Inverse agonism at the benzodiazepine site of α5 subunit-containing GABAA receptors is an attractive approach for the development of putative cognition-enhancing compounds, which are still far from clinical application. Several ligands with binding and/or functional selectivity for α5 GABAA receptors have been synthesized and tested in a few animal models. PWZ-029 is an α5 GABAA selective inverse agonist whose memory enhancing effects were demonstrated in the passive avoidance task in rats and in Pavlovian fear conditioning in mice. In the present study we investigated the effects of PWZ-029 administration in novel object recognition test and Morris water maze, in normal and scopolamine-treated rats. All the three doses of PWZ-029 (2, 5 and 10 mg/kg) improved object recognition after the 24-h delay period, as shown by significant differences between the exploration times of the novel and old object, and the respective discrimination indices. PWZ-029 (2 mg/kg) also successfully reversed the 0.3 mg/kg scopolamine-induced deficit in recognition memory after the 1-h delay. In the Morris water maze test, PWZ-029 (5, 10 and 15 mg/kg) did not significantly influence swim patterns, either during five acquisition days or during the treatment-free probe trial. PWZ-029 (2, 5 and 10 mg/kg) also proved to be ineffective in the reversal of the 1 mg/kg scopolamine-induced memory impairment in the water maze. The present mixed results encourage use of a variety of tests and experimental conditions in order to increase the predictability of preclinical testing of selective α5 GABAA inverse agonists.

Benzodiazepines negatively affect motor coordination and balance and produce myorelaxation. The aim of the present study was to examine the extent to which populations of GABAA receptors containing α1 and α5 subunit contribute to these motor-impairing effects in rats. We used the nonselective agonist diazepam and the α1-selective agonist zolpidem, as well as nonselective, α1- and α5-subunit-selective antagonists flumazenil, βCCt and XLi093, respectively. Ataxia and muscle relaxation were assessed by rotarod and grip strength tests performed 20 minutes after i.p. treatment. Diazepam (2 mg/kg) induced significant ataxia and muscle relaxation which were completely prevented by pretreatment with flumazenil (10 mg/kg) and βCCt (20 mg/kg). XLi093 antagonized the myorelaxant, but not ataxic actions of diazepam. All three doses of zolpidem (1, 2 and 5 mg/kg) produced ataxia, but only the highest dose (5mg/kg) significantly decreased the grip strength. These effects of zolpidem were reversed by ßCCt at doses of 5 and 10 mg/kg, respectively. The present study demonstrates that α1 GABAA receptors mediate ataxia and indirectly contribute to myorelaxation in rats, while α5 GABAA receptors contribute significantly, although not dominantly, to muscle relaxation but not ataxia.

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Despite significant advances in understanding the role of benzodiazepine (BZ)-sensitive populations of GABAA receptors, containing the α1, α2, α3 or α5 subunit, factual substrates of BZ-induced learning and memory deficits are not yet fully elucidated. It was shown that α1-subunit affinity-selective antagonist β-CCt almost completely abolished spatial learning deficits induced by diazepam (DZP) in the Morris water maze. We examined a novel, highly (105 fold) α1-subunit selective ligand - WYS8 (0.2, 1 and 10 mg/kg), on its own and in combination with the non-selective agonist DZP (2 mg/kg) or β-CCt (5 mg/kg) in the water maze in rats. The in vitro efficacy study revealed that WYS8 acts as α1-subtype selective weak partial positive modulator (40% potentiation at 100 nM). Measurement of concentrations of WYS8 and DZP in rat serum and brain tissues suggested that they did not substantially cross-influence the respective disposition. In the water maze, DZP impaired spatial learning (acquisition trials) and memory (probe trial). WYS8 caused no effect per se, did not affect the overall influence of DZP on the water-maze performance and was devoid of any activity in this task when combined with β-CCt. Nonetheless, an additional analysis of the latency to reach the platform and the total distance swam suggested that WYS8 addition attenuated the run-down of the spatial impairment induced by DZP at the end of acquisition trials. These results demonstrate a clear difference in the influence of an α1 subtype-selective antagonist and a partial agonist on the effects of DZP on the water-maze acquisition.

The abrupt discontinuation of prolonged benzodiazepine treatment elicits a withdrawal syndrome with increased anxiety as a major symptom. The neural mechanisms underlying benzodiazepine physical dependence are still insufficiently understood. Flumazenil, the non-selective antagonist of the benzodiazepine binding site of GABAA receptors was capable of preventing and reversing the increased anxiety during benzodiazepine withdrawal in animals and humans in some, but not all studies. On the other hand, a number of data suggest that GABAA receptors containing α1 subunits are critically involved in processes developing during prolonged use of benzodiazepines, such are tolerance to sedative effects, liability to physical dependence and addiction. Hence, we investigated in the elevated plus maze the level of anxiety 24 h following 21 days of diazepam treatment and the influence of flumazenil or a preferential α1-subunit selective antagonist βCCt on diazepam withdrawal syndrome in rats. Abrupt cessation of protracted once-daily intraperitoneal administration of 2 mg/kg diazepam induced a withdrawal syndrome, measured by increased anxiety-like behavior in the elevated plus maze 24 h after treatment cessation. Acute challenge with either flumazenil (10 mg/kg) or βCCt (1.25, 5 and 20 mg/kg) alleviated the diazepam withdrawal-induced anxiety. Moreover, both antagonists induced an anxiolytic-like response close, though not identical, to that seen with acute administration of diazepam. These findings imply that the mechanism by which antagonism at GABAA receptors may reverse the withdrawal-induced anxiety involves the α1 subunit and prompt further studies aimed at linking the changes in behavior with possible adaptive changes in subunit expression and function of GABAA receptors.