Chronic repeated predatory stress induces resistance to quinine adulteration of ethanol in male mice

Abstract

Background: Trauma related psychiatric disorders, such as posttraumatic stress disorder (PTSD), and alcohol use disorder (AUD) are highly comorbid illnesses that separately present an opposing, sex-specific pattern, with increased prevalence of PTSD in females and increased prevalence of AUD diagnoses in males. Likewise, PTSD is a risk factor in the development of AUD, with conflicting data on the impact of sex in the comorbid development of both disorders. Because the likelihood of experiencing more than one traumatic event is high, we aim to utilize chronic repeated predatory stress (CRPS) to query the extent to which sex interacts with CRPS to influence alcohol consumption, or cessation of consumption.

Methods: Male (n = 16) and female (n = 15) C57BL/6 J mice underwent CRPS or daily handling for two weeks during adolescence (P35-P49) and two weeks during adulthood (P65-P79). Following the conclusion of two rounds of repeated stress, behavior was assessed in the open field. Mice subsequently underwent a two-bottle choice intermittent ethanol access (IEA) assessment (P90-131) with the options of 20 % ethanol or water. After establishing drinking behavior, increasing concentrations of quinine were added to the ethanol to assess the drinking response to adulteration of the alcohol.

Results: CRPS increased fecal corticosterone concentrations and anxiety-like behaviors in the open field in both male and female mice as compared to control mice that had not been exposed to CRPS. Consistent with previous reports, we observed a sex difference in alcohol consumption such that females consumed more ethanol per gram of body mass than males. In addition, CRPS reduced alcohol aversion in male mice such that higher concentrations of quinine were necessary to reduce alcohol intake as compared to control mice. CRPS did not alter alcohol-related behaviors in female mice.

Conclusion: Collectively, we demonstrate that repeated CRPS can induce anxiety-like behavior in both sexes but selectively influences the response to ethanol adulteration in males.

Keywords: Adolescence; Alcohol; Anxiety; Chronic stress; Sex difference; Stress.

Figure 1:. Experimental Design and Timeline.

A) Experimental timeline. B) 31 mice were used in this experiment, 16 males and 15 females. Each sex was separated equally into non stress and chronic repeated predatory stress (CRPS) groups. Mice subject to CRPS were chosen at random at PND 35 before the beginning of CRPS.

Figure 2:. CRPS Increases Corticosterone Concentrations.

Corticosterone concentrations collected following the 15^th (PND 49) and 30^th (PND 79) exposure to handling (control) or chronic repeated predatory stress (CRPS group) were collected from fecal boli. Corticosterone concentrations were higher in the CRPS groups of both sexes at both timepoints and females had higher corticosterone concentrations in fecal boli than males. Reported values depict mean ± SEM. **p < 0.01, ****p < 0.0001.

Figure 3:. CRPS increases anxiety-like behaviors in the open field test.

A) Chronic repeated predatory stress (CRPS) decreased time spent in the center regardless of sex when compared to controls. B-D) Likewise, a history of CRPS increased locomotor activity in the open field. Reported values depict mean ± SEM. **p < 0.01, ***p < 0.001.

Figure 4:. CRPS does not alter alcohol intake.

A) Baseline ethanol intake by weight shows a sex difference between ethanol consumption (p < 0.0001), with females consuming more ethanol by weight than males regardless of chronic repeated predatory stress (CRPS) history. B) There were no baseline difference in ethanol preference in terms of sex or CRPS history. C) Total fluid intake shows a steady intake of fluid over time (p < 0.0001) as well as a significant sex difference with females consuming more total fluid than males (p < 0.0001). D) Males with a history of CRPS consumed more quinine adulterated ethanol than male controls. Within females, there was no significant difference in quinine adulterated ethanol consumption between the CRPS backgrounds. E) Males with a history of CRPS had a higher quinine adulterated 20% ethanol preference than control males. Control males significantly decreased quinine adulterated ethanol consumption at 100mg/L quinine; whereas, males with a history of CRPS significantly decreased at 200mg/L quinine adulterated ethanol. Within females, there was no significant difference in quinine adulterated ethanol preference between the controls and mice with a history of CRPS. F) Total fluid intake displays a significant effect of sex (p < 0.0001) and time (p < 0.0001). An interaction between time and sex show that females consumed more total fluid on average than males at baseline, 5mg/L, 150mg/L, and 200mg/L quinine. Reported values depict mean ± SEM. #p = 0.06, *p < 0.05, **p < 0.01, ****p < 0.0001.