Reverse-translational investigation of gut microbial contributions to alcohol sensitivity and tolerance. - ABSTRACT The goal of this proposal is to investigate the role of the gut microbiota in alcohol sensitivity and tolerance. Alcohol is the leading risk factor for premature death and disability for individuals between 15 and 49 years old, accounting for 3 million deaths worldwide each year. Yet, current treatment options for alcohol use disorder (AUD) are inadequate. Low initial sensitivity to the sedative or ataxic intoxicating effects of alcohol is a major risk factor for future development of AUD. Tolerance is a core feature of AUD and is associated with greater alcohol intake, preference, and reinforcing effects. Furthermore, most pharmacotherapies for AUD enhance sensitivity or reduce tolerance. Thus, alcohol sensitivity and tolerance are prominent targets for AUD treatment development. AUD is associated with shifts in the gut microbiota, the community of microbes that populates the gastrointestinal tract. Mounting evidence indicates that the gut microbiota drives AUD-associated alcohol intake, preference, withdrawal, craving, or relapse. Strikingly, initial studies have demonstrated a causal role for AUD- associated gut microbiota in these phenotypes through fecal microbiota transplant (FMT), the gold standard for isolating effects driven by the microbiota. However, no studies to date have assessed a causal role for the gut microbiota in sensitivity and functional tolerance to alcohol sedation. Here, we will use our previously collected fecal samples from individuals with AUD, but without liver disease, to perform FMT into alcohol-naïve mice. These humanized microbiota-associated (HMA) mice will be used to investigate the role of AUD-specific gut microbiota in sensitivity and functional tolerance to alcohol sedation, a reverse-translational design that will maximize the translational implications of findings. We hypothesize that AUD-HMA mice will take on a chronic tolerance-like phenotype, both in terms of behavioral manifestations and molecular adaptations. We will first replicate prior reports (n = 2) of elevated alcohol intake and preference in AUD-HMA mice and determine features of the microbiome that predict these behaviors (Aim 1). We will then test for a causal role of AUD gut microbiota in sensitivity and tolerance to alcohol sedation in the loss of righting reflex assay and determine features of the microbiome that predict these behaviors (Aim 2). Importantly, the version of this assay used is specifically designed to distinguish between effects of alcohol metabolism versus functional adaptations. Finally, we will measure a panel of multi-organ biomarkers associated with alcohol tolerance and microbiota-mediated effects of alcohol to identify candidate drivers of AUD microbiota-associated tolerance (Aim 3). Specifically, we will measure microbial metabolites, intestinal permeability, immune signaling, key excitatory and inhibitory genes in the brain, and select neural markers of tolerance that are modulated by the gut microbiota. Completion of these aims will bolster prior findings regarding the role of AUD-associated microbiota in alcohol intake, determine the causal role of the AUD microbiota in tolerance, and identify key biomarkers of these processes for future treatment development.
