Sedative and anticonvulsant effects of zolpidem in adult and aged mice

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1 J Neural Transm (2008) 115: DOI /s BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - ORIGINAL ARTICLE Sedative and anticonvulsant effects of zolpidem in adult and aged mice Danka Peričić Æ Josipa Vlainić Æ Dubravka Švob Štrac Received: 3 September 2007 / Accepted: 7 January 2008 / Published online: 24 January 2008 Ó Springer-Verlag 2008 Abstract To evaluate the possible age-related differences in the behavioral effects of zolpidem, a widely used hypnotic, we compared the effects of zolpidem on the locomotor activity and on the seizure threshold for pentylenetetrazole (PTZ) and picrotoxin (given by i.v. infusion) between adult (3 months) and aged (13 months) mice. Zolpidem (10 mg/kg) produced similar enhancements of the seizure threshold in adult and aged mice. The drug was more potent against PTZ- than against picrotoxin-induced seizures. Diazepam (1 mg/kg), which was taken for comparison, had a weaker effect on picrotoxin-induced tonic seizures in aged than in adult mice. Sedative effect of zolpidem (10 mg/kg), as assessed by its effect on the locomotor activity, was very strong in both groups of mice. The results suggest that sedative and antiseizure effects of zolpidem are not changed in aged mice. Keywords Zolpidem Aging Seizure threshold Picrotoxin Pentylenetetrazole Locomotor activity Introduction Zolpidem is a widely used hypnotic drug known to produce its effects via benzodiazepine binding sites on the GABA A receptors (Depoortere et al. 1986; Sanger et al. 1994). Although the imidazopyridine zolpidem and benzodiazepines use the same binding sites, most (Depoortere et al. 1986; Lloyd and Zivkovic 1988; Perrault et al. 1990; D. Peričić (&) J. Vlainić D. Š. Štrac Laboratory for Molecular Neuropharmacology, Division of Molecular Medicine, Rud er Bošković Institute, P.O. Box 180, Zagreb, Croatia pericic@irb.hr Sanger et al. 1996; Sanger 2004) but not all studies (Fahey et al. 2006), have demonstrated that the pharmacological profile of this drug is different from that of classical benzodiazepines. Unlike benzodiazepines, zolpidem has very pronounced sedative and mild anxiolytic and anticonvulsant properties (Depoortere et al. 1986; Perrault et al. 1990; Sanger 2004). This difference in the behavioral properties of drugs was explained by the fact that zolpidem binds with highest affinity to sites located on a 1 -containing GABA A receptors (Sanger et al. 1994; Crestani et al. 2000). It has been demonstrated that the sedative, amnesic and partially the anticonvulsant, but not the anxiolytic effects of benzodiazepines are mediated by a 1 -containing GABA A receptors (Rudolph et al. 1999; McKernan et al. 2000; Kralic et al. 2002). Moreover, Cui et al. (2007) have suggested that calcium channel modulation may be involved in the anxiolytic-like properties of zolpidem. Structural determinants for high-affinity zolpidem binding to GABA A receptors have recently been determined (Sancar et al. 2007; Wulff et al. 2007). Older people suffer more often from insomnia than young people. They also take anxiolytic and anticonvulsant drugs more often. However, most preclinical studies are performed on adult or young animals, while the studies on aged and old animals are relatively scarce, and their results are often contradictory (Bickford and Breiderick 2000; Wikinski et al. 2001). The aim of this study was to compare the sedative and anticonvulsant effects of zolpidem between adult (3 months) and aged (13 months) mice. As in the study of Fahey et al. (2006), sedative and anticonvulsant effects of zolpidem against pentylenetetrazole (PTZ)-induced seizures were studied in the same animal, while the effects of zolpidem on the seizure threshold for picrotoxin were studied in a separate group of animals. In the latter case,

2 796 J Neural Transm (2008) 115: the classic benzodiazepine, diazepam, was taken for comparison. According to previous findings (Wikinski et al. 2001), its effects on aged animals might be different. Sedative activity was assessed by measuring the decreases in locomotor activity. Seizures were produced by an i.v. infusion of picrotoxin or PTZ, both the compounds are supposed to be non-competitive GABA A receptor antagonists. We measured drug-induced changes in the threshold for picrotoxin-induced clonic and tonic, and PTZ-induced myoclonic, clonic and tonic seizures. Materials and methods Animals Adult (3 months) and aged (13 months) male CBA mice, bred in our institute, were used. They were housed at a constant temperature (22 C) and under a light cycle of 12-h light/12-h darkness (lights on at 7.00 a.m.). Food and water were freely available. The animals were deprived of food only the night before the experiment. As in most other studies, before the experiment, mice were not habituated