PROJECT SUMMARY While several medications have been approved for alcohol use disorder (AUD), many patients fail to respond or comply with the treatments. Relapse triggered by reminders of alcohol use is a particular challenge to prevent, as the underlying memories exert a powerful motivational influence over behavior and represent a lifelong relapse risk factor. Learning of these associations is supported by structural plasticity in dendritic spines, driven by training-induced actin polymerization. Memory stability is subsequently achieved by arresting actin dynamics, stabilizing the cytoskeleton. As a result, memory is impervious to actin depolymerization within minutes of learning. However, prior work in the lab discovered that the actin cytoskeleton supporting methamphetamine and amphetamine memories remains uniquely dynamic in the amygdala long after training. This enables selective, retrieval-independent disruption of these memories and associated drug seeking with a single administration of an actin depolymerizer. Because actin’s critical roles in the body limit its therapeutic potential, focus shifted to nonmuscle myosin II (NMII), a direct driver of learning-stimulated actin polymerization in spines. Genetic and pharmacologic targeting of NMII established it is a viable therapeutic target and an NIH- funded medication development project for a clinically safe NMII inhibitor is underway, currently at the stage of IND-enabling studies (UH3 NS096833). The central hypothesis in the current proposal is that a single administration of an NMII inhibitor will produce a long-lasting disruption of alcohol seeking. Aim 1 will determine the retrieval-independent ability of NMII inhibition to disrupt associations that trigger alcohol seeking, like methamphetamine and amphetamine. Interestingly, the retrieval-independent effect of NMII inhibition does not extend to memories for fear, food, spatial memory or several other drugs of abuse. However, it does disrupt the reconsolidation of memories associated with drugs of abuse, including cocaine. Aim 2 will test the effect of NMII inhibition on reconsolidation of alcohol-associated memories and seeking. However, the relatively unique approach of reactivating memory with the unconditioned stimulus (US; a small amount of alcohol), rather than the conditioned stimuli (CS; associated context and cues) will be utilized. This US-based approach circumvents the limitation inherent to CS-based reactivation strategies, which require that potentially hundreds of associations be recalled in a clinical setting to enable disruption. As proof-of-principle, preliminary data indicate that US-based reactivation renders cocaine (COC)-associated memory susceptible to NMII inhibition. The proposed work is expected to identify a new therapeutic approach to the prevention of relapse to alcohol seeking with the potential for rapid translation through the use of a first in class compound whose NIH- funded development is on track f...