Anna K. Radke

Introduction: Alcohol use disorders (AUDs) and excessive alcohol use are a growing public health burden across the world, with sex differences in their presentation. Rodent models of drinking have revealed consistent sex differences in ethanol consumption, preference, and compulsive-like intake. Previous studies have found that ovariectomy reduces ethanol drinking. Increased consumption has also been observed in male, but not female, mice with XY sex chromosome complement. These results and others suggest an interaction between gonadal and genetic sex, but the influence of circulating gonadal hormones has not been assessed. Methods: Three experiments used removal of the gonads and the Four Core Genotypes (FCG) mouse line to further investigate interactions between gonadal hormones and sex chromosome complement on alcohol drinking. Experiment 1: ovariectomy (OVX) was conducted before the onset of drinking in XX/ Sry - and XY/ Sry - FCG females. Experiment 2: orchiectomy (ORX) was performed in C57BL/6J male mice. Experiment 3: ORX was conducted in XX/ Sry + and XY/ Sry + FCG males. Voluntary, binge-like intake of 15% ethanol was measured in a 4-h, two-bottle choice drinking-in-the-dark (DiD) paradigm followed by tests of quinine-resistant drinking (0-500 µM) and, in Experiments 2 and 3 only, ethanol vs. sucrose (1.5-12%) choice. Results: Removal of the testes increased 15% ethanol consumption and preference in male C57BL/6J mice and consumption in FCG mice. Conversely, there was evidence suggesting that ORX reduced quinine-resistance and ethanol vs. sucrose choice in C57BL/6J, but not FCG, males. Previously observed increases in binge-like consumption following OVX were not replicated in FCG female mice. Sex chromosome complement did not interact with gonadal status for any measure. Discussion: These studies demonstrate that testicular hormones suppress voluntary binge-like ethanol consumption in male mice and provide some evidence for facilitatory effects on two measures of inflexible drinking – quinine resistance and ethanol vs. sucrose choice. These findings are interesting to consider alongside clinical data demonstrating positive associations between testosterone and alcohol drinking in men. There was no evidence for interactions between circulating gonadal hormones and sex chromosome complement, which may suggest gonadal sex-dependent effects of sex chromosome complement on drinking rely on organizational changes earlier in development.

Background Studying sex differences in alcohol drinking behaviors in rodents can reveal molecular and physiological differences that may be relevant to addiction. Prior work with the Four Core Genotypes (FCG) mouse line suggests that sex chromosome complement and gonadal type contribute to ethanol consumption in male vs. female mice. Here, we confirmed an effect of sex chromosome complement on alcohol intake in Sry+ male mice using an intermittent access two-bottle choice model. Methods Mice consumed 20% ethanol or water for 13 sessions over 4.5 weeks. Three days after drinking, tissue from the nucleus accumbens was collected and analyzed using RNASeq. Results Robust transcriptional changes were identified in male (XY/Sry+) mice drinking ethanol vs. water but not in females (XX/Sry-). There were also limited effects of gonad type (Sry- vs. Sry+) and sex chromosome complement (XX vs. XY) on differential gene expression, suggesting that neither of these factors serves as the primary driver of the transcriptional response to alcohol. Conclusions Both alcohol drinking and RNASeq analyses demonstrate that sex chromosome complement and gonad type interact to produce distinct behavioral and molecular profiles in males and females.

Background: In humans, early life stress (ELS) is associated with an increased risk for developing both alcohol use disorder (AUD) and posttraumatic stress disorder (PTSD). We previously used an infant footshock model in rats that produces stress‐enhanced fear learning (SEFL) and increases aversion‐resistant alcohol drinking to explore this shared predisposition. The goal of the current study was to test the viability of this procedure as a model of comorbid PTSD and AUD in male and female C57BL/6J mice.Methods: Acute ELS was induced using 15 footshocks on postnatal day (PND) 17. In adulthood, alcohol drinking behavior was tested in one of three two‐bottle choice drinking paradigms. In continuous access, mice were given 24 h access to 5% and 10% ethanol and water for five consecutive drinking sessions each. In limited access drinking in the dark, mice were given 2 h of access to 15% ethanol and water across 15 sessions 3 h into the dark cycle. In intermittent access, mice were presented with 20% ethanol and water Monday, Wednesday, and Friday, for four consecutive weeks. In a fifth week of intermittent access drinking, increasing concentrations of quinine (10, 100, and 200 mg/L) were added to the ethanol to test aversion‐resistant drinking. Intermittent access drinking was tested with and without a period of adolescent drinking (PND 35).Results: Infant footshock did not alter drinking in the continuous or limited access tasks. In the intermittent access task, adult consumption and preference were lower in shocked mice when adolescent drinking was included. Aversion resistance was greater in females following infant footshock and adolescent drinking.Conclusions: Our results demonstrate that ELS, in the form of infant footshock on PND 17, must be followed by a period of adolescent drinking to affect adult alcohol consumption in mice.

