Behavioral effects of flumazenil in the social conflict test in mice

Rationale Flumazenil, a competitive antagonist of benzodiazepine receptors (BZRs), has been used as a probe to detect effects of putative endogenous ligands for BZRs in anxiety. Flumazenil is renowned for its highly inconsistent behavioral effects.Objective To ascertain effects of flumazenil in the social conflict test in mice, which provides complex measures for prediction of anxiolytic and anxiogenic activity of drugs in behaviorally different groups of animals.Methods Singly housed male mice treated with flumazenil (5, 20 or 80 mg/kg i.p.) or vehicle were paired with untreated non-aggressive group-housed male mice in a novel cage. Behavior was analyzed from video tapes of the social interactions in three populations of mice: timid (n=21), aggressive (n=11), and sociable (n=7). Levels of -aminobutyric acid (GABA) were measured in vivo in the prefrontal cortex.Results Flumazenil reduced timid (defensive-escape) and increased locomotor activities in timid mice. The drug reduced aggressive and increased sociable (social investigation) activities in aggressive mice. These behavioral changes were produced at the lowest dose of flumazenil tested (5 mg/kg) and were not increased further by higher doses of the drug (20 mg/kg or 80 mg/kg). A tendency to increased timidity was found after flumazenil in sociable mice. Concentrations of GABA were markedly higher in the prefrontal cortex of sociable mice than in timid or aggressive mice.Conclusions Flumazenil produced moderate anxiolytic-like behavioural changes and a slight anxiogenic-like effect. The present data might be reflecting antagonism of corresponding endogenous BZR ligands. However, these putative ligands seem to exert only modest modulatory influence.     

Beta-CCE and FG 7142 increase defensiveness during intraspecies encounters in mice

The effects were ascertained of two partial inverse agonists at benzodiazepine receptors (beta-CCE and FG 7142) on the incidence of timid (defensive-escape), aggressive, sociable and locomotor activities in both timid and aggressive singly-housed male mice, treated with drugs in paired interactions with untreated non-aggressive males. FG 7142 (5 mg/kg) and beta-CCE (8 mg/kg) increased defenses and escapes without producing other behavioral changes in timid mice. FG 7142 (20 mg/kg) and beta-CCE (1–8 mg/kg) moderately increased defenses and alert postures in aggressive mice and this effect was associated with marked reduction of aggressive behavior. FG 7142 (20 and 80 mg/kg) also decreased walking across cage in aggressive mice. It is concluded that beta-CCE and FG 7142 produced behavioral changes which could be interpreted as anxiogenic.     

Timid singly-housed mice: their value in prediction of psychotropic activity of drugs.

1 About 45% of singly-housed male mice showed timidity (alert postures, running away, defensive postures) instead of aggression on interactions in pairs with group-housed male mice, though their partners did not show any aggression. The isolation-induced timidity was stable in repeated interactions. Timid mice also showed locomotion (walking across cage and rearing) and a small amount of sociable activity (sniffing, following partners and climbing over them). 2 Diazepam (5 mg/kg), chlordiazepoxide (20 mg/kg), chlorpromazine (7.5 mg/kg) and barbitone (60 mg/kg) given orally inhibited the isolation-induced timidity without reducing other motor activities in the timid mice. Imipramine lessened timidith only in a dose (80 mg/kg) which also decreased other components of behaviour in the timid isolates. (+)-Amphetamine and lysergic acid diethylamide (LSD) increased the timid response. 3 Comparison of the inhibition of timid activities with changes in other behaviour occurring at the same time seems a better measure of selective timidity-reducing effects of drugs than the rota-rod test. 4 Diazepam (5 mg/kg) increased sociable and locomotor activities. Barbitone (20 and 60 mg/kg) increased sociable activities; however, the higher dose also evoked some aggression in timid mice. 5 Behaviour of timid singly housed male mice seems to be a good measure for prediction of activity of drugs in relieving anxiety as well as for detection of aggression-evoking and sociability-increasing effects of drugs.     

