Female Rats Are More Susceptible To High-Risk Addictive Behaviors Than Male Rats

Background: Opioid addiction is a chronic, relapsing disorder, characterized by bouts of drug taking and drug seeking The ongoing opioid epidemic in the United States has resulted in nearly half a million deaths since 1999 (NCHS, National Vital Statistics System, Mortality), and overdose-related deaths during the early months of COVID-19 increased more than 40% from the same time in 2019 (ODMAP) Although men have historically been more likely to die of a fatal opioid overdose, the death rate has been rising faster in women than men, though whether this is due to longerterm transitions from prescription opioids to highly potent opioids (e g , fentanyl) or innate differences in vulnerability to the addictive properties of opioids is unknown Of note, reports suggest women may escalate drug intake at a faster rate and be more prone to relapse than men Importantly, however, only a subset of individuals transitions from casual drug use to addiction, so whether women are generally more susceptible to addiction or whether a greater subset of women progress to addiction is unknown The historical exclusion of female subjects from preclinical research has contributed to the incomplete understanding of sex differences in addiction;moreover, recent efforts to begin studying sex differences have focused almost exclusively on psychostimulant addiction Addiction develops in part from disruptions within the cortico-basal ganglia circuit (C-BG), a network integral for learning, decisionmaking, and motivation However, whether these disruptions differ between females and males and contribute to differences in addictive behaviors remains to be studied Methods: To examine the impact of sex differences on vulnerability to opioid addiction, separate groups of female and male Sprague-Dawley rats were tested in classical addiction models In experiment 1, rats (n=54) were trained to selfadminister heroin (0 075mg/kg per infusion) using an intermittent-access procedure that produces variability in addiction severity and allows extraction of six notable addictive behaviors: drug intake, intake consistency, drug seeking, motivation, extinction, and cue-induced-reinstatement Individualaddictive behaviors were combined into an overall severity score that was used to classify individual subjects as low-risk or high-risk for addiction In experiment 2, rats (n=34) were trained on a heroin conditioned place preference (CPP) procedure to assess sensitivity to heroin reward After an initial day of exploration (15min), rats received four days of conditioning, with saline in the morning and heroin (1mg/kg or 3mg/kg) in the afternoon The following day, rats were again allowed to freely explore the box to assess preference for the two contexts In experiment 3, rats (n=32) underwent locomotor sensitization to assess sensitivity to the sensitizing effects of heroin After three days of habituation (30min, saline), rats received nine days of sensitization (30min, 2mg/kg heroin) in chambers equipped with infrared beam-breaks to monitor locomotor activity Immediately after completion of behavioral testing, brains were extracted and processed for cFos immunohistochemistry (experiment 1: cue-induced reinstatement;experiment 2: post-test) in key nuclei of the C-BG Results: Intermittent-access heroin self-administration produced subsets of low-risk and high-risk rats that significantly differed in daily heroin intake, total heroin consumption, drug seeking, motivation, extinction, and cue-induced reinstatement (all p 0 05) However, female rats were significantly more likely to be classified as high-risk than male rats (p \u003c 0 05), suggesting greater vulnerability to the development of heroin taking and heroin seeking behaviors in females Conversely, females and males did not differ in their development of heroin CPP at either dose tested, nor did they differ in development of heroin sensitization (all p \u003c 0 05) Interestingly, cFos immunoh stochemistry revealed divergent activation in the nucleus accumbens and the prefrontal cortex between females and males, suggesting a sex difference in the encoding of heroin cues Conclusions: Epidemiological research has shown women to be more vulnerable to the negative consequences of opioid addiction, yet preclinical research has focused almost exclusively on male subjects for decades The data presented here demonstrates increased vulnerability to high-risk addictive behaviors in female rats as well as non-overlapping patterns of neuronal activation following presentation of drug-paired cues and contexts Experiments are currently underway to further explore sex differences in drug- and cue-encoding by the C-BG, including fiber photometry recordings from the nucleus accumbens during heroin CPP and proteomic analysis of nucleus accumbens synaptosomes following heroin sensitization Together, these studies will clarify how C-BG network dynamics shift during the development of heroin addictive behaviors and will explore whether the encoding of heroin cues follows divergent pathways in females and males