Project Summary/Abstract The goal of this proposal is to capture and then characterize engrams for ethanol tolerance in Drosophila. Engrams are the neurons and their molecular changes that encode lasting behavioral change. Engrams described for ethanol in mammals are limited to specific brain regions, encoding factors, and behavioral states, and little is known about effectors in ethanol engrams. Drosophila offer significantly higher throughput, facilitating whole brain capture of engrams, testing of multiple types of potential engrams, and molecular discovery of the mechanisms of encoding. Further, we developed behavioral paradigms for rapid, chronic, and repeated exposure tolerance, and we discovered that they are encoded by distinct immediate early gene transcription factors. We will develop a new method to permanently tag engrams in the brain. Permanent tagging lets us define molecular changes in engram neurons, explore their function as tolerance is acquired and then expressed, and to define their function in brain circuits. Comparing the anatomy and effector molecules for different forms of tolerance will unveil the complex actions of ethanol on the brain. Our focus on evolutionarily conserved factors provides a means for future development of diagnostics and therapeutics for alcohol use disorders.