Transcriptional Profile of Exercise-Induced Protection Against Relapse to Cocaine Seeking in a Rat Model.

Background: Exercise has shown promise as a treatment for cocaine use disorder; however, the mechanism underlying its efficacy has remained elusive. Methods: We used a rat model of relapse (cue-induced reinstatement) and exercise (wheel running, 2 hours/day) coupled with RNA sequencing to establish transcriptional profiles associated with the protective effects of exercise (during early withdrawal [days 1-7] or throughout withdrawal [days 1-14]) versus noneffective exercise (during late withdrawal [days 8-14]) against cocaine-seeking and sedentary conditions. Results: As expected, cue-induced cocaine seeking was highest in the sedentary and late-withdrawal exercise groups; both groups also showed upregulation of a Grin1-associated transcript and enrichment of Drd1-Nmdar1 complex and glutamate receptor complex terms. Surprisingly, these glutamate markers were also enriched in the early- and throughout-withdrawal exercise groups, despite lower levels of cocaine seeking. However, a closer examination of the Grin1-associated transcript revealed a robust loss of transcripts spanning exons 9 and 10 in the sedentary condition relative to saline controls that was normalized by early- and throughout-withdrawal exercise, but not late-withdrawal exercise, indicating that these exercise conditions may normalize RNA mis-splicing induced by cocaine seeking. Our findings also revealed novel mechanisms by which exercise initiated during early withdrawal may modulate glutamatergic signaling in dorsomedial prefrontal cortex (e.g., via transcripts associated with non-NMDA glutamate receptors or those affecting signaling downstream of NMDA receptors), along with mechanisms outside of glutamatergic signaling such as circadian rhythm regulation and neuronal survival. Conclusions: These findings provide a rich resource for future studies aimed at manipulating these molecular networks to better understand how exercise decreases cocaine seeking.

Role of nucleus accumbens dopamine 2 receptors in motivating cocaine use in male and female rats prior to and following the development of an addiction-like phenotype.

A hallmark of cocaine use disorder (CUD) is dysfunction of dopamine signaling in the mesolimbic pathway, including impaired dopamine 2 (D2) receptor signaling. One of the most replicated findings in human imagining studies is decreased striatal D2 receptor binding in individuals with a substance use disorder relative to healthy controls; however, the vast majority of the data is from males, and findings in smokers suggest this molecular shift may not translate to females. The goal of this study was to determine whether there are sex differences in the role of D2 receptors in motivating cocaine use prior to and following the development of an addiction-like phenotype (defined by an enhanced motivation for cocaine relative to the short-access, ShA, group). Here, male and female rats were given ShA (20 infusions/day, 3 days) or extended-access (ExA; 24h/day, 96 infusions/day, 10 days) to cocaine self-administration and then following 14 days of withdrawal, were tested under a progressive-ratio schedule to assess motivation for cocaine use. Once a stable level of motivation was established, the effect of NAc-infusions of the D2 receptor antagonist eticlopride (0-3.0 µg/side) were examined. We found that in males, eticlopride was less effective at decreasing motivation for cocaine following ExA versus ShA self-administration, particularly at low eticlopride doses. In contrast, in females, there were no differences in the effectiveness of eticlopride between ExA and ShA. These findings indicate that males, but not females, become less sensitive to NAc-D2 receptor antagonism with the development of an addiction-like phenotype.

Estradiol Enhances the Development of Addiction-Like Features in a Female Rat Model of Opioid Use Disorder.

INTRODUCTION: Women are more vulnerable than men in many aspects of opioid use disorder (OUD); a major theory of sex differences in substance use disorders is that these differences are due to ovarian hormones with estradiol enhancing vulnerability in females. However, most of this evidence is for psychostimulants and alcohol; evidence with opioids is sparse. Thus, the goal of this study was to determine the impact of estradiol on vulnerability in females in a rat model of OUD. METHOD: Following self-administration training, ovariectomized (OVX) females with (E) or without (V) estradiol replacement were given extended (24 h/day), intermittent access (2, 5-min trials/h) to fentanyl for 10 days. Then, the development of three key features of OUD were assessed, including physical dependence, defined by the magnitude and time course of weight loss during withdrawal; an enhanced motivation for fentanyl, assessed using a progressive-ratio schedule; and relapse vulnerability, assessed using an extinction/cue-induced reinstatement procedure. These later two characteristics were examined following 14 days of withdrawal when the phenotypes are known to be highly expressed. RESULTS: OVX+E females self-administered markedly higher levels of fentanyl under extended, intermittent-access conditions and showed a longer time course of physical dependence, a greater increase in motivation for fentanyl, and an enhanced sensitivity to the reinstating effects of fentanyl-associated cues compared to OVX+V rats. Severe health complications were also observed in OVX+E, but not OVX+V females, during withdrawal. CONCLUSION: These results indicate that, as with findings with psychostimulants and alcohol, estradiol enhances vulnerability in females to developing opioid addiction-like features and serious opioid-related health complications.

