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

综述：美国阿片使用障碍和阿片类药物过量死亡率创历史新高 新冠肺炎大流行期间的水平。了解内源性阿片活性如何影响自然奖赏 寻找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 涉及中脑边缘多巴胺通路，指的是从腹侧投射的多巴胺神经元 被盖区(VTA)至伏核(NAC)，在奖赏和成瘾中。最近的研究表明， VTA(VTAGABA)内含GABA的神经元对多巴胺有局部抑制作用 突触到NAC的神经元，因此在调节奖赏行为方面发挥作用。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(MOR)。 然而，这些受体的确切作用和它们的信号在VTAGABA神经元和随后的 对自然奖励的监管尚不清楚。内源性阿片神经肽对这些MORS的来源，以及 这些影响如何影响信号和活动还没有被描述。这项提议的中心假设是 VTAGABA神经元上的µ-阿片受体信号受传入内源性阿片肽的调节，结果 去抑制VTAGABA神经元的兴奋性。这导致了对多巴胺神经元活动的控制，最终 自然寻赏的表现。该提案直接涉及NIDA的优先科学领域1，即 目的是为了进一步了解药物使用引起的分子、神经药理学和电路变化。目标 1将分离内源性阿片肽在VTA中对多巴胺信号和自然奖赏的作用- 寻找。Aim1A使用体外双光子成像和神经药理学方法显示VTAGABA和 VTA DA动态。Aim1B将研究MOR消融VTA对多巴胺活动和 行为。AIM 2将在自然奖赏期间分离VTA内源性阿片类药物的来源和动态 行为。在目标2a中，我将使用病毒跟踪技术来解剖可视化来自外侧的输入 下丘脑(Lh)至VTA。目的2B将测试内源性阿片信号对VTAGABA和VTAGABA的影响 寻找奖赏行为过程中NAC的DA活动。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微阿片生物传感器(µMass1)来理解时空 在寻找奖赏过程中VTAGABA神经元中MOR信号的特点。这个职业发展培训和 一系列实验将深入了解内源性MOR信号在多巴胺能活动中的作用 以及自然而然的追求回报。对于这项提议，我将培训切片电生理学，双光子切片成像， 基因编辑、分子神经药理学和行为学方法，以了解 神经肽调节奖赏回路。这份F31提案将极大地推进我的职业发展计划，并 为我的职业生涯做好准备，成为一名独立的、学术的神经药剂师。

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.