ABSTRACT Despite the enormous harms of alcohol use disorders (AUDs), many mechanisms, as well as effective prevention or treatment strategies remain elusive. Genetic factors dictate a majority of AUD risk. These risk factors can manifest as reduced naïve sensitivity to alcohol’s intoxicating effects and increased functional tolerance, i.e., brain-mediated decreases in sensitivity upon repeat exposure. The underlying neurobiology of how AUD-associated genes alter these endophenotypes remains poorly understood. Genes implicated in AUDs include epigenetic modifiers, such as histone demethylases, including Kdm3 . We previously showed that whole-body and neuronal Kdm3 strongly affect ethanol sensitivity and tolerance in Drosophila . Here, we investigate the mechanisms of these effects, and, by extension, mechanisms of sensitivity and tolerance. RNA-seq and pathway analysis on Kdm3 KO flies revealed disproportionate upregulation of genes involved in amino acid metabolism, including 1-carbon pathways. We show that acute amino acid feeding modulates sensitivity and tolerance in a Kdm3 -dependent manner. Global manipulation of 1-carbon genes, especially glycine N -methyltransferase ( Gnmt ), glycine decarboxylase ( Gldc ), and sarcosine dehydrogenase ( Sardh ), alters alcohol sensitivity and tolerance. These changes in alcohol responses are likely mediated by global glycine levels (a substrate of these enzymes) rather than by 1-carbon input. Conversely, neuronal manipulations of 1-carbon pathways change alcohol sensitivity and tolerance in a pattern that suggests a mechanism through S -adenosyl methionine (SAM), a 1-carbon metabolite that is the universal methyl donor required for epigenetic methylation. Increasing SAM production specifically in glutamatergic neurons increases sensitivity and tolerance. Together, these findings reveal distinct mechanisms affecting alcohol sensitivity and tolerance globally (via glycine) and neuronally (via SAM), thus revealing an important and complex role of 1-carbon metabolism in mediating AUD phenotypes.

One-carbon Pathways and Methylation Potential in Glutamatergic Neurons Regulate Behavioral Alcohol Responses