Facilitated intermale aggression in the mouse after 6-hydroxydopamine administration

The effect of 5 and 50 μg of 6-hydroxydopamine on brain catecholamines and behaviour of singly-housed male mice on paired interactions with group-housed partners was studied. 6-Hydroxydopamine produced a significant and dose-dependent depletion of noradrenaline and dopamine in the brain. No significant behavioural changes were found after 5 μg of 6-hydroxydopamine. The higher dose of 6-hydroxydopamine (50 μg) significantly increased the frequency of attacks, aggressive unrest and of tail-rattles in the initially non-aggressive singly-housed mice without changing their sociable. timid or locomotory activities. Significant negative correlations were found between the individual stimulation of aggressive activities and depletion of brain catecholamines. The present results suggest that 6-hydroxydopamine can selectively facilitate intermale spontaneous aggression in the mouse. This facilitation seems to he related to the effect of 6-hydroxydopamine on catecholamine neurones.     

Differential effects of six structurally related benzodiazepines on some ethological measures of timidity,aggression and locomotion in mice

The effects were compared of three 2' chloro-phenyl-benzodiazepines (triazolam, clonazepam and lorazepam) and three corresponding 2' deschloro-phenylderivatives (alprazolam, nitrazepam and oxazepam, respectively) on the incidence of six ethological elements in both timid and aggressive singly-housed male mice, treated with drugs in paired interactions with untreated non-aggressive males. Alprazolam and oxazepam reduced defensive upright postures and escapes at doses which did not reduce rearing and actually increased walking, while their chlorinated counterparts (triazolam and lorazepam, respectively) decreased incidence of defenses and escapes mostly at doses decreasing locomotor acts involving a similar movement (rears and walks, respectively). Alprazolam and oxazepam also reduced attacks at doses not reducing rears, in contrast to triazolam and lorazepam which reduced attacks only at doses suppressing rearing. Nitrazepam stimulated sniffing partners much more than its chlorinated counterpart clonazepam. The 2 deschloro-phenyl-benzodiazepines were more potent in reducing defensive-escape activities than attacks or locomotion. Yet, none of the benzodiazepines tested produced a complete inhibition of timid defensive-escape behavior at non-sedative doses. The present study suggests that 2 deschloro-phenyl-benzodiazepines are less sedative with respect to their anxiolytic activity.     

Differential effects of modafinil, methamphetamine, and MDMA on agonistic behavior in male mice

The aim of the present study was to compare the behavioral effects of modafinil, an atypical psychostimulatory acting and cognitive-function improving drug, with the effects of the psychostimulants methamphetamine (MET) and MDMA (3,4-methylenedioxymethamphetamine, or “ecstasy”) in a model of mouse agonistic behavior. This model enables the observation of ethologically well-defined sociable, timid, aggressive, and locomotor behavioral acts and postures. Singly-housed male mice (isolates) were separated into 4 groups. The observations were performed in 4 sessions, 1 week apart. In each interaction, singly-housed mice were paired with non-aggressive group-housed partners for 4 min in a neutral environment. The isolates received, in a Latin square design, either a) a vehicle or modafinil at doses 2.0, 10.0, or 50.0 mg/kg; or b) a vehicle or MET at doses 1.0, 5.0, or 10.0 mg/kg; or c) a vehicle or MDMA at doses 2.5, 10.0, or 30.0 mg/kg. The isolates were categorized as timid or aggressive according to their behavior in the control interaction (vehicle pre-treatment). Elements of locomotor, sociable, aggressive, and timid behavior were evaluated (one-way ANOVA).In the aggressive mice, no change in the sum of aggressive behavior was measured following modafinil administration, while both methamphetamine and MDMA produced dose-dependent inhibition of aggression (p < 0.01). The substantial difference in the tested drug effects on agonistic behavior was an increased occurrence of sociable acts (p < 0.01) accompanied by a simultaneous increase of timid acts (p < 0.01) recorded after MDMA, but not after modafinil or methamphetamine administration. In the timid mice, at least some doses of modafinil decreased timidity (p < 0.01) and increased aggression (p < 0.01) with no effect on sociability. Administration of MDMA increased timid activities (p < 0.01). Both MDMA and MET decreased sociability (p < 0.01).     