either to intraperitoneal (i.p.) or intravenous (i.v.) injections. Each mouse was used only once. To better evaluate the existence of possible age-related differences in response to drugs, adult and aged mice were tested in the same experiment. Hence, the experiments shown in Figs. 1, 2 and 3 consisted of four experimental groups. To evaluate the effect of drug on the seizure threshold, the data obtained from drug treated (adult and aged) mice are being compared to the data obtained from vehicle treated (adult and aged) mice. To assess the effect of age on the same parameter, the data obtained from aged (vehicle and drug treated) mice are being compared to the data obtained from adult (vehicle and drug treated) mice. All animal care and experimental procedures were carried out in accordance with the principles of laboratory animal care (NIH publication No , revised 1985), and the Croatian law on animal welfare. Drugs Zolpidem hemitartrate (kindly provided by Pliva, Zagreb, Croatia), diazepam (Apaurin ampullae, Krka, Novo mesto, Slovenia), picrotoxin and pentylenetetrazole (PTZ, Sigma, St Louis, MO) were used. Zolpidem was dissolved in distilled water, diazepam was diluted with saline, and picrotoxin and PTZ were dissolved in saline. Picrotoxin and PTZ were given by constant i.v. infusion into a tail vein, while other drugs were administered i.p. in the volume 1 ml per 100 g body weight. Control animals received Fig. 1 Effect of zolpidem on the threshold for clonic (a) and tonic (b) seizures induced in adult (3 months) and aged (13 months) CBA mice by i.v. infusion of picrotoxin. Zolpidem (10 mg/kg i.p.) was administered 30 min before the infusion of picrotoxin was started. Control animals received i.p. injections of vehicle. Bars represent mean ± SD from 6 to 8 animals per group. * P \ 0.01, ** P \ versus the corresponding vehicle treated group. w P \ 0.05 or 0.01, ww P \ versus identically treated group of adult mice (ANOVA followed by Newman Keuls test) i.p. injection of vehicles. The timing of zolpidem administration was chosen based on previous studies (e.g. Depoortere et al. 1986; Fahey et al. 2006). Seizure threshold determination The threshold for picrotoxin-induced clonic and tonic and PTZ-induced myoclonic, clonic and tonic seizures was determined by inserting a butterfly infusion needle (length 20 mm, gauge 27) into the tail vein and infusion of picrotoxin (0.75 mg/ml) and PTZ (4 mg/ml), respectively, at a constant rate of 1.1 ml/min. The animal was observed throughout the infusion, and the time between the start of infusion and the onset of seizures (latency) was recorded, mainly as previously described (Kosobud and Crabbe 1990; Peričić et al. 2001, 2006). Clonic seizures were characterized by whole-body clonus, including running

3 J Neural Transm (2008) 115: Fig. 2 Effect of diazepam on the threshold for clonic (a) and tonic (b) seizures induced in adult (3 months) and aged (13 months) CBA mice by i.v. infusion of picrotoxin. Diazepam (1 mg/kg i.p.) was administered 30 min before the infusion of picrotoxin was started. Control animals received i.p. injections of vehicle. Bars represent means ± SD from 7 to 10 animals per group. * P \ 0.05 or P \ 0.01, ** P \ versus the corresponding vehicle treated group. ww P \ versus identically treated group of adult mice (ANOVA followed by Newman Keuls test) and explosive jumps, while tonic seizures were characterized by extreme rigidity, with forelimbs and hindlimbs extended caudally. For PTZ, the first convulsive sign was myoclonus (a sudden involuntary muscle jerk, usually accompanied by a head twitch). For each animal, the threshold dose of convulsant (mg/kg of body weight) required to elicit myoclonic, clonic and tonic seizures was calculated from the time of infusion (latency), infusion rate, concentration of convulsant and body weight. All experiments were carried out between 9:00 and 13:00 h. Statistical analysis Statistical analysis of the results was by two-way analysis of variance (ANOVA) because the effects of two different factors (drug 9 age or drug 9 experimental procedure) were studied in the same experiment. Post-hoc Fig. 3 Effect of zolpidem on the threshold for myoclonic (a), clonic (b) and tonic (c) seizures induced in adult (3 months) and aged (13 months) CBA mice by i.v. infusion of PTZ. Zolpidem (10 mg/kg i.p.) was administered 30 min before the infusion of PTZ was started. To test the locomotor activity, the same animals spent 10 min (15 25 min following zolpidem administration) in Ugo-Basile Activity Cage. Bars represent mean ± SD from 5 to 8 animals per group. * P \ versus the corresponding vehicle treated group (ANOVA followed by Newman Keuls test) analysis was done by the Newman Keuls multiple comparison test. The analyses were performed using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, CA, USA. P-values of \0.05 were considered significant.

