Antiaggressive Effects of Zolpidem and Zopiclone in Agonistic Encounters Between Male Mice

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1 AGGRESSIVE BEHAVIOR Volume 28, pages (2002) Antiaggressive Effects of Zolpidem and Zopiclone in Agonistic Encounters Between Male Mice Mercedes Martín-López, José Francisco Navarro n Department of Psychobiology, Faculty of Psychology, University of Málaga, Spain : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : The effects of benzodiazepines on various types of aggression have been extensively studied. These substances produce their pharmacological effects by allosterically modulating the action of GABA via specific recognition sites on the GABA A receptor called omega 1 and omega 2. The antiaggressive profile of non-benzodiazepine compounds that also act at omega sites, such as zopiclone (a non-selective omega 1 and 2 ligand) and zolpidem (a selective omega 1 ligand) has been scarcely explored. In this study, we examined the action of zolpidem ( mg/kg, intraperitoneally) and zopiclone (1.5-6 mg/ kg), administered acutely or subchronically for 10 days, on agonistic behavior elicited by isolation in male mice. Individually housed mice were exposed to anosmic standard opponents 30 min after drug administration, and the encounters were videotaped and evaluated using an ethologically based analysis. Acute treatment with zopiclone produced a marked antiaggressive effect, reducing offensive behaviors (threat and attack) at all doses used (1.5, 3, and 6 mg/kg) without affecting immobility. Likewise, the intermediate dose of zolpidem (1.5 mg/kg) significantly decreased aggression in a specific manner, without altering immobility, whereas the highest dose (3 mg/kg) provoked a reduction of aggression accompanied by a weak (but significant) increase of immobility. With repeated treatment, no tolerance to the antiaggressive effects of zopiclone and zolpidem was developed. It is concluded that omega sites at the GABA A receptor could be involved in the control of aggression. Aggr. Behav. 28: , r 2002 Wiley-Liss, Inc. : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : Keywords: aggression; zolpidem; zopiclone; GABA; subchronic treatment; mice INTRODUCTION Aggressive behavior of animals is controlled or modulated by a variety of different neurotransmitter systems in the brain, such as serotonine [Bell and Hobson, 1994; Navarro and Maldonado, 1999; Sa nchez et al., 1993], dopamine [Manzaneque and Navarro, 1999; Navarro and Manzaneque, 1997; Navarro et al., 2000], opiates [Espert et al., 1993; Navarro and Da vila, 1997], or nitric oxide [Nelson et al., 1995; Navarro et al., 1997]. However, it is now recognized that aggression may be also influenced by the GABAergic system. Thus, many substances that act on the GABA A /BDZ/Cl receptor complex provoke marked effects on aggressive behavior. Benzodiazepines produce their pharmacological effects by n Correspondence to: José Francisco Navarro, Department of Psychobiology, Faculty of Psychology, University of Málaga, Campus de Teatinios, Málaga, Spain. navahuma@uma.es Received 13 May 2001; amended version accepted 26 June 2001 r 2002 Wiley-Liss, Inc. DOI /ab.80013