Sex/Gender Differences in the Time-Course for the Development of Substance Use Disorder: A Focus on the Telescoping Effect.

Sex/gender effects have been demonstrated for multiple aspects of addiction, with one of the most commonly cited examples being the "telescoping effect" where women meet criteria and/or seek treatment of substance use disorder (SUD) after fewer years of drug use as compared with men. This phenomenon has been reported for multiple drug classes including opioids, psychostimulants, alcohol, and cannabis, as well as nonpharmacological addictions, such as gambling. However, there are some inconsistent reports that show either no difference between men and women or opposite effects and a faster course to addiction in men than women. Thus, the goals of this review are to evaluate evidence for and against the telescoping effect in women and to determine the conditions/populations for which the telescoping effect is most relevant. We also discuss evidence from preclinical studies, which strongly support the validity of the telescoping effect and show that female animals develop addiction-like features (e.g., compulsive drug use, an enhanced motivation for the drug, and enhanced drug-craving/vulnerability to relapse) more readily than male animals. We also discuss biologic factors that may contribute to the telescoping effect, such as ovarian hormones, and its neurobiological basis focusing on the mesolimbic dopamine reward pathway and the corticomesolimbic glutamatergic pathway considering the critical roles these pathways play in the rewarding/reinforcing effects of addictive drugs and SUD. We conclude with future research directions, including intervention strategies to prevent the development of SUD in women. SIGNIFICANCE STATEMENT: One of the most widely cited gender/sex differences in substance use disorder (SUD) is the "telescoping effect," which reflects an accelerated course in women versus men for the development and/or seeking treatment for SUD. This review evaluates evidence for and against a telescoping effect drawing upon data from both clinical and preclinical studies. We also discuss the contribution of biological factors and underlying neurobiological mechanisms and highlight potential targets to prevent the development of SUD in women.

Sex- and Dose-Dependent Differences in the Development of an Addiction-Like Phenotype Following Extended-Access Fentanyl Self-Administration.

Opioid use disorder (OUD) is a major epidemic in the United States, and fentanyl is a major culprit. The National Institute on Drug Abuse has highlighted an urgent need for research on the risks and outcomes of OUD with fentanyl; a better understanding of sex/gender differences is also critically needed given that the opioid epidemic has been particularly impactful on women. In response to this need, we developed a rat model of OUD with fentanyl and showed that sex impacts relapse vulnerability following extended-access self-administration under a low fentanyl dose. Here, our goal was to determine sex differences across a broad dose range, including high doses expected to maximize the expression of addiction-like features (e.g., vulnerability to relapse and physical dependence). Male and female rats were assigned to self-administer one of four fentanyl doses (0.25, 0.75, 1.5, and 3.0 µg/kg/infusion), and once they acquired, they were given extended (24-h/day), intermittent access (2, 5 min trials/h, fixed-ratio 1) to fentanyl for 10 days. Physical dependence (spontaneous weight loss) was assessed during early withdrawal, and relapse vulnerability was assessed on withdrawal day 15 using an extinction/cue-induced reinstatement procedure. Despite markedly higher intake in the high- versus low-dose groups, each group responded similarly during relapse testing (extinction and cue-induced reinstatement). However, number of infusions, or frequency of use, during extended access was predictive of later vulnerability to relapse, whereas total intake impacted physical dependence given that weight loss only occurred following the discontinuation of fentanyl self-administration at the three highest doses. Females self-administered more fentanyl each day and within each binge (active trial), and had longer lasting weight loss during withdrawal than males. Relapse vulnerability was also higher in females than males and highest in females tested during estrus. These findings indicate that sex is an important risk factor for patterns and levels of fentanyl intake, relapse, and physical dependence, and while fentanyl intake predicts physical dependence, frequency of use predicts relapse.

Sex differences in the neuroadaptations associated with incubated cocaine-craving: A focus on the dorsomedial prefrontal cortex.