Mu-opioid receptors (MORs) in the amygdala and striatum are important in addictive and rewarding behaviors. The transcription factor Foxp2 is a genetic marker of intercalated (ITC) cells in the amygdala and a subset of striatal medium spiny neurons (MSNs), both of which express MORs in wild-type mice and are neuronal subpopulations of potential relevance to alcohol-drinking behaviors. For the current series of studies, we characterized the behavior of mice with genetic deletion of the MOR gene Oprm1 in Foxp2 -expressing neurons (Foxp2-Cre/Oprm1 ^fl/fl ). Male and female Foxp2-Cre/Oprm1 ^fl/fl mice were generated and heterozygous Cre+ (knockout) and homozygous Cre-(control) animals were tested for aversion-resistant alcohol consumption using an intermittent access (IA) task, operant responding for a sucrose reward, conditioned place aversion (CPA) to morphine withdrawal, and locomotor sensitization to morphine. The results demonstrate that deletion of MOR on Foxp2 -expressing neurons renders mice more sensitive to quinine-adulterated ethanol (EtOH). Mice with the deletion (vs. Cre-controls) also consumed less alcohol during the final sessions of the IA task, responded less for sucrose under an FR3 schedule, and were less active at baseline and following morphine injection. Foxp2 -MOR deletion did not impair the ability to learn to respond for reward or develop a conditioned aversion to morphine withdrawal. Together, these investigations demonstrate that Foxp2 -expressing neurons may be involved in escalation of alcohol consumption and the development of compulsive-like alcohol drinking.

: Heavy alcohol use and binge drinking are important contributors to alcohol use disorder (AUD). The endogenous opioid system has been implicated in alcohol consumption and preference in both humans and animals. The mu opioid receptor (MOR) is expressed on multiple cells in the striatum, however little is known about the contributions of specific MOR populations to alcohol drinking behaviors. The current study used mice with a genetic deletion of MOR in cholinergic cells (ChAT- Cre/Oprm1 ^fl/fl ) to examine the role of MORs expressed in cholinergic interneurons (CINs) in home cage self-administration paradigms. Male and female ChAT-Cre/Oprm1 ^fl/fl mice were generated and heterozygous Cre+ (knockout) and Cre- (control) mice were tested for alcohol and nicotine consumption. In Experiment 1, binge-like and quinine-resistant drinking was tested using 15% ethanol (EtOH) in a two- bottle, limited-access Drinking in the Dark paradigm. Experiment 2 involved a six-week intermittent access paradigm in which mice received 20% EtOH, nicotine, and then a combination of the two drugs. Experiment 3 assessed locomotor activity, sucrose preference, and quinine sensitivity. Deleting MORs in cholinergic cells did not alter consumption of EtOH in Experiment 1 or 2. In Experiment 1, the MOR deletion resulted in greater consumption of quinine-adulterated EtOH in male Cre+ mice (vs. Cre-). In Experiment 2, Cre+ mice demonstrated a significantly lower preference for nicotine but did not differ from Cre- mice in nicotine or nicotine + EtOH consumption. Overall fluid consumption was also heightened in the Cre+ mice. In Experiment 3, Cre+ females were found to have greater locomotor activity and preference for sucrose vs. Cre- mice. These data suggest that cholinergic MORs are not required for EtOH, drinking behaviors but may contribute to aversion resistant EtOH drinking in a sex- dependent manner.

Rationale Female rodents consume more ethanol (EtOH) than males and exhibit greater aversion-resistant drinking in some paradigms. Ovarian hormones promote EtOH drinking but the contribution of ovarian hormones to aversion-resistant drinking has not been assessed. Objectives We aimed to investigate the role of ovarian hormones to aversion-resistant drinking in female mice in a drinking in the dark (DID) task. Methods Female C57BL/6 J mice first underwent an ovariectomy (OVX, n = 16) or sham (SHAM, n = 16) surgery. Four weeks following surgery, mice underwent a DID paradigm where they were given access to water and 15% EtOH 3 h into the dark cycle for up to 4 h across 15 drinking sessions. To assess frontloading behavior, bottles were weighed at 30 min, 2 h, and 4 h. Aversion-resistance was tested by adding escalating concentrations of quinine (0, 100, 250, and 500 µM) to the 15% EtOH bottle on sessions 16 – 19. Results Removal of the ovaries reduced EtOH consumption in OVX subjects. When assessing aversion-resistant EtOH drinking, mice with ovarian hormones (SHAM) reduced consumption of 250 and 500 µM quinine in EtOH, while OVX subjects exhibited aversion-resistance at all quinine concentrations. OVX mice had greater frontloading for quinine + EtOH at higher concentrations of quinine. Conclusions These results indicate that circulating ovarian hormones may be protective against the development of aversion-resistant EtOH drinking and call for further investigation of the role of ovarian hormones in models of addictive behavior.

Withdrawal from opioids involves a negative affective state that promotes maintenance of drug-seeking behavior and relapse. As such, understanding the neurobiological mechanisms underlying withdrawal from opioid drugs is critical as scientists and clinicians seek to develop new treatments and therapies. In this review, we focus on the neural systems known to mediate the affective and somatic signs and symptoms of opioid withdrawal, including the mesolimbic dopaminergic system, basolateral amygdala, extended amygdala, and brain and hormonal stress systems. Evidence from preclinical studies suggests that these systems are altered following opioid exposure and that these changes mediate behavioral signs of negative affect such as aversion and anxiety during withdrawal. Adaptations in these systems also parallel the behavioral and psychological features of opioid use disorder (OUD), highlighting the important role of withdrawal in the development of addictive behavior. Implications for relapse and treatment are discussed as well as promising avenues for future research, with the hope of promoting continued progress toward characterizing neural contributors to opioid withdrawal and compulsive opioid use.