Engineering optogenetic tools for studying neuropeptide activity

7.项目总结/摘要 阿片受体（Opioid receptor，ORs）是一种神经肽受体，包括阿片受体、阿片受体和阿片肽受体，广泛参与阿片受体的形成， 镇痛情绪奖励和运动协调阿片类信号传导相应地涉及 包括抑郁症、毒瘾、压力和运动障碍在内的各种行为障碍。 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 治疗抑郁症和成瘾症由于这些神经回路深深地嵌入在一起， 高密度的大脑，需要非常高分辨率的工具来调制和研究单个电路， 详细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，包括OR在内的神经肽受体是G蛋白偶联受体（GPCR）， 当被激活时，会在细胞内触发多个生化信号级联。这些信号级联影响 从膜电位和放电率到DNA转录和生长，神经元中的各种过程。 因此，理解OR信号的一个关键障碍是缺乏可以完全复制的高分辨率工具。 OR激活的膜电生理和生物化学后果。 燕麦光、氧、电压感应结构域2（LOV 2）是一个光遗传学平台， 通过将肽融合到 以将肽结合反应与LOV 2中的光依赖性构象变化偶联的方式与LOV 2结合。 该LOV 2肽笼化策略将用于该提案中，以克服目前对阿片类药物的障碍。 通过以下具体目标进行信号研究：1）开发快速设计的筛选平台， 基于LOV 2的光遗传阿片肽调节剂，和2）开发LOV 2-阿片肽（LOV-OP）融合物 能够在细胞培养物和脑切片模型中光依赖性激活OR的蛋白质。或激活 将使用高通量GPCR cAMP活化试验在目标1中评价LOV-OP蛋白。目标2将 通过诱变优化LOV-OP蛋白以显示光依赖性OR激活， 研究OR激活的工具。神经元光依赖性抑制的电生理测量 通过LOV-OP激活的OR的放电将验证LOV-OP在原代神经元培养物和脑切片中的功能。 这项提案的广泛的长期目标是使阿片类药物的高时空分辨率研究成为可能。 肽活性的大脑，导致突破性的理解如何具体阿片肽 回路导致认知和行为障碍。高通量筛选方法开发于 这项建议也可以迅速扩展到研究大脑中其他神经肽的回路。

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.