The synthetically produced predator odor 2,5-dihydro-2,4,5-trimethylthiazoline increases alcohol self-administration and alters basolateral amygdala response to alcohol in rats

Abstract

Post-traumatic stress disorder (PTSD) is a psychiatric illness that can increase the risk for developing an alcohol use disorder (AUD). While clinical data has been useful in identifying similarities in the neurobiological bases of these disorders, preclinical models are essential for understanding the mechanism(s) by which stressors increase the risk for escalated alcohol consumption. The purpose of these studies was to examine if exposure of male Long-Evans rats to the synthetically derived predator odor 2,5-dihydro-2,4,5-trimethylthiazoline (TMT; a component of fox feces) would increase sweetened alcohol self-administration, potentially by facilitating transfer of salience towards cues, and alter neuronal response to alcohol as measured by the immediate early gene c-Fos. In experiment 1, rats exposed to repeated (4×) TMT showed reductions in port entries in Pavlovian conditioned approach and increases in sweetened alcohol self-administration. In experiment 2, rats exposed to repeated TMT showed blunted basolateral amygdala c-Fos response to alcohol. In experiment 3, rats exposed to single, but not repeated TMT, showed increases in sweetened alcohol self-administration, and no change in anxiety-like behavior or hyperarousal. In experiment 4, rats continued to show a significant corticosterone response to TMT after repeated exposures. In summary, exposure of male rats to TMT can cause escalations in sweetened alcohol self-administration and reduction in BLA response to alcohol. These studies outline and utilize a novel preclinical model that can be used to further neurobiological understanding of the emergence of escalated alcohol consumption following stress exposure.

Keywords: Alcohol; Amygdala; Glucocorticoids; Predator odor; Stress; c-Fos.

Fig. 1

Timelines for Experiments 1–4. (a) In Experiment 1, male rats (n = 6/group) were trained on PCA for 8 sessions prior to 4 TMT exposures. Rats then returned to PCA for 8 additional sessions and underwent an open field test 48 hours after the final TMT exposure. After PCA rats were trained to self-administer sweetened alcohol. (b) In Experiment 2, male rats (n = 6/dose control group and 10/dose TMT group) were exposed to TMT 4 times. 7 days later, rats received 2 g/kg alcohol or water (i.g.) and were sacrificed 90 minutes later to examine brain regional expression of c-Fos. (c) In Experiment 3, male rats (n = 12/group) were exposed 0, 1, or 4 times to TMT and examined for anxiety-like behavior and hyperarousal 7 days later. Rats were then trained to self-administer sweetened alcohol. (d). In Experiment 4, male rats (n = 8/group) were exposed to TMT 4 times and blood was drawn 30 minutes post-exposure on days 1 and 4 for analysis of plasma corticosterone

Fig. 2

Effects of TMT exposure on PCA behavior. (a,b) Rats (n = 6/group) exposed to TMT showed no change in lever presses (sign-tracking) and significant reductions in port-entry elevation score (goal-tracking) compared to controls. (c,d) Rats exposed to TMT showed no change in latency to first lever press, and a trend for increased latency to first port-entry as compared to controls. (e) PCA indices for TMT exposed rats did not differ significantly from controls. Dotted line represents 4 TMT exposures across 7 days, between PCA sessions 8 and 9. * - p < 0.05 versus control

Fig. 3

Acquisition and maintenance of sweetened alcohol self-administration following TMT exposure in Experiment 1. (a) Rats (n = 6/group) exposed to TMT showed increased alcohol lever responses across sucrose fading compared to controls. (b) TMT-exposed rats consumed significantly more alcohol than controls across sucrose fading. (c) TMT-exposed rats showed increased alcohol lever responses during maintenance compared to controls. (d) There was no significant difference in total alcohol intake across the maintenance phase between TMT-exposed animals and controls. * - p < 0.05 versus control

Fig. 4

Alcohol induced c-Fos in naïve (n = 4–6/group) and TMT-exposed (n = 10/group) rats. (a,b) Following an alcohol (2 g/kg, IG) injection, there were significant reductions in PL and IL c-Fos IR. (c) Alcohol treated rats showed increased CeA c-Fos IR regardless of TMT history. (d) Alcohol reduces BLA c-Fos IR in naïve, but not TMT-exposed rats. The shaded area on the brain atlas illustrations show the quantified region. (e) Representative images of BLA c-Fos immunoreactivity at 10X magnification. * - p < 0.05 versus water, + - p < 0.05 vs control – water

Fig. 5

Acquisition and maintenance of sweetened alcohol self-administration following TMT exposure in Experiment 3 (n = 11–12/group). (a) There were no significant differences between groups in alcohol lever responses during sucrose fading. (b) The sTMT group showed significantly higher alcohol intake than the control group during sucrose fading. (c) The sTMT group showed significantly higher alcohol lever responses during maintenance than controls. (d) The sTMT group had significantly higher alcohol intake than both the control and rTMT groups across the maintenance phase. sTMT: single TMT exposure; rTMT: repeated TMT exposures. * - p < 0.05 versus control, + - p < 0.05 versus rTMT

Fig. 6

Acute physiological and neuroendocrine stress responses to TMT across repeated exposures. (a) On days 1–3 the rTMT group produced significantly more fecal boli than the control group and sTMT group (control exposure days). On day 4 both rTMT and sTMT (TMT exposure day) groups produced significantly more fecal boli than controls (n = 12/group). (b) Plasma corticosterone 30 minutes after exposure (at onset of dark cycle 7:30 – 9:00 pm) was significantly elevated in TMT exposed animals compared to controls on both exposure day 1 and 4 (n = 8/group). TMT exposures. + - p < 0.05 versus control and sTMT, * - p < 0.05 versus control