Stress sensitization of ethanol withdrawal-induced reduction in social interaction: inhibition by CRF-1 and benzodiazepine receptor antagonists and a 5-HT1A-receptor agonist

Abstract

Repeated withdrawals from chronic ethanol sensitize the withdrawal-induced reduction in social interaction behaviors. This study determined whether stress might substitute for repeated withdrawals to facilitate withdrawal-induced anxiety-like behavior. When two 1-h periods of restraint stress were applied at 1-week intervals to rats fed control diet, social interaction was reduced upon withdrawal from a subsequent 5-day exposure to ethanol diet. Neither this ethanol exposure alone nor exposure to three restraint stresses alone altered this measure of anxiety. Further, the repeatedly stressed singly withdrawn rats continued to exhibit a reduction in social interaction 16 days later, upon withdrawal from re-exposure to 5 days of chronic ethanol, consistent with a persistent adaptation by the multiple-stress/withdrawal protocol. Weekly administration of corticosterone in place of stress induced no significant change in social interaction upon withdrawal from the single chronic ethanol exposure, indicative that corticoid release is not responsible for the stress-induced reduction in anxiety-like behavior during withdrawal. In the multiple-withdrawal protocol, stress applied during withdrawal from voluntary ethanol drinking by P-rats facilitated ethanol drinking sufficiently, to induce a withdrawal-induced reduction in social interaction. Administration of a CRF-1 receptor antagonist, a benzodiazepine receptor antagonist, or a 5-HT(1A) receptor agonist prior to each stress minimized sensitization of the withdrawal-induced reduction in anxiety-like behavior. Since these pharmacological consequences on the induction of anxiety-like behavior following the stress/withdrawal protocol are like those previously seen when these drug treatments were given prior to multiple withdrawals, evidence is provided that repeated stresses and multiple withdrawals sensitize the withdrawal reduction in social interaction by similar central adaptive mechanisms.

Figure 1

Sensitization of the withdrawal-induced reduction in social interaction by multiple stresses: comparison with multiple withdrawals. (a) Social interaction following repeated stresses: Male rats were exposed to either continuous CD or to stress applied at 6 and 11 days while on CD, followed by a 5-day cycle of 4.5% ethanol liquid diet (2STR-ETOH-Cy1) prior to withdrawal. The social interaction testing was performed between 5 and 6 h after the final ethanol diet treatment was terminated, or 5 days after the second stress. 2STR-ETOH-Cy1 = two stresses + ethanol + withdrawal. CD = control diet. CD-3STR = stressed at 6, 11, and 16 days. ETOH-CONT-2STR = stress at 6 and 11 days during continuous ethanol diet + withdrawal. BECs are presented in Figure 3. The social interaction is expressed from data from individual rats in the pairs; however, in Table 1, these results with individual animal values are compared with the determination obtained with pairs of rats. Social interaction was measured 5–6 h after withdrawal from ethanol diet. **P < 0.01 compared to CD or CD-3STR. (b) Social interaction after single and multiple withdrawals: Male rats were exposed to CD, 5 days of 4.5% ethanol diet for only 1 cycle (ETOH-Cy1), 15 consecutive days of 4.5% ethanol diet (ETOH-CONT), or three cycles of 5 days of 4.5% ethanol diet with 2 days interruption of diet after withdrawal from the first two cycles (ETOH-Cy3). Social interaction was measured 5–6 h after the final withdrawal from ethanol diet. The social interaction is expressed from data presented as rat pairs; however, data based upon presentation of determinations of social interaction on individual rats are provided in Table 1. BECs are presented in Figure 3. **P < 0.01 when compared to any of the other groups.

Figure 2

(a) Locomotor activity during withdrawal in the presence of stress: Experimental groups are the same as those in Figure 1a. *P < 0.05; **P < 0.01 compared to CD. CD-3STR is not significantly different from CD (P > 0.05). (b) Locomotor activity in rats undergoing repeated withdrawals in the absence of stress. Experimental groups are the same as those in Figure 1b. *P < 0.05 compared to the activity for any of the other groups.

Figure 3

BECs in male rats undergoing repeated chronic ethanol exposures and repeated stresses. Rats were exposed continuously to diet for 15 days, or received the diet in three cycles where ethanol was provided in three 5-day exposures to the 4.5% diet with 2-day periods of CD between the fifth and sixth days and the 10th and 11th days of the protocol. Blood was withdrawn from the tail tip during the last hour of darkness on days 1 and 5, on days 6 and 10, day 11, and on day 15 prior to withdrawal. On day 15, BECs were determined at 0, 2, and 4 h after the removal of the ethanol diet from the two groups of the protocol. An ANOVA revealed no significant differences among the sampling times, except for the expected reduction at 2 and 4 h after withdrawal from the chronic ethanol exposures. The ethanol intake of these groups of animals was not significantly different. On the right side of the figure (single-withdrawal stress experiment), rats were administered ethanol either continuously for 15 days or in a single 5-day cycle with 1 h of stress given at days 6 and 11 with continuous ethanol group or at days 6 and 11 when on CD followed by a single 5-day cycle of ethanol diet. Blood sampling during withdrawal then proceeded as above. ^†Treatment day (hour of withdrawal).