Effect of alcohol on behaviour of pairs of rats

The effects of alcohol (ethanol 1.2 and 3 g/kg orally) on the occurrence of 13 social and 5 non-social acts and postures in alcohol-treated male rats and their untreated partners in a monosexual dyadic interaction were investigated. Alcohol decreased all social activities in the treated rats even in the lower dose of 1.2 g/kg which hardly affected ataxia. Some of the social activities (social sniffing, social grooming and all social acts involving walking) were reduced quite selectively as corresponding non-social activities involving similar movements (self-grooming, walking across cage) were not influenced even after 3 g/kg of alcohol. On the other hand, both social and non-social activities involving upright movement (mounting, aggressive posture, boxing and rearing) were reduced which may have been due to impaired motor function. Some social activities (e.g. defensive postures) may have been reduced because appropriate provoking activities (aggressive postures) in the untreated partners were decreased.     

Effects of drugs on behaviour of aggressive mice

1 The occurrence of 11 aggressive and non-aggressive activities was observed in aggressive male mice treated with drugs in paired interactions with non-aggressive males given water. Effects of chlordiazepoxide, diazepam, barbitone, chlorpromazine, imipramine, (+)-amphetamine, lysergic acid diethylamide (LSD) all given orally and of intraperitoneal scopolamine were investigated.2 Scopolamine (0.25 and 0.75 mg/kg), (+)-amphetamine (0.25 and 1 mg/kg), chlorpromazine (2.5 mg/kg), diazepam (10 mg/kg) and chlordiazepoxide (50 mg/kg) reduced aggressive activities (attacks, aggressive unrest) without inhibiting walking across the cage or rearing in the aggressive mice. Thus, the inhibition of aggression induced by these drugs does not seem to be due to neuromuscular impairment and seems to this extent specific. On the other hand, imipramine lessened aggressive activities only at a dose (80 mg/kg) which also decreased walking across the cage and rearing. Barbitone or LSD did not change aggression at either dose tested (20 and 60 or 0.01 and 1 mg/kg, respectively). Aggressive activities were increased significantly only by chlordiazepoxide at a dose of 5 mg/kg.3 (+)-Amphetamine (0.25 mg/kg) and scopolamine (0.75 mg/kg) increased escapes and alert postures, respectively, in the aggressive mice.4 Diazepam and chlordiazepoxide decreased tail rattling at 1 and 5 mg/kg, respectively, doses 10 times lower than those inhibiting attacks. The other drugs tested inhibited tail rattling only at doses reducing attacks. Tail rattling appears to be a convenient measure for testing effects of drugs on behavioural conflict.5 Diazepam (5 and 10 mg/kg), chlordiazepoxide (20 and 50 mg/kg), barbitone (60 mg/kg) and scopolamine (0.25 and 0.75 mg/kg) increased sociable activities (sniffing, following partners and climbing over them) whereas (+)-amphetamine, chlorpromazine, imipramine and LSD did not. Effects of the drugs on sociable activities in aggressive mice seem to correlate with their action on punished responding and other types of suppressed behaviour.     

Effect of repeated administration of the antioxidant stobadine on the behavior of singly-housed male mice in intraspecies conflict

The aim of the present study was to assess the effect of repeated oral administration of stobadine (70 mg/kg) on the occurrence of selected behavioral elements during exposure to an intraspecies conflict between singly-housed and group-housed male mice. Isolation induced timidity (defensive-escape behavior without attacks) in most mice (87%). This isolation-induced timidity was reduced after stobadine treatment. In the stobadine-treated group, sociable activities (following, climbing) were also decreased. After discontinuation of the treatment (18 days), aggressive behavior tended to increase in the stobadine-treated group. The results of this study are indicative of an inhibitory effect of repeated administration of stobadine on some behavioral activities of singly-housed male mice in an intraspecies conflict.     

Lack of increased intermale fighting behavior in mice after low ethanol doses.

The effect of ethanol on intermale fighting behavior, measured mainly as the total fighting time, was studied using Swiss-Webster mice in 5-min encounters in a neutral arena (i.e., not the home cage). Ethanol treatment compared to control treatment had no statistically significant effect on fighting behavior when given to both equal-sized members of a pair of males socially isolated for a) 5 or 10 days at a dose of 0.4 g/kg IP; b) 4 weeks at 0.8 g/kg IP; and c) 38 weeks at 0.4 g/kg IP. Moreover, no significant effect was found when ethanol was given only to the expected dominant member of a pair, that is, to: a) a male isolated for 48 weeks confronting a younger and smaller group-housed male at 0.4 g/kg PO; and b) a male that had been pair housed with a female conspecific for 5 weeks confronting a group-housed male of equal age and weight at 0.4 g/kg IP. The results suggest that under these conditions ethanol does not lead to increased fighting behavior in Swiss-Webster male mice.     

