Project Summary/Abstract A key issue in the alcohol field is the lack of knowledge on the discreet neuronal ensembles that are responsible for excessive alcohol drinking in alcohol-dependent subjects. This is a major obstacle for the alcohol field because investigations of the neuronal ensembles that mediate excessive alcohol drinking would provide a comprehensive understanding of the neuronal circuits that causally contribute to alcohol dependence. The recent development of pharmacogenetic and optogenetic tools that allow selective targeting of specific neuronal ensembles is a tremendous opportunity to bridge this gap in the literature. The central hypothesis of this proposal is that activation of the parabrachial nucleus (PBN) and central nucleus of the amygdala (CeA) during withdrawal is responsible for the recruitment of a set of discreet neuronal ensembles that are scattered throughout the brain and ultimately responsible for excessive drinking and the emergence of negative emotional states in dependent rats. We obtained robust preliminary results that show that selectively targeting these neuronal ensembles produces long-lasting reversal of excessive alcohol drinking in dependent rats, identifies a causal relationship between these ensembles, and reveals novel neuronal pathways that contribute to alcohol dependence. Specific Aim 1 characterizes the role of the CeA and PBN withdrawal neuronal ensembles in excessive alcohol drinking in dependent rats. Specific Aim 2 dissects the role of the different CeA CRF pathways in the recruitment of the neuronal ensembles and excessive alcohol drinking in alcohol dependence. Results from these studies have the potential to have a strong and lasting impact in the field because our approach will improve our understanding of the neurobiological mechanisms of alcohol dependence and identify novel neuronal populations and circuits that specifically control behaviors associated with alcohol dependence.