PROJECT SUMMARY: Alcohol is the widely abused drug in the world, yet our understanding of the molecular mechanisms by which it regulates brain function and behavior is rudimentary. Many of the molecules implicated in alcohol-induced behaviors have broad roles in regulating diverse processes such as cell signaling, transcription, and neuronal plasticity. The complexity of these processes has led to confusion about alcohol’s molecular underpinnings, which has been further compounded by lack of the ability to manipulate gene expression with precise temporal and spatial control within a well-defined neural circuit. The temporal and spatial specificity for manipulating gene expression afforded in the fruit fly, Drosophila melanogaster, provides the ability to define where and when and where alcohol is acting in the brain to influence both naive and learned behavior. Our unbiased forward genetics approach in Drosophila has revealed the importance of the Notch signaling pathway in regulating formation of memories of a sensory cue associated with the intoxicating properties of alcohol. Notch is a highly conserved cell-signaling mechanism that involves cell-to-cell contact and initiates a transcriptional cascade important for determining cell fate and function. Notch signaling also plays a key role in multiple forms of cancer and immune disorders, as well as cardiovascular, kidney, liver, respiratory and neurodegenerative disease. We recently demonstrated that rewarding doses of alcohol activate Notch signaling in memory-encoding neurons, and this affects expression of novel Notch transcriptional targets such as dopamine receptors. What remains to be understood are the mechanisms through which alcohol activates Notch, and how this ultimately affects dynamics of memory circuits, both acutely and with chronic alcohol use. We hypothesize that Notch plays an important role in activity-dependent transcription required for memory formation and thus ultimately shapes future reward seeking. The goal of the proposed work is to use a genes-to-circuits-to-behavior approach to provide a causal Notch-dependent mechanism through which alcohol-induced cellular activity can guide a transcriptional cascade that influences future alcohol seeking. This increased molecular understanding of the lasting sensory memories for intoxication will ultimately inform more effective targets for pharmacological treatment of alcohol use disorder.