Flunitrazepam in combination with alcohol engenders high levels of aggression in mice and rats

Rationale

Higher doses of benzodiazepines and alcohol induce sedation and sleep; however, in low to moderate doses these drugs can increase aggressive behavior.

Objectives

To assess firstly the effects of ethanol, secondly the effects of flunitrazepam, a so-called club drug, and thirdly the effects of flunitrazepam plus alcohol on aggression in mice and rats.

Methods

Exhaustive behavioral records of confrontations between a male resident and a male intruder were obtained twice a week, using CF-1 mice and Wistar rats. The salient aggressive and non-aggressive elements in the resident's repertoire were analyzed. Initially, the effects of ethanol (1.0 g/kg), and secondly flunitrazepam (0; 0.01; 0.1; and 0.3 mg/kg) were determined in all mice and rats; subsequently, flunitrazepam or vehicle, given intraperitoneally (0; 0.01; 0.1; and 0.3 mg/kg) was administered plus ethanol 1.0 g/kg or vehicle via gavage.

Results

The most significant finding is the escalation of aggression after a moderate dose of ethanol, and a low dose of flunitrazepam. The largest increase in aggressive behavior occurred after combined flunitrazepam plus ethanol treatment in mice and rats.

Conclusions

Ethanol can heighten aggressive behavior and flunitrazepam further increases this effect in male mice and rats.     

The effects of repeated doses of ethanol on exploration and its habituation

The effects of ethanol (0.8–2.4 g/kg) on exploratory behavior and its habituation were investigated by testing DBA/2 mice in a holeboard apparatus. Mice familiar with the holeboard had lower levels of exploration than animals with no previous experience in the apparatus. If the animals received ethanol during their first exposure to the holeboard they did not show the same degree of habituation. Ethanol (0.8 g/kg) increased the exploration of animals naive to the holeboard, but failed significantly to increase the exploration of animals familiar with the apparatus. An increase in locomotor activity was observed following treatment with ethanol (2.4 g/kg). This was potentiated if animals received a single treatment with ethanol 3 days earlier, but only if they were tested in the apparatus following the initial treatment. These results have important implications for the design of experiments investigating tolerance and sensitization to ethanol's effects on locomotor activity and exploration.     

The effects of novelty,isolation, light and ethanol on the social behavior of mice

The social behavior of pairs of male NIH Swiss mice was assessed under a variety of experimental conditions. Increasing periods of isolation increased both the total time spent in social interaction and also increased the incidence of aggressive behavior. Familiarity with the testing arena tended to increase social behavior, but the magnitude of this effect was considerably less than that previously observed in rats. High light levels reduced social interaction. Ethanol (0.8–2.4 g/kg) caused a dose-related decrease in the total time spent in social interaction, a biphasic effect on aggressive behavior and a dose-related increase in locomotor activity. While the social interaction test in this form may not be a suitable model of anxiety in NIH Swiss mice, it should provide a useful method of assessing drug effects and investigating genetic influences on social and aggressive behavior.     

Testosterone modulates the effects of ethanol on male mouse aggression

In a series of three experiments, we evaluated the degree to which the effects of acutely administered ethanol on aggressive behavior of male CFW mice toward a male intruder interact with, and depend on, androgen levels. In the first experiment, mice were tested at 15 min after 0, 0.1, 0.3, 1.0, 1.7, or 3.0 g/kg of ethanol PO. The highest dose (3.0 g/kg) significantly suppressed aggression by the male residents. In the second experiment, aggressive behavior was suppressed from 5 to 60 min after 3.0 g/kg ethanol administration PO. The third experiment evaluated the role of testosterone in these effects in another set of male mice that were castrated and then implanted with a 7.5-mm or 2.5-mm silastic capsule of testosterone (T) or a silastic capsule containing cholesterol as a control. The castrated mice with 2.5-mm T capsules or cholesterol capsules had lower baseline levels of aggression than intact mice or castrated males with 7.5-mm T capsules, but they demonstrated an alcohol dose-response pattern similar to that of the intact males. The castrated males with 7.5-mm T capsules had a different dose-response curve than the other males. Doses of 1.0 and 1.7 g/kg ethanol PO significantly enhanced aggression; 5.6 g/kg was required to suppress aggression. Ethanol effects on aggressive behavior did not require T or changes in T level, but T levels altered behavioral sensitivity to ethanol.     