2 Zolpidem, Zopiclone, and Aggression in Mice 417 allosterically modulating the action of GABA via specific recognition sites on the GABA A receptor called omega 1 and omega 2 [Langer and Arbilla, 1988]. In animal studies, benzodiazepines such as diazepam [Martı n-lo pez and Navarro, 1997], clobazam [Martín- Lo pez and Navarro, 1996], midazolam [Martı n-lo pez and Navarro, 1999], and bentazepam [Martı n-lo pez and Navarro, 1998] have been demonstrated to possess antiaggressive properties using different animal models of aggression. This antiaggressive activity seems to be reverted by flumazenil administration [Miczek et al., 1994; Olivier et al., 1991]. Overall, these data underlie a significant role for the GABA A receptor in modulating of aggressive behavior. During the past few years, several compounds chemically unrelated to benzodiazepines that also act at omega sites associated with GABA A receptor have been developed, such as zopiclone and zolpidem. Zopiclone is a new class of hypnotic agent belonging to the cyclopyrrolone family that shows pharmacological properties similar to those of benzodiazepines (anxiolytic, sedative/hypnotic, anticonvulsant, anticonflict). This compound interacts with high affinity and efficacy at both omega 1 and omega 2 receptors, differing from hypnotic benzodiazepines by its lower miorelaxant activity and its propensity to induce physical dependence [Goa and Heel, 1986; Piot et al., 1990]. In concordance with the behavioral profile of benzodiazepines, zopiclone exhibits an antiaggressive action in rats [Ueki, 1987] and mice [Julou et al., 1983, Martı n-lo pez et al., 1994]. On the other hand, zolpidem is an imidazapyridine with a selective affinity only for the omega 1 receptor subtype, which corresponds with GABA A receptors containing a1b2g2 subunits [McKernan and Whiting, 1996]. In contrast to benzodiazepines, which produce sedative effects with equal or lower potency than anticonvulsant and miorelaxant effects, zolpidem is strongly sedative, showing lower anxiolytic, miorelaxant, and anticonvulsant activity [Holm and Goa, 2000]. Likewise, zolpidem develops less tolerance to their sedative and anticonvulsant effects after repeated administration in rodents [Sanger and Depoortere, 1998]. In this sense, it has been reported that increased convulsant sensitivity occurs during spontaneous and flumazenilprecipitated withdrawal after repeated administration of benzodiazepines but not zolpidem, suggesting that this compound may not produce benzodiazepine-like physical dependence [Perrault et al., 1992]. Although the antiaggressive profile of zopiclone (a non-selective omega 1 and 2 ligand) has been explored [Julou et al., 1983; Martı n-lo pez et al., 1994; Ueki, 1987], there is no evidence with respect to the antiaggressive properties of zolpidem (a selective omega 1 ligand). Therefore, this study was designed to assess the acute and subchronic effects of zopiclone (experiment 1) and zolpidem (experiment 2) on agonistic encounters in isolated male mice using an ethopharmacological approach. The term agonistic behavior encompasses threats and aggressive acts as well as defensive, submissive, and flight behaviors. The ethological analyses of these social encounters seems to be an appropriate technique to distinguish between specific and non-specific drug-induced changes. MATERIALS AND METHODS Animals A total of 336 albino male mice of the OF.1 strain (provided by CRIFFA, Barcelona, Spain) weighing 25 to 30 g were used. Animals were housed under standardized lighting conditions (white lights on: 20:00-8:00) at a constant temperature (211C), and food and tap

3 418 Martín-López and Navarro water were available ad libitum, except during behavioral trials. On arrival in the laboratory, the subjects were allocated to two different categories. Half were housed individually in transparent plastic cages ( cm) as experimental animals. The remainder were housed in groups of five to be used as standard opponents and were rendered temporally anosmic by intranasal lavage with 4% zinc sulfate solution (Sigma Laboratories) on both 1 and 3 days before testing. Fighting in mice, as in most rodents, is closely related to olfaction. We used this type of opponent because it elicits attack but never initiates such behavior [Brain et al., 1981]. These animals very rarely direct spontaneous attacks toward the test animals, and, consequently, fighting is always unidirectional, being easily quantified. All the experimental animals underwent an isolation period of 30 days before the behavioral test (isolation-induced aggression model). Social isolation is an effective form of increasing the level of aggressiveness in different species of animals. This phenomenon is particularly well demonstrated in laboratory mice [Navarro, 1997; Valzelli, 1969]. Experimental Design Seven groups of mice were used in each experiment. Individually housed animals were allocated randomly to one control group receiving vehicle and six experimental groups (n ¼ 12 each) receiving acute or subchronic zopiclone (experiment 1) or zolpidem (experiment 2) injections. Drug administration consisted of (1) acute treatment: each animal received vehicle for 9 consecutive days and zopiclone or zolpidem on day 10; (2) subchronic treatment: each animal received a daily injection of zopiclone or zolpidem for 10 consecutive days, and (3) vehicle: each animal received a daily injection of vehicle for 10 consecutive days (control group). Drugs Zopiclone and zolpidem (Sigma Laboratories) were diluted in physiological saline to provide appropriate doses for injections. Zopiclone was administered either acutely or subchronically (for 10 days) in three doses: 1.5, 3, and 6 mg/kg. Zolpidem was also administered either acute or subchronically (for 10 days) in three doses: 0.75, 1.5, and 3 mg/ kg. The control groups received physiological saline. Drugs and vehicles were injected intraperitoneally in a volume of 10 ml/kg. The doses used in both experiments were chosen on the basis of a pilot study carried out previously in our laboratory. Procedure and Behavioral Analysis Thirty minutes after the last injection, an isolated animal and a standard opponent (marked with fur dye for identification) were confronted in a neutral area for 10 min. This neutral cage consisted of an all-glass area, measuring cm, with a fresh sawdust substrate. Before the encounter, the animals were allowed 1 min of adaptation to the neutral cage, remaining separated by means of a plastic barrier throughout this time. The social encounters were videotaped using a Sony-V8 camera. All tests were conducted under white light between the second and seventh hours of the dark phase of the artificial cycle of the animals. After each encounter, the neutral cage was washed out and the sawdust bedding was replaced.

