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Background Sex is an important factor in the progression and treatment of alcohol addiction, and therapeutic approaches may have to be tailored to potential sex differences. This highlights the importance of understanding sex differences in behaviors that reflect key elements of clinical alcohol addiction, such as continued use despite negative consequences (“compulsive use”). Studies in experimental animals can help provide an understanding of the role sex plays to influence these behaviors. Methods Large populations of genetically heterogeneous male and female Wistar rats were tested in an established model of compulsive alcohol self-administration, operationalized as alcohol responding despite contingent foot shock punishment. We also tested baseline (fixed ratio, unpunished) operant alcohol self-administration, motivation to self-administer alcohol (progressive ratio), and temporal discounting for alcohol reward. In search of predictors of compulsivity, animals were screened for novelty-induced place preference, anxiety-like behavior, pain sensitivity and corticosterone levels. The estrous cycle was monitored throughout the study. Results Unpunished self-administration of alcohol did not differ between males and females when alcohol intake was corrected for body weight. Overall, females showed higher levels of compulsive responding for alcohol. Compulsive response rates showed bimodal distributions in male but not in female rats when intermediate shock intensities were used (0.2 and 0.25 mA); at higher shock intensities, responding was uniformly suppressed in both males and females. We also found less steep discounting in females when alcohol was devalued by delaying its delivery. Males exhibited a stronger motivation to obtain alcohol under unpunished conditions, while females showed higher corticosterone levels at baseline. Factor analysis showed that an underlying dimension related to stress and pain predicted compulsivity in females, while compulsivity in males was predicted by a reward factor. We did not find differences in alcohol-related behaviors throughout the various stages of the estrous cycle. Conclusions Our results suggest that mechanisms promoting compulsivity, a key feature of alcohol addiction, likely differ between males and females. This underscores the importance of considering sex as a biological variable in both preclinical and clinical research, and has potential treatment implications in alcohol addiction. Plain language summary Sex plays an important role in the progression and treatment of alcohol addiction. While men show a higher prevalence of alcohol addiction, women are more susceptible to the adverse effects of excessive alcohol consumption. Additionally, women often rely on heavy drinking as a maladaptive coping mechanism to alleviate stress and anxiety, driven by negative affect. On the other hand, men are more likely to report heavy drinking and relapse in response to positive emotions and social influences. These sex-based differences underline the importance of understanding how vulnerability to alcohol addiction and its treatment varies in males and females. We used genetically heterogeneous rats to explore the behavioral traits that contribute to compulsivity, a key clinical feature of alcohol addiction. We found that motivation to self-administer alcohol was higher in males, while females showed higher compulsive alcohol self-administration. In males, motivation to self-administer alcohol showed a significant correlation with compulsivity, while in females compulsivity was predicted by higher basal corticosterone levels. These findings underlie the importance of sex-specific factors in compulsive alcohol self-administration, with potential prevention and treatment implications in alcohol addiction. Highlights Male rats showed a higher motivation to obtain alcohol. Females showed higher levels of compulsive responding for alcohol and a less steep discounting when alcohol was devalued by delaying its delivery. In males compulsivity was predicted by a reward factor, while in females by stress-pain factors.

Alcohol addiction (hereafter equated to alcohol dependence according to the WHO ICD 10 classification, or moderate-to-severe alcohol use disorder, AUD, according to the DSM classification of the American Psychiatric Association), is a complex psychiatric illness with an approximate global lifetime prevelance (of AUD) of 14.1 % and 3.4 % for men and women respectively. Only a subset of alcohol users develop addiction, suggesting that research to discover novel treatments should consider individual differences in susceptibility for clinically relevant behaviors. Continued use of alcohol despite negative consequences, commonly referred to as “compulsive use”, is a hallmark of the transition from recreational to addictive use of alcohol. Research in animal models has begun to identify mechanisms behind compulsive alcohol taking, but the neural basis of individual differences in this behavior remains poorly understood.The main aim of this thesis is to investigate the neural mechanisms of individual susceptibility to compulsive alcohol use, a key feature of alcohol addiction, and the potential role of sex as a biological variable.In paper I,we characterized susceptibility to developing compulsive alcohol self-administration in rats. We identified an ensemble of neurons in the central nucleus of the amygdala (CeA) that promoted compulsive self-administration. We identified these neurons as PKCδ neurons, one of two major subpopulations in the central nucleus. Lastly, we investigated the causal role of PKCδ itself in compulsive self-administration by knocking down its expression and found that this reduced compulsive self-administration. In paper II,we studied the role of the GABABreceptor agonist, baclofen, in compulsive alcohol self-administration, and on activity of neurons in the centrolateral amygdala (CeL). This study provides a mechanistic rationale for developing improved alcohol addiction medications that target GABABreceptors and PKCδ+ neurons in the CeL. In paper IIIwe characterized sex differences in animal models of alcohol addiction, including compulsive alcohol self-administration. We found that female rats consumed equal amount of alcohol as males in unpunished conditions, but that they were more resistant to aversive consequences when alcohol rewards were paired with either footshock or quinine adulteration. We investigated potential predictors of compulsive self-administration in both sexes and found that for male rats, compulsivity was predicted by motivation to obtain alcohol, whereas for females, compulsivity was promoted by stress-pain factors. Lastly, in paper IV,we characterize a novel tool for studying the role of PKCδ+ neurons, a transgenic rat line that expresses Cre-recombinase under the control of the PKCδ+ promoter, allowing selective access to, and control of PKCδ+ neurons.Collectively, these studies highlight PKCδ-expressing neurons in the CeA as critical players in punishment-resistant alcohol self-administration, pointing to a new avenue for developing targeted treatments. The findings also emphasize the need for sex-specific approaches in both preclinical models and clinical interventions.

Alcohol use despite negative consequences is a core phenomenon of alcohol addiction. We recently used alcohol self-administration that is resistant to footshock punishment as a model of this behavior, and found that activity of PKCδ + GABAergic neurons in the central amygdala (CeA) is a determinant of individual susceptibility for punishment resistance. In the present study, we examined whether activation of GABAB receptors in CeA can attenuate the activity of PKCδ + neurons in this region, and whether this will result in suppression of punishment- resistant alcohol self-administration in the minority of rats that show this behavior. Systemic administration of the clinically approved GABAB agonist baclofen (1 and 3 mg/kg) dose- dependently reduced punishment-resistant alcohol self-administration. Bilateral microinjections of baclofen into CeA (64 ng in 0.3 µl/side) reduced the activity of PKCδ + neurons, as measured by Fos expression. This manipulation also selectively suppressed punished alcohol self-administration in punishment-resistant rats. Expression analysis indicated that virtually all CeA PKCδ + neurons express the GABAB receptor. Using in vitro electrophysiology, we found that baclofen induced hyperpolarization of CeA neurons, reducing their firing rate in response to depolarizing current injections. Together, our findings provide a potential mechanism that contributes to the clinical efficacy of baclofen in alcohol addiction. Therapeutic use of baclofen itself is limited by problems of tolerance and need for dose escalation. Our findings support a mechanistic rationale for developing novel, improved alcohol addiction medications that target GABAB receptors, and that lack these limitations, such as e.g., GABAB positive allosteric modulators (PAM:s).