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Introduction GABA dysfunction is associated with a number of psychiatric conditions including schizophrenia, autism and depression. Blocking cortical GABAA receptors in rodents causes behavioral deficits, including impaired attention and sociability, that are consistent with the symptoms of these conditions. The subunit composition of GABAA receptors is diverse and can affect receptor function. The current experiment examined the role of GABAA receptors containing different α-subunits in social behavior and attention. Methods Male Sprague-Dawley rats were administered FG7142 (0.0–5.0 mg/kg; a non-selective GABAA receptor inverse agonist), L-655,708 (0–1.0 mg/kg; a low efficacy inverse agonist at α 5-containing GABAA receptors), MRK-016 (0.0–2.0 mg/kg; a high efficacy inverse agonist at α5-containing GABAA receptors), or L-838,417 (0.0–3.0 mg/kg; an antagonist at α1-containing receptors and a partial agonist at α2, α3, α5-containing GABAA receptors) and either tested on the social interaction and social preference tests or the 5-choice serial reaction time task. Results FG7142 decreased social interactions and impaired attention. MRK-016 impaired attention but did not affect social behavior. Neither L-655,708 nor L-838,417 significantly affected either social behavior or attention. Discussion Systemic reduction in GABAA receptor signaling decreased sociability and attention, a result consistent with past research demonstrating cortical GABAA receptor blockade impairs social behavior and attention. Overall, the effects of the receptor subtype selective ligands were minimal; α5-containing GABAA receptors may contribute to the attentional deficit but do not contribute to the decrease in sociability. Further research is needed to determine the GABAA receptor subunits that contribute to social behavior and attention.

The extracellular α(+)/γ2(-) interface in the α1,2,3,5βγ2 GABAA receptor harbours the allosteric binding site targeted by benzodiazepines and newer generations of subtype-selective modulators. We have probed the molecular determinants for the affinity/potency-based α1-preference exhibited by the hypnotic zolpidem (Ambien®, Stilnox®) and the efficacy-based α3-over-α1 selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type, concatenated, mutant and chimeric α1,3β2γ2S receptors expressed in tsA201 cells and Xenopus oocytes by [3H]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of Gly201 in α1 with the corresponding Glu in α3 completely eliminated the α1-over-α3 preference exhibited by zolpidem. In contrast, the reverse α3-E225G mutation did not yield corresponding increases in the binding affinity or modulatory potency of zolpidem at α3β2γ2S, and two additional molecular elements in the extracellular domain of the α-subunit were found also to contribute to its α1-preference. Interestingly, the α1-Gly201/α3-Glu225 residue was also a key determinant of the efficacy-based α3-over-α1 selectivity exhibited by NS11394, and a pronounced correlation existed between the side-chain bulkiness of this residue and the modulatory efficacy of NS11394 at the receptor. The subtype-selectivity determinants identified for zolpidem and NS11394 were found also to apply in different degrees to the α1-preferring modulator indiplon and the α3-over-α1 selective modulator L-838,417, respectively. In conclusion, the molecular origins of subtype-selectivity exhibited by benzodiazepine-site modulators at the α1,2,3,5βγ2 GABAA receptor seem more complex than previously appreciated, and the importance of the α1-Gly201/α3-Glu225 residue for both potency- and efficacy-based subtype-selective modulation through this site is likely to be rooted in different molecular mechanisms.

Previous studies have demonstrated that classical benzodiazepines decrease hypothalamic-pituitary-adrenocortical cortex (HPA) axis activity. Paradoxically, high doses of benzodiazepines also stimulate basal circulating corticosterone levels in some conditions. Because benzodiazepine agonists display little selectivity to any of the alpha subtypes of the gamma-amino butyric acid (GABA)(A) receptor to which they bind, we propose that the unequivocal results are due to an alpha subtype-dependent modulation of the hypothalamic-pituitary-adrenocortical cortex axis output. To test this, basal hormonal output and induction of Fos in the hypothalamic paraventricular nucleus were measured after administration of various benzodiazepine ligands in mice. Zolpidem, a selective alpha1 subtype agonist, produced a very strong increase in plasma adrenocorticotropic hormone and corticosterone whereas the inverse agonist FG7142 induced a small rise in plasma corticosterone. More surprisingly, the non-selective full agonists diazepam and zopiclone induced a lower increase in circulating corticosterone than after zolpidem. In contrast, the alpha(2,3,5)-selective benzodiazepine agonist and alpha1 antagonist L-838,417 had no effect on corticosterone levels. Strong induction of Fos in the paraventricular nucleus was found in response to zolpidem, diazepam, and zopiclone, but not after L-838,417. Finally, pre-administration of L-838,417 prior to zolpidem strongly inhibited the effect of zolpidem on corticosterone. Likewise, the non-selective agonists diazepam and zopiclone at a dose that alone had no effect on corticosterone also inhibited the effect of zolpidem. Taken together, these results suggest that benzodiazepine ligands modulate the hypothalamic-pituitary-adrenocortical cortex axis through partly opposite mechanisms; and that the net effect is dependent on the composition of the GABA(A) receptor subunits to which they bind.

