Project Summary/Abstract Repeated alcohol (ethanol) exposure results in cellular adaptations that are thought to be critical for the transition from low/moderate drinking to excessive alcohol consumption, which is a prerequisite for the development of Alcohol Use Disorder (AUD). Local protein synthesis (translation) in dendrites and axons plays a central role in synaptic plasticity and neuroadaptations to environmental challenges including exposure to drugs. Acute and chronic alcohol changes RNA abundance in synaptic terminals, suggesting a change in local protein synthesis, but we do not know which specific mRNAs undergo active translation at the synapse (the synaptic translatome) in response to alcohol. Actively translated synaptic mRNAs are mechanistically linked to neuronal functions and provide primary mechanistic candidates for alcohol-induced neuroadaptations. Here, we propose to use a combination of synaptoneurosome preparations and polysome profiling followed by RNA sequencing (RNA-Seq) to determine changes in the synaptic translatome after chronic alcohol drinking in mice. Synaptoneurosomes have been widely used to study molecular functions at the synapse in health and disease and polysome profiling is the gold standard for studying cellular translatomes. Because the synaptoneurosome/polysome profiling combination has not been used in alcohol studies, our initial goal is to develop/optimize the existing methodology for alcohol research. Alcohol-preferring C57BL/6J mice will be exposed to alcohol for several weeks using a 2-bottle choice chronic intermittent alcohol drinking paradigm that is used as a model of escalating alcohol consumption. The prefrontal cortex, a critical brain region implicated in AUD, will be dissected and subjected to the synaptoneurosome/polysome profiling followed by RNA-Seq. We hypothesize that chronic alcohol intake will affect the synaptic translatome and change local protein synthesis in the prefrontal cortex, which may contribute to neuroadaptations underlying high alcohol drinking. These initial experiments will identify alcohol-sensitive actively translated synaptic mRNAs (genes) that may eventually be targeted to reduce alcohol intake via manipulation of synaptic functions. The long-term goal of this research is to determine the role of local protein synthesis in brain mechanisms underlying AUD traits. This knowledge may be used to develop new therapies for the prevention and treatment of AUD.