Pharmacological modulation of repeated ethanol withdrawal-induced anxiety-like behavior differs in alcohol-preferring P and Sprague-Dawley rats

Abstract

Previous work with Sprague-Dawley (SD) rats indicated that subjecting these rats to multiple episodes of ethanol diet could provoke anxiety-like responses. Because alcohol-preferring P rats have been reported to have neurochemical alterations in many systems shown to modulate anxiety-like responses, P rats were compared to SD rats. Rats were subjected to one or three cycles of 5 days' exposure to 4.5% or 7% ethanol diet to assess anxiety-like behavior. The social interaction test was conducted 5 h after ethanol was removed. Other groups of P and SD rats were injected with flumazenil (5 mg/kg), a benzodiazepine (BZD) receptor antagonist, CP-154,526 (10 mg/kg), CRF1 receptor antagonist, SB243,213, a 5-HT2C receptor inverse agonist, or vehicle during the 1st and 2nd withdrawals but not the third. After a single 5-day cycle of ethanol exposure, SD rats did not exhibit a change in social interaction, but P rats exhibited a decrease after exposure to the 7% ethanol. Both strains of rats exhibited anxiety-like behavior following three cycles of exposure to ethanol and the concentration of ethanol in the diet did not influence the response. It was confirmed that flumazenil, CP-154,523, and SB243,213 had prophylactic effects on anxiety-like behavior in the SD rats. Neither flumazenil nor SB243,213 was as effective in the P rats, while the CRF1 receptor antagonist completely counteracted the reduced social interaction in repeatedly withdrawn P rats. A small study showed that buspirone, a 5-HT1A agonist, also had prophylactic effects in P rats. These findings show that alcohol-preferring P rats exhibit anxiety-like behavior more readily following exposure to ethanol-containing diets and that this behavior is counteracted more readily by pretreatment with a CRF1 receptor antagonist than with BZD or 5-HT2C receptor antagonists.

Fig. 1

Repeated exposures to 7% ethanol diet in P and SD rats. Rats were exposed to control liquid diet continuously or 7% ethanol diet (ED) in three cycles of 5 days, with 2 days of withdrawal after each cycle. The social interaction test was carried out after the 1st or 3rd cycles. Upper panel: data represent the mean sec±S.E.M. for 8–10 rats; lower panel: data represent the mean line crosses±S.E.M. Groups with different letters are significantly different, p < 0.01, according to 2-way ANOVA and Tukey’s protected t tests.

Fig. 2

Repeated exposures to 4.5% ethanol diet in P and SD rats. Rats were exposed to control liquid diet continuously or 4.5% ethanol diet (ED) in three cycles of 5 days, with 2 days of withdrawal after each cycle. The social interaction test was carried out after the 1st or 3rd cycles. Upper panel: data represent the mean sec±S.E.M. for 8–10 rats. Lower panel: data represent the mean line crosses±S.E.M. Groups with different letters are significantly different, p < 0.01, according to 2-way ANOVA and Tukey’s protected t tests.

Fig. 3

Alcohol intake in P or SD rats exposed to three cycles of 4.5 or 7% ethanol diets. Data represent the mean g/kg±S.E.M. ethanol intake. *Significant difference, p < 0.01, from SD rats of the same condition, t test.

Fig. 4

Prophylactic effects of flumazenil in P or SD rats subjected to three cycles of exposure to 7% ethanol diet. Rats were exposed continuously to control diet or to three cycles of 5 days’ exposure to 7% ethanol, with 2 days of withdrawal between cycles. Flumazenil (5 mg/kg) or CMC vehicle were injected i.p. 4 h into the 1st and 2nd withdrawals only. The social interaction test was conducted 5 h into the 3rd withdrawal. Upper panel: data represent the mean±S.E.M sec of social interaction for 8–10 rats. Lower panel mean line crosses±S.E.M. Groups with different letters are significantly different, p < 0.01, according to 2-way ANOVA and Tukey’s protected t tests.

Fig. 5

The prophylactic effects of SB243,213, 5-HT_2C receptor inverse agonist, on anxiety-like behavior in P and SD rats induced by repeated ethanol withdrawals. Rats were exposed to control diet (CD) continuously or to three cycles of 5 days’ exposure to 4.5% ethanol, with two days of withdrawal between cycles. Injections of CMC vehicle (EDV) or 3 mg/kg SB243,213 (EDSB) were given at 4 h into withdrawal from the 1st and 2nd cycles. The social interaction test was conducted 5 h into the 3rd withdrawal. Upper panel: data represent the mean±S.E.M. sec of social interaction for 6 – 8 rats. Lower panel: mean line crosses±S.E.M. Groups with different letters are significantly different, p < 0.01, according to 2-way ANOVA and Tukey’s protected t tests.

Fig. 6

Prophylactic effects of CP-154,526, CRF₁ receptor antagonist, on anxiety-like behavior of P Rats exposed to repeated cycles of ethanol exposure. Rats were exposed to control diet (CD) continuously or three cycles of 5 days’ exposure to 4.5% ethanol diet (EDV or EDCP). Injections of the vehicle CMC or CP-154,526 (10 mg/kg) were given 4 h into the 1st and 2nd withdrawals only. The social interaction test was conducted 5 h into the 3rd withdrawal. Upper panel: data represent the mean±S.E.M. sec of social interaction for 8 – 10 rats. Lower panel: data represent the mean line crosses±S.E.M. *Significantly different from CD group, p < 0.01, Tukey’s protected t test. +Significantly different from EDV group, p < 0.01, Tukey’s protected t test. Groups: CDP—control diet in P rats; EDPV—ethanol diet in P rats given vehicle; EDPCP—ethanol diet in P rats given CP-154526.

Fig. 7

Counteraction of restraint stress facilitation of anxiety-like behavior induced by ethanol withdrawal in P rats by CP-154,526, CRF₁ receptor antagonist. Rats were exposed to control diet (CDP) throughout or to a final period of 5 days of 4.5% ethanol diet (ED). The rats exposed to ED were either unstressed (EDP) or subjected to 1 h of restraint stress 1 and 6 days prior to being exposed to ethanol. One group received CMC vehicle (EDPST) 30 prior to restraint and the other received CP-154,526 (EDPSTCP, 10 mg/kg). Upper panel: data represent the mean±S.E.M. sec of social interaction for 5–8 rats. Lower panel: data represent the mean line crosses±S.E.M. *Significantly different from CD group, p < 0.01, Tukey’s protected t test. +Significantly different from EDV group, p < 0.01, Tukey’s protected t test.