Hypofunction of the N-methyl- d -aspartic acid receptor (NMDAr) has been considered to play a crucial role in the pathophysiology of schizophrenia. In rodent electroencephalogram (EEG) studies, non-competitive NMDAr antagonists have been reported to produce aberrant basal gamma band oscillation (GBO), as observed in schizophrenia. Aberrations in GBO power have attracted attention as a translational biomarker for the development of novel antipsychotic drugs. However, the neuronal mechanisms as well as the pharmacological significance of NMDAr antagonist-induced aberrant GBO power have not been fully investigated. In the present study, to address the above questions, we examined the pharmacological properties of MK-801 (0.1 mg/kg)-increased basal GBO power in rat cortical EEG. Riluzole (3–10 mg/kg), a glutamate release inhibitor, reduced the MK-801-increased basal GBO power. In contrast, L-838,417 (1–3 mg/kg), an α2/3/5 subunit-selective GABA A receptor-positive allosteric modulator, enhanced the GBO increase. Antipsychotics such as haloperidol (0.05–0.3 mg/kg) and clozapine (1–10 mg/kg) dose-dependently attenuated the MK-801-increased GBO power. Likewise, LY379268 (0.3–3 mg/kg), an metabotropic glutamate 2/3 receptor (mGlu2/3 receptor) agonist, reduced the GBO increase in a dose-dependent manner, which was antagonized by an mGlu2/3 receptor antagonist LY341495. These results suggest that an increase in cortical GBO power induced by NMDAr hypofunction can be attributed to the aberrant activities of both excitatory pyramidal neurons and inhibitory interneurons in local circuits. The aberrant cortical GBO power reflecting cortical network dysfunction observed in schizophrenia might be a useful biomarker for the discovery of novel antipsychotic drugs.

receptors containingα2/3 subunits are current targets in the battle to develop new pain medications, as they are expressed in the spinal cord where increasing inhibitory drive should result in analgesia. However, this approach is prone to a range of side effects including sedation, cognitive impairment, and abuse as a consequence of the widespread influence of GABA. The ability to make subtype selective low-efficacy benzodiazepine compounds, which potentiate the action of GABA at specificαsubunits, has the potential to reduce this side effect profile. In this study, we have investigated the effects of the medium-efficacy positive allosteric modulator (PAM) L-838,417 and the low-efficacy PAM TPA023 in a number of preclinical inflammatory and neuropathic pain models. We conclude that either the higher level of efficacy atα2/3 or efficacy atα5 is required for compounds to have a significant analgesic effect in a range of models, and, therefore, although the side-effect profile of compounds can be reduced compared to typical benzodiazepines, it is unlikely that it can be completely eliminated.

