Investigating individual variability and therapeutic targets in alcohol and opioid use disorders

Alcohol use disorder (AUD) is a highly heterogeneous disorder marked by substantial inter- individual differences in vulnerability, disease progression, comorbidity, and response to treatment. These differences arise from the interaction of genetic background, environmental influences, biological sex, and neurobiological processes within reward and stress-related circuits. In this thesis, vulnerability to AUD and opioid use disorder (OUD) was investigated using a combination of genetically selected and genetically heterogeneous animal models, together with behavioral, molecular, and pharmacological approaches. In Chapter 2, we used the Marchigian Sardinian alcohol-preferring (msP) rat line, a genetically selected model characterized by high alcohol intake and emotional dysregulation, to determine whether this phenotype is preserved when animals are bred and tested in an experimental facility different from the historical site at the University of Camerino (UNICAM). Since their behavior have traditionally been studied exclusively at UNICAM, this chapter aimed to validate the reproducibility of their behavioral phenotype in a new research environment. msP rats housed and tested in The Scripps Research Institute (TSRI) displayed elevated voluntary alcohol consumption and increased anxiety-like behavior in the light-dark box compared to Wistar controls, demonstrating that their characteristic phenotype is maintained despite environmental relocation. Building on this behavioral validation, Chapter 3 investigated therapeutic modulation in a genetically vulnerable population by assessing the effects of the GLP-1 receptor agonist semaglutide on alcohol intake and affective behavior in msP rats. Semaglutide robustly reduced alcohol consumption in a dose-dependent manner and exerted anxiolytic-like effects during alcohol withdrawal, supporting GLP-1 receptor signaling as a promising target for AUD in susceptible individuals. We then examined, in Chapter 4, the impact of early-life stress by assessing the effects of adolescent social isolation. This manipulation produced limited and sex-dependent effects, with modest increases in alcohol seeking observed in females, highlighting the interaction between genetic vulnerability and biological sex. Extending the investigation to polysubstance vulnerability, in Chapter 5, we demonstrated that msP rats exhibit increased heroin self-administration associated with elevated μ-opioid receptor (Oprm1) expression in the ventral tegmental area. Targeted downregulation of Oprm1 reduced heroin intake, establishing a causal link between mesolimbic opioid signaling and opioid vulnerability in an alcohol-preferring genetic background. 10 Finally, to address the translational limitations of homogeneous models, we employed the NIH Heterogeneous Stock rats in Chapter 6 to capture human-like variability in AUD. This approach successfully predicted differential responses to pharmacological treatments, distinguishing responder and non-responder subpopulations and revealing sex-dependent treatment effects. Together, these findings highlight vulnerability to substance use disorders as a multidimensional construct shaped by genetic background, environmental exposure, sex, and neurobiological mechanisms. By integrating complementary animal models, this thesis provides a translational framework for understanding individual variability in addiction and for improving preclinical prediction of treatment efficacy.