Modulation of multiple ethanol withdrawal-induced anxiety-like behavior by CRF and CRF1 receptors

Abstract

Previous work demonstrated that rats subjected to multiple withdrawals from chronic ethanol exhibit a sensitization of anxiety-like behavior compared to animals withdrawn from treatment with an equal but continuous amount of ethanol. This study sought to examine whether corticotropin-releasing factor (CRF) could modulate this ethanol-withdrawal-induced anxiety-like behavior. Initially, rats were administered with CRF (1 microg) or vehicle intraventricularly on two occasions 5 days apart while on control diet (CD) followed by exposure to 7% ethanol diet (ED) for 5 days, with social interaction assessed 5 h into withdrawal. Social interaction was significantly reduced in the CRF-treated animals compared to vehicle-treated rats and vehicle- and CRF-treated rats maintained on CD, indicative that CRF given before ethanol exposure was capable of inducing an adaptive change that sensitized withdrawal-induced anxiety-like behavior. Next, the CRF(1) receptor antagonist CRA1000 (3 mg/kg, systemically), the CRF(2) receptor antagonist antisauvagine-30 (20 microg intraventricularly), or vehicle was injected 4 h after the ethanol was removed following the first and second cycles of chronic ethanol exposure and the effect on the multiple-withdrawal-induced anxiety-like behavior determined after the third withdrawal cycle. The CRF(1) receptor antagonist blocked the reduced social interaction behavior, whereas the CRF(2) receptor antagonist was without effect. Similar pretreatment with another CRF(1) receptor antagonist CP-154,526 (10 mg/kg systemically) during the first and second withdrawals also counteracted anxiety-like behavior. These findings indicate that the CRF system and CRF(1) receptors play key roles in the adaptive change responsible for the anxiety-like behavior induced by repeated withdrawals from chronic ethanol.

Fig. 1

Effects of acute intraventricular injection of CRF or vehicle on social interaction behavior. One week after rats were cannulated into the cerebral ventricles, the rats were infused with 1 μg CRF (n = 16) or artificial cerebrospinal fluid (n = 14). Some pairs of rats with the same treatment were placed in the open field arena 30 min later for the measurement of social interaction behavior. Other rats (8 of each) were placed in the open field with untreated control rats as their partners. CRF-and vehicle-treated rats exhibited similar levels of social interaction behavior in the two conditions, so the data were combined. The data represent the mean seconds ± S.E.M. of time spent in social interaction. The CRF-treated group (CRF–ICV) spent significantly less time in social interaction than either the vehicle-treated group (VEH–ICV) or the two partner groups (PART-C; PART-V), according to Tukey’s protected t tests (P < .01).

Fig. 2

Effects of prior treatment with CRF or vehicle on social interaction behavior in rats maintained on CD or withdrawn from a 5-day exposure to 7% ethanol. Artificial cerebrospinal fluid or CRF (1 μg) were infused 1 and 6 days before exposure to 5 consecutive days of 7% ED. The social interaction test was carried out 5 h after withdrawal from ethanol or at the same time in the afternoon 5 days after the last CRF treatment in the rats maintained on CD. The data represent the mean seconds ± S.E.M. of time spent in social interaction. The group pretreated with CRF and subsequently exposed to ethanol (ED – CRF) engaged in significantly less social interaction behavior than the group pretreated with vehicle and exposed to ethanol (ED–VEH) or the groups maintained throughout on CD (CD–CRF; CD–VEH), according to Tukey’s protected t tests (P < .01).

Fig. 3

Effects of CRA1000 on social interaction behavior of rats subjected to repeated withdrawals from ethanol. Rats were exposed to CD throughout (n = 8) or three cycles of 5 days of an ED (7% w/v). The rats were maintained on CD during the 2 days of withdrawal between the first and second, and the second and third cycles, and between ethanol withdrawal and behavior testing after the third cycle. One group was injected with CMC vehicle at 4 h into the first and second withdrawal (ED –VEH); one was pretreated with 3 mg/kg CRA1000 at the same times (ED–CRA/P); the final group was injected acutely with 1 mg/kg CRA1000 30 min before the social interaction test on the third withdrawal, 4.5 h after the ethanol was removed (ED–CRA/A). The other groups exposed to ED were also tested in the social interaction arena 5 h after removal of ethanol. The data represent the mean seconds ± S.E.M. of time spent in social interaction for eight rats per group. A one-way ANOVA revealed significant group differences (P < .01). Groups with different letters are significantly different according to Tukey’s test (P < .01).

Fig. 4

Effects of CRA1000 on line crosses of rats subjected to repeated withdrawals from ethanol. See legend of Fig. 3 for description of procedure. The data represent the mean ± S.E.M. line crosses for eight rats per group. A one-way ANOVA revealed significant group differences (P < .001). Groups with different letters are significantly different according to Tukey’s test (P < .01).