Effects of ethanol on fight- or swim-stressed mice in Porsolt's swim test

The effects of ethanol in Porsolt's swim test on mice preexposed to fight- or swim-stressors were investigated. The control mice did not change their behavior in the swim test after an acute injection of 0.4 or 0.8 g/kg ethanol; 1.2 g/kg ethanol increased their immobility in one but not in another experiment. The mice exposed to continuous fight-attacks in their home cage by one dominant mouse shortened immobility after 0.8 g/kg ethanol as well as tended to shorten it after 0.4 g/kg ethanol. The mice that were forced to swim in the water twice before the actual swim test responded to 0.4 g/kg ethanol by shortening immobility; 0.8 g/kg tended to have the same effect; 1.2 g/kg ethanol just failed to lengthen immobility of the fight-stressed mice and had no effect on the swim-stressed mice. Because antidepressant drugs decrease and stressors increase immobility in the swim test, the test may serve as a putative animal model of depression. The present findings showed that low doses of ethanol reverse lengthened immobility of mice preexposed to a stressor. This suggests that ethanol either has antidepressant-like properties, or it improves animal's ability to cope with a stressful situation, or both.     

Investigation of a possible sensitization development to a challenge dose of ethanol after 2 weeks following the single injection in mice

In the present study, a possible sensitization development to a single injection of ethanol in mice was investigated. Subjects were adult male Swiss-Webster mice. Ethanol (0.5-4 g/kg) or saline (control) was intraperitoneally injected to mice. Horizontal, vertical and ambulatory locomotor activities were recorded for 30 min immediately following the ethanol or saline injections. After 2 weeks, each group of mice was randomly assigned to two groups. A single challenge dose of ethanol (1 g/kg) was administered to the first group, and saline was injected to the second group. Then, the locomotor activities were recorded for 30 min. In the first experiment, ethanol significantly increased the horizontal and ambulatory activities of the mice at the doses of 0.5 and 1 g/kg, but not at 2 g/kg, while they were decreased at the dose of 4 g/kg. Ethanol (0.5 g/kg) also significantly increased the vertical activity. After 2 weeks, the challenge injection of ethanol (1 g/kg) produced some significant increases in the horizontal and ambulatory activities of the group pretreated with ethanol (2 g/kg). It did not cause any significant change on the locomotor activities of the other three groups treated with lower (stimulant) or higher (depressant) doses of ethanol. In addition, there was no significant difference between locomotor activities of the groups challenged with saline. However, a two-way ANOVA of the data on the challenge injections did not indicate any sensitization development to the effects of ethanol on locomotor activities of the mice. Our results suggest that a locomotor sensitization did not develop to a single injection of ethanol after 2 weeks following the first injection in mice.     

Ethanol-induced depression of aggression in mice antagonized by hyperbaric exposure

The present study investigated the effect of hyperbaric exposure on ethanol-induced depression of aggressive behavior measured by resident-intruder confrontations. Adult male CFW mice (residents) were paired with females and housed together for 26 days. Then, resident mice were intubated with either ethanol (2 g/kg) or water (20 ml/kg) and were exposed to 1 atmosphere absolute (ATA) air, 1 ATA helium oxygen (heliox) or 12 ATA heliox using a within-subjects counterbalanced design. Thirty minutes after intubation an intruder was introduced. Ethanol significantly decreased aggressive behaviors (attack latency, attack bites, sideways threats, tail rattles and pursuit) in 1 ATA-treated animals. Pressure completely antagonized the depression of aggression induced by ethanol. Ethanol alone and pressure alone did not significantly affect nonaggressive behaviors. There were no statistically significant differences between groups in blood ethanol concentrations 50 minutes after intubation. These results suggest that ethanol's effects on aggressive behavior result from the same membrane actions leading to loss of righting reflex, depression of locomotor activity, tolerance and dependence.     

