Isolating the role of endogenous mu-opioid activity in the VTA during natural reward

摘要：美国阿片类药物使用障碍和阿片类药物过量死亡率达到前所未有的水平 于2019冠状病毒病大流行期间的水平。了解内源性阿片活性如何影响自然奖励 寻求对于理解阿片类药物使用障碍的神经药理学基础至关重要。以前的研究 暗示了中脑边缘多巴胺通路，这是指多巴胺神经元从中脑边缘投射到中脑边缘。 被盖区（VTA）到伏隔核（NAc），在奖励和成瘾中。近年来研究表明 腹侧被盖区（VTAGABA）中含有γ-氨基丁酸（GABA）的神经元提供多巴胺的局部抑制 神经元突触到NAc，从而在调节奖励行为中发挥作用。重要的是这些 VTAGABA神经元含有多种G蛋白偶联受体，特别是μ-阿片受体（莫尔）。 然而，这些受体及其信号传导在VTAGABA神经元中的确切作用和随后的结果是未知的。 自然奖励的规则是未知的。内源性阿片神经肽的来源到这些MORs， 没有描述这些如何影响信号和活性。这一提议的核心假设是， VTAGABA神经元上的μ-阿片受体信号由传入的内源性阿片肽调节， VTAGABA神经元兴奋性解除抑制。这导致多巴胺神经元活动的控制，并最终 自然奖赏的表现。该提案直接涉及NIDA的优先科学领域1， 旨在进一步了解药物使用引起的分子，神经药理学和电路变化。目的 1将分离VTA中内源性μ-阿片肽对多巴胺信号传导和自然奖赏的作用- 寻找Aim 1A使用离体双光子成像和神经药理学方法来可视化VTAGABA， VTA DA动力。Aim 1B将研究腹侧被盖区莫尔消融对多巴胺活性的影响， 行为目标2将在自然奖赏期间分离VTA中内源性μ-阿片类药物的来源和动力学 行为在Aim 2A中，我将使用病毒追踪技术从解剖学上可视化来自外侧的输入， 下丘脑（LH）至腹侧被盖区。目的2B将测试内源性μ-阿片样物质信号传导对VTAGABA和 在奖赏寻求行为中NAc中的DA活性。在目标2C中，我将使用分子方法，包括 CRISPR基因编辑和最近开发的µ-阿片类生物传感器（µ MASS 1），以了解时空 在奖赏寻求过程中VTAGABA神经元中的莫尔信号方面。职业发展培训和 一系列的实验将提供深入了解内源性莫尔信号转导对多巴胺能活性的作用 和自然的奖赏。对于这个提议，我将在切片电生理学，双光子切片成像， 基因编辑，分子神经药理学和行为方法，以了解如何具体 神经肽调节奖赏回路。这个F31提案将大大推进我的职业发展计划， 为我成为一名独立的学术神经药理学家做好准备。

SUMMARY: Opioid use disorder and opioid overdose death rates in the United States reached unprecedented levels during the COVID-19 pandemic. Understanding how endogenous opioid activity affects natural reward seeking is crucial to understanding the neuropharmacological basis of opioid use disorder. Previous research implicates the mesolimbic dopamine pathway, which refers to dopamine neurons projecting from the Ventral Tegmental Area (VTA) to the Nucleus Accumbens (NAc), in reward and addiction. Recent studies show that gamma-aminobutyric acid (GABA) containing neurons in the VTA (VTAGABA) provide local inhibition of dopamine neurons that synapse onto the NAc, thus playing a role in regulating reward behaviors. Importantly, these VTAGABA neurons contain a variety of G-protein coupled receptors, specifically the µ-opioid receptor (MOR). However, the exact role of these receptors and their signaling play in VTAGABA neurons and consequent regulation of natural reward is unknown. The source of endogenous opioid neuropeptide onto these MORs, and how these impacts signaling and activity has not been described. The central hypothesis of this proposal is that µ-opioid receptor signaling on VTAGABA neurons is regulated by afferent endogenous opioid peptides, resulting in disinhibition of VTAGABA neuron excitability. This results in control of dopamine neuron activity, and ultimately the expression of natural reward-seeking. This proposal directly addresses NIDA's Priority Scientific Area 1 that aims to further understand the molecular, neuropharmacological and circuit changes induced by drug use. Aim 1 will isolate the role of endogenous µ-opioid peptides in the VTA on dopamine signaling and natural reward- seeking. Aim1A uses ex vivo two-photon imaging and neuropharmacology approaches to visualize VTAGABA and VTA DA dynamics. Aim1B will investigate the effects of MOR ablation in the VTA on dopamine activity and behavior. Aim 2 will isolate the source and dynamics of endogenous µ-opioids in the VTA during natural reward behavior. In Aim 2A, I will use viral tracing techniques to anatomically visualize inputs from the lateral hypothalamus (LH) to the VTA. Aim 2B will test the effects of endogenous µ-opioid signaling on VTAGABA and DA activity in the NAc during reward seeking behaviors. In Aim 2C, I will use molecular approaches including CRISPR gene-editing and a recently developed µ-opioid biosensor (µMASS1) to understand the spatiotemporal aspects of MOR signaling in VTAGABA neurons during reward seeking. This career development training and series of experiments will provide insight into the role of endogenous MOR signaling on dopaminergic activity and natural reward-seeking. For this proposal, I will train in slice electrophysiology, two-photon slice imaging, gene-editing, molecular neuropharmacology, and behavioral approaches to understand how specific neuropeptides regulate reward circuits. This F31 proposal will greatly advance my career development plan and prepare me for a career as an independent, academic neuropharmacologist.