GABAC ρ1 Subunits Form Functional Receptors But Not Functional Synapses in Hippocampal Neurons

Abstract

The ability to control the physiological and pharmacological properties of synaptic receptors is a powerful tool for studying neuronal function and may be of therapeutic utility. We designed a recombinant adenovirus to deliver either a GABAC receptor ρ1 subunit or a mutant GABAA receptor β2 subunit lacking picrotoxin sensitivity [β2(mut)] to hippocampal neurons. A green fluorescent protein (GFP) reporter molecule was simultaneously expressed. Whole cell patch-clamp recordings demonstrated somatic expression of both bicuculline-resistant GABAC receptor-mediated and picrotoxin-resistant GABAA receptor-mediated GABA-evoked currents in ρ1- and β2(mut)-transduced hippocampal neurons, respectively. GABAergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of 6-cyano-7-nitroquinoxalene-2,3-dione, Mg2+, and TTX revealed synaptic events with monoexponential activation and biexponential decay phases. Despite the robust expression of somatic GABAC receptors in ρ1-neurons, no bicuculline-resistant mIPSCs were observed. This suggested either a kinetic mismatch between the relatively brief presynaptic GABA release and slow-activating ρ1 receptors or failure of the ρ1 subunit to target properly to the subsynaptic membrane. Addition of ruthenium red, a presynaptic release enhancer, failed to unmask GABACreceptor-mediated mIPSCs. Short pulse (2 ms) application of 1 mM GABA to excised outside-out patches from ρ1 neurons proved that a brief GABA transient is sufficient to activate ρ1 receptors. The simulated-IPSC experiment strongly suggests that if postsynaptic GABACreceptors were present, bicuculline-resistant mIPSCs would have been observed. In contrast, in β2(mut)-transduced neurons, picrotoxin-resistant mIPSCs were observed; they exhibited a smaller peak amplitude and faster decay compared with control. Confocal imaging of transduced neurons revealed ρ1immunofluorescence restricted to the soma, whereas punctate β2(mut) immunofluorescence was seen throughout the neuron, including the dendrites. Together, the electrophysiological and imaging data show that despite robust somatic expression of the ρ1 subunit, the GABACreceptor fails to be delivered to the subsynaptic target. On the other hand, the successful incorporation of β2(mut) subunits into subsynaptic GABAA receptors demonstrates that viral transduction is a powerful method for altering the physiological properties of synapses.