4 798 J Neural Transm (2008) 115: Results Effect of zolpidem on the seizure threshold for picrotoxin in adult and aged mice The ability of zolpidem to affect the threshold for picrotoxin-induced seizures was tested in adult (3 months) and aged (13 months) mice. As shown by the interaction plots (Fig. 1), zolpidem (10 mg/kg), given 30 min before the beginning of picrotoxin infusion, increased in both adult and aged mice the threshold for picrotoxin-induced clonic (Fig. 1a) and tonic (Fig. 1b) seizures. The increases were of similar intensity for both types of seizures and in both groups of mice. They ranged from 41 to 53%. The observed distance between the lines (adult, aged), which in Fig. 1a, b denote the change of the seizure threshold from its control values to values obtained after treatment with zolpidem, suggested that the age of the animals also has an effect on the seizure threshold for picrotoxin. As indicated by two-way ANOVA with factors drug (zolpidem vs. vehicle) and age (aged vs. adult mice), the effects of zolpidem on threshold doses of picrotoxin producing clonic [F(1,25) = 53.3] and tonic [F(1, 25) = 45.7] seizures were highly significant (P \ ). There was also a significant effect of age on the clonic [F(1, 25) = 32.2; P \ ] and tonic seizure threshold [F(1, 25) = 8.6; P \ 0.01]. There was no significant interaction between the two factors (effect of drug, effect of age), suggesting that in aged animals the anticonvulsant effect of zolpidem was as potent as in adult animals. Effect of diazepam on the seizure threshold for picrotoxin in adult and aged mice To test whether the anticonvulsant effect of a classic benzodiazepine on the seizure threshold for picrotoxin will also be unaffected by aging, we treated adult and aged mice with diazepam (1 mg/kg). As shown in Fig. 2a, the threshold for picrotoxin-induced clonic seizures was again lower in aged than in adult mice, and diazepam produced similar anticonvulsant effects on both groups of mice. The effect of diazepam against picrotoxin-induced tonic seizures (Fig. 2b) was weaker in aged than in adult mice. Two-way ANOVA confirmed a highly significant (P \ ) effect of diazepam on the threshold for picrotoxin-induced clonic [F(1, 32) = 47.1] and tonic seizures [F(1, 32) = 39.2]. There was also a significant effect of age on the clonic [F(1, 32) = 84.3; P \ ] and tonic seizure threshold [F(1, 32) = 11.3; P \ 0.002], and a significant (P \ 0.04) drug 9 age interaction in tonic [F(1, 32) = 4.45], but not in clonic seizures [F(1, 32) = 2.68]. This result suggested that the anticonvulsant effect of diazepam against picrotoxin-induced tonic seizures is agedependent. Effect of zolpidem on the seizure threshold for PTZ in adult and aged mice The anticonvulsant properties of zolpidem (10 mg/kg) in adult and aged mice were also tested against convulsions produced by PTZ. This testing was done 5 min after measurement of locomotor activity for 10 min in Ugo- Basile Activity Cage and 30 min after zolpidem administration. The effect of zolpidem on the threshold for PTZinduced myoclonus, clonic and tonic seizures in two groups of mice is presented in Fig. 3a c. Zolpidem produced a marked effect on all types of PTZ-induced seizures. The two-way ANOVA confirmed that the drug had a highly significant effect (P \ ) on threshold doses of PTZ required to induce myoclonus [F(1, 24) = 59.90], clonic [F(1, 24) = ] and tonic seizures [F(1, 21) = ]. The effect of aging on PTZ-induced seizures was smaller than on picrotoxin-induced seizures. Nevertheless, there was a significant effect of age on the tonic seizure threshold [F(1, 21) = 8.22; P \ 0.009], and close to the significant effect of age [F(1, 24) = 4.08; P \ 0.055] on myoclonus. There was no significant drug 9 age interaction for neither of the seizure types (suggested also by the lines in the interaction plots that are parallel or close to parallel), although the interaction for clonic seizure threshold was close to significance [F(1, 24) = 4.03; P \ 0.056]. The lack of significance in drug 9 age interaction suggested that the anticonvulsant effect of zolpidem in mice is the same regardless of age. Effect of zolpidem on the seizure threshold for PTZ in