4 Zolpidem, Zopiclone, and Aggression in Mice 419 The tapes were analyzed using a microprocessor and a custom-developed program [Brain et al., 1989], which facilitated estimation of time allocated to 10 broad behavioral categories. The names of the categories and their constituent elements are as follows: 1. Body care (abbreviated groom, self-groom, wash, shake, scratch). 2. Digging (dig, kick dig, push dig). 3. Non-social exploration (explore, rear, supported rear, scan). 4. Exploration from a distance (approach, attend, circle, head orient, stretched attention). 5. Social investigation (crawl over, crawl under, follow, groom, head groom, investigate, nose sniff, sniff, push past, walk around). 6. Threat (aggressive groom, sideways offensive, upright offensive, tail rattle). 7. Attack (charge, lunge, attack, chase). 8. Avoidance/flee (evade, flinch, retreat, ricochet, wheel, startle, jump, leave, wall, clutch). 9. Defense/submission (upright defensive, upright submissive, sideways defensive). 10. Immobility (squat, cringe). A detailed description of all elements can be found in Brain et al. [1989]. This ethoexperimental procedure allows a complete quantification of the behavioral elements shown by the subject during the agonistic encounters. Only the behavior of the isolated animal was assessed. The analysis was carried out by a trained experimenter, unaware of the treatment of the groups. Statistical Analysis Nonparametric Kruskal-Wallis tests were used to assess the variance of the behavioral measures over different treatment groups. Subsequently, appropriate paired comparisons were carried out using Mann-Whitney U-tests. The analyses were performed using nonparametric statistics since the criteria for parametric statistics were not met by the data. The criterion for statistical significance for all the tests was Po.05. RESULTS The effects of acute and subchronic administration of different doses of zopiclone and zolpidem are shown in Tables I and II, respectively. Kruskal-Wallis analysis showed that zopiclone administration had significant effects on non-social exploration (Po.05), threat (Po.05), and attack (Po.02). Post-hoc Mann-Whitney U-tests revealed that, after acute treatment, zopiclone significantly reduced the time spent in threat and attack behaviors in comparison with the control group (1.5 and 3 mg/kg, Po.02; 6 mg/kg, Po.001). Non-social exploration was significantly increased with the highest dose used (Po.001). After subchronic treatment, zopiclone (1.5 and 6 mg/kg) produced a significant reduction of threat (Po.02) and attack (3 and 6 mg/kg, Po.02) compared with the control group. Kruskal-Wallis analysis revealed significant treatment effects in mice treated with zolpidem on exploration from a distance, threat, attack, and immobility (Po.05). Further statistical analysis with Mann-Whitney U-tests showed that acute treatment with zolpidem produced a significant increase in exploration from a distance (0.75, 1.5, and 3 mg/kg, Po.02) and immobility (3 mg/kg, Po.02), as well as a significant decrease in threat (1.5 and 3 mg/kg,

5 420 Martín-López and Navarro TABLE 1. Median Values (With Ranges) for Times (In Seconds) Allocated to Broad Behavioral Categories in Animals Receiving Acute and Subchronic Treatment With Zopiclone Dose of zopiclone, mg/kg Acute treatment Subchronic treatment Behavioral categories Vehicle Body care ( ) (0 19.9) ( ) (0 20.1) (0 15.9) ( ) (0 10.7) Digging (0 49.5) (0 60.7) (0 40.8) (0 24.1) (0 19.4) (0 33.2) (0 2.5) Non social exploration n nnn ( ) ( ) ( ) ( ) ( ) ( ) ( ) Explore from a distance (10 310) (14 107) ( ) ( ) ( ) (14 91) ( ) Social investigation (29 203) (30 212) (31 332) (39 198) (57 440) (55 317) (47 236) Threat n nnnn nnnn 0 nnn 52 nnnn nnnn (61 168) (0 147) (0 112) (0 108) (0 115) (0 168) (0 156) Attack nn nnnn 20.4 nnnn 0 nnn **** 3.25 nnnn (20 60) (0 54) (0 61) (0 42) (0 85) (0 51) (0 71) Avoidance/flee (0 0.33) (0 1.06) (0 2.3) (0 4.43) (0 1.29) (0 0.23) (0 0.55) Defense/submission (0 0.31) (0 17.5) (0 27.8) (0 21) (0 56) (0 4.5) (0 21.5) Immobility (0 0) (0 0) (0 0) (0 0) (0 0) (0 0) (0 0) Kruskal Wallis test showed significant variance: n Po.05; nn Po.02

