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.
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