition is essential for an understanding of GABA/A receptor function throughout the central nervous system.