The long term goal of these studies is to identify molecular events and signaling cascades involved in regulating GABAA receptor subunit expression during cerebellar granule neuron differentiation. Cerebellar granule neurons undergo extensive postnatal maturation. During this process they cease dividing in the external germinal layer (EGL), migrate to their mature positions in the internal granule layer (IGL), and receive synaptic input. One phenotypic change accompanying this maturation process is the increased expression of six mRNAs encoding subunits of the GABAA receptor. This multisubunit, ligand-gated ion channel receptor mediates the actions of gamma-aminobutyric acid, the major inhibitory neurotransmitter in the brain. Despite the temporally concurrent increases in subunit transcript levels in vivo, studies using cultured granule neurons have led to the hypothesis that subunit expression is differentially regulated and dependent on multiple regulatory cues. Signals in the cerebellar environment which selectively induce GABAA receptor beta2 and delta subunit expression in granule neurons in culture have recently been identified. To investigate the actions of these signals, the following studies are proposed. First, the actions of neuregulin and cerebellar EGL extracts in regulating beta2 subunit expression and GABAA receptor function will be examined in cultures of granule neurons using Western blotting, immunocytochemistry, and RT-PCR. The possibility that the effects of the EGL extract are mediated by neuregulin will be determined. Electrophysiological studies will then investigate whether alterations in subunit expression are correlated with changes in response to GABA. Second, the mechanisms of action of neuronal activity and glutamate receptor activation in regulating delta subunit expression and GABAA receptor function will be examined using similar experimental approaches. Studies will be performed on cerebellar slices grown in the presence or absence of pons explants, a source of excitatory mossy fiber input found in vivo. Third, signal transduction pathways mediating the changes in subunit expression will be identified. Together, these studies will yield novel information concerning environmental signals involved in directing the developmental expression and regulation of the GABAA receptor in a neuronal population.
