ACCELERATED COMMUNICATION Tonically Activated GABAA Receptors in Hippocampal Neurons Are High-Affinity, Low-Conductance Sensors for Extracellular GABA

In the hippocampus, two distinct forms of GABAergic inhibition have been identified, phasic inhibitory postsynaptic currents that are the consequence of the vesicular release of GABA and a tonic conductance that is activated by low ambient concentrations of extracellular GABA. It is not known what accounts for the distinct properties of receptors that mediate the phasic and tonic inhibitory conductances. Moreover, the physiological role of the tonic inhibitory conductance remains uncertain because pharmacological tools that clearly distinguish tonic and phasic receptors are lacking. Here, we demonstrate that GABAA receptors that generate a tonic conductance in cultured hippocampal neurons from embryonic mice have different pharmacological properties than those in cerebellar granule neurons or pyramidal neurons in the dentate gyrus. The tonic conductance in cultured hippocampal neurons is enhanced by the benzodiazepine, midazolam, and is insensitive to the inhibitory effects of the competitive antagonist, gabazine ( 10 M). We also identify penicillin as an uncompetitive antagonist that selectively inhibits the synaptic but not tonic conductance. GABA was applied to hippocampal neurons to investigate the properties of synaptic and extrasynaptic receptors. GABA-evoked current was composed of two components: a rapidly desensitizing current that was blocked by penicillin and a nondesensitizing current that was insensitive to penicillin blockade. The potency of GABA was greater for the penicillin-insensitive nondesensitizing current. Single-channel studies show that the gabazine-insensitive GABAA receptors have a lower unitary conductance (12 pS) than that estimated for synaptic receptors. Thus, specialized GABAA receptors with an apparent higher affinity for GABA that do not readily desensitize mediate the persistent tonic conductance in hippocampal neurons. The receptors underlying tonic and phasic inhibitory conductances in hippocampal neurons are pharmacologically and biophysically distinct, suggesting that they serve different physiological roles. The inhibitory neurotransmitter GABA is thought to regulate point-to-point communication between neurons by activating GABAA receptors clustered in postsynaptic densities. However, GABA also serves a “paracrine” function by diffusing away from the synaptic cleft and activating extrasynaptic GABAA and GABAB receptors that reside beyond subsynaptic domains (for review, see Isaacson, 2000; Mody, 2001). Ambient GABA can also arise by the reverse operation of GABA cotransporters in neurons and astrocytes (Liu et al., 2000; Wu et al., 2001). Thus, GABAA receptors need not be restricted to synapses to serve physiological functions. Moreover, GABAA receptors responsible for the tonic inhibitory conductance in some brain regions may be of clinical importance as targets for anesthetics and sedative drugs (Bai et al., 2001). Abnormal regulation of the tonic conductance may also play a role in hyperexcitatory disorders such as epilepsy. Anticonvulsants that increase the extracellular concentration of GABA may primarily increase the tonic inhibitory conductance (Overstreet and Westbrook, 2001). Despite their This work was supported by the Ontario Ministry of Health, Epilepsy Canada, Canadian Institutes of Health Research, and Natural Sciences and Engineering Research Council of Canada. ABBREVIATIONS: mIPSC, miniature inhibitory postsynaptic current; GBZ, gabazine; BIC, bicuculline; PEN, penicillin-G; VGB, vigabatrin; TBPS, tert-butyl-bicyclo[2.2.2]phosphorothionate; MIDZ; midazolam. 0026-895X/03/6301-2–8$7.00 MOLECULAR PHARMACOLOGY Vol. 63, No. 1 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2075/1033631 Mol Pharmacol 63:2–8, 2003 Printed in U.S.A. 2 at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from probable importance, the physiological role of tonic GABAA receptors in specific brain regions, including the hippocampus, remains to be elucidated. At least 19 different GABAA subunits that confer distinct pharmacological and biophysical properties have been identified (Rudolph et al., 2001). Subunit composition influences agonist affinity, receptor kinetics, and segregation of receptors to subcellular regions of the neuron (Nusser et al., 1998). For example, extrasynaptic GABAA receptors in cerebellar granule neurons contain 6 and subunits. The properties of native and recombinant GABAA receptors suggest that these subunits confer a high affinity for GABA, low single-channel conductance, and slow kinetics of desensitization (Saxena and Macdonald, 1994; Brickley et al., 1999; Haas and Macdonald, 1999; Mellor et al., 2000). Receptors containing subunits may also underlie a benzodiazepine-insensitive tonic conductance in dentate granule neurons (Nusser and Mody, 2002). Tonic inhibitory conductances have also been identified in the thalamus, the CA1 hippocampal region, and the cortex (Valeyev et al., 1998; Liu et al., 2000; Bai et al., 2001). However, the subunit composition and the pharmacological characteristics of GABAA receptors responsible for the tonic GABAergic conductance (referred to here as tonic receptors) remain to be elucidated in these brain regions. We first showed that the tonic and synaptic conductances in embryonic hippocampal neurons displayed different sensitivities to the high-affinity competitive antagonist gabazine (SR-95531) (Bai et al., 2001). Also, noise analysis indicated the tonic receptors have a lower unitary channel conductance (7 pS) than that reported for synaptic receptors (25 pS). Here, we further characterized the properties of the tonic and synaptic (phasic) receptors in hippocampal neurons and identified penicillin-G as a selective antagonist of phasic receptors. Single-channel studies provided evidence that low-conductance, high-affinity channels mediated the tonic conductance. Materials and Methods Cell Culture and Electrophysiological Techniques. Cultures of hippocampal neurons were prepared from embryonic Swiss White mice as described previously (MacDonald et al., 1989). Conventional whole-cell currents were recorded under voltage-clamp ( 60 mV) using an Axopatch 200 amplifier (Axon Instruments Inc., Union City, CA) that was interfaced to a Digidata 1200 (Instrutech Corp., Elmont, NY). Records were filtered (2 kHz) and digitized at 10 kHz using pClamp6 software (Axon Instruments Inc.) for off-line analy-