NEUROCHEMICAL CORRELATES OF CEREBELLAR DEVELOPMENT

本提案的目的是研究γ-氨基丁酸 牛和小鼠小脑的GABA/BZ受体 在发展过程中。 GABA/BZ受体结合位点将是 用受体特异性配体放射自显影定位， [3 H]蝇蕈醇和[3 H]氟硝西泮; GABA/BZ受体蛋白将被 使用de las 62- 3G 1抗受体单克隆抗体进行解剖学定位 抗体，并且GABA/BZ受体α和β亚基mRNA将被 通过互补[35 S]寡核苷酸的原位杂交检测 probes. 这些技术也将用于研究 GABA/BZ结合位点、GABA/BZ受体免疫反应性和GABA/BZ 在来自发育中的正常C57 BL/6小鼠的细胞培养物中的受体mRNA。 在 此外，我们将确定是否在细胞中发现的特定细胞缺陷， 小脑发育中的“织布者”、“浦肯野细胞变性”和 “staggerer”突变小鼠影响GABA/BZ α和β的表达 亚基mRNA。 将提出以下实验问题：（1） 编码α和β的mRNA的解剖学定位是什么 GABA/BZ受体复合物的亚基，GABA/BZ受体蛋白， 和GABA/BZ配体结合位点在成年牛小脑？(2)什么 是GABA/BZ配体的获得与 结合位点、GABA/BZ受体蛋白和编码GABA/BZ受体的mRNA。 GABA/BZ受体复合物的α和β亚单位在 牛小脑的发育(3)什么是实验 在小鼠中使用牛寡核苷酸探针所需的条件 小脑？[35 S]标记的放射自显影分布是什么？ 探针在正常成人和发育中的小鼠小脑？(4)是 颗粒细胞靶点浦肯野细胞的存在， 持续维持颗粒细胞GABA/BZ受体mRNA？(5)是 突触接触浦肯野细胞所需的初始表达 颗粒细胞GABA/BZ受体mRNA？(6)是颗粒细胞的诱导 GABA/BZ受体mRNA，由于内在的定时机制， 颗粒细胞，还是迁移环境的某些成分？(7)做 在与神经胶质细胞结合之前从小脑分离的颗粒细胞 或浦肯野细胞表达GABA/BZ受体结合位点， 编码受体α和β的蛋白质分子或mRNA 亚单位？上述研究的数据将用于确定 一个工作假设的有效性，其中的初始表达 GABA/BZ受体独立于与其他受体的突触接触而发生。 细胞 随后这些受体的稳定和维持， 然而，依赖于突触的形成和持续的轴突接触， 与传出靶细胞的联系 在这个假设中，传出输入 从GABA能神经元的诱导，稳定， GABA/BZ受体的维持。

The aim of this proposal is to study the expression gamma-aminobutyric acid/benzodiazepine (GABA/BZ) receptors in bovine and murine cerebellum during development. GABA/BZ receptor binding sites will be autoradiographically localized with the receptor specific ligands, [3H]muscimol and [3H]flunitrazepam; the GABA/BZ receptor protein will be anatomically localized with the de las 62-3G1 anti-receptor monoclonal antibody, and the GABA/BZ receptor alpha and beta subunit mRNAs will be detected by in situ hybridization of complementary [35S]oligonucleotide probes. These techniques will also be used to study the expression of GABA/BZ binding sites, GABA/BZ receptor immunoreactivity and GABA/BZ receptor mRNA in cell cultures from developing normal C57BL/6 mice. In addition we will ascertain if the specific cellular deficits found in the cerebella of developing "weaver", "Purkinje cell degeneration" and "staggerer" mutant mice affect the expression of GABA/BZ alpha and beta subunit mRNAs. The following experimental questions will be asked: (1) What is the anatomical localization of mRNAs coding for the alpha and beta subunits of the GABA/BZ receptor complex, the GABA/BZ receptor protein, and the GABA/BZ ligand binding sites in adult bovine cerebellum ? (2) What is the temporal relationship between the acquisition of GABA/BZ ligand binding sites, the GABA/BZ receptor protein, and mRNAs coding for the alpha and beta subunits of the GABA/BZ receptor complex during the development of the bovine cerebellum ? (3) What are the experimental conditions required for the use of bovine oligonucleotide probes in mouse cerebellum ? What is the autoradiographic distribution of [35S]-labeled probes in the normal adult and developing mouse cerebellum ? (4) Is the presence of the granule cell target, the Purkinje cell, required for continued maintenance of granule cell GABA/BZ receptor mRNAs ? (5) Is synaptic contact with Purkinje cell required for the initial expression of granule cell GABA/BZ receptor mRNAs ? (6) Is the induction of granule cell GABA/BZ receptor mRNAs due to an intrinsic timing mechanism within the granule cell, or to some component of the migratory environment ? (7) Do granule cells isolated from the cerebellum prior to association with glial or Purkinje cells express GABA/BZ receptor binding sites, the receptor protein molecule, or mRNAs which code for the receptor alpha and beta subunits ? The data from the above studies will be used to determine the validity of a working hypothesis in which the initial expression of GABA/BZ receptors occurs independently of synaptic contact with other cells. Subsequent stabilization and maintenance of these receptors is, however, dependent upon synapse formation and continued axonal contact with the efferent target cells. In this hypothesis, the efferent input from Gabaergic neurons is not required for induction, stabilization of maintenance of GABA/BZ receptors.