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

项目总结 阿片类药物和可卡因滥用在美国的流行率飙升，助长了目前的 服药过量死亡。尽管阿片类药物和可卡因对公共健康造成影响，但我们仍然缺乏基本的 了解这些药物的作用机制，特别是在细胞和电路水平上。 进一步了解阿片类药物和可卡因奖赏背后的神经回路的神经解剖学是 在靶向和阐明其机制方面迈出关键的第一步。然而，全面可视化相关 药物奖励中的回路一直受到将这些回路及其对药物的反应联系起来的方法的限制 在整个大脑中的滥用。我们开发了一种以三维方式快速成像整个大脑的方法 (3D)使用超高速高分辨率带状扫描共聚焦显微镜的空间。我们的带状扫描共焦 成像方法可以在不到24小时的时间内对整个啮齿类动物的大脑进行成像和可视化，而更传统的 方法(例如，光片)目前需要数天甚至数周的时间。此外，我们的带状扫描共聚焦 该方法达到了衍射限制的分辨率(~200-300 nm)，使我们能够可视化 大脑及其超微结构。我们可以应用这些独特的工具来开始解决基本问题：1) 定义毒品奖励的精确电路是什么？2)可卡因和可卡因的不同作用是什么 这个电路上有阿片类药物吗？像许多滥用药物一样，可卡因和阿片类药物依赖于 腹侧被盖区的多巴胺(DA)神经元。然而，直到最近，解析 DA神经元的独特亚群及其在药物奖赏中的潜在作用一直是困难的。我们开发了 一套交叉的遗传工具来明确地解剖这些 同一个大脑中的不同亚群。我们将整合我们对DA的3D带状扫描共聚焦成像 神经元活性标记物免疫标记的神经元亚群，以准确显示哪些DA神经元 对可卡因和阿片类药物的反应被激活。使用全脑免疫标记和成像，我们还将 可视化并绘制整个大脑中药物依赖神经活动的变化，从而有可能揭示新的 神经元群体对可卡因和阿片类药物的反应不同。我们的总体目标是： 全面绘制包括DA/谷氨酸共传递细胞在内的DA神经元亚群的分布 相对于整个大脑内的整个DA系统(目标1)；并确定可卡因和阿片类药物如何 不同地影响这些DA神经元亚群的活动(目标2)。我们将生成一个全面的 3D脑图谱，以确定与可卡因和阿片类药物高度相关的独特DA神经元亚群的作用， 这将作为我们实施超高速高分辨率3D带状扫描的原则证明 共聚焦显微镜。我们的建议将促进未来第一个3D高分辨率综合 研究成瘾的整个大脑内部的神经传递图。

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.