Abstracts

Rationale: Tonic inhibition mediated by extrasynaptic GABA_A receptors influences cellular and network excitability in the hippocampus, including epileptic seizures. We have recently shown that postsynaptic GABA_B receptors enhance the function of delta subunit-containing extrasynaptic GABA_A­ receptors in dentate gyrus granule cells (DGGCs) (Tao et al., 2013). This effect is G protein dependent, occurs within minutes, and is specific for delta subunit-containing GABA_A receptors. However, the molecular mechanisms underlying the enhancement of tonic inhibition by GABA_B receptors is not known. Methods: Patch clamp recordings were made from rat DGGCs in acute hippocampal brain slices using standard techniques under conditions designed to isolate GABA_A currents. Total and plasma membrane expression of GABA_A receptor subunits was assayed using biotinylation and western blotting techniques with delta and gamma2 subunit specific antibodies. Results: Consistent with prior results, the GABA_B receptor agonist baclofen increased the amplitude of endogenous tonic currents and currents evoked with exogenous GABA. Treatment of acute hippocampal brain slices with the GABA_B receptor agonist baclofen increased the plasma membrane expression of GABA_A receptor delta subunits, without affecting total delta subunit protein level. Surface expression of GABA_A receptor gamma2 subunits was not affected by baclofen. Estimates of single channel conductance and open probability, made by stationary noise analysis of whole cell currents, were unaffected by baclofen treatment. PKA activation with Sp-cAMP reduced tonic GABA currents and surface expression of delta subunits. Activation of PKC with PDBU had the opposite effect, and increased tonic GABA current and delta subunit surface expression. Furthermore, the enhancement of GABA currents by baclofen was prevented by both Sp-cAMP and PDBU. Inhibition of G protein beta/gamma subunits with gallein or inhibition of PLC with u73122 also occluded baclofen-induced enhancement of GABA currents. Conclusions: Our results indicate that GABA_B receptors enhance extrasynaptic GABA_A receptor function in DGGCs by increasing the number of plasma membrane receptors, without altering receptor properties. These data suggest multiple signaling pathways regulate the trafficking of extrasynaptic GABA_A receptors. Our data is consistent with a model in which GABA_B receptors regulate surface expression of delta subunit-containing GABA_A receptors, and this regulation requires signaling through both a G_i/o-cAMP-PKA pathway and a G_βγ-PLC-PKC pathway.