Project summary Alcohol use disorder (AUD) is an extremely common and serious condition that is associated with numerous somatic diseases, early mortality, personal and interpersonal hardship, and direct and indirect economic costs to society. Although there are several effective pharmacological treatments for AUD, their limited effectiveness contributes to the ongoing burden of AUD on society. A major impediment to better treatments for AUD is the lack of understanding surrounding fundamental molecular mechanisms associated with alcohol’s acute and chronic effects on the brain. Ongoing work in our lab has demonstrated that genetic and pharmacological manipulation of the enzyme Glyoxalase 1 (GLO1) can reduce ethanol drinking in mice and rats. GLO1 is an evolutionarily conserved enzyme that metabolizes methylglyoxal (MG), which is a non- enzymatic side product of glycolysis and is thus present in all animal cells. Transgenic overexpression of GLO1 decreases MG (GLO1’s substrate) in the brain. Reciprocally, direct administration of MG, genetic knockdown of Glo1 or pharmacological inhibition of GLO1 increase MG concentrations in the brain. We showed that MG is a selective agonist at GABA-A receptors. More recently, we discovered that GLO1 and MG modulate ethanol drinking behavior, which we hypothesize is due to MG’s effects at GABA-A receptors. In this supplement request, we are extending on the studies funded by the parent R01 to pursue a new line of experimentation. Specifically, we are exploring the possibility that ethanol may be directly converted into MG on a timescale that is consistent with the acute effects of ethanol. This exciting possibility raises the tantalizing possibility that some of ethanol’s effects on GABA-A receptors could be due to MG (rather than ethanol), directly influencing GABA-A signaling. We will test this possibility by measuring MG levels at various time points following administration of ethanol. In subsequent experiments, we will explore the possibility that ethanol is directly converted into MG (as opposed to altering MG in an indirect manner) by administering stable-labeled ethanol in which the hydrogen atoms have been replaced with deuterium. We will use mass- spec to assess MG levels, which will allow us to distinguish unlabeled MG from MG labeled with one or more deuterium atoms. These studies have the potential to greatly enhance our mechanistic understanding of ethanol’s actions. The mentoring plan is also described; the primary goal is to prepare the trainee to enter a Ph.D. program.