Introduction: Women have a shorter course from initial cocaine use to meeting the criteria for cocaine use disorder as compared to men. Preclinical findings similarly indicate that females develop key features of an addiction-like phenotype faster than males, including an enhanced motivation for cocaine and compulsive use, indicating that this phenomenon is biologically based. The goals of this study were to determine whether cocaine-craving, another key feature of addiction, also develops sooner during withdrawal in females than males and to determine whether there are sex differences in the molecular mechanisms associated with its development focusing on markers known to mediate cocaine-craving in males (i.e., dorsomedial prefrontal cortex, dmPFC, expression of brain-derived neurotrophic factor exon-IV, Bdnf-IV, and NMDA receptor subunits, Grin2a, Grin2b, and Grin1). Methods: Cocaine-craving was assessed following extended-access cocaine self-administration and 2, 7, or 14 days of withdrawal using an extinction/cue-induced reinstatement procedure. Tissue was obtained from the dmPFC immediately after reinstatement testing and gene expression changes were analyzed using real-time qPCR. Results: In males, cocaine-craving (total extinction and cue-induced reinstatement responding) progressively increased from early to later withdrawal time-points whereas in females, cocaine-craving was already elevated during early withdrawal (after 2 days) and did not further increase at later withdrawal time-points. Levels of cocaine-craving, however, were similar between the sexes. Gene expression changes differed markedly between the sexes such that males showed the expected relapse- and withdrawal-associated changes in Bdnf-IV, Grin2a, Grin2b, and Grin1 expression, but females only showed a modest increase Grin1 expression at the intermediate withdrawal timepoint. Discussion: These findings indicate that cocaine-craving is similarly expressed in males and females although the time-course for its incubation appears to be accelerated in females; the molecular mechanisms also likely differ in females versus males.

Females develop features of an addiction-like phenotype sooner during withdrawal than males.

RATIONALE: Women meet criteria for substance use disorder after fewer years of drug use than men; this accelerated time course, or telescoping effect, has been observed for multiple drugs, including cocaine. Preclinical findings similarly indicate an enhanced vulnerability in females to developing an addiction-like phenotype; however, it is not yet known if this phenotype develops faster in females versus males. OBJECTIVES: The goal of this study was to determine using a rat model whether two key features of addiction in humans, an enhanced motivation for cocaine and compulsive use, emerge sooner during withdrawal from extended access cocaine self-administration in females versus males. METHODS: Motivation for cocaine, as assessed under a progressive-ratio reinforcement schedule, was determined prior to and following extended access cocaine self-administration (24 h/day, 96 infusions/day, 10 days) and after 7, 14, or 60 days of withdrawal. Compulsive use, or use despite punishment, was evaluated once progressive-ratio responding stabilized by adding histamine, an aversive stimulus, to the cocaine solutions. RESULTS: Motivation for cocaine increased from baseline sooner during withdrawal in females than males (at 7 versus 14 days); motivation was also highest in the 60-day group. Histamine decreased progressive-ratio responding for cocaine in both sexes, although effects were greatest in males in the 7-day withdrawal group; males reached the female-level of resistance to histamine punishment by 14 days of withdrawal. CONCLUSIONS: Female rats developed addition-like features sooner during withdrawal than male rats indicating that the telescoping effect observed in humans is biologically based. Additionally, like drug-seeking/craving, motivation for cocaine and measures of compulsive use incubate over withdrawal.

A buprenorphine-validated rat model of opioid use disorder optimized to study sex differences in vulnerability to relapse.

RATIONALE: Opioid use disorder (OUD) is a major epidemic in the USA. Despite evidence indicating that OUD may be particularly severe for women, preclinical models have yet to establish sex as a major factor in OUD. OBJECTIVES: Here, we examined sex differences in vulnerability to relapse following intermittent access fentanyl self-administration and protracted abstinence and used buprenorphine, the FDA-approved treatment for OUD, to test the validity of our model. METHODS: Following acquisition of fentanyl self-administration under one of two training conditions, male and female rats were given extended, 24-h/day access to fentanyl (0.25 µg/kg/infusion, 10 days) using an intermittent access procedure. Vulnerability to relapse was assessed using an extinction/cue-induced reinstatement procedure following 14 days of abstinence; buprenorphine (0 or 3 mg/kg/day) was administered throughout abstinence. RESULTS: Levels of drug-seeking were high following extended-access fentanyl self-administration and abstinence; buprenorphine markedly decreased drug-seeking supporting the validity of our relapse model. Females self-administered more fentanyl and responded at higher levels during subsequent extinction testing. Buprenorphine was effective in both sexes and eliminated sex and estrous phase differences in drug-seeking. Interestingly, the inclusion of a time-out during training had a major impact on later fentanyl self-administration in females, but not males, indicating that the initial exposure conditions can persistently impact vulnerability in females. CONCLUSIONS: These findings demonstrate the utility of this rat model for determining sex and hormonal influences on the development and treatment of OUD.