ACTIVITY-DEPENDENT REGULATION OF GABA-A RECEPTOR FUNCTION IN RAT NEOCORTICAL NEURONS

M. L. GUTIÉRREZ; M- C. FERRERI; D. H. FARB; M. C. GRAVIELLE

Nueva Orleans

Congreso; XLII Annual Meeting of the Society for Neuroscience; 2012

Society for Neuroscience

Several lines of evidence suggest the existence of homeostatic mechanisms to control synaptic inhibition by changes in the number and/or function of post-synaptic GABAA receptors. Chronic in vivo administration of GABAA receptor positive allosteric modulators, such as ethanol and benzodiazepines, in rodents has been reported to produce adaptive alterations in receptor function. In a previous work we have demonstrated that exposure of rat neocortical cultures to GABA for 5-10 min (t1/2= 3.2 min) initiates a process that results in a reduced interaction between GABA and benzodiazepine binding sites, named uncoupling, hours later (t1/2= 12 h), without changes in GABAA receptor number. Uncoupling was accompanied by a decrease in α1, α3, and β1-3 subunit mRNA levels with no change in α2, α4, α5, γ1 and γ2 mRNAs. These alterations in mRNA levels were associated with corresponding changes in subunit protein expression. The strength of allosteric coupling between GABA and benzodiazepine binding sites depends on the α subunit subtype present in the GABAA receptor with the following rank order: α3 > α1/2. As a first step to determine whether uncoupling produces a change in the percentage of receptors containing these subunits, we performed in this work immunoprecipitation experiments followed by western blot assays. Neocortical cultures were incubated with GABA for 10 min, washed and cells were collected 48 h later. Receptor immunoprecipitation assays were carried out using an antibody against γ2 subunits that are present in most of the receptors. Results from western blot experiments using antibodies against α3 and α1 subunits showed a decrease in the percentage of receptors containing α3 with no changes in receptors containing α1. We next performed a biotinylation assay followed by western blot, to measure the cell surface expression of α3 subunits. In agreement with the previous results, GABA induced a reduction in cell surface levels of α3. In conclusion, these results suggest that GABA-induced uncoupling involves a change in the subunit composition, resulting in receptors with a reduced allosteric coupling between GABA and benzodiazepine binding sites.