Blockade of corticotropin-releasing factor-1 receptors in the infralimbic cortex prevents stress-induced reinstatement of alcohol seeking in male Wistar rats: Evidence of interaction between CRF1 and orexin receptor signaling

Abstract

Alcohol use dysregulates responsivity to stress, which is mediated by corticotropin-releasing factor (CRF). With repeated cycles of alcohol use, the hypothalamic-pituitary-adrenal axis becomes hyporesponsive, rendering individuals vulnerable to the reinstatement of alcohol-seeking behavior during stressful episodes. Orexin (Orx; also called hypocretin) plays a well-established role in regulating diverse physiological processes, including stress, and interacts with CRF. The infralimbic cortex (IL) is a CRF-rich region. Anatomical evidence suggests that CRF and Orx interact in this area. To test the behavioral implication of CRF and Orx transmission in the IL during the stress-induced reinstatement of alcohol-seeking behavior, male Wistar rats were trained to self-administer 10% alcohol for 3 weeks. The rats then underwent two weeks of extinction training (identical to the alcohol self-administration sessions, but alcohol was withheld). The day after the last extinction session, the rats received a bilateral intra-IL injection of the CRF₁ receptor antagonist CP154,526 (0.6 μg/0.5 μl/side), the dual Orx receptor antagonist TCS1102 (15 μg/0.5 μl/side), or their combination and then were tested for the footshock stress-induced reinstatement of alcohol-seeking behavior. CP154,526 significantly prevented reinstatement, but TCS1102 did not produce such an effect. Interestingly, the co-administration of TCS1102 and CP154,526 reversed the effect of CP154,526 alone, and footshock stress induced a significant increase in Crhr1 and Hcrtr2 mRNA expression in the IL. These results demonstrate a functional interaction between Orx receptor and CRF₁ receptor signaling and suggest that CRF₁ receptor antagonism may ameliorate stress-induced alcohol-seeking behavior.

Keywords: Alcohol; CP154,526; Corticotropin-releasing factor; Orexin; TCS1102.

Fig. 1.

Timeline of the experimental procedures. (A) Behavioral procedure for stress-induced reinstatement and representation of injection sites in animals that received bilateral IL and PL cannulations. (B) Spontaneous locomotion testing procedure and representation of injection sites. o, Rats with correct injection sites. ×, Rats with missed injection sites. (C) Molecular procedure (qPCR).

Fig. 2.

Time course of alcohol self-administration over 21 sessions of training and 10 extinction sessions. (A) Rats acquired alcohol self-administration over the 21 training sessions. (B) Extinction of alcohol-seeking behavior rats over the 10 sessions. The data are expressed as mean + SEM. ***p < 0.001, vs. inactive lever presses.

Fig. 3.

Effects of CP154,526 (CP), TCS1102 (TCS), and CP154,526+TCS1102 (CP+TCS) on the stress-induced reinstatement of alcohol-seeking behavior. (A) Intermittent footshock stress precipitated alcohol-seeking behavior in rats that received vehicle (VEH). The administration of CP154,526 in the IL prevented the stress-induced reinstatement of alcohol seeking, an effect that was blocked by the co-administration of CP154,526+TCS1102. The administration of CP154,526 in the PL had no effect on the stress-induced reinstatement of alcohol-seeking behavior. (B) Co-administration of CP154,256 and TCS1102 increased inactive lever responses following administration in the IL. The data are expressed as mean + SEM. ⁺p < 0.05, vs. respective sham injection; *p < 0.05, vs. VEH.

Fig. 4.

Effects of CP154,526+TCS1102 on spontaneous locomotion. (A) CP154,526+TCS1102 administration increased the total distance traveled compared with VEH administration. (B) No significant difference in speed was found between VEH and CP154,526+TCS1102. The data are expressed as mean + SEM. *p < 0.05.

Fig. 5.

Effects of alcohol extinction and intermittent footshock stress on the gene expression of Crhr1, Hcrtr1, and Hcrtr2. (A) Rats acquired alcohol self-administration over the 21 training sessions. (B) Extinction of alcohol-seeking behavior over the 10 extinction sessions. (C) Stress increased Crhr1 mRNA expression compared with extinction and naive rats but decreased following extinction. (D) Increase in Hcrtr1 mRNA expression following extinction and stress. (E) Increase in Hcrtr2 mRNA expression by stress. The data are expressed as mean + SEM. ***p < 0.001, vs. inactive lever; ⁺p < 0.05, vs. extinction (EXT). *p < 0.05, vs. naive.