The role of central amygdala corticotropin-releasing factor in predator odor stress-induced avoidance behavior and escalated alcohol drinking in rats

Abstract

Post-traumatic stress disorder (PTSD) is characterized by avoidance of trauma-associated stimuli and amygdala hyperreactivity, and is highly co-morbid with alcohol use disorder (AUD). Our lab uses a predator odor (bobcat urine) stress model that produces conditioned avoidance of an odor-paired context in a subset of rats, mirroring avoidance symptoms that manifest in some but not all humans exposed to trauma. We previously showed that after predator odor stress, Avoiders exhibit escalated operant alcohol self-administration (SA), higher aversion-resistant operant alcohol responding, hyperalgesia, and greater anxiety-like behavior compared to unstressed Controls. We also showed previously that systemic antagonism of corticotropin-releasing factor-1 receptors (CRFR1) reduced escalation of operant alcohol SA in rats not indexed for avoidance, that corticotropin-releasing factor (CRF) infusions into the central amygdala (CeA) produced conditioned place avoidance in stress-naïve rats, and that intra-CeA infusion of a CRFR1 antagonist reduced hyperalgesia in Avoiders. Here, we show that avoidance behavior is persistent after repeated predator odor exposure. In addition, Avoiders showed lower weight gain than Controls after predator odor re-exposure. In the brain, higher avoidance was correlated with higher number of c-Fos + cells and CRF immunoreactivity in the CeA. Finally, we show that intra-CeA CRFR1 antagonism reversed post-stress escalation of alcohol SA and reduced avoidance behavior in Avoiders. Collectively, these findings suggest that elucidation of the mechanisms by which CRFR1-gated CeA circuits regulate avoidance behavior and alcohol SA may lead to better understanding of the neural mechanisms underlying co-morbid PTSD and AUD.

Keywords: Corticotropin-releasing factor; Operant alcohol self-administration; Predator odor stress.

Figure 1.

Predator odor stress produces avoidance behavior that is stable and changes in weight gain. (A) Timeline for testing the effect of repeated predator odor exposure on avoidance behavior and weight gain. (B) Time in odor-paired chamber during Posttests 1 (solid bars) and 2 (hatched bars) (after 1^st and 2^nd predator odor exposure). *p = 0.01, Avoiders vs. Non-Avoiders. (C) Change in body weight 1 day after predator odor exposure (*p’s < 0.001) and (D) 7 days after predator odor exposure (*p’s ≤ 0.05). (E) Change in body weight 1 day after 2^nd predator odor exposure (*p = 0.04). (F) Adrenal and (G) thymus weights of Controls, Non-Avoiders, and Avoiders.

Figure 2.

Avoiders have more c-Fos+ cells in CeA than Non-Avoiders. (A) Timeline for testing the effect of predator odor exposure on c-Fos induction. (B) c-Fos+ cells in CeA, (C) CeM, and (D) CeL (*p ≤ 0.05). (E) Correlation between avoidance and number of c-Fos+ cells in CeA. (F) Representative images of c-Fos staining in Control, (G) Single Stress, (H) Non-Avoider, and (I) Avoider rats. Scale bar = 100 μm.

Figure 3.

Avoiders have greater CRF immunoreactivity in CeA than Non-Avoiders. (A) Timeline for testing the effect of predator odor exposure on CRF immunoreactivity. (B) CRF immunoreactivity in CeA, (C) CeM, and (D) CeL (*p’s < 0.05). (E) Correlation between avoidance and CeA CRF immunoreactivity. (F) Representative images of CRF staining in CeA of Control, (G) Single Stress, (H) Non-Avoider, and (I) Avoider rats. Scale bar = 100 μm.

Figure 4.

Intra-CeA CRFR1 antagonism reduces escalated alcohol drinking in Avoiders and avoidance behavior. (A) Timeline for testing the effect of intra-CeA CRFR1 antagonism on alcohol drinking and avoidance behavior. (B) Number of alcohol presses across 5 × 30 min operant alcohol oral self-administration test sessions. Rats were treated with R121919 (0.25 μg/side) or vehicle immediately before test sessions 1–5. All rats were treated with vehicle immediately before test session 0. (C) Number of alcohol presses averaged across test sessions 1–5. ^#p = 0.008, Avoiders vs. Controls in the Vehicle group. *p = 0.001, R121919 vs. Vehicle treatment in the Avoider group. (D) Time spent in odor-paired chamber during Posttests 1 and 2 by Non-Avoiders and (E) Avoiders. All rats received intra-CeA vehicle infusions 1 day prior to Posttest 1 and were treated repeatedly with intra-CeA R121919 or vehicle during the 5 intervening operant alcohol self-administration sessions between Posttests 1 and 2 (last infusion occurred 1 day prior to Posttest 2). (F) Time spent in odor-paired chamber during Posttests 1 and 3 by Non-Avoiders and (G) Avoiders. Rats were treated with intra-CeA R121919 or vehicle immediately before Posttest 3. *p = 0.003, Avoiders treated with R121919, Posttest 1 vs. 3. (H) Schematic of infusion sites within the CeA.