Prior multiple ethanol withdrawals enhance stress-induced anxiety-like behavior: inhibition by CRF1- and benzodiazepine-receptor antagonists and a 5-HT1a-receptor agonist

Abstract

Repeated withdrawals from chronic ethanol induce a persistent adaptive change. Further, stress substitutes for the initial two withdrawals of a multiple-withdrawal protocol to sensitize rats to withdrawal-induced anxiety-like behavior ('anxiety'). Therefore, it was tested whether the persistent adaptation induced by multiple-withdrawal exposures allows stress to elicit anxiety after a period of abstinence. Social interaction was used to assess the degree of anxiety induced by 45 min of restraint stress 3, 7, or 14 days after rats were exposed to multiple withdrawals from a chronic 4.5% ethanol diet. Restraint stress reduced social interaction (ie anxiety-like behavior) at 3, but not at 7 or 14 days, after the multiple withdrawals. No anxiety response was observed in animals that received multiple withdrawals without stress or in animals that received stress when exposed only to control liquid diet. Drugs (ie a CRF1-receptor antagonist, a benzodiazepine receptor antagonist, and a 5-HT1A-receptor agonist) previously demonstrated to block the cumulative adaptation, when administered during repeated withdrawals, prevented stress-induced anxiety-like behavior during abstinence. Additionally, these drugs applied prior to stress in the rats previously exposed to the repeated withdrawal protocol, likewise, minimized stress-induced anxiety. The anxiety following stress during abstinence from previous chronic ethanol exposure is indicative of an interaction of stress with the persistent adaptive change caused by repeated withdrawals. Stress eliciting anxiety-like behavior during abstinence from previous ethanol exposures in rats is consistent with stress inducing anxiety during recovery (sobriety) in the alcoholic, a circumstance that can facilitate craving and relapse.

Figure 1

Duration of restraint stress to reduce social interaction. Restraint stress was applied to rats (8–10/group) for 15, 30, 45, or 60 min, and social interaction, a measure of anxiety-like behavior, was assessed 30 min after the stress exposure. Only the group exposed to 60 min of restraint stress exhibited significant reduction in social interaction (F(4,33) = 6.73). *p < 0.001 compared to the groups that received no stress (No Stress). No change in locomotor activity was recorded after any of the applications of stress (F(4,33) = 1.48; p > 0.1).

Figure 2

Effect of stress on anxiety-like behavior in animals previously exposed to multiple withdrawals. Rats (8–10/group) were exposed to restraint stress for 45 min 3 days after receiving control diet (Control Diet Stress) or multiple withdrawals from 4.5% ethanol diet (Ethanol Diet Stress). A control diet without stress group (Control Diet, No Stress) or a multiple withdrawal without stress group (Ethanol Diet, No Stress) were included for comparison. Social interaction was assessed 30 min after receiving restraint stress (F(3,27) = 3.83). *p < 0.05 when Ethanol Diet Stress group is compared to the other treatments. No significant change (F(3,27) = 2.28; p > 0.1) in locomotor activity was observed in the repeatedly withdrawn rats 30 min after stress application—the time social interaction was measured.

Figure 3

Effect of stress on social interaction in animals 3, 7, and 14 days after the repeated withdrawal protocol. Animals were tested for social interaction 30 min after being stressed for 45 min at 3, 7, or 14 days after removal of the ethanol diet. *p < 0.0001 (F(3,34) = 14.9) compared to control diet (Control Diet, No Stress).

Figure 4

Effect of drug treatments during repeated withdrawals on the reduction in social interaction induced by restraint stress. The 5-HT_1A agonist, buspirone, (Busp; 0.6 mg/kg), flumazenil (Flumaz; 5 mg/kg), a benzodiazepine antagonist, or the CRF₁-receptor antagonist, CP154,526 (10 mg/kg), were administered during the first two withdrawal periods, but not the final withdrawal, of the multiple-withdrawal protocol. Vehicle was given to the group that received ethanol diet and stress (ET Diet Stress Veh). All drug treatments significantly prevented the reduction in social interaction induced by stress applied 3 days following the repeated withdrawals. *p < 0.05 (F(6,48) = 2.77) compared to Ethanol Diet No Stress, Control Diet Stress, and Control Diet No stress groups and all drug groups stressed (Ethanol Diet Stress—buspirone (Busp), flumazenil (Flumaz), and CP-154,526 groups). Locomotor activity was not reduced following stress in any of the drug treatment groups compared to Ethanol No Stress (F(6,48) = 2.00; p > 0.05).

Figure 5

Effect of drug treatments prior to the stress-induced reduced social interaction during abstinence from repeated withdrawals. The buspirone, (Busp; 0.6 mg/kg), flumazenil (Flumaz; 5 mg/kg), or the CP-154,526 (10 mg/Kg) were administered 30 min prior to the application of 45 min of restraint stress applied 3 days after the repeated withdrawals. Vehicle (Veh) was given to the group that received ethanol diet and stress (ET Diet Stress Veh). Drug treatments significantly attenuated the reduction in social interaction induced by stress in the rats exposed to the repeated withdrawals. (*p < 0.01 (F(4,32) = 5.19) compared to Control Diet group with no stress and to all drug groups stressed (Ethanol Stress buspirone, flumazenil, and CP154,526 groups).) Locomotor activity was significantly reduced (p < 0.01; (F(4,33) = 4.93) following stress in the group pretreated with flumazenil, but not in any other group (p > 0.1). As noted in Table 1, flumazenil, buspirone, and the CP-154,526 were without effect on social interaction when tested in rats 3 days after repeated withdrawals in the absence of stress.