Ultra-fast high-resolution imaging of whole mouse brain for the study of drug addiction

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PROJECT SUMMARY Opioid and cocaine abuse prevalence has skyrocketed in the United States, fueling the current epidemic of overdose deaths. Despite the public health impact of opioids and cocaine, we still lack a fundamental understanding of the mechanisms by which these drugs work, particularly across cellular and circuit levels. Further understanding of the neuroanatomy of the neural circuitry underlying opioid and cocaine reward is a critical initial step in targeting and elucidating their mechanisms. However, comprehensively visualizing relevant circuits in drug reward has been limited by approaches to contextualize these circuits and their response to drugs of abuse in the whole brain. We developed an approach to rapidly image the whole brain in three-dimensional (3D) space using ultra-fast high-resolution ribbon-scanning confocal microscopy. Our ribbon-scanning confocal imaging approach can image and visualize an entire rodent brain in less than 24 hours, where more conventional approaches (e.g., light-sheet) currently require days or even weeks. Furthermore, our ribbon-scanning confocal approach reaches diffraction-limited resolutions (~200-300nm), enabling us to visualize individual cells in the brain and their ultrastructure. We can apply these unique tools to begin solving the fundamental questions: 1) What is the precise circuitry that defines drug reward? And 2) What are the differential effects of cocaine and opioids on this circuitry? Like many drugs of abuse, cocaine and opioids rely on neurotransmission from dopamine (DA) neurons in the ventral tegmental area (VTA). However, until recently, parsing the connectivity of unique subpopulations of DA neurons and their potential roles in drug reward has been difficult. We developed a suite of intersectional genetic tools to definitively dissect the anatomical and functional properties of these different subpopulations within the same brain. We will integrate our 3D ribbon-scanning confocal imaging of DA neuron subpopulations with immunolabeling of neuronal activity markers to visualize precisely which DA neurons are activated in response to cocaine and opioids. Using whole brain immunolabeling and imaging, we will also visualize and map drug-dependent neuronal activity changes in the whole brain with the potential to reveal new populations of neurons differentially response to cocaine and opioids. Our overall objectives are to: Comprehensively map the distribution of DA neuron subpopulations including DA/glutamate co-transmitting cells relative to the overall DA system within whole brain (Aim 1); and to determine how cocaine and opioids differentially affect the activity of these DA neuron subpopulations (Aim 2). We will generate a comprehensive 3D brain atlas to identify the roles of unique subpopulations of DA neurons highly relevant to cocaine and opioids, which will serve as a proof of principle for the implementation of our ultra-fast high-resolution 3D ribbon-scanning confocal microscopy. Our proposal will foster future development of the first 3D high-resolution comprehensive maps of neurotransmission within in whole brain to study addiction.