5-HT(3) receptors, alcohol and aggressive behavior in mice

Alcohol is a positive modulator at the 5-HT(3) receptor, which has been implicated in alcohol drinking, anxiety and aggression. The reported experiments explored the role of the 5-HT(3) receptor in aggressive behavior and alcohol-heightened aggression. Male, CFW mice were trained to self-administer 1.0 g/kg of alcohol, after which they confronted an intruder. Half of the CFW mice exhibited consistently increased aggressive behavior after alcohol and were designated as showing alcohol-heightened aggression, the others showed no increase and were designated as showing alcohol non-heightened aggression. The 5-HT(3) antagonist, ondansetron (0.01-1.0 mg/kg), significantly reduced aggression in both groups of CFW mice without affecting non-aggressive behaviors. Zacopride also reduced aggression effectively in both groups of mice, but at high doses began to affect walking. Male B6SJL/F2 transgenic 5-HT(3) over-expressing mice (TG) and wild-type mice (WT) were tested for aggressive behavior in their home cage. In those individuals that fought in tests of resident-intruder aggression, no differences were found in aggression after alcohol intake. In tests of aggression without alcohol intake, zacopride reduced aggression in both TG and WT mice at a dose of 56 mg/kg. Antagonism of 5-HT(3) receptors shows promising anti-aggressive effects, although these effects depend on the genetic background of the mice.     

NMDA receptor antagonism: escalation of aggressive behavior in alcohol-drinking mice

Rationale

Memantine is a potential treatment for alcoholic patients, yet few studies investigate the effect of concurrent treatment with memantine and ethanol on aggression. We evaluated aggressive behavior following ethanol consumption and treatment with glutamatergic drugs to characterize interactions between these compounds.

Objective

This study aimed to use rodent models of aggression to examine interactions between glutamatergic compounds and ethanol.

Materials and methods

Once male CFW mice reliably self-administered 1 g/kg ethanol or water, they were assessed for aggression in resident–intruder confrontations. Alternatively, aggression was evaluated following a social-instigation procedure. Animals were then injected with memantine, ketamine, neramexane, MTEP, or LY379268 before aggressive confrontations. Effects of the pharmacological manipulations on salient aggressive and non-aggressive behaviors were analyzed.

Results

Moderate doses of memantine, neramexane, and MTEP interacted with ethanol to increase the frequency of attack bites while ketamine did not. The highest dose of LY379268, an mGluR_2/3 agonist, reduced both aggressive and non-aggressive behaviors after water and ethanol self-administration. Attack bites increased with social instigation and decreased with administration of high doses of MTEP and LY379268. Memantine and MTEP both reduced attack bite frequency in the instigation condition without reducing locomotor behavior.

Conclusions

Memantine and neramexane interacted with ethanol to heighten aggression. The binding characteristics of these compounds allow for ‘partial trapping’ by which some NMDARs are unblocked between depolarizations. We propose that this feature may contribute to the differential aggression-heightening interactions between these compounds and ethanol. MTEP also interacted with ethanol to escalate aggression, possibly through inhibition of mGluR₅ modulation of NMDARs.     

Differences among nine 1,4-benzodiazepines: an ethopharmacological evaluation in mice

The present study explored whether the profiles of action of benzodiazepines on intraspecies conflict behavior in mice are different. The occurrence of seven behavioral elements was observed in aggressive and timid singlyhoused male mice treated with drugs in paired interactions with untreated non-aggressive males. At low doses, some benzodiazepines (alprazolam, oxazepam and diazepam) inhibited defenses, escapes, or attacks, but did not reduce other activities (social sniffing, walking, rearing), and actually increased most of them. At comparable doses, other benzodiazepines (flunitrazepam, nitrazepam, clonazepam and chlordiazepoxide) stimulated only social sniffing, but reduced rearing or walking. Further benzodiazepines (triazolam and lorazepam) reduced defenses, escapes and attacks only at doses that suppressed most of the remaining activities as well. Thus, the nine benzodiazepines tested exhibited different profiles of action in the present study. Alprazolam, oxazepam and diazepam appeared least sedative, while triazolam and lorazepam were most sedative.