Next-gen Opto-GPCRs: spatiotemporal simulation of neuromodulator signaling

项目摘要/摘要：光遗传学的新兴领域-使用光参与生物系统 - 为神经回路的解剖、细胞信号和操纵 神经生理学系统的研究。然而，实施的技术限制 光遗传学在解剖清醒的神经调质，自由移动的行为是明确的，而工作与 需要受体信号传导的离散时空控制的范例， 电路具有非常小的多样性，“难以到达”的架构，例如异质的脑核。到 神经药理学受体底物，几乎每个领域的神经科学家都使用套管（简单 金属管），并且最近已经采用拴系光纤用于体内光遗传学，以控制局部 释放神经调节剂单胺或神经肽。不幸的是，这些当前的方法相当 因为它们严重限制了对受体信号传导的时空控制 在离散细胞类型中的通路。此外，目前的技术缺乏一个完整的工具箱，用于多路复用，亚细胞， G蛋白偶联受体信号传导的时空控制， 大脑中的交流。由于这些原因，一项将神经科学与生物化学相结合的创新努力 和药理学是必要的，以使时空在体外和体内控制GPCR- 神经调质信号因此，我们在此直接讨论本RFA-NS-16-775的中心目标， 遵循方式。该提案的中心目标是开发一种尖端的v2.0 Opto-XR受体， 在体外和自由移动动物中空间和时间控制神经调节剂信号传导。我们有 提出了一种独特的综合方法来实现这一目标，使药理学家，生理学家， 生物化学家和神经科学家以一种独特的平行方式结合在一起。在这两个具体目标中，我们将制定 并在体外和体内测试这些新工具：1）为了开发突变的Gi和Gs，Opto-XR v2.0受体， 使用结构-功能分析实现更强的信号动力学和改变的色谱和灵敏度， 彻底的体外表征;和2）开发实用性并表征Opto-XR的Gi和Gs版本 v2.0使用传统和无线光遗传学在体内和自由移动行为模型中构建 接近。该提案的成功完成将为更广泛的神经科学界提供一个 期待已久的GPCR时空操纵-清醒状态下神经回路内的神经调制信号 行为自由的动物。这项新技术还将进一步拓宽能够 的离散控制和脑组织中神经调节功能的光动力学模拟。

Project Summary/Abstract: The emerging field of optogenetics — using light to engage biological systems — holds tremendous promise for dissection of neural circuits, cellular signaling and manipulating neurophysiological systems in awake, behaving animals. However, the technological limits for implementing optogenetics in dissecting neuromodulators in awake, freely-moving behavior is clear while working with paradigms that require discrete spatiotemporal control of receptor signaling and when investigating neural circuits that have very small diverse, “hard to reach” architecture, such as heterogeneous brain nuclei. To engage neuropharmacological receptor substrates, neuroscientists in nearly every field use cannulas (simple metal tubes) and have more recently adopted tethered fiber optics for in vivo optogenetics to control local release of neuromodulator monoamine or neuropeptides. Unfortunately, these current methods are rather limited and difficult to implement because they severely limit the spatiotemporal control over receptor signaling pathways in discrete cell types. Moreover, current technology lacks a full tool box for multiplexed, subcellular, spatiotemporal control of G protein coupled receptor signaling, the predominant means for neuromodulator communication in the brain. For these reasons, an innovative effort combining neuroscience with biochemistry and pharmacology was necessary in order to bring spatial-temporal in vitro and in vivo control over GPCR- neuromodulator signaling. Therefore, here we directly address the central goals of this RFA-NS-16-775 in the following manner. The central goal of this proposal is to develop a cutting-edge v2.0 Opto-XR receptors that spatially and temporally control neuromodulator signaling in vitro and in freely moving animals. We have proposed an uniquely integrated approach to achieve this goal that brings pharmacologists, physiologists, biochemists, and neuroscientists together in a unique parallel manner. In the two specific aims we will develop and test these novel tools in vitro and in vivo: 1) To develop mutant Gi and Gs, Opto-XR v2.0 receptors with greater signaling dynamics and altered color spectra and sensitivity using structure-function analyses and thorough in vitro characterization; and 2) To develop utility and characterize Gi and Gs versions of Opto-XR v2.0 constructs in vivo and in models of freely-moving behavior using both traditional and wireless optogenetic approaches. Successful completion of the proposal will provide the wider community of neuroscience with a long awaited spatiotemporal manipulation of GPCRs – neuromodulator signaling within neural circuits in awake freely behaving animals. This new technology will also further widen the field for approaches that are capable of discrete control and optodynamic simulation of neuromodulator function in brain tissue.