mice disturbed by preceding measurement of locomotor activity To determine whether a lesser effect of zolpidem on the picrotoxin than on the PTZ seizure threshold might be due at least in part to the procedure that in the latter case preceded the testing (the measurement of locomotor activity in an unfamiliar environment), we tested in the same experiment the effect of zolpidem on the PTZ seizure threshold in mice previously undisturbed and those previously disturbed by placement in the activity cage (Fig. 4). The two-way ANOVA showed a highly significant effect of drug (P \ ) on the threshold for PTZ-induced myoclonus [F(1, 28) = 72.15], clonic [F(1, 28) = ] and tonic seizures [F(1, 28) = ]. The experimental procedure had a significant effect on PTZ-induced myoclonus [F(1, 28) = 5.37; P \ 0.03] and tonic [F(1, 28) =

5 J Neural Transm (2008) 115: ,76; P \ 0.02], but not clonic seizure threshold [F(1, 28) = 0.19]. There was a significant drug 9 procedure interaction for myoclonus [F(1, 28) = 5.11; P \ 0.03], but not for clonic [F(1, 28) = 0.21] and tonic seizure threshold [F(1, 28) = 3.61]. Post hoc tests have shown that zolpidem had a greater effect (P \ 0.01) on the threshold for PTZinduced myoclonic (Fig. 4a) and tonic (Fig. 4c) convulsions in mice, previously subjected to measurement of locomotor activity. However, these differences were not very pronounced and they can by no means explain the presence of considerable differences in the anticonvulsant activity of zolpidem against the two convulsants, picrotoxin and PTZ. Effect of zolpidem on locomotor activity in adult and aged mice As shown in Table 1, zolpidem (10 mg/kg i.p.) blocked almost completely the locomotor activity of adult and aged mice. The two-way ANOVA [F(1, 28) = ] confirmed the high significance (P \ ) of this result. The age [F(1, 28) = 0.51] and the interaction drug 9 age [F(1, 28) = 0.71] did not have a significant effect on the locomotor activity. Discussion The results of this study have demonstrated a lack of agerelated differences in the sedative and anticonvulsant effects of zolpidem against PTZ- and picrotoxin-induced seizures. They also show that zolpidem was more potent against PTZ- than against picrotoxin-induced seizures. To our knowledge, neither the anticonvulsant activity of zolpidem against the picrotoxin-induced seizures, nor the behavioral effects of zolpidem in aged animals, have been studied. We found that the effect of zolpidem (10 mg/kg) on the threshold for both clonic and tonic seizures induced by picrotoxin was of similar intensity in adult and aged mice. The classic benzodiazepine, diazepam (1 mg/kg), was also approximately equally potent against picrotoxininduced clonic seizures in both groups of mice. However, in aged mice, the same drug was less active against picrotoxin-induced tonic seizures (Fig. 2b). This difference in the effects of diazepam against two types of seizures was confirmed by a positive interaction between the effects of diazepam and age, obtained for tonic (but not clonic) seizures in the two-way ANOVA. Wikinski et al. (2001) have demonstrated even the lack of anxiolytic effect of diazepam in 24 months old rats, and suggested that this agedependent change could partially be explained by pharmacodynamic changes in cortical GABA A receptors. Fig. 4 Effect of zolpidem on the threshold for myoclonic (a), clonic (b) and tonic (c) seizures induced by i.v. infusion of PTZ in adult mice disturbed by preceding measurement of locomotor activity. Zolpidem (10 mg/kg i.p.) was administered 30 min before the infusion of PTZ was started. Before testing of convulsive activity, one group of mice spent 10 min (15 25 min following zolpidem administration and 5 min before starting the infusion of convulsant) in Ugo-Basile Activity Cage. Another group of mice was left undisturbed in the cage until the beginning of infusion. Bars represent mean ± SD from 8 animals per group. * P \ versus the corresponding vehicle treated group. w P \ 0.01 versus zolpidem treated group of mice previously disturbed by measurement of locomotor activity (ANOVA followed by Newman Keuls test) Additionally, as shown in Fig. 3, zolpidem (10 mg/kg) had a strong anticonvulsant effect against PTZ-induced myoclonic, clonic and tonic seizures in both adult and aged

