Structure guided design of photoselectable channelrhodopsins

项目总结： 这项提案概述了一种全新的光遗传技术的发展 灵活地操纵数千个神经元的活动，这些神经元参与了动态活动 具有单神经元分辨率的分布式神经电路。 目前甚至不存在任何方法 远程满足灵活、选择性地控制分布在 大量的大脑。填补这一方法论空白是 大脑倡议，因为这样做将改变我们研究神经如何紧张的能力 系统对信息进行编码、处理、利用、存储和检索。 本应用程序的总体目标是获取感光剂的关键结构知识 红移通道的状态和视紫质，并用它们来设计可光选择的通道 原型，展示了我们的方法在行为方面未来发展的潜力 动物。这将允许视蛋白表达神经元被灵活地选择、激活和 用光取消选择。通过利用新的结构知识，我们预计我们可以 开发一种全新的光遗传学方法，使我们超越基因 有针对性的控制和进入一个功能有针对性的、灵活控制任何神经集合的时代。 我们的研究目的是获得红移通道视紫红质的第一个原子结构 三个通道态的突变体，设计一个高开放的三态ReaChR突变体 电导和优化的作用光谱，并展示了可逆光选择控制 体内具有PReaChR原型的神经元。 我们预计，完成这些目标将产生以下预期结果。首先，它 将产生关于潜在的结构转换的新知识 通道视紫红质光态，将使光选择的高效计算设计成为可能 光遗传工具。其次，它将产生第一个光选择的例子 对神经兴奋有用的通道视紫红质。第三，它将评估这些新的 用于灵活控制分布的神经元集合的OPTINS。总体而言，这些将提供一个 将光选择性的变革性新特性扩展到广泛的 现有的用于兴奋、抑制和调节神经活动的光遗传工具。进一步 这一方向的研究最终应该能够灵活地控制空间复杂性 头部固定和自由活动动物在行为过程中神经元的分布 加深我们对错综复杂的神经动力学的理解，这些神经动力学是我们思想、感觉、 以及神经紊乱是如何扰乱电路动力学的。

Project Summary: This proposal outlines the development of a fundamentally new optogenetic technology capable of flexibly manipulating the activity of thousands of neurons contributing to the dynamic activity of distributed neural circuits with single neuron resolution. No method that currently exists even remotely meets the need of flexible, selective control of thousands of neurons distributed across large volumes of the brain. Filling this methodological gap is a central research objective of the BRAIN Initiative, because doing so will transform our ability to investigate how the nervous system encodes, processes, utilizes, stores, and retrieves information. The overall objective for this application is to acquire critical structural knowledge of photoactive states of a red-shifted channelrhodopsin and use these to engineer a photoselectable channel prototype that demonstrates the potential of our approach for future development in behaving animals. This would allow opsin-expressing neurons to be flexibly selected, activated, and deselected with light. By leveraging new structural knowledge, we anticipate that we can develop a fundamentally new approach to optogenetics that takes us beyond genetically targeted control and into an era of functionally targeted, flexible control of any neural ensemble. The aims of our research are to obtain the first atomic structures of red-shifted channelrhodopsin mutants in three channel states, engineer a three-state ReaChR mