Enhanced heroin self-administration and distinct dopamine adaptations in female rats

Abstract

Increasing evidence suggests that females are more vulnerable to the harmful effects of drugs of abuse, including opioids. Additionally, rates of heroin-related deaths substantially increased in females from 1999 to 2017 [1], underscoring the need to evaluate sex differences in heroin vulnerability. Moreover, the neurobiological substrates underlying sexually dimorphic responding to heroin are not fully defined. Thus, we evaluated male and female Long Evans rats on acquisition, dose-responsiveness, and seeking for heroin self-administration (SA) as well as using a long access model to assess escalation of intake at low and high doses of heroin, 0.025 and 0.1 mg/kg/inf, respectively. We paired this with ex vivo fast-scan cyclic voltammetry (FSCV) in the medial nucleus accumbens (NAc) shell and quantification of mu-opioid receptor (MOR) protein in the ventral tegmental area (VTA) and NAc. While males and females had similar heroin SA acquisition rates, females displayed increased responding and intake across doses, seeking for heroin, and escalation on long access. However, we found that males and females had similar expression levels of MORs in the VTA and NAc, regardless of heroin exposure. FSCV results revealed that heroin exposure did not change single-pulse elicited dopamine release, but caused an increase in dopamine transporter activity in both males and females compared to their naïve counterparts. Phasic-like stimulations elicited robust increases in dopamine release in heroin-exposed females compared to heroin-naïve females, with no differences seen in males. Together, our results suggest that differential adaptations of dopamine terminals may underlie the increased heroin SA behaviors seen in females.

Fig. 1. Females show increased responding, intake, and seeking for heroin across doses, but similar rates of acquisition.

A Experimental timeline of behavioral procedure. B Female and male rats did not differ in total number of days to reach acquisition criteria. C Comparison of dose-responsivity for heroin revealed that females have greater responding for and D intake of heroin across various doses. E Responding during a seeking probe test was greater in female rats. F Correlation of responding during the seeking probe and total intake during the dose–response curve demonstrates a positive interaction between the two measures. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. n = 8–10 per group.

Fig. 2. Females exhibit increased responding and escalation for a low dose, 0.025 mg/kg/inf, of heroin during long access.

A Responding for heroin at 0.025 mg/kg/inf was greater in females rats compared to male rats during the second half of long access (sessions 6–10). B Average responding during the final 3 days of long access was increased in females. C Analysis of escalation ratios, responding during individual sessions compared to session 1, during long access demonstrate increased escalation of responding in female rats across sessions 6–10. D The slope of responding during the ten long access sessions was increased in female rats. *p < 0.05; **p < 0.01; ***p < 0.001. n = 8–10 per group.

Fig. 3. Female rats also have increased responding for a high dose, 0.1 mg/kg/inf, of heroin on long access.

A Experimental timeline of the behavioral procedure. B Female rats display increased lever press responding for heroin during the second half of long access (sessions 6–10). C Females show a trend for increased heroin responding during the final 3 days of long access (p = 0.0519). D Analysis of escalation ratios during long access demonstrate increased escalation of responding in female rats across sessions 6–10. E The slope of responding during the ten long access sessions was increased in female rats. *p < 0.05; **p < 0.01; ****p < 0.0001. n = 5 per group.

Fig. 4. Mu opioid receptor (MOR) protein levels do not differ between drug naïve or heroin-exposed male and female rats in the NAc or VTA.

A Representative blots of all four treatment groups. B MOR expression in the nucleus accumbens (NAc) did not differ between the groups. C No significant difference in MOR expression in the VTA across groups.

Fig. 5. Heroin self-administration increased the rate of dopamine reuptake in both sexes and phasic-like dopamine release in the medial NAc shell of female rats.

A Representative traces of electrically-evoked dopamine demonstrating increased uptake in heroin-exposed males and females compared to their respective heroin-naïve counterparts. B Stimulated dopamine release did not differ between any treatment groups. C The maximal rate of dopamine uptake (Vmax) was increased in heroin-exposed males and females. D Heroin-exposed and heroin-naïve males did not differ in dopamine elicited by multi-pulse stimulation trains at increasing frequencies; however, heroin females have greater dopamine release across frequencies than heroin-naïve females (E). F A comparison of both sexes and conditions reveal that the increase in phasic dopamine release is specific to female rats after heroin. G Comparison of average phasic/tonic ratios was increased in heroin females compared to their naïve counterparts, as depicted by the concentration of stimulated dopamine release at ten pulses over one pulse. *p < 0.05; **p < 0.01. n = 5–6 rats per group.