Sex differences in the long-term effects of past stress on alcohol self-administration, glucocorticoid sensitivity and phosphodiesterase 10A expression

Abstract

Stress responses differ by sex, and females are more susceptible to developing mental illnesses because of past stress, including alcohol use disorder. Investigation of neuroadaptations governing the interaction between past stress and future alcohol intake remains understudied in females. A history of footshock stress previously was shown to increase alcohol self-administration under relapse-like conditions in male rats, associated with elevated phosphodiesterase 10A (PDE10A) mRNA expression in the dorsomedial prefrontal cortex and basolateral amygdala. To identify sex differences in long-term stress effects, male and female Wistar rats were exposed to light-cued footshock stress prior to alcohol self-administration training. While past stress did not alter acquisition or extinction, reacquisition self-administration was oppositely impacted by past stress. Stress history slightly increased reacquisition self-administration in males, but reduced alcohol self-administration in females, relative to same-sex controls. Control females self-administered less alcohol following glucocorticoid receptor inhibition by mifepristone, which did not significantly alter alcohol consumption in the other groups. PDE10A expression in synaptically enriched fractions also differed by sex and stress history in a brain region-specific manner. Females expressed more synaptic PDE10A than males in basolateral amygdala and dorsolateral striatum, regardless of stress history, whereas dorsomedial prefrontal cortex PDE10A protein levels matched group differences in reacquisition drinking, but also were expressed at much lower levels than all other regions examined. Together, these data show stress history differentially impacts alcohol self-administration and PDE10A expression by sex, with control females consuming alcohol in a glucocorticoid receptor-sensitive fashion that may relate to sex differences in PDE10A expression.

Keywords: Ethanol; Footshock; Mifepristone; Relapse; Sex differences; Stress history.

Figure 1.. Stress history differentially alters alcohol-reinforced operant behavior by sex.

Male (A,C) and female (B,D) rats without (Control, open shapes) and with (Stress History, filled shapes) previous exposure to light-cued footshocks performed operant responses over 1-h sessions to earn alcohol reinforcers. Presses on the active lever (A,B) were reinforced as labeled in panels A-B, with alcohol delivery after 1 (fixed ratio 1, FR1) or 3 (FR3) lever presses, or with no alcohol delivery during extinction (EXT). Data are presented separately by sex for ease of visualization. Presses on the inactive lever (C,D) were never reinforced. Data are displayed as mean ± S.E.M. and shown across all 39 operant sessions. * p < 0.05, Control vs. Stress History. n = 10, Control Males; n = 12, Stress History Males; n = 11, Control Females; n = 8, Stress History Females.

Figure 2.. Alcohol intake during FR3-reinforced operant sessions differs by sex, and stress history bi-directionally changes reacquisition alcohol intake in male vs. female rats.

Male (A,C,E,F) and female (B,D,E,F) rats without (Control, open shapes) and with (Stress History, filled shapes) previous exposure to light-cued footshock self-administered 0.1-mL 10% (v/v) alcohol reinforcers in 1-h operant sessions. (A,B) Alcohol intake, expressed as g alcohol consumed normalized to body weight in kg, is shown for all FR3 sessions across the experiment. (C,D) Change in alcohol intake after extinction training, displayed as g/kg alcohol intake normalized to each rat’s pre-extinction average g/kg intake, is shown for each reacquisition session. (E) Corticosterone levels were measured from plasma collected at euthanasia, expressed as ng corticosterone per ml plasma. (F) Average alcohol intake across the reacquisition period was positively related to corticosterone levels at euthanasia. Data are presented as mean ± S.E.M. (A-E) or individual values with sex indicated by shape (circles – males; squares – females) and group indicated by fill (open – Control; filled – Stress History) (F). Multi-session data are displayed on separate panels by sex for ease of visualization (A-D). * p < 0.05, Control vs. Stress History; ** p < 0.01, Male vs. Female; ⁺⁺⁺ p < 0.001, Control vs. Stress History (main effect of group across the reacquisition period). n = 8–12 per group.

Figure 3.. Alcohol intake is selectively reduced by mifepristone in the highest-drinking, footshock stress-naïve females.