6 Zolpidem, Zopiclone, and Aggression in Mice 421 TABLE 2. Median Values (With Ranges) For Times (in Seconds) Allocated to Broad Behavioral Categories in Animals Receiving Acute and Subchronic Treatment With Zolpidem Dose of zolpidem, mg/kg Acute treatment Subchronic treatment Behavioral categories Vehicle Body care (1.3 19) (0.5 30) ( ) (0 20) (0.2 24) (1 13.1) ( ) Digging (0 44) (0 27) (0 67) (0 25.5) (0 8.37) (0 34) (0 17) Non social exploration ( ) ( ) ( ) ( ) ( ) ( ) ( ) Explore from a distance n nn nn nn nn nn (14 34) (15 108) (18 128) (35 121) (10 70) (18 150) ( ) Social investigation (42 139) ( ) ( ) ( ) ( ) ( ) (11 287) Threat n ; nn nnn nnnn nnnn; (32 191) ( ) (0 149) (0 134) (0 201) ( ) (0 156) Attack n nn 7.72 nn nn nn (13 97) (0.2 84) (0 59) (0 94) (0 99) (0 53) (0 116) Avoidance/flee (0 0.3) (0 3.8) (0 4) (0 2.13) (0 0.5) (0 0.94) (0 1.33) Defense/submission (0 19.2) (0 16.5) (0.68 4) (0 15.5) (0 10.4) (0 14.5) (0 7.41) Immobility n nn nnnnn (0 0) (0 7.3) (0 4.7) ( ) (0 0) (0 0) (0 0) Kruskal Wallis test showed significant variance: n Po.05. Differs from controls on Mann Whitney U tests: nnnn Po.05; nn Po.02; nnn Po.001. Differs from acute treatment on Mann Whitney U test: nnnnn Po.02.

7 422 Martín-López and Navarro Po.02 and Po.001, respectively) and attack (1.5 and 3 mg/kg, Po.02) behaviors, in comparison with the control group. Furthermore, after subchronic treatment with the drug, exploration from a distance was significantly increased (1.5 and 3 mg/kg; Po.02) whereas threat (1.5 and 3 mg/kg; Po.05) and attack (1.5 and 3 mg/kg; Po.02) behaviors were significantly decreased, as compared with the control group. DISCUSSION The results obtained in the present study indicate that zopiclone (a non-selective omega 1 and 2 ligand) and zolpidem (a selective omega 1 ligand) exhibit an antiaggressive activity in isolated male mice. As Table I shows, acute treatment with zopiclone produced a marked antiaggressive effect, reducing offensive behaviors (threat and attack) at all doses used (1.5, 3, and 6 mg/kg) without affecting immobility. Our findings are in concordance with others studies using this drug [Julou et al., 1983; Martı n-lo pez et al., 1994; Ueki, 1987]. Thus, in an experiment in which the same animal model of aggression was employed, it was found that a low dose of zopiclone (1 mg/kg) did not modify aggression in mice, whereas a high dose of the drug (8 mg/kg) provoked a significant reduction of aggressive behaviors, without altering immobility [Martı n-lo pez et al., 1994]. These findings suggest that 1.5 mg/kg might be the minimal effective dose to produce an antiaggressive action, at least when an animal model of isolation-induced aggression is used. In addition, the failure of zopiclone to produce motor impairment agrees with several studies in which are required higher doses of zopiclone to reduce spontaneous locomotor activity (11.5 mg/kg, intraperitoneally) [Perrault et al., 1990] and to produce ataxia in the rotarod test (25 mg/kg, intraperitoneally) in mice [Sanger and Zivkovic, 1992]. On the other hand, our results are similar to those described with benzodiazepines. However, whereas 1 to 4 benzodiazepines usually reduced aggression only at doses that produced pronounced muscle relaxation and sedation [Martı n-lo pez and Navarro, 1997], zopiclone decreased aggressive behavior selectively, without affecting immobility. In sum, the results of this experiment indicate that zopiclone exhibits an ethopharmacological profile characterized by specific suppression of aggression and no evident impairment of motor activity. In different animal models of anxiety, zopiclone has been demonstrated to possess an anxiolytic-like activity [Carlson et al., 2001; Griebel et al., 1998; Ueki, 1987]. In our study, although zopiclone increased the time spent in social investigation behaviors, a parameter commonly used to assess the anxiety-changing properties of drugs [Brain et al., 1991; Maldonado and Navarro, 2001], no significant differences were reached. This lack of action of the drug on social investigation behaviors may be related to a possible ceiling effect (91.05 sec in controls). On the other hand, a significant increase in the behavioral category of non-social exploration was observed with the highest dose used. A possible explanation for this effect might be that animals devote more time to these exploratory behaviors to compensate for the reduction in the offensive behaviors (threat and attack) (see Table I). With repeated treatment, no tolerance to the antiaggressive effects of zopiclone was developed. This finding is in concordance with that of Julou et al. [1983], who also found a lack of tolerance to this action of the drug using a shock-induced aggression model in mice. Repeated administration of various benzodiazepines has been demonstrated to produce sensitivity changes in the efficacy spectrum of compounds acting at the GABA A receptor, and

