Neuronal activity and its influence on developmentally regulated GABA sub(A) receptor expression in cultured mouse cerebellar granule cells

Mouse cerebellar granule cells (CGCs) were cultured in either non-depolarising (5 mM KCl, '5K') or depolarising (25 mM KCl, '25K') media. CGCs at 5K developed an elaborate network of processes and formed compact cell aggregates, whilst at 25K, cell aggregation was rarely observed. GABA sub(A) receptor (GABAR) expression was significantly affected by the culture conditions. Depolarisation resulted in a similar to 55% reduction in the expression of total specific [ super(3)H]Ro15-4513 binding sites, largely due to a >85% loss of the flunitrazepam-insensitive (BZ-IS) subtype of binding sites. The number of flunitrazepam-sensitive (BZ-S) [ super(3)H]Ro15-4513 binding sites expressed and the K sub(D) of [ super(3)H]Ro15-4513 to either GABAR subtype were not significantly affected. The BZ-S subtype expressed by 5K CGCs was essentially all type I, as expected of mature CGCs, however at 25K, these were predominantly type II ( similar to 70%) and zolpidem-insensitive ( similar to 30%) - pharmacological finger-prints of immature CGCs. By immunoblotting we determined that CGCs cultured at 25K expressed GABAR alpha 1, alpha 6 and beta 3 subunits at 14, 3 and 167% of 5K levels, respectively. GABAR beta 2 subunits, however, were barely detectable. The changes in GABAR subunit expression were paralleled by similar changes in the steady-state levels of the subunit mRNAs. The switch from type I to type II BZ-S pharmacology upon depolarisation was mirrored by a switch in gene expression from alpha 1 (12% of 5K) to alpha 3 (371% of 5K). Interestingly, although depolarisation reduced beta 2 subunit mRNA to 25% of 5K, beta 2 protein was undetectable. Thus, we have shown that electrically active (5K) mouse CGCs differentiate in vitro to express a GABAR profile expected of adult CGCs in vivo. Chronically depolarised, electrically silent CGCs appear to be developmentally arrested, expressing some GABAR characteristics of prenatal CGCs. This model system should prove invaluable for elucidating signalling pathways orchestrating GABAR differentiation.