The aim of this proposal is to examine the cellular interactions underlying the expression of granule cell GABAA (gamma-aminobutyric acid) receptors of the mouse cerebellum during development. The general hypothesis to be tested is that the initial expression of granule cell GABAA receptors in the granule cell membrane occurs independently of synaptic contact with other cells. Subsequent stabilization and maintenance of these receptors is dependant, however, on synapse formation and continued axonal contact with the efferent target, the Purkinje cell. In this hypothesis, the afferent input to the granule cell from the GABAergic Golgi cell is not required for GABAA receptor induction, stabilization or maintenance. Autoradiographic techniques for the visualization of (3H)muscimol binding sites (indicative of high affinity GABAA receptors), and immunocytochemical methods for the visualization of AB131 (a polyclonal anti-GABA antibody) and mAb62-3G1 (a monoclonal anti- GABAA receptor antibody) will be used to localize cerebellar GABA- containing cells and GABAA receptors, respectively, at the light microscopic level. The following experimental studies will be undertaken: The binding characteristics and autoradiographic distribution of (3H)muscimol, and the distribution of mAb62-3G1, will be examined in developing and mutant mouse cerebella, to determine if changes in autoradiographic grain density represent alterations in GABAA receptor affinity, or in the number of receptors. In additions the appearance of (3H)muscimol, mAb62-3G1 and AB131 binding will be examined in developing normal, weaver and in alpha-difluoromethylornithine treated cerebella, to determine whether GABAA receptor appearance is due to an intrinsic timing mechanism, or to induction by an extracellular factor, possibly GABA. (3H)muscimol, mAb62-3G1 and AB131 binding will also be studied in the developing staggerer mouse cerebellum, to examine if there is an initial expression of GABAA receptors which later disappears due to the failure of granule cells to form contacts with their targets. Finally (3H)muscimol and mAb62-3G1 binding will be studied in the aging Purkinje cell degeneration mutant, to determine whether the presence of target cell is required for the long term maintenance of granule cell GABAA receptors. This work will lead to a fundamental understanding of GABAA receptor regulation by cellular interactions.
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