8 Zolpidem, Zopiclone, and Aggression in Mice 423 it has been suggested that these changes may be underlying the development of tolerance or dependence. Zopiclone has been shown to possess a limited ability to produce receptor sensitivity changes [Piot et al., 1990]. Perhaps this fact is related to the capacity of zopiclone to bind to a site close to, but probably distinct from, that of the benzodiazepine hypnotics and that zopiclone, whose binding is not sensitive to GABA, may not able to induce the conformational state from which receptor sensitivity changes are triggered [Doble et al., 1995]. After acute treatment with zolpidem, a clear dose-dependent reduction of aggression was observed (see Table II). While the lowest dose used (0.75 mg/kg) reduced the time spent in offensive behaviors (threat and attack), no significant differences were found. The intermediate dose (1.5 mg/kg) significantly decreased aggression in a specific manner, without altering immobility, whereas the highest dose (3 mg/kg) provoked a reduction in aggression accompanied by a weak (but significant) increase in immobility. This slight motor impairment is in agreement with the generalized observation that this compound has a sedative activity [Holm and Goa, 2000; Sanger and Depoortere, 1998]. To our knowledge, this is the first report in which an antiaggressive action of zolpidem is described. Therefore, the lack of experimental studies about the antiaggressive action of zolpidem in animals does not permit us to compare our results with other works. The antiaggressive activity of this substance is foreseeable since other omega receptor agonists (benzodiazepine and non-benzodiazepine compounds) also display remarkable antiaggressive properties in several animal models. Zolpidem, in contrast with other omega ligands, shows a different pattern of behavioral actions in rodents, and these differences have been explained by the selectivity of zolpidem for the omega 1 receptor subtype. This aspect seems to be of particular importance in mediating the sedative/hypnotic effects of this drug [Sanger 1997; Sanger and Depoortere, 1998]. Our results indicate that the antiaggressive action of zolpidem is selective only at 1.5 mg/kg, since with a slightly higher dose (3 mg/kg) a weak sedative action is evident. On the other hand, with repeated treatment, no tolerance to the antiaggressive effects of zolpidem was developed. As Table II shows, no significant differences in the offensive behaviors were found when subchronically and acutely treated groups were compared. A similar absence of tolerance to antiaggressive action has also been described with other omega agonists such as clobazam [Martı n-lo pez and Navarro, 1996], bentazepam [Martín- Lo pez and Navarro, 1998], and midazolam [Martı n-lo pez and Navarro, 1999]. In contrast, after repeated treatment with zolpidem for 10 consecutive days, a significant decrease in immobility was observed, and, consequently, tolerance to the motor effects of the drug was developed. This divergence in the temporal course of tolerance to antiaggressive and motor effects of zolpidem has been also reported with neuroleptic drugs, such as haloperidol [Navarro et al., 1993; Puigcerver et al., 1996] and tiapride [Navarro and Manzaneque, 1997], suggesting that these actions are mediated through different neurophysiological mechanisms. Likewise, animals chronically treated with classical benzodiazepines, such as diazepam or lorazepam, show changes in GABA A receptor subunit genes [Heninger et al., 1990; Impagnatiello et al., 1996], several of these changes being region specific [Galpern et al., 1990; Kang and Miller, 1991]. In this context, recently it has been described as a significant decrease in the level of a 1 subunit mrna in the rat cortex after 14 days of treatment with zolpidem [Holt et al., 1997]. In sum, the development of tolerance may depend not only on pharmacokinetic and pharmacological factors but also on the manner in which different drugs interact with GABA A receptors.

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