Opioid withdrawal produces sex-specific effects on fentanyl-vs.-food choice and mesolimbic transcription

Abstract

Background: Opioid withdrawal is a key driver of opioid addiction and an obstacle to recovery. However, withdrawal effects on opioid reinforcement and mesolimbic neuroadaptation are understudied and the role of sex is largely unknown.

Methods: Male (n=13) and female (n=12) rats responded under a fentanyl-vs.-food "choice" procedure during daily 2h sessions. In addition to the daily choice sessions, rats were provided extended access to fentanyl during 12h self-administration sessions. After two weeks of this self-administration regimen, the nucleus accumbens (NAc) and ventral tegmental area (VTA) of a subset of rats were subjected to RNA sequencing. In the remaining rats, a third week of this self-administration regimen was conducted, during which methadone effects on fentanyl-vs.-food choice were determined.

Results: Prior to opioid dependence, male and female rats similarly allocated responding between fentanyl and food. Abstinence from extended fentanyl access elicited similar increases in somatic withdrawal signs in both sexes. Despite similar withdrawal signs and extended access fentanyl intake, opioid withdrawal was accompanied by a maladaptive increase in fentanyl choice in males, but not females. Behavioral sex differences corresponded with a greater number of differentially expressed genes in the NAc and VTA of opioid-withdrawn females relative to males. Methadone blocked withdrawal-associated increases in fentanyl choice in males, but failed to further decrease fentanyl choice in females.

Conclusions: These results provide foundational evidence of sex-specific neuroadaptations to opioid withdrawal, which may be relevant to the female-specific resilience to withdrawal-associated increases in opioid choice and aid in the identification of novel therapeutic targets.

Keywords: Opioid withdrawal; RNA sequencing; decision making; nucleus accumbens; self-administration; sex differences.

Figure 1

Opioid withdrawal promotes sex-specific effects on fentanyl-vs.-food choice: decreased fentanyl choice in female relative to male rats. (A–C) Escalating self-administration of fentanyl and its effects on somatic withdrawal sign expression and bodyweight in male (n = 13, dotted line) and female (n = 12, solid line) rats. Abscissas: Experimental day. (A) Number of fentanyl injections (3.2 μg/kg/infusion unit dose) during each 12-hour session. (B) Number of somatic withdrawal signs (maximum of nine) observed 8 hours after the conclusion of each overnight self-administration session. (C) Change in bodyweight expressed as a percentage of baseline. Initial average weights (± SEM): male, 470 ± 12 g; female, 290 ± 5 g. (D–I) Sex comparison of opioid-withdrawal effects on fentanyl-vs.-food choice assessed 8 hours after overnight (6:00 pm–6:00 am) fentanyl self-administration. Abscissas: Intravenous unit fentanyl dose in μg/kg/injection. (D, F, H) Percentage of completed ratio requirements on the fentanyl-associated lever. (E, G, I) Number of choices completed per component. Points represent mean ± SEM. Data of week 1 and week 2 show averaged results of the choice sessions within the particular week. ∗Denotes significant difference relative to day 1A or baseline (B, C), which are placed above the symbol for female rats and below the symbol for male rats. #Denotes a significant main effect of sex. ^Denotes a sex × time interaction. Significance is defined as p < .05. See Table S1 in Supplement 1 for statistics relevant to each panel.

Figure 2

Withdrawal from overnight fentanyl self-administration produces opposing, sex-specific effects on fentanyl-vs.-food choice. Fentanyl-vs.-food choice results assessed 8 hours after overnight (6:00 pm–6:00 am) fentanyl self-administration sessions in male (n = 13, left) and female (n = 12, right) rats. Abscissas: Intravenous unit fentanyl dose in μg/kg/injection. (A, B) Percentage of completed ratio requirements on the fentanyl-associated lever. (C, D) Number of choices completed per component. Points represent mean ± SEM. ∗Denotes a significant difference at a unit fentanyl dose relative to baseline. Significance is defined as p < .05. See Table S1 in Supplement 1 for statistics relevant to each panel.

Figure 3

Female and male rats experience distinct transcriptional activity within the nucleus accumbens following repeated fentanyl withdrawal. Volcano plots depicting the DEGs from microdissected nucleus accumbens identified in (A) male (n = 4) and (B) female (n = 4) rats. DEGs were normalized to sex-matched rats subjected to saline-vs.-food protocol. DEG cutoffs for volcano plots are Wald test–adjusted p value < .05 and absolute log₂ fold change >1. (C) Union heatmap comparing female transcripts organized from downregulated to upregulated (top) to male transcripts of the same identity (bottom). Significance cutoffs for this and subsequent analyses are raw p value < .05 and absolute log₂ fold change >1.3, to enable broad pattern recognition. Venn diagrams depicting the degree of overlap in (D) upregulated and (E) downregulated transcripts across males and females. (F–H) GO terms enriched within the sex-specific regions of the Venn diagrams. ATP, adenosine triphosphate; DEG, differentially expressed gene; GO, Gene Ontology; miRNA, microRNA.

Figure 4

Female and male rats experience distinct transcriptional activity within the ventral tegmental area following repeated fentanyl withdrawal. Volcano plots depicting the DEGs from microdissected ventral tegmental area identified in (A) male (n = 3) and (B) female (n = 3) rats. DEGs were normalized to sex-matched rats subjected to saline-vs.-food protocol. DEG cutoffs for volcano plots are Wald test–adjusted p value < .05 and absolute log₂ fold change >1. (C) Union heatmap comparing female down- and upregulated transcripts (top) to male transcripts (bottom). Significance cutoffs for this and subsequent analyses are raw p value < .05 and absolute log₂ fold change >1.3, to enable broad pattern recognition. Degree of overlap in (D) upregulated and (E) downregulated transcripts across males and females. (F–I) GO terms enriched within the sex-specific regions of the Venn diagrams. DEG, differentially expressed gene; GO, Gene Ontology.

Figure 5

Acute methadone decreases fentanyl choice in male, but not female, opioid-withdrawn rats. Effects of acute methadone treatment on fentanyl choice in male (n = 6) and female (n = 5) rats during week 3 of overnight (6:00 pm–6:00 am) fentanyl self-administration. Top abscissas: Experimental day. Middle and bottom abscissas: Unit dose of fentanyl. Top ordinate: Number of fentanyl injections (3.2-μg/kg unit dose) during each 12-hour session in (A) male and (B) female rats. Middle ordinate: Percentage of completed ratio requirements on the fentanyl-associated lever in (C) male and (D) female rats. Bottom ordinate: Number of choices completed per component in (E) male and (F) female rats. Points represent mean ± SEM. ∗Denotes significant difference at a unit fentanyl dose relative to day 1 in panel (A). +Denotes a main effect of time in panel (A). ∗Denotes difference from saline within a sex in panels (B, C). Significance is defined as p < .05. See Table S1 in Supplement 1 for statistics relevant to each panel.