The long term goal of this project is to understand how GABAA receptor expression is regulated in the CNS. This multisubunit, ligand-gated ion channel is the site of action for GABA, the main inhibitory neurotransmitter in the brain. Recent molecular biologic studies have documented that each receptor subunit is encoded by a family of genes which exhibit distinct regional distributions. In cerebellar granule neurons, at least 8 subunit RNAs are expressed, many of which rise several-fold in the second postnatal week. These changes coincide temporally with extensive cerebellar differentiation and granule cell migration from a germinal zone (the EGL) to their mature positions. Studies on cultured granule neurons have shown that increases in these transcript levels is not preprogrammed, but are dependent on various aspects of cerebellar development. These findings suggest that GABAA receptor mRNA in cerebellar granule neurons is influenced by environmental cues received during postnatal differentiation. This proposal will test this hypothesis and identify signals that regulate receptor subunit mRNA expression. To gain insight into the regulation of GABAA receptor subunit mRNAs in cerebellar granule neurons, several experiments are proposed. First, age and stage of cerebellar maturation at which granule neurons become committed and/or competent to express receptor mRNAs will be determined. The importance of cell age for subunit transcript expression will be examined using RT-PCR in cultures prepared at several postnatal ages. Because such cultures contain cells at multiple stages of maturation, subunit transcripts will also be examined in developmentally homogeneous cultures prepared by immunoisolating cells with antisera to stage- specific markers. Second, the role of pre- and postsynaptic interactions in regulating granule neuron subunit mRNAs will be investigated. The influence of afferent input will be determined in cocultures of pontine nuclei explants and granule neurons by RT-PCR; the importance of granule neuron interaction with target Purkinje neurons will be determined with in situ hybridization in two mouse mutants which undergo Purkinje cell degeneration at different stages. Third, the role of environmental signals (such as neurotrophins or glutamate) in modulating GABAA receptor mRNAs will be determined by maintaining cells in conditions mimicking the cerebellar milieu. Finally, the relationship between subunit mRNA and polypeptide expression will be examined using subunit- specific antisera. The proposed studies will yield important new information concerning the developmental expression and regulation of GABAA receptor mRNAs and polypeptides in an identified neuronal population. Moreover, these studies will provide insight into the relationship between cerebellar differentiation and the development of the GABAA receptor system.