Male and female rats were treated with mifepristone (0, 30 mg/kg, subcutaneous) 90 min prior to reacquisition self-administration sessions using a within subjects design, counterbalanced by group across treatment days. Mifepristone treatment significantly reduced active lever presses (A) and weight-normalized alcohol intake (B) in Control females, but minimally change inactive lever pressing (C). Data are expressed as mean ± S.E.M., with sex, group and dose as indicated. (D) Rats consuming more alcohol (g/kg) on vehicle treatment day showed greater reductions in active lever pressing after mifepristone treatment, expressed as percent change in active lever presses, [(active lever presses, mifepristone) – (active lever presses, vehicle)] / (active lever presses, vehicle). Individual data points by rat are displayed with sex indicated by shape (circles – males; squares – females) and group indicated by fill (open – Control; filled – Stress History). * p < 0.05, Vehicle vs. Mifepristone or Control, Vehicle vs. Stress History, Vehicle, as indicated. n = 8 per group.

Figure 4.. Striatal PDE10A near the synapse differs by sex in a subregion-specific pattern, with DLS PDE10A correlated with corticosterone and expressed at higher levels in females.

Tissue punches from dorsolateral striatum (DLS), dorsomedial striatum (DMS), and nucleus accumbens (NAc) were collected 48 h after the final alcohol self-administration session and were enriched for synaptic content via centrifugal fractionation. Western blot membranes were probed for PDE10A, then Coomassie stained for total protein normalization. Data are calculated as a ratio of PDE10A to total protein load as measured by Coomassie staining, normalized across blots via a 10 μg rat brain lysate standard, and expressed in histograms as mean ± S.E.M. (in arbitrary units, a.u.), with individual data points overlaid. Representative images are included in the top panels, displaying PDE10A and Coomassie staining, as labeled. Samples are designated as control (Ct) or stress history (SH), as indicated above the blot images. (A) DLS PDE10A expression was higher in females compared to males, regardless of Stress History. (B) DLS PDE10A levels significantly correlated with serum corticosterone levels at the time of brain collection, with data presented as individual data points by rat, with sex indicated by shape (circles – males; squares – females) and group indicated by fill (open – Control; filled – Stress History). (C) PDE10A expression was slightly, but not significantly, lower in DMS female vs. male samples. (D) No differences were observed in NAc samples. **p < 0.005 Male vs. Female, all n’s as indicated on histograms. Full size blot images can be found in Supplemental Figure 1.

Figure 5.. Basolateral (BLA), but not central (CeA), amygdala PDE10A is elevated adjacent to the synapse in females, relative to males, independent of stress history.

Synaptically enriched protein samples processed from tissue punches collected 48 h after the final alcohol self-administration session were resolved by Western blotting. Membranes were probed for PDE10A, then Coomassie stained for total protein normalization. Data are calculated as a ratio of PDE10A to total protein load as measured by Coomassie staining, normalized across blots via a 10 μg rat brain lysate standard, and expressed in histograms as mean ± S.E.M. (in arbitrary units, a.u.), with individual data points overlaid. Representative images (top panels) are included for each region to show PDE10A protein bands and Coomassie staining, as labeled. Samples are designated as control (Ct) or stress history (SH), as indicated above the blot images. Quantification (bottom panels) shows higher PDE10A expression in females compared to males for BLA (A), a pattern not seen in CeA (B), independent of Stress History. * p < 0.05, Male vs. Female. All n’s as indicated on histograms. Full size blot images can be found in Supplemental Figure 2.

Figure 6.. Prefrontal cortex PDE10A expression differs by sex and stress exposure in the dorsomedial (dmPFC) but not the ventromedial (vmPFC) subdivision.

Synaptically-enriched protein samples (A1,B1) or whole lysate (A2,B2) processed from tissue punches collected 48 h after the final alcohol self-administration session were resolved by Western blotting. Membranes were probed for PDE10A, then Coomassie stained for total protein normalization. Data are calculated as a ratio of PDE10A to total protein load, as measured by Coomassie staining, normalized to a 10 μg rat brain lysate standard, and expressed in histograms as mean ± S.E.M. (in arbitrary units, a.u.), with individual data points overlaid. Representative images (top panel) show PDE10A and Coomassie staining, as labeled. Samples are designated as control (Ct) or stress history (SH), as indicated above the blot images. Quantification of PDE10A (bottom panels) in synaptically enriched dmPFC fractions (A1) shows very low levels overall, with higher expression in females vs. males in the Control group, as well as reduced PDE10A levels in Stress History females relative to same-sex Controls. This difference was not observed in dmPFC lysates from the same rats (A2), where PDE10A levels were somewhat higher overall and showed a trend towards higher expression in females. In the neighboring vmPFC, PDE10A did not significantly differ by Sex or Stress History in either the synaptically enriched fraction (B1) or the total lysate (B2), and PDE10A levels were more prominent after synaptic enrichment than in lysates. *p < 0.05, vs. Control Female. All n’s as indicated on histograms. Full size blot images can be found in Supplemental Figure 3.