Imaging and Neural Circuits Core Summary Opioid abuse and related deaths are rising exponentially, with synthetic opioids dominating the recent increase in overdoses. The Imaging and Neural Circuits Core (INCC) is ideally situated to collaborate across UW and the broader NIDA research community by developing publicly available imaging and modern circuit neuroscience resources, alongside unique training opportunities focused on the high-resolution investigation of opioid circuits and neurons that play a role in naturalistic motivated behavior, drug seeking, stress, anhedonia, and reinstatement/relapse. Here, we propose to continue the existing capabilities and expand our resources for investigating single cells and circuits in motivation, drug-seeking, and opioid neurobiology by integrating cutting- edge genetic, electrophysiological, and imaging technologies. The central goals of this proposal are to i) facilitate 2-photon imaging and simultaneous holographic optogenetics in awake and behaving animals; ii) generate an experimental pipeline for single-cell gene expression profiling of intact tissue volumes; iii) develop instrumentation and training protocols for high-resolution Neuropixels electrophysiology and to further enhance existing fiber photometry, 1p calcium imaging, and optogenetics instrumentation. The continuation of the existing INCC infrastructure will integrate leading animal models of motivated behavior, non-contingent and contingent administration of opioids together with high-resolution neural circuit analysis to provide critical resources that enhance and facilitate basic science and training in addiction research at the University of Washington and partnered Historically Black Colleges and Universities (HBCUs). This INCC proposal also includes the expansion of cutting-edge high-density in vivo electrophysiology and light sheet microscopy coupled with molecular feature identification pipelines. Together, the implementation of existing and new technologies in neural circuit dissection and reward behavior will provide unique opportunities for undergraduate, graduate, and post-doctoral trainees, as well as early-stage investigators across many disciplines and backgrounds to further our understanding of the neurobiology of addiction.