Predator odor (TMT) exposure potentiates interoceptive sensitivity to alcohol and increases GABAergic gene expression in the anterior insular cortex and nucleus accumbens in male rats

Abstract

Post-traumatic stress disorder (PTSD) confers enhanced vulnerability to developing comorbid alcohol use disorder (AUD). Exposure to the scent of a predator, such as the fox odor TMT, has been used to model a traumatic stressor with relevance to PTSD symptomatology. Alcohol produces distinct interoceptive (subjective) effects that may influence vulnerability to problem drinking and AUD. As such, understanding the lasting impact of stressors on sensitivity to the interoceptive effects of alcohol is clinically relevant. The present study used a 2-lever, operant drug discrimination procedure to train male Long-Evans rats to discriminate the interoceptive effects of alcohol (2 g/kg, i.g. [intragastrically]) from water. Upon stable performance, rats underwent a 15-min exposure to TMT. Two weeks later, an alcohol dose-response curve was conducted to evaluate the lasting effects of the TMT stressor on the interoceptive effects of alcohol. The TMT group showed a leftward shift in the effective dose (ED₅₀) of the dose-response curve compared to controls, reflecting potentiated interoceptive sensitivity to alcohol. TMT exposure did not affect response rate. GABAergic signaling in both the anterior insular cortex (aIC) and the nucleus accumbens (Acb) is involved in the interoceptive effects of alcohol and stressor-induced adaptations. As such, follow-up experiments in alcohol-naïve rats examined neuronal activation (as measured by c-Fos immunoreactivity) following TMT and showed that TMT exposure increased c-Fos expression in the aIC and the nucleus accumbens core (AcbC). Two weeks after TMT exposure, Gad-1 gene expression was elevated in the aIC and Gat-1 was increased in the Acb, compared to controls. Lastly, the alcohol discrimination and alcohol-naïve groups displayed dramatic differences in stress reactive behaviors during the TMT exposure, suggesting that alcohol exposure may alter the behavioral response to predator odor. Together, these data suggest that predator odor stressor results in potentiated sensitivity to alcohol, possibly through GABAergic adaptations in the aIC and Acb, which may be relevant to understanding PTSD-AUD comorbidity.

Keywords: AUD; Gad-1; Gat-1; PTSD; c-Fos; operant drug discrimination.

Figure 1 –. Experimental Timelines.

(A) Operant drug discrimination experiment (Experiment 1). (B) c-Fos experiment (Experiment 2). (C) Gene expression experiment (Experiment 3).

Figure 2 -. Operant lever response is under the control of the alcohol training dose (2 g/kg).

Prior to TMT exposure, (A) rats showed appropriate stimulus control with high alcohol-appropriate lever responses following alcohol administration and low alcohol-appropriate lever responses following water. (B) Response rate was lower following alcohol than water administration. Dotted lines at 80% indicate full substitution for the 2 g/kg alcohol training dose. *p<0.05

Figure 3 -. TMT exposure potentiates interoceptive sensitivity to alcohol 2 weeks after stressor exposure.

(A) 2 weeks after stressor, the TMT group showed increased alcohol-appropriate responses. (B) ED₅₀ of the dose-response curve for alcohol-appropriate responses was lower in the TMT group. (C) TMT did not affect response rate, but alcohol did affect response rate. Dotted lines at 80% indicate full substitution for the 2 g/kg alcohol training dose. *p<0.05

Figure 4 –. Molecular adaptations of TMT exposure in the anterior insular cortex and the nucleus accumbens.

The TMT group showed increased c-Fos immunoreactivity compared to controls in the (A) aIC and (B) AcbC. The TMT group showed increased gene expression of Gad-1 in the (C) aIC and Gat-1 in the (D) Acb compared to controls. *p<0.05

Figure 5 –. TMT exposure produces stress-reactive behaviors in alcohol-naïve and alcohol-experienced rat cohorts during the TMT exposure.

Stress reactive behaviors during the TMT exposure for the alcohol-experienced cohort (Experiment 1, A-F). (A) TMT exposure increased time spent digging, but (B) did not affect immobility. (C) TMT exposure decreased grooming behavior. (D) TMT increased distance traveled and (E) did not affect time spent on the TMT side. (F) TMT increased the number of midline crossings. Stress reactive behaviors during the TMT exposure for the alcohol-naive cohort (Experiment 3, G-L). (G) TMT exposure increased digging and (H) immobility. (I) TMT did not affect grooming. (J) TMT exposure decreased distance traveled, (K) time spent on the TMT side, and (L) the number of midline crossings. *p<0.05