The Journal of Neurophysiology Vol. 86 No. 5 November 2001, pp. 2605-2615
Copyright ©2001 by the American Physiological Society

GABA_{C 1} Subunits Form Functional Receptors But Not Functional Synapses in Hippocampal Neurons

 ¹Department of Pharmacology/Physiology and  ²Department of Anesthesiology, University of Rochester Medical Center, Rochester, New York 14641

Cheng, Qing, Paul M. Burkat, John C. Kulli, and Jay Yang. GABA_{C 1} Subunits Form Functional Receptors But Not Functional Synapses in Hippocampal Neurons. J. Neurophysiol. 86: 2605-2615, 2001. 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 GABA_C receptor ₁ subunit or a mutant GABA_A receptor ₂ subunit lacking picrotoxin sensitivity [2(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 GABA_C receptor-mediated and picrotoxin-resistant GABA_A receptor-mediated GABA-evoked currents in ₁- and ₂(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 GABA_C receptors in ₁-neurons, no bicuculline-resistant mIPSCs were observed. This suggested either a kinetic mismatch between the relatively brief presynaptic GABA release and slow-activating ₁ receptors or failure of the ₁ subunit to target properly to the subsynaptic membrane. Addition of ruthenium red, a presynaptic release enhancer, failed to unmask GABA_C receptor-mediated mIPSCs. Short pulse (2 ms) application of 1 mM GABA to excised outside-out patches from ₁ neurons proved that a brief GABA transient is sufficient to activate ₁ receptors. The simulated-IPSC experiment strongly suggests that if postsynaptic GABA_C receptors were present, bicuculline-resistant mIPSCs would have been observed. In contrast, in ₂(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 ₁ immunofluorescence restricted to the soma, whereas punctate ₂(mut) immunofluorescence was seen throughout the neuron, including the dendrites. Together, the electrophysiological and imaging data show that despite robust somatic expression of the ₁ subunit, the GABA_C receptor fails to be delivered to the subsynaptic target. On the other hand, the successful incorporation of ₂(mut) subunits into subsynaptic GABA_A receptors demonstrates that viral transduction is a powerful method for altering the physiological properties of synapses.