Next Generation Opto-GPCRs for Neuromodulatory Control

项目摘要/摘要：光遗传学领域--利用光参与生物系统--是 广泛用于解剖神经回路、细胞信号和操纵神经生理系统 在清醒的，有行为的动物中。然而，尽管许多新的视蛋白已经被开发并被积极使用， 挑战依然存在，目前的技术缺乏一个完整的工具箱，用于亚细胞、时空控制 信号传递--大脑中神经调节剂交流的主要方式。在这里，我们建议，一个 将神经科学与结构生物学和高通量药理学相结合的创新成果 开发一系列尖端的新型光学GPCR，将允许时空精确和 在体外和自由活动的动物中神经调节剂信号的路径选择性控制。在四个目标中 五个领先的实验室，我们将在体外和体内开发和测试这些新工具。具体来说，我们将 致力于1)开发和全面优化OptoGPCR-v3.0(GI耦合)受体，以增强光谱 利用结构-功能分析和突变体文库HTS着陆进行多路复用和改变敏感性 PAD系统；2)开发和全面优化OptoGPCR-v3.0-Gq受体，用于选择性偶联Gq信号 使用结构制导和HTS着陆台系统的路径；3)使用较少探索的数据库 测试、筛选和进一步开发具有独特轮廓的新光学工具的自然产生的视光-GPCR；以及 4)评估在体内同时使用OptoGPCR-v3.0构建物的光谱兼容性，以及 使用光度学、2p成像和体内并行行为测量的生物传感器。成功完成 将为广泛的神经科学界提供期待已久的 在体内外神经回路中GPCR-神经调节剂信号的时空操作 唤醒自由行为的动物，可用于广泛的各种应用。这项新技术将 还进一步拓宽了允许离散控制和光动力学的独特光学方法的领域 脑组织中神经调质功能的模拟。因此，我们认为，通过实现这些目标 我们直接针对此RFA-NS-21-027呼叫的中心目的。

Project Summary/Abstract: The field of optogenetics — utilizing light to engage biological systems — is widely used for the dissection of neural circuits, cellular signaling and manipulating neurophysiological systems in awake, behaving animals. However, while many new opsins have been developed and are actively used, challenges still remain, and the current technology lacks a full toolbox for sub-cellular, spatiotemporal control of signaling — the predominant means for neuromodulator communication in the brain. Here we propose, an innovative effort combining neuroscience with structural biology and high-throughput pharmacology for the development of a series of cutting-edge novel Opto-GPCRs that will allow spatiotemporally precise and pathway-selective control of neuromodulator signaling in vitro and in freely moving animals. In four aims across five leading laboratories, we will develop and test these novel tools in vitro and in vivo. Specifically, we will work to 1) Develop and fully optimize OptoGPCR-v3.0 (Gi coupled) receptors for enhanced spectral multiplexing and altered sensitivity using structure-function analysis together with mutant-library HTS landing pad system; 2) Develop and fully optimize OptoGPCR-v3.0-Gq receptors for selective coupling to Gq signaling pathways using structure-guidance and the HTS landing pad system; 3) Utilize databases of less-explored naturally-occurring opsin-GPCRs to test, screen and further develop new optical tools with unique profiles; and 4) Assess the spectral compatibility for simultaneous use of OptoGPCR-v3.0 constructs in vivo, together with biosensors using photometry, 2p imaging and concurrent behavioral measures in vivo. Successful completion of the proposal will provide the wide neuroscience community with the long awaited capabilities of spatiotemporal manipulation of GPCR – neuromodulator signaling within neural circuits in vitro and in vivo, in awake freely behaving animals, and could be used for a wide variety of applications. This new technology will also further widen the field for unique optical approaches that allow discrete control and optodynamic simulation of neuromodulator function in brain tissue. We therefore believe that by the fulfilment of these goals we directly address the central purpose of this RFA-NS-21-027 call.