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

用于药物成瘾研究的小鼠全脑超快高分辨率成像

• 批准号: 10359049
• 负责人: ZACHARY FREYBERG
• 金额: $ 19.8万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10359049
• 关键词: 3-Dimensional; Acute; Address; Affect; Anatomy; Atlases; Behavior; Behavioral; Biological Assay; Brain; Brain imaging; Brain region; Cells; Chemicals; Cocaine; Cocaine Abuse; Color; Complex; Corpus striatum structure; Development; Disease; Dopamine; Drug Addiction; Epidemic; Exhibits; Fentanyl; Fostering; Future; Genetic; Global Change; Glutamates; Heterogeneity; Hour; Image; Imaging technology; Individual; Laboratories; Laser Scanning Confocal Microscopy; Light; Maps; Medial; Midbrain structure; Morphologic artifacts; Mosaicism; Movement; Mus; Neuroanatomy; Neurons; Opioid; Pharmaceutical Preparations; Population; Prevalence; Process; Property; Public Health; Resolution; Rewards; Rodent; Role; Scanning; Site; Societies; Speed; System; Techniques; Technology; Time; Tyrosine 3-Monooxygenase; United States; Ventral Tegmental Area; Work; activity marker; addiction; base; cell type; combinatorial; conditioned place preference; confocal imaging; dopamine system; dopaminergic neuron; drug of abuse; drug reinforcement; drug reward; effective therapy; family burden; genetic approach; high resolution imaging; imaging approach; neural circuit; neuronal patterning; neurotransmission; new therapeutic target; opioid abuse; overdose death; recombinase; regional difference; response; tool; vesicular glutamate transporter 2

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.

项目摘要 阿片类药物和可卡因滥用的流行率在美国飙升，助长了目前的流行病， 过量死亡尽管阿片类药物和可卡因对公共卫生产生了影响，但我们仍然缺乏一个基本的 了解这些药物的工作机制，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励背后的神经回路的神经解剖学是一个重要的研究方向。 这是确定目标和阐明其机制的关键初始步骤。然而，全面可视化相关 药物奖赏中的回路受到了将这些回路及其对药物的反应置于情境中的方法的限制 虐待的迹象我们开发了一种方法来快速成像整个大脑的三维 (3D)空间使用超快速高分辨率带扫描共聚焦显微镜。我们的带式扫描共聚焦 成像方法可以在不到24小时的时间内对整个啮齿动物大脑进行成像和可视化， 方法（例如，光片）目前需要数天甚至数周。此外，我们的带扫描共聚焦 这种方法达到了衍射极限分辨率（~200- 300 nm），使我们能够可视化单个细胞在 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） 是什么精确的电路定义了药物奖励？2）可卡因和可卡因的不同作用是什么？ 阿片类药物在这个电路上吗像许多滥用药物一样，可卡因和阿片类药物依赖于神经传递， 腹侧被盖区（VTA）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 一套交叉的遗传工具，以明确解剖这些器官的解剖和功能特性， 同一个大脑中的不同亚群。我们将整合我们的3D带状扫描共聚焦成像的DA 神经元亚群与神经元活性标记物的免疫标记，以精确地可视化哪些DA神经元 在可卡因和阿片类药物的作用下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性神经元活动的变化，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DA神经元亚群的分布，包括DA/谷氨酸共传递细胞 相对于整个大脑中的整个DA系统（目标1）;并确定可卡因和阿片类药物如何 差异地影响这些DA神经元亚群的活性（目的2）。我们将提供一个全面的 3D脑图谱，以确定与可卡因和阿片类药物高度相关的DA神经元独特亚群的作用， 这将作为我们实现超快速高分辨率3D带状扫描的原理证明 共聚焦显微镜我们的建议将促进第一个3D高分辨率综合的未来发展， 全脑神经传递的地图来研究成瘾。

项目成果

Roles of VGLUT2 and Dopamine/Glutamate Co-Transmission in Selective Vulnerability to Dopamine Neurodegeneration.

• DOI: 10.1021/acschemneuro.1c00741
• 发表时间: 2022-01-19
• 期刊: ACS CHEMICAL NEUROSCIENCE
• 影响因子: 5
• 作者: Buck, Silas A.;Erickson-Oberg, M. Quincy;Bhatte, Sai H.;McKellar, Chase D.;Ramanathan, Vishan P.;Rubin, Sophie A.;Freyberg, Zachary
• 通讯作者: Freyberg, Zachary

Relevance of interactions between dopamine and glutamate neurotransmission in schizophrenia.

• DOI: 10.1038/s41380-022-01649-w
• 发表时间: 2022-09
• 期刊: MOLECULAR PSYCHIATRY
• 影响因子: 11
• 作者: Buck, Silas A.;Erickson-Oberg, M. Quincy;Logan, Ryan W.;Freyberg, Zachary
• 通讯作者: Freyberg, Zachary