Baclofen blocks expression and sensitization of anxiety-like behavior in an animal model of repeated stress and ethanol withdrawal

Abstract

Background: Repeated exposures to forced ethanol diets (EDs) or restraint stress sensitize anxiety-like behavior during a future ethanol withdrawal. The present investigation assessed whether pretreatment of rats with agents targeting receptor systems thought to be important in treating relapse in alcoholic patients would prevent sensitization of anxiety-like behavior.

Methods: Groups of rats were exposed to either (1) three 5-day cycles of ED with 2 days of withdrawal between cycles, (2) continuous ED, or (3) 5 days of ED in a single cycle preceded by 2 episodes of restraint stress 6 days apart. Drugs [baclofen, acamprosate, naloxone, lamotrigine, ifenprodil, dizocilpine (MK-801), CGS19755, diazepam, flumazenil, or 6-methyl-2-(phenylethynyl)pyridine] were given prophylactically during the first and second withdrawal periods only or, in separate baclofen experiments, acutely during the third withdrawal or during withdrawal from continuous ED. Baclofen administration preceded each stress session in the stress-withdrawal protocols. Anxiety-like behavior was assessed in the social interaction (SI) test 5 hours after the ethanol was removed or after 3 days of abstinence.

Results: Baclofen (1.25, 2.5, and 5 mg/kg), flumazenil (5 mg/kg), and diazepam (1 mg/kg) blocked the reduction in SI induced by ethanol withdrawal. Among the drugs that alter glutamate function, only acamprosate (300 mg/kg) was effective. In the stress protocols, baclofen (5 mg/kg) given before each of the 2 restraint stress sessions before ethanol exposure or before stress during abstinence also attenuated SI deficits.

Conclusions: These findings suggest that GABAB and GABAA, but not glutamate or opioid mechanisms, are involved in adaptive changes associated with anxiety-like behavior induced by these repeated ethanol-withdrawal and stress-withdrawal paradigms. The lack of action of agents attenuating different aspects of glutamate function suggests that acamprosate's action is related to some other, as yet undetermined, mechanism.

Fig. 1

Acute actions of baclofen, flumazenil, and diazepam on social interaction (SI) during withdrawal from prolonged 7% ethanol diet (ED). Rats were exposed to control diet (CD) throughout or 23 days of 7% ED before withdrawal. Rats exposed to ED were injected with 1 mg/kg diazepam (ED-Diaz), 5 mg/kg flumazenil (ED-Flu), or 1.25 or 2.5 mg/kg baclofen (ED-Bac1.25 or ED-Bac5.0) 30 min (10 for Flu) before the SI test. SI behavior and line crossings were recorded 5 h after withdrawal. Bars represent the means ± SEM. Groups with different letters are significantly different from each other (Tukey’s protected t-tests).

Fig. 2

Prophylactic actions of acamprosate but not ifenprodil on the reduced social interaction (SI) time induced by repeated chronic exposures to 7% ethanol diet (ED). Rats were exposed to control diet (CD) throughout or 3 cycles of 5 days’ exposure to 7% ED. Rats exposed to ED were treated with vehicle (ED-Veh), 300 mg/kg acamprosate (ED-Acamp), or 10 mg/kg ifenprodil (ED-Ifen) 4 h into the first and second withdrawals. Social interaction behavior and line crossings were recorded 5 h after the last withdrawal. Data represent the means ± SEM. Groups with different letters are significantly different from each other (Tukey’s protected t-tests).

Fig. 3

Failure of lamotrigine, dizocilpine (MK-801), or CGS19755 to prevent reduced social interaction (SI) time induced by repeated chronic exposures to 7% ethanol diet (ED). Rats were exposed to control diet (CD) throughout or 3 cycles of 5 days’ exposure to 7% ED. Rats exposed to ED were injected with vehicle (ED-V), 30 mg/kg lamotrigine (ED-Lamot), 0.3 mg/kg dizocilpine (ED-MK), or 10 mg/kg CGS19755 (ED-CGS) at 4 h into the first and second withdrawals. Social interaction behavior and line crossings were recorded 5 h into withdrawal after the third ethanol exposure was stopped. Bars represent the means ± SEM. Groups with different letters are significantly different from each other (Tukey’s protected t-tests).

Fig. 4

Inactivity of 6-methyl-2-(phenylethynyl)pyridine (MPEP) against reduced social interaction (SI) time in rats repeatedly withdrawn from chronic 4.5% ethanol diet (ED). Rats were exposed to control diet (CD) throughout or 3 cycles of 5 days’ exposure to 4.5% ED. The rats exposed to ED were injected with vehicle (ED-Veh) or 10 mg/kg MPEP 4 h into the first and second withdrawal. Social interaction behavior and line crossings were recorded 5 h after the last ethanol exposure was stopped. Bars represent the means ± SEM. Groups with different letters are significantly different from each other (Tukey’s protected t-tests).

