Midazolam impairs acquisition and retrieval, but not consolidation of reference memory in the Morris water maze.

Amnesia is one of the most discussed properties of the benzodiazepine class of drugs. The effects of benzodiazepines on human memory are usually anterograde, while changes in retrograde memory functions were seldom reported. Such inconsistent findings have prompted numerous animal studies investigating the influences of these positive modulators of inhibitory neurotransmission on different stages of memory. Among the benzodiazepines, memory effects of midazolam are of special interest due to its many and varied clinical applications. The present Morris water maze study in adult male Wistar rats was performed in three experiments in which midazolam was administered at doses of 0.5, 1 and 2 mg/kg intraperitoneally, before or immediately after each of five daily learning sessions, with two trials in a session, as well as before the probe test. Midazolam impaired acquisition and subsequent retention of spatial learning of the position of the hidden platform even at a pre-training dose of 0.5 mg/kg. This low dose was not associated with impairment of the procedural component of learning, manifested by increased time spent in the periphery of the pool. The lack of midazolam effect on consolidation has not been confounded by the observed below-chance performance of the control group since our additional experiment using diazepam also administered immediately after each of five learning sessions has revealed a similar pattern of results. Finally, midazolam administered before the probe test impaired retrieval of reference memory at all tested doses. Hence, induction of retrograde, besides anterograde amnesia should be kept in mind as a possibility when midazolam is used in clinical settings.

PWZ-029, an inverse agonist selective for α5 GABAA receptors, improves object recognition, but not water-maze memory in normal and scopolamine-treated rats.

Inverse agonism at the benzodiazepine site of α(5) subunit-containing GABA(A) 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 α(5) GABA(A) receptors have been synthesized and tested in a few animal models. PWZ-029 is an α(5) GABA(A) 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 1mg/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 α(5) GABA(A) inverse agonists.

Benzodiazepine-induced spatial learning deficits in rats are regulated by the degree of modulation of α1 GABA(A) receptors.

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 ß-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 ß-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 100nM). 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 ß-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.

ßCCT, an antagonist selective for α(1)GABA(A) receptors, reverses diazepam withdrawal-induced anxiety in rats.

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 GABA(A) 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 GABA(A) receptors containing α(1) 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 α(1)-subunit selective antagonist ßCCt 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 (10mg/kg) or ßCCt (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 GABA(A) receptors may reverse the withdrawal-induced anxiety involves the α(1) subunit and prompt further studies aimed at linking the changes in behavior with possible adaptive changes in subunit expression and function of GABA(A) receptors.

Tolerance liability of diazepam is dependent on the dose used for protracted treatment.

BACKGROUND: Behavioral effects of benzodiazepines following repeated exposure vary according to the intrinsic efficacy of the benzodiazepine studied, treatment schedule and the behavioral parameters evaluated. METHODS: We applied the behavioral paradigms of spontaneous locomotor activity, elevated plus maze and grip strength to investigate the sedative, anxiolytic and myorelaxant effect of acute challenge with 2 mg/kg diazepam administered after 14 days of protracted treatment with 0.5, 2 or 10 mg/kg of diazepam. In addition, we studied the effects of everyday handling and intraperitoneal (ip) administration on animal behavior. RESULTS: Tolerance to the sedative effect of 2 mg/kg diazepam ensued after 14 days of protracted treatment with 2 and 10 mg/kg of diazepam. In contrast, treatment with the lowest dose (0.5 mg/kg) of diazepam resulted in potentiation of the sedative effect of acute challenge with 2 mg/kg diazepam thus confounding the detection of the anxiolytic effect of diazepam. Asensitization-like response to the anxiolytic action of 2 mg/kg diazepam was seen after protracted treatment with the intermediate dose (2 mg/kg); however, anxiolytic effect was absent after protracted administration of the highest dose. Partial tolerance to the muscle relaxant effect of 2 mg/kg diazepam ensued after protracted treatment with diazepam regardless of the dose. Daily handling or ip administration did not alter the behavioral response to acute challenge with 2 mg/kg diazepam in all the three behavioral paradigms studied. CONCLUSION: The presented results showed that behavioral effects of acute challenge with diazepam were differently affected by the dose administered during protracted treatment.

The role of α1 and α5 subunit-containing GABAA receptors in motor impairment induced by benzodiazepines in rats.

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 γ-aminobutyric acid A (GABAA) receptors containing α1 and α5 subunits 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-subunit and α5-subunit-selective antagonists flumazenil, ßCCt, and XLi093, respectively. Ataxia and muscle relaxation were assessed by rotarod and grip strength tests performed 20 min after intraperitoneal treatment. Diazepam (2 mg/kg) induced significant ataxia and muscle relaxation, which were completely prevented by pretreatment with flumazenil (10 mg/kg) and ßCCt (20 mg/kg). XLi093 antagonized the myorelaxant, but not the ataxic actions of diazepam. All three doses of zolpidem (1, 2, and 5 mg/kg) produced ataxia, but only the highest dose (5 mg/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, whereas α5 GABAA receptors contribute significantly, although not dominantly, to muscle relaxation but not ataxia.

Behavioural characterization of four endemic Stachys taxa.

We performed a basic behavioral characterization of methanol extracts of four Balkan endemic Stachys taxa: S. anisochila (SA), S. beckeana (SB), S. plumosa (SP) and S. alpina subsp. dinarica (SAD). The behavioral activity of extracts dosed intraperitoneally in the range 100-400 mg/kg was examined in adult male Wistar rats, in the elevated plus maze, spontaneous locomotor activity, and grip strength tests, mainly predictive of anxiolytic, sedative and myorelaxant actions, respectively. All investigated Stachys extracts lacked anxiolytic or myorelaxant activities, while SB at 400 mg/kg exerted an anxiogenic-like effect. The study with the selective antagonist beta-CCt showed that the sedative effect of SAD was only partially mediated by GABAA receptors containing the alpha1-subunit. While discernible, the behavioral effects of SA and SP were not distinct. In all extracts, chlorogenic acid and verbascoside were identified. In SA, SB, and SAD the flavonoid fraction was constituted of isoscutellarein and hypolaetine glycosides, while in SP chrysoeriol and apigenin glycosides were present. The results reveal the psychotropic potential of four endemic Stachys taxa, of which SAD appeared most promising as a natural sedative.