Previous reports suggest that gamma-aminobutyric acid type A (GABA(A)) receptors containing alpha1 subunits may play a pivotal role in mediating the discriminative stimulus effects of benzodiazepines (BZs). L-838,417 (7-tert-Butyl-3-(2,5-difluoro-phenyl)-6-(2-methyl-2H-[1,2,4]triazol-3-ylmethoxy)-[1,2,4]triazolo[4,3-b]pyridazine) is a GABA(A) receptor modulator with intrinsic efficacy in vitro at alpha2, alpha3, and alpha5 subunit-containing GABA(A) receptors, and little demonstrable intrinsic efficacy in vitro at alpha1 subunit-containing GABA(A) receptors. The present study evaluated the discriminative stimulus effects of L-838,417 in order to determine the extent to which the alpha2, alpha3, and alpha5 subunit-containing GABA(A) receptors contribute to the interoceptive effects of BZ-type drugs. Squirrel monkeys (Saimiri sciureus) were trained to discriminate L-838,417 (0.3 mg/kg, i.v.) from vehicle under a 5-response fixed-ratio schedule of food reinforcement. Under test conditions, L-838,417 administration resulted in dose-dependent increases in drug-lever responding that were antagonized by the BZ-site antagonist, flumazenil. Administration of non-selective BZs, compounds with 10-fold greater affinity for alpha1 subunit-containing GABA(A) receptors compared to alpha2, alpha3, and alpha5 subunit-containing GABA(A) receptors, barbiturates and ethanol (which modulate the GABA(A) receptor via a non-BZ site), all resulted in a majority of responses on the L-838,417-paired lever (65-100% drug-lever responding). betaCCT, an antagonist that binds with 20-fold greater affinity for alpha1 subunit-containing GABA(A) receptors relative to alpha2, alpha3, and alpha5-containing GABA(A) receptors, had no significant effect on the discriminative stimulus effects of L-838,417 or the L-838,417-like effects of diazepam or zolpidem. These data suggest that efficacy at alpha2, alpha3, and/or alpha5 subunit-containing GABA(A) receptors likely are sufficient for engendering BZ-like discriminative stimulus effects.

The control of the corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) is balanced by excitatory and inhibitory inputs. The GABA-A receptor, which is a major target for the inhibitory control, is composed of five subunits. The presence of an alpha(1)-, alpha(2)-, alpha(3)-, or alpha(5)-subunit in the GABA-A receptor protein complex is necessary for benzodiazepines to exert their potentiating effect on the receptor. We postulate that the effect of nonselective benzodiazepines on the hypothalamo-pituitary adrenocortical (HPA) axis is critically dependent on the composition of the GABA-A receptor subunits through which they act. We show here that positive modulators of alpha(1)-subtype containing GABA-A receptors with zolpidem (10 mg/kg) increase HPA activity in terms of increase in plasma corticosterone and induction of Fos in the PVN, whereas activation of non-alpha(1)-subtype GABA-A receptors using L-818,417 (10 mg/kg) likely inhibits the activity. We also show that the alpha(2)-subunit gene is highly expressed in the PVN, but its expression is not affected by chronic mild stress. These results show that the stimulatory effect on HPA activity after positive modulation of GABA-A receptors composed of alpha(1)-subunit(s) affects a selective afferent system than the PVN, which is distinct from another afferent system(s) activated by non alpha(1)-containing GABA-A receptors.

The promise of subtype-selective GABA(A) receptor drugs with anxiolytic properties but with a much reduced side-effect burden (compared to benzodiazepines) is an attainable goal. However, its achievement necessitates the availability of in vivo preclinical assays capable of demonstrating differences as well as similarities between subtype-selective agents and non-selective benzodiazepines. In this study, we have compared three mouse strains (NMRI, C57BL/6J and DBA/2) in four models of anxiety-like behaviour (plus-maze, zero-maze, light-dark, and Vogel conflict). Furthermore, in each model, we have contrasted in detail the behavioural responses of each strain to the non-selective benzodiazepine chlordiazepoxide (CDP; 5-20 mg/kg), and the subtype-selective agents L-838,417 (GABA(A)-alpha(2/3/5); 3-30 mg/kg) and zolpidem (GABA(A)-alpha1; 0.3-3.0 mg/kg). The data show a complex mouse strainxmodelxpharmacological agent interaction. Most importantly, not all mouse strainxmodel test systems showed a positive response to CDP or predicted the response to L-838,417. This dissociation between CDP and L-838,417 opens up opportunities for preclinical test systems that differentiate subtype-selective and non-selective GABA(A) receptor agents, an attribute that might well be important in providing the necessary confidence for further drug development. Present findings suggest the need for a much greater focus on defining test systems appropriate for screening novel chemical entities, rather than self-selection of models or genotypes based on responses to known pharmacological agents. For example, if current data with L-838,417 are confirmed with compounds showing similar selectivity profiles, such agents may in future be best identified and characterised using test systems comprising NMRI mice in the zero-maze and/or C57 mice in the Vogel conflict and/or light-dark tests.

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.