Multi-Scale Imaging Core (MSIC)

点击翻译按钮获取中文摘要

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 difficult to evaluate by standard brain section staining. Therefore, we will exploit our experience in ScaleS methodology combined with optimized 2P imaging of the entire mouse brain. This approach will be used to determine the impact of developmental exposure to THC and other drugs on the integrity and trajectory of identified long-range axons. Because prenatal cannabis exposure modifies human neural circuits and rodent studies found that developing long-range glutamatergic axons are particularly sensitive to THC, we will initially determine the impact of perinatal THC exposure on glutamatergic axons originating from medial prefrontal cortex to various brain regions. Aim 3. Use in vitro and in vivo 2P sensor imaging to determine the mesoscale physiological changes in brain circuits elicited by chronic exposure to drugs of abuse. Recent advances in genetically encoded sensors for Ca2+, endocannabinoids, and monoamines provide excellent tools to visualize dynamic changes of these signaling molecules in a specific cell-type-specific manner in real-time. By combining our established and comprehensive methodology for Ca2+_imaging in acute brain slices or awake behaving mice (as young as ten days old) extending from the surgical procedure through the data analysis pipeline with High Performance Computing together with GRAB-eCB2.1 and GRABDA sensor imaging, we will support center and affiliated scientists to perform longitudinal 2P imaging to examine endocannabinoid, dopamine, and network activity changes in their relevant models of substance use disorders. We will also determine if perinatal THC exposure perturbs the development of endocannabinoid signaling in association with Ca2+-spike patterns in the primary somatosensory cortex of awake behaving mouse pups from early postnatal to weaning ages. Aim 4. Develop in vivo protocols for Fluorescence Lifetime Imaging Microscopy (FLIM) in addiction research. Drugs of abuse evoke substantial metabolic changes and perturb astrocyte-neuron interactions. We will use 2P-FLIM imaging to develop in vivo applications using FLIM-based sensors to monitor energy metabolism, signaling cascades, protein-protein interactions and to estimate the proximity between astrocytes and neurons in the substance use disorder models established by local and affiliate researchers of the imaging core.