Sensitivity and Resilience to Predator Stress-Enhanced Ethanol Drinking Is Associated With Sex-Dependent Differences in Stress-Regulating Systems - PubMed (original) (raw)
Sensitivity and Resilience to Predator Stress-Enhanced Ethanol Drinking Is Associated With Sex-Dependent Differences in Stress-Regulating Systems
Mehrdad Alavi et al. Front Behav Neurosci. 2022.
Abstract
Stress can increase ethanol drinking, and evidence confirms an association between post-traumatic stress disorder (PTSD) and the development of alcohol use disorder (AUD). Exposure to predator odor is considered a traumatic stressor, and predator stress (PS) has been used extensively as an animal model of PTSD. Our prior work determined that repeated exposure to intermittent PS significantly increased anxiety-related behavior, corticosterone levels, and neuronal activation in the hippocampus and prefrontal cortex in naïve male and female C57BL/6J mice. Intermittent PS exposure also increased subsequent ethanol drinking in a subgroup of animals, with heterogeneity of responses as seen with comorbid PTSD and AUD. The present studies built upon this prior work and began to characterize "sensitivity" and "resilience" to PS-enhanced drinking. Ethanol drinking was measured during baseline, intermittent PS exposure, and post-stress; mice were euthanized after 24-h abstinence. Calculation of median and interquartile ranges identified "sensitive" (>20% increase in drinking over baseline) and "resilient" (no change or decrease in drinking from baseline) subgroups. Intermittent PS significantly increased subsequent ethanol intake in 24% of male (↑60%) and in 20% of female (↑71%) C57BL/6J mice in the "sensitive" subgroup. Plasma corticosterone levels were increased significantly after PS in both sexes, but levels were lower in the "sensitive" vs. "resilient" subgroups. In representative mice from "sensitive" and "resilient" subgroups, prefrontal cortex and hippocampus were analyzed by Western Blotting for levels of corticotropin releasing factor (CRF) receptor 1, CRF receptor 2, CRF binding protein, and glucocorticoid receptor, vs. separate naïve age-matched mice. In prefrontal cortex, CRF receptor 1, CRF receptor 2, CRF binding protein, and glucocorticoid receptor levels were significantly higher in "sensitive" vs. naïve and "resilient" mice only in females. In hippocampus, CRF receptor 1, CRF receptor 2 and glucocorticoid receptor levels were significantly lower in "resilient" vs. naïve and "sensitive" mice across both sexes. These results indicate that sex strongly influences the effects of ethanol drinking and stress on proteins regulating stress and anxiety responses. They further suggest that targeting the CRF system and glucocorticoid receptors in AUD needs to consider the comorbidity of PTSD with AUD and sex of treated individuals.
Keywords: C57BL/6J mice; CRH; corticosterone; hippocampus; hypothalamic-pituitary-adrenal axis; predator odor; prefrontal cortex.
Copyright © 2022 Alavi, Ryabinin, Helms, Nipper, Devaud and Finn.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Figures
FIGURE 1
Experimental timeline. Male and female C57BL/6J mice were treated as described. Ethanol drinking (10% ethanol vs. water) was measured for 23 h per day in the home cage. Predator stress (PS) exposure occurred twice per week for 2 weeks, where mice were exposed to dirty rat bedding for 30 min. After the fourth PS exposure, post-stress drinking was measured for approximately 1 1/2 weeks. Mice were euthanized at 24 h after the final drinking session (and consumption of water for 24 h). For the Western Blot study, tissue from age-matched naïve mice was used as the comparator group. mPFC, medial prefrontal cortex.
FIGURE 2
Identification of “sensitive” and “resilient” subgroups. (A) Horizontal scatterplot of the distribution of percent (%) change in baseline (BL) ethanol drinking after four predator stress (PS) exposures, with median and interquartile ranges for male (triangles) and female (circles) C57BL/6J mice. Mice chosen for the “sensitive” (red) and “resilient” (blue) subgroups were the extremes of the distribution and did not include animals in the middle (gray, “intermediate”). (B) Frequency distribution histogram for the % change in BL ethanol drinking after intermittent PS further illustrate the distinct “sensitive” and “resilient” subgroups. (C) Mean ± SEM % change in ethanol intake data for the three subgroups, with the number of animals in each subgroup shown in parentheses. The proportion of mice in the “sensitive” subgroup is shown below the mean data for this group. Data for “intermediate” group are shown for informational purposes. Analyses were conducted across the “sensitive” and “resilient” subgroups. ***p < 0.001 vs. resilient subgroup.
