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Among hypnotic agents that enhance GABA A receptor function, etomidate is unusual because it is selective for ␤ 2 /␤ 3 compared with ␤ 1 subunit-containing GABA A receptors. Mice incorporating an etomidate-insensitive ␤ 2 subunit (␤ 2N265S ) revealed that ␤ 2 subunitcontaining receptors mediate the enhancement of slow-wave activity (SWA) by etomidate, are required for the sedative, and contribute to the hypnotic actions of this anesthetic. Although the anatomical location of the ␤ 2 -containing receptors that mediate these actions is unknown, the thalamus is implicated.We have characterized GABA A receptor-mediated neurotransmission in thalamic nucleus reticularis (nRT) and ventrobasalis complex (VB) neurons of wild-type, ␤ 2 Ϫ/Ϫ , and ␤ 2N265S mice. VB but not nRT neurons exhibit a large GABA-mediated tonic conductance that contributes ϳ80% of the total GABA A receptor-mediated transmission. Consequently, although etomidate enhances inhibition in both neuronal types, the effect of this anesthetic on the tonic conductance of VB neurons is dominant. The GABA-enhancing actions of etomidate in VB but not nRT neurons are greatly suppressed by the ␤ 2N265S mutation. The hypnotic THIP (Gaboxadol) induces SWA and at low, clinically relevant concentrations (30 nM to 3 M) increases the tonic conductance of VB neurons, with no effect on VB or nRT miniature IPSCs (mIPSCs) or on the holding current of nRT neurons. Zolpidem, which has no effect on SWA, prolongs VB mIPSCs but is ineffective on the phasic and tonic conductance of nRT and VB neurons, respectively. Collectively, these findings suggest that enhancement of extrasynaptic inhibition in the thalamus may contribute to the distinct sleep EEG profiles of etomidate and THIP compared with zolpidem.

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Because GABA A receptors containing α2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltageclamp recordings from NAcc medium spiny neurons of mice with α2 gene deletion showed reduced synaptic GABA A receptor-mediated responses. Behaviorally, the deletion abolished cocaine's ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of α2-GABA A receptors (α2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In α2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of α2−GABA A receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.GABRA2 | behavioral sensitization | nucleus accumbens | mutant mouse | human genetics

Thalamic ventrobasal (VB) relay neurones express multiple GABA A receptor subtypes mediating phasic and tonic inhibition. During postnatal development, marked changes in subunit expression occur, presumably reflecting changes in functional properties of neuronal networks. The aims of this study were to characterize the properties of synaptic and extrasynaptic GABA A receptors of developing VB neurones and investigate the role of the α 1 subunit during maturation of GABA-ergic transmission, using electrophysiology and immunohistochemistry in wild type (WT) and α 1 0/0 mice and mice engineered to express diazepam-insensitive receptors (α 1H101R , α 2H101R ). In immature brain, rapid (P8/9-P10/11) developmental change to mIPSC kinetics and increased expression of extrasynaptic receptors (P8-27) formed by the α 4 and δ subunit occurred independently of the α 1 subunit. Subsequently (≥ P15), synaptic α 2 subunit/gephyrin clusters of WT VB neurones were replaced by those containing the α 1 subunit. Surprisingly, in α 1 0/0 VB neurones the frequency of mIPSCs decreased between P12 and P27, because the α 2 subunit also disappeared from these cells. The loss of synaptic GABA A receptors led to a delayed disruption of gephyrin clusters. Despite these alterations, GABA-ergic terminals were preserved, perhaps maintaining tonic inhibition. These results demonstrate that maturation of synaptic and extrasynaptic GABA A receptors in VB follows a developmental programme independent of the α 1 subunit. Changes to synaptic GABA A receptor function and the increased expression of extrasynaptic GABA A receptors represent two distinct mechanisms for fine-tuning GABA-ergic control of thalamic relay neurone activity during development.

No abstract

The sedative and hypnotic agent THIP is a GABA A receptor agonist that preferentially activates δ-subunit containing GABA A receptors (δ-GABA A Rs). To clarify the role of δ-GABA A Rs in mediating the sedative actions of THIP, we have utilised mice lacking the α1 or δ subunit in a combined electrophysiological and behavioural analysis.Whole-cell patch-clamp recordings were obtained from thalamic ventrobasal (VB) neurones at a holding potential of −60mV. Application of bicuculline to wild type (WT) VB neurones revealed a GABA A R-mediated tonic current of 92 ± 19 pA, which was greatly reduced (13 ± 5 pA) for VB neurones of δ 0/0 mice. Deletion of the δ, but not the α1 subunit, dramatically reduced the THIP (1µM)-induced inward current in these neurones (WT = −309 ± 23 pA; δ 0/0 = −18 ± 3 pA; α1 0/0 = −377 ± 45 pA). Furthermore, THIP selectively decreased the excitability of WT and α1 0/0 , but not δ 0/0 VB neurones. THIP did not affect the properties of mIPSCs in any of the genotypes. No differences in rotarod performance and locomotor activity were observed across the three genotypes. In WT mice, performance of these behaviours was impaired by THIP in a dosedependent manner. The effect of THIP on rotarod performance was blunted for δ 0/0 , but not α1 0/0 mice. We previously reported deletion of the α1 subunit to abolish synaptic GABA A responses of VB neurones. Therefore, collectively, these findings suggest that extrasynaptic δ-GABA A Rs vs synaptic α1-GABA A Rs of thalamocortical neurones represent an important molecular target underpinning the sedative actions of THIP.

As neuronal development progresses, GABAergic synaptic transmission undergoes a defined program of reconfiguration. For example, GABAA receptor (GABAAR)-mediated synaptic currents, (miniature inhibitory postsynaptic currents; mIPSCs), which initially exhibit a relatively slow decay phase, become progressively reduced in duration, thereby supporting the temporal resolution required for mature network activity. Here we report that during postnatal development of cortical layer 2/3 pyramidal neurons, GABAAR-mediated phasic inhibition is influenced by a resident neurosteroid tone, which wanes in the second postnatal week, resulting in the brief phasic events characteristic of mature neuronal signalling. Treatment of cortical slices with the immediate precursor of 5α-pregnan-3α-ol-20-one (5α3α), the GABAAR-inactive 5α-dihydroprogesterone, (5α-DHP), greatly prolonged the mIPSCs of P20 pyramidal neurons, demonstrating these more mature neurons retain the capacity to synthesize GABAAR-active neurosteroids, but now lack the endogenous steroid substrate. Previously, such developmental plasticity of phasic inhibition was ascribed to the expression of synaptic GABAARs incorporating the α1 subunit. However, the duration of mIPSCs recorded from L2/3 cortical neurons derived from α1 subunit deleted mice, were similarly under the developmental influence of a neurosteroid tone. In addition to principal cells, synaptic GABAARs of L2/3 interneurons were modulated by native neurosteroids in a development-dependent manner. In summary, local neurosteroids influence synaptic transmission during a crucial period of cortical neurodevelopment, findings which may be of importance for establishing normal network connectivity.