Figure 4

Effect of multiple corticosterone treatments instead of stresses on the withdrawal-induced deficit in social interaction. (a) Social interaction: In two of the groups, animals received either two stresses (2STR-ETOH-Cy1) or two injections of 15 mg/kg of corticosterone (CORT-ETOH-Cy1) at 6 and 11 days prior to withdrawal from 5 days of 4.5% ethanol diet. See corticosterone levels after these treatments are shown in Table 2. Social interaction was determined 5–6 h following withdrawal from the chronic ethanol diet. Since the CD group did not differ from a group that received vehicle (Veh-ETOH-Cy1) (P > 0.1), these data were combined. **P < 0.01 compared to Veh-ETOH-Cy1 and CD groups. The 2STR-ETOH-Cy1 group is not significantly different from Veh-ETOH-Cy1 or CD groups (P > 0.05). (b) Locomotor activity: The groups are the same as those in (a) above. *P < 0.05 compared to CD.

Figure 5

Effect of stress during withdrawal in the multiple-withdrawal protocol on voluntary drinking in P-rats: Relation to withdrawal-induced anxiety-like behavior. (a) Alcohol intake: P-rats were given either continuous voluntary access to water and an ethanol solution (10%) to drink (CONT) or three cycles of voluntary access to water and ethanol solution exposure with 2 days of abstinence between the initial two cycles (CYCLE). As noted in Methods, the baseline intake for the first 5 days is subtracted from the intake for the third 5-day exposure to voluntary ethanol. A final set of animals undergoing multiple cycles of exposure was stressed during the initial two periods of withdrawal (4 h) from 5 days of ethanol drinking (CYCLE + 2STR). *P < 0.05 compared to CONT for the final 5 days of exposure. ^#P < 0.05 compared to CYCLE for the final 5 days of voluntary ethanol exposure. (b) Social interaction: Following the treatments outlined in Figure 5a, social interaction was assessed in each of the groups following final withdrawal from the voluntary drinking of the P-rats. When social interaction was measured 5–6 h after removal of the ethanol, only the P-rats that received the multiple stresses during the first two withdrawals had a significant reduction in social interaction upon removal from the final exposure to ethanol compared to CONT and CYCLE groups (**P < 0.01).

Figure 6

Effect of selected receptor agents on the stress sensitization of the withdrawal deficit in social interaction. (a) Social interaction: CD = control-liquid diet. 2STR-ETOH-Veh = two cycles of restraint stress (1 h) at 6 and 11 days while on liquid diet, followed by withdrawal from 4.5% ethanol diet. 2STR-CD-VEH = vehicle at 6 and 11 days prior to stress administered when rats on CD (ie no ethanol). 2STR-ETOH-Veh = rats received two stresses with pretreatment with vehicle for drugs plus chronic ethanol diet followed by withdrawal. All other groups received a selected drug prior to each of the restraint stresses. 2STR-ETOH-CRA = rats received 3 mg/kg of the CRF-1 receptor antagonist CRA-1000 prior to stress. 2STR-ETOH-FLU = rats received 5 mg/kg of flumazenil, a benzodiazepine receptor antagonist, prior to stress. 2STR-ETOH-BUS = rats received 0.6 mg/kg of the 5-HT_1A-receptor agonist buspirone prior to stress. The social interaction testing was performed between 5 and 6 h after the final ethanol diet treatment was terminated. When drugs were administered in the absence of stress while rats drank liquid diet, there was no influence on social interaction behavior with withdrawal from the chronic ethanol exposure (Overstreet et al, 2003; unpublished data). **P < 0.01 compared to CD or 2-STR-CD-Veh. 2STR-ETOH-CRA, 2STR-ETOH-FLU, and 2STR-ETOH-BUS do not differ from CD or 2STR-CD-Veh (P > 0.1). (b) Locomotor activity: *P < 0.05; **P < 0.01 compared to 2STR-ETOH-VEH or CD groups. Pretreatment with flumazenil (2STR-ETOH-FLU) and buspirone (2STR-ETOH-BUS) blocked the reduced activity induced by 2-STR-ETOH-Veh (P < 0.05), whereas pretreatment with CRA-1000 (2STR-ETOH-CRA) enhanced the reduction seen with 2-STR-ETOH-Veh (P < 0.05).