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Neuronal activity and its influence on developmentally regulated GABAA receptor expression in cultured mouse cerebellar granule cells.

Ives, J. H. and Drewery, D. L. and Thompson, C. L. (2002) 'Neuronal activity and its influence on developmentally regulated GABAA receptor expression in cultured mouse cerebellar granule cells.', Neuropharmacology., 43 (4). pp. 715-725.


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. GABAA receptor (GABAR) expression was significantly affected by the culture conditions. Depolarisation resulted in a ˜55% reduction in the expression of total specific [3H]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) [3H]Ro15-4513 binding sites expressed and the KD of [3H]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 (˜70%) and zolpidem-insensitive (˜30%)—pharmacological finger-prints of immature CGCs. By immunoblotting we determined that CGCs cultured at 25K expressed GABAR α1, α6 and β3 subunits at 14, 3 and 167% of 5K levels, respectively. GABAR β2 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 α1 (12% of 5K) to α3 (371% of 5K). Interestingly, although depolarisation reduced β2 subunit mRNA to 25% of 5K, β2 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.

Item Type:Article
Keywords:Mouse, Benzodiazepine, Receptor regulation, Subunit-specific antibodies, RT-PCR, Differentiation.
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Record Created:10 Feb 2009
Last Modified:08 Apr 2009 16:32

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