6 800 J Neural Transm (2008) 115: Table 1 Effect of zolpidem on locomotor activity (counts/10 min) in adult and aged mice Adult Aged Vehicle ± 30.6 (7) ± 44.1 (8) Zolpidem (10 mg/kg) 5.3 ± 1.7* (9) 3.4 ± 1.6* (8) Zolpidem was administered i.p. 15 min before placing the animals in the apparatus (Hugo-Basile). The horizontal locomotor activity was measured for 10 min * P \ versus the corresponding vehicle treated group (ANOVA and Newman Keuls test) mice. The effect against the first type of seizures was weaker than against the two other seizure types. Previous studies failed to show anticonvulsant effectiveness of zolpidem against PTZ-induced myoclonic seizures (Crestani et al. 2000). Hence, the results of this study suggest that the seizure threshold determination following i.v. infusion of PTZ is a more sensitive method than the use of a lethal dose of PTZ. The presence of an insignificant interaction between the effect of zolpidem and age suggested that the anticonvulsant effect of zolpidem against PTZ-induced convulsions is independent of age. The observed dissimilarities in the intensity of anticonvulsant response between the seizure types are in accordance with the suggestion that the convulsive responses, which appear following the administration of convulsants represent qualitatively distinct seizure components mediated by separable and independent anatomical circuits located in the forebrain and hindbrain (Gale 1988). The lack of age-related differences in the behavioral effects of zolpidem was also seen in the experiment used to assess the sedative activity of drug. The locomotor activity of vehicle treated aged mice was not different from that of identically treated adult mice, and zolpidem (10 mg/kg) inhibited this activity in both groups of mice almost completely. Ruano et al. (1993) reported that aging did not modify the parameters of [ 3 H]-zolpidem binding in cortical membranes from Wistar rats. In addition, it did not modify the alpha 1 protein expression and the number and affinity of [ 3 H]zolpidem binding sites in the rat cerebral cortex and cerebellum of Sprague-Dawley rats (Gutiérrez et al. 1997). However, while some data suggest that in clinical studies zolpidem was effective and safe in people aged over 70 (Cotroneo et al. 2007), other reports indicate that pharmacokinetic properties of zolpidem can be altered in the elderly (Olubodun et al. 2003), suggesting the need of dosage reductions in elderly patients. Because of the limited number of aged animals we tested only one dose of drug, which under our experimental conditions produced a very strong effect. However, the likelihood of existence of more subtle age-related differences in the sedative and anticonvulsant effects of zolpidem, which might possibly be discovered using lower doses of drug, cannot be excluded. Early studies have reported that sedation with zolpidem occurs at doses 10 and 20 times lower than those inducing anticonvulsant effect (Depoortere et al. 1986). The same study demonstrated that zolpidem shows a weak anticonvulsant activity against PTZ, while its effect against picrotoxin has not been studied. In the present study, in adult mice zolpidem (10 mg/kg) enhanced by 53 and 50% and diazepam (1 mg/kg) by 32 and 31%, respectively, the threshold for picrotoxin-induced clonic and tonic seizures. However, in our study zolpidem was much more active against PTZ- than against picrotoxin-induced convulsions. The enhancements of PTZ seizure threshold were previously obtained following exposure to well known stressors e.g. swim stress (Soubrie et al. 1980; Peričić et al. 2001) and restraint (Homayoun and Dehpour 2004). Although measurement of locomotor activity enhanced the threshold for PTZ-induced myoclonic and tonic seizures suggesting that the procedure was stressful for the animals, this increase could not justify a much greater anticonvulsant effect of zolpidem against PTZ than against picrotoxininduced seizures. In adult previously undisturbed mice, zolpidem (10 mg/kg) elevated the threshold for PTZinduced clonic and tonic seizures by 284 and 393%, respectively. Fahey et