Neuronal activity and its influence on developmentally regulated GABA(A) receptor expression in cultured mouse...

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(A) receptor (GABAR) expression was significantly affected by the culture conditions. Depolarisation resulted in a approximately 55% reduction in the expression of total specific [(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) [(3)H]Ro15-4513 binding sites expressed and the K(D) of [(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 (approximately 70%) and zolpidem-insensitive (approximately 30%)-pharmacological finger-prints of immature CGCs. By immunoblotting we determined that CGCs cultured at 25K expressed GABAR alpha1, alpha6 and beta3 subunits at 14, 3 and 167% of 5K levels, respectively. GABAR beta2 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 alpha1 (12% of 5K) to alpha3 (371% of 5K). Interestingly, although depolarisation reduced beta2 subunit mRNA to 25% of 5K, beta2 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.