NEUROCHEMICAL CORRELATES OF CEREBELLAR DEVELOPMENT

这项建议的目的是研究伽玛-氨基丁酸的表达 牛和小鼠小脑的酸/苯二氮(GABA/BZ)受体 在开发过程中。GABA/BZ受体结合位点将是 放射自显影定位于受体特定的配体， [3 H]蝇草酚和[3 H]氟硝西潘；GABA/BZ受体蛋白将是 De LAS 62-3G1抗受体单抗的解剖定位 抗体，GABA/BZ受体的α和β亚基mRNAs将 互补的[35S]寡核苷酸原位杂交检测 探测器。这些技术也将被用来研究表达 GABA/BZ结合部位、GABA/BZ受体免疫反应性及GABA/BZ 发育正常C57BL/6小鼠细胞培养中受体基因的表达。在……里面 此外，我们将确定特定的细胞缺陷是否在 发育中的“织女”、“浦肯野细胞变性”和小脑 “交错”突变小鼠对GABA/BZα和β表达的影响 亚基mRNAs将提出以下试验性问题：(1) 编码α和β基因的mRNAs的解剖学定位是什么？ GABA/BZ受体复合体的亚单位，GABA/BZ受体蛋白， 成年牛小脑内的GABA/BZ配体结合部位？(2)什么？ 是GABA/BZ配体获得之间的时间关系 结合部位、GABA/BZ受体蛋白和编码 GABA/BZ受体复合体的α和β亚基 牛小脑的发育？(3)什么是实验 牛寡核苷酸探针在小鼠体内使用的条件 小脑？[35S]标记的放射自显影分布是什么 在正常成人和发育中的小鼠小脑中的探针？(4)是 颗粒细胞靶标Purkinje细胞的存在 颗粒细胞GABA/BZ受体mRNAs的持续维持？(5) 与浦肯野细胞的突触接触是其最初表达所必需的 颗粒细胞GABA/BZ受体mRNAs？(6)是颗粒细胞的诱导 GABA/BZ受体mRNAs在细胞内的内在时序机制 颗粒细胞，还是迁移环境的某些成分？(7) 小脑分离的颗粒细胞与神经胶质细胞的联系 或浦肯野细胞表达GABA/BZ受体结合部位，该受体 蛋白质分子，或编码受体α和β的mRNAs 亚单位？上述研究的数据将被用来确定 一个工作假设的有效性，在该假设中，初始表达式 GABA/BZ受体独立于与神经细胞的突触接触而发生 细胞。随后这些受体的稳定和维持是， 然而，依赖于突触的形成和持续的轴突接触 传出的靶细胞。在这个假设中，传出输入 GABA能神经元不需要诱导、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.