al. (2006) described similar increases of the PTZ seizure threshold following 10 mg/kg of zolpidem. As shown in Results (Figs. 1, 2, 3), decreases in the seizure threshold, observed in the aged vehicle treated mice, were more obvious after picrotoxin than after PTZ administration. Although PTZ is a widely used convulsant in animal seizure models, the mechanism by which it elicits its action is not entirely clear. PTZ is thought to act at the picrotoxin site of the GABA A receptor (Ramamjaneyulu and Ticku 1984; Squires et al. 1984), and it is generally accepted that both the compounds are noncompetitive GABA A receptor antagonists. It has also been proposed that PTZ-induced convulsions may be associated with an action at benzodiazepine 2 receptor subtypes (Griebel et al. 1999), i.e. GABA A receptors containing a 2, a 3 or a 5 subunits. A different sensitivity of mice to picrotoxin and PTZ might presumably be explained by results of Huang et al. (2001), who reported that the two convulsants interact with overlapping but distinct domains of the GABA A receptor. It has been reported that zolpidem is about seven times less potent against PTZ than against isoniazid (Depoortere et al. 1986; Perrault et al. 1990). Sanger et al. (1996) demonstrated that zolpidem (10 mg/kg) increased approximately threefold the latency to clonic seizures induced by isoniazid. In our study, the same dose of zolpidem increased approximately threefold the latency to PTZ-

7 J Neural Transm (2008) 115: induced clonic seizures, and consequently to the calculated seizure threshold for PTZ-induced clonic seizures. While in this study, as well as in the study of Davies et al. (1994), zolpidem (10 mg/kg) inhibited almost completely the locomotor activity, Sanger et al. (1996) could not obtain an analogue effect with doses of zolpidem lower than 100 mg/kg. Elliot and White (2001) compared acute effects of zolpidem, diazepam and lorazepam in rats using radiotelemetry and found that maximal decrements in locomotor activity produced by zolpidem (2.5, 5 and 10 mg/kg) were comparable to those produced by twofold greater doses of diazepam and 2.5-fold greater doses of lorazepam. Moreover, Fahey et al. (2006), who studied in the same animal the effect of zolpidem on locomotor activity and PTZ-induced seizure threshold, obtained an increase in the anticonvulsant activity even with a dose (2 mg/kg) that failed to diminish locomotor activity. This suggests that in addition to sedative, zolpidem also has a respectable anticonvulsant activity. Differences in the effects of two structurally different ligands of benzodiazepine binding sites, diazepam and zolpidem, should not be surprising. Early binding experiments with this drug have demonstrated that zolpidem displays a high affinity to GABA A receptors containing a 1 subunit (Arbilla et al. 1986). Unlike diazepam, which has a similar affinity for a 1 3 and a 5 containing GABA A receptors, zolpidem shows a low affinity to receptors containing the a 5 subunit (Pritchett and Seeburg 1990; Graham et al. 1996). Already in 1988, Perrault et al. (1988) suggested that hypnoselective properties of zolpidem might be linked to its selectivity for the omega (BZ-1) site of the GABA A receptor. This suggestion was confirmed by Crestani et al. (2000), who demonstrated that sedative action and activity of zolpidem against PTZ-induced tonic convulsions are mediated by a 1 -containing GABA A receptors. A recent study demonstrated that binding of zolpidem requires a phenylalanine residue (Phe77) in the c 2 subunit (Wulff et al. 2007). In conclusion, our results demonstrate that sedative and anticonvulsant effects of zolpidem are not changed in aged mice. In addition, we have demonstrated the pronounced potency of zolpidem against PTZ-induced seizures, and for the first time the anticonvulsant effect of zolpidem against picrotoxin-induced convulsions. The results suggest that the anticonvulsant properties of zolpidem should not be neglected. Acknowledgments This study was supported by the Croatian Ministry of Science, Education and Sports (Project: Stress, GABA A receptors and mechanisms of action of neuropsychoactive drugs). 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