SPECIFIC AIMS-Multiscale Imaging Core (MSIC) Addictive substances trigger plasticity at the molecular, cellular and circuit levels that manifest as persistent behavioral changes that may cause substance use disorders. Targeting these changes may lead to novel strategies for preventing or treating substance use disorders. However, our knowledge of the molecular changes, the cellular processes and the abnormal circuit activity patterns that underlie various aspects of substance use disorders including compulsion, loss of intake control, withdrawal, and relapse is rather limited. To facilitate a better understanding of the molecular to circuit level plasticity accompanying drug abuse, the C3A multi- scale imaging core will support center investigators, affiliates from the Midwest and beyond, and trainees at different career stages to acquire the conceptual and technical know-how, and to access state-of-the-art equipment for nanoscale molecular measurements, for microscale anatomical analysis of subcellular and cellular profiles and for mesoscale physiological imaging of brain circuits. The C3A multi-scale imaging core will provide unprecedented imaging opportunities to examine models of substance use disorders at multiple levels, including: (1) molecular and cellular level imaging with internationally unique cell-type- and subcellular compartment-specific correlated STORM super-resolution imaging, and its recently developed PharmacoSTORM extension for nanoscale pharmacology; (2) circuit level 2P imaging to examine selective neural circuits and cell-type-specific dynamic physiological changes among large cell populations. Aim 1. Determine the cell- and subcellular compartment-specific nanoscale molecular and microscale cellular alterations triggered by chronic exposure to drugs of abuse. By employing fluorescent small molecule-based PharmacoSTORM single-molecule nanoscale pharmacology and antibody-based ImmunoSTORM super-resolution imaging, we and C3A-affiliated researchers will determine if chronic drug exposure and/or withdrawal elicit persistently altered nanoscale distribution and abundance of important signaling proteins in the cell types and brain circuits that are most relevant for substance use disorders. By correlating the nanoscale molecular measurements with microscale confocal microscopy data, we will also establish the associated morphological changes in identified subcellular compartments. Particular attention will be devoted to CB1 cannabinoid and D3 dopamine receptors that have essential roles in all phases of the addiction cycle and whose antagonists/negative allosteric modulators are among NIDA’s ten highest medication development priorities. Aim 2. Characterize the mesoscale circuit rewiring of long-range glutamatergic, dopaminergic and serotonergic axons induced by developmental or chronic exposure to drugs of abuse. Axon tracts connecting distant brain regions follow irregular trajectories, thus white matter morphology is difficu...