FIGURE 3
Different pattern of ethanol intake (A,B) and preference (C,D) after intermittent predator stress (PS) in “sensitive” vs. “resilient” subgroups. Depicted are mean ± SEM intake of 10% ethanol (g/kg/23 h) intake (A,B) and 10% ethanol preference (C,D) for the “sensitive” and “resilient” animals depicted in Figure 2. Individual data points are shown for the sexes combined (males = triangles; females = circles). Ethanol intake and preference were averaged into five blocks: baseline (BL), PS1 (after first PS, 2 days), PS2 (after second PS, 3 days), PS3 (after third PS, 2 days), and PS4 (after fourth PS, 3 days). (A,B) Ethanol intake began increasing significantly vs. BL after PS1 in the “sensitive” subgroup, whereas ethanol intake decreased significantly vs. BL after PS1-4 in the “resilient” subgroup. Following PS4, ethanol intake increased over BL by 62% (males) and 59% (females) in the “sensitive” subgroup, and decreased from BL by 30% (males) and 27% (females) in the “resilient” subgroup. (C,D) Ethanol preference increased significantly vs. BL after PS2-4 in the “sensitive” subgroup, whereas ethanol preference decreased significantly vs. BL after PS1-4 in the “resilient” subgroup. Following PS4, ethanol preference increased over BL by 44% (males) and 61% (females) in the “sensitive” subgroup, and decreased from BL by 18% (males) and 16% (females) in the “resilient” subgroup. **p < 0.01, ***p ≤ 0.001 vs. respective BL.
FIGURE 4
Predator stress (PS) significantly increases plasma corticosterone (CORT) levels, with greater increases in the “resilient” vs. “sensitive” subgroup. Depicted are mean ± SEM plasma CORT levels for male and female mice following exposure to the first and fourth predator stress (PS1 and PS4) vs. values in naïve mice. Individual data points are shown collapsed across sex (males = triangles, females = circles) and PS. Group size: naïve (15 male, 21 female), “sensitive” subgroup (25 male, 11 female per PS), “resilient” subgroup (17 males, 10 females per PS). Plasma CORT levels increased significantly over naïve values following PS, and they were significantly higher in the “resilient” (↑124%) subgroup vs. values in the “sensitive” (↑88%) subgroup. *p < 0.05 vs. “sensitive” subgroup; ###p < 0.001 vs. naïve.
FIGURE 5
Sex- and subgroup-dependent changes in protein levels related to stress-regulating systems in the medial prefrontal cortex (mPFC) after a history of ethanol drinking and intermittent predator stress exposure. There were divergent, treatment- and subgroup-induced alterations in the proteins examined, with sex differences observed for CRF-R1, CRF-R2, and GR. Representative animals from the “sensitive” and “resilient” subgroups depicted in Figures 2, 3 were chosen for the Western blot analyses, and values were compared to separate groups of naïve mice. Values are mean ± SEM levels that were normalized to β-actin and then normalized to the respective naïve group (dashed line) for male (A) and female (B) mice. Changes in relative protein levels were compared using normalized optical density measurements. Representative bands from the immunoblots for each protein are included beneath each panel on the graph. Representative immunoblots for each protein, brain region, and sex are depicted in Supplementary Figures 1–4. Group size: naïve (10–11 male, 8–10 female), “sensitive” subgroup (7 male, 5 female), “resilient” subgroup (10 males, 8 females). *p ≤ 0.05, ***p ≤ 0.001 vs. respective “resilient” subgroup; ##p ≤ 0.01, ###p ≤ 0.001 vs. respective naïve. The results depicted reflect either separate analyses for each sex (CRF R1, CRF R2, GR) or analyses with the sexes combined (CRF BP). See Table 2 for ANOVAs. CRF R1, corticotropin releasing factor receptor 1; CRF R2, corticotropin releasing factor receptor 2; CRF BP, corticotropin releasing factor binding protein; GR, glucocorticoid receptor.
FIGURE 6
Sex- and subgroup-dependent changes in protein levels related to stress-regulating systems in the hippocampus after a history of ethanol drinking and intermittent predator stress exposure. There were treatment- and subgroup-induced alterations in the proteins examined, with sex differences only observed for CRF-BP. Representative animals from the “sensitive” and “resilient” subgroups depicted in Figures 2, 3 were chosen for the Western blot analyses, and values were compared to separate groups of naïve mice. Values are mean ± SEM levels that were normalized to β-actin and then normalized to the respective naïve group (dashed line) for male (A) and female (B) mice. Changes in relative protein levels were compared using normalized optical density measurements. Representative bands from the immunoblots for each protein are included beneath each panel on the graph. Representative immunoblots for each protein, brain region, and sex are depicted in Supplementary Figures 1–4. Group size is the same as in Figure 5. *p ≤ 0.05, ***p ≤ 0.001 vs. respective “resilient” subgroup; ##p ≤ 0.01, ###p ≤ 0.001 vs. respective naïve. The results depicted reflect either separate analyses for each sex (CRF BP) or analyses with the sexes combined (CRF R1, CRF R2, GR). See Table 2 for ANOVAs. CRF R1, corticotropin releasing factor receptor 1; CRF R2, corticotropin releasing factor receptor 2; CRF BP, corticotropin releasing factor binding protein; GR, glucocorticoid receptor.
References
- Albrechet-Souza L., Hwa L. S., Han X., Zhang E. Y., DeBold J. F., Miczek K. A. (2015). Corticotropin releasing factor binding protein and CRF2 receptors in the ventral tegmental area: modulation of ethanol binge drinking in C57BL/6J mice. Alcohol. Clin. Exp. Res. 39 1609–1618. 10.1111/acer.12825 - DOI - PMC - PubMed
- American Psychiatric Association (2013). Diagnostic and Statistical Manual of Mental Disorders, 5th Edn. Arlington, TX: American Psychiatric Publishing.
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