Fig. 5

Prophylactic effects of flumazenil but not naloxone on reduced social interaction (SI) behavior in rats repeatedly withdrawn from 4.5% ethanol diet (ED). Rats were exposed to control diet (CD) throughout or 3 cycles of 5 days’ exposure to 4.5% ED. Rats exposed to ED were injected with vehicle (ED-Veh), 20 mg/kg naloxone (ED-NLX), or 5 mg/kg flumazenil (ED-Flu) 4 h into the first and second withdrawals. Social interaction behavior and line crossings were recorded 5 h after the third ethanol exposure was stopped. Bars represent the means ± SEM. Groups with different letters are significantly different from each other (Tukey’s protected t-tests).

Fig. 6

Acute and prophylactic effects of baclofen on reduced social interaction (SI) in rats repeatedly withdrawn from chronic 4.5% ethanol diet (ED). Rats were exposed to control diet (CD) throughout or 3 cycles of 5 days’ exposure to 4.5% ED. The rats exposed to ED were injected with vehicle (ED-Veh) or 2.5 mg/kg baclofen (ED-Bac2.5) 4 h into the first and second withdrawal or with 1.25 mg/kg baclofen (ED-Bac1.25) 4.5 h into the third withdrawal. Social interaction behavior and line crossings were recorded 5 h into withdrawal after the third ethanol exposure was stopped. Bars represent the means ± SEM. Groups with different letters are significantly different from each other (Tukey’s protected t-tests).

Fig. 7

Prophylactic effects of baclofen against reduced social interaction (SI) in rats repeatedly withdrawn from 4.5% ethanol diet (ED). Rats were exposed to control diet (CD) throughout or 3 cycles of 5 days’ exposure to 4.5% ED. The rats exposed to ED were injected with vehicle (ED-Veh) or 1.25 or 5 mg/kg baclofen, or 5 mg/kg naloxone 4 h into the first and second withdrawal periods. Social interaction behavior and line crossings were recorded 5 h after the last ethanol exposure was stopped. Bars represent the means ± SEM. Groups with different letters are significantly different from each other (Tukey’s protected t-tests).

Fig. 8

Actions of flumazenil and baclofen, but not naloxone, against stress-enhanced ethanol withdrawal anxiety. Rats were exposed to control diet (CD), restraint stressed twice (6 days apart; 45 min each session), and then given 4.5% ED for 5 days before social interaction (SI) testing. Thirty minutes before each stress (10 min for flumazenil), rats were treated with either vehicle, flumazenil (5 mg/kg), naloxone (20 mg/kg), or baclofen (2.5 mg/kg), respectively. Social interaction testing was carried out 5 h after ethanol exposure was stopped. Bars represent the means ± SEM. Groups with different letters are significantly different from each other (Tukey’s protected t-tests).

Fig. 9

Baclofen (1.25 and 5 mg/kg) actions against stress-enhanced ethanol withdrawal anxiety: additional doses. Rats were treated as in Fig. 8, except that subgroups received either 1.25 or 5.0 mg/kg baclofen 30 min before the stress sessions. An additional group was not subjected to stress. Bars represent the means ± SEM. Groups with different letters are significantly different from each other (Tukey’s protected t-tests). CD, control diet.

Fig. 10

Acute actions of baclofen against stress-elicited social interaction (SI) deficits during abstinence from ethanol diet (ED) but no effects of prophylactic baclofen in animals not exposed to ED. Groups of rats were exposed to control diet (CD) and given either vehicle (CD-Veh) or baclofen (Bac-CD) twice or were similarly exposed to CD and baclofen, followed by one 5-day cycle of 4.5% ED (Bac-ED). Two additional groups were exposed to ED for 3 cycles, behaviorally tested to confirm the sensitization of anxiety (ED-Pre Veh stress and ED-Pre Bac stress) during withdrawal, and then retested immediately after administration of a single restraint stress (45 min) 3 days into abstinence. Baclofen had no effect on rats given only CD, tended to increase anxiety in animals subsequently exposed to 1 ED cycle (Bac-ED), and completely reversed stress-elicited anxiety in the multiply withdrawn abstinent rats (ED-Post Bac stress). Bars represent the means ± SEM. Groups with different letters are significantly different from each other (Tukey’s protected t-tests). *Significantly different from the stressed Veh-treated group (ED-Post Veh stress).