Engineering optogenetic tools for studying neuropeptide activity

7.项目摘要/摘要 阿片受体(ORs)由-，-和-ORs组成，是一种涉及广泛的神经肽受体 在调节止痛、情绪、奖励和运动协调方面。阿片类信号相应地牵涉到 在各种行为障碍中，包括抑郁、毒瘾、压力和运动障碍。 因此，了解OR回路活动如何影响行为对于开发更好的治疗方法至关重要 治疗抑郁症和成瘾等疾病。因为这些神经电路深深地嵌在一起 需要高密度、高分辨率的大脑工具来调制和研究 细节。目前用于解剖神经元回路的高分辨率光学方法仅限于改变膜 离子梯度。然而，神经肽受体，包括ORs，是G蛋白偶联受体(GPCRs)， 激活时，在细胞内触发多个生化信号级联。这些信号级联影响 神经元中的不同过程，从膜电位和放电率到DNA转录和生长。 因此，理解OR信号的一个关键障碍是缺乏能够完全复制的高分辨率工具 OR激活对细胞膜的电生理和生化影响。 燕麦的光、氧、电压传感结构域2(LOV2)是一个光遗传平台，已经 通过融合多肽成功地用于光调节多肽及其伴侣之间的相互作用 LOV2以一种将多肽结合反应与LOV2中依赖于光的构象变化相耦合的方式。 这项提议将使用LOV2-多肽笼化策略来克服目前阿片类药物的障碍 通过以下具体目标进行信令研究：1)开发一个快速设计的信令筛选平台 基于LOV2的光遗传阿片肽调节剂，以及2)开发LOV2-阿片肽融合(LOV-OP) 在细胞培养和脑切片模型中能够依赖光激活ORs的蛋白质。或激活方式 LOV-OP蛋白将在AIM 1中使用高通量GPCRcAMP激活试验进行评估。目标2将 优化LOV-OP蛋白以显示光依赖或通过突变激活，创造高分辨率 学习或激活的工具。神经元光依赖性抑制的电生理测量 LOV-OP激活的ORs将在原代神经元培养和脑片上验证LOV-OP的功能。 这项提议的广泛、长期目标是使阿片类药物的高时空分辨率研究成为可能 大脑中的多肽活性，导致对特定阿片肽如何 神经回路导致认知和行为障碍。中开发的高通量筛选方法 这项提议还可以迅速扩展到研究大脑中其他神经肽的电路。

7. Project Summary/Abstract Opioid receptors (ORs), consisting of -, -, and -ORs, are neuropeptide receptors that are broadly involved in regulating analgesia, mood, reward, and motor coordination. Opioid signaling is correspondingly implicated in a variety of behavioral disorders, including depression, drug addiction, stress, and dyskinesia. Understanding how OR circuit activity influences behavior is therefore critical to developing better treatments for disorders such as depression and addiction. Since these neural circuits are deeply embedded together in the brain at high density, very high resolution tools are needed for modulating and studying individual circuits in detail. Current high resolution optical methods for dissecting neuronal circuits are limited to altering membrane ion gradients. However, neuropeptide receptors, including ORs, are G-protein coupled receptors (GPCRs) that trigger multiple biochemical signaling cascades inside the cell when activated. These signaling cascades affect diverse processes in the neuron from membrane potential and firing rates to DNA transcription and growth. Thus, a critical barrier to understanding OR signaling is the lack of high-resolution tools that can fully replicate both membrane electrophysiological and biochemical consequences of OR activation. The Avena sativa Light, Oxygen, Voltage sensing domain 2 (LOV2) is an optogenetic platform that has been successfully utilized to photoregulate interactions between peptides and their partners by fusing peptides to LOV2 in a way that couples the peptide binding reaction to a light-dependent conformational change in LOV2. This LOV2-peptide caging strategy will be used in this proposal to overcome the current barriers to opioid signaling research through the following specific aims: 1) develop a screening platform for the rapid design of LOV2-based optogenetic opioid peptide modulators, and 2) develop a LOV2-opioid peptide (LOV-OP) fusion protein capable of light-dependent activation of ORs in cell culture and brain slice models. OR activation by LOV-OP proteins will be evaluated in Aim 1 using a high-throughput GPCR cAMP activation assay. Aim 2 will optimize a LOV-OP protein to show light-dependent OR activation via mutagenesis, creating a high resolution tool for studying OR activation. Electrophysiological measurements of light-dependent depression of neuron firing by ORs activated by LOV-OP will validate LOV-OP function in primary neuron culture and brain slice. The broad, long-term objectives of this proposal are to enable high spatiotemporal resolution studies of opioid peptide activity in the brain that lead to breakthroughs in the understanding of how specific opioid peptidergic circuits contribute to cognition and behavioral disorders. The high-throughput screening methods developed in the proposal can also be rapidly extended to studying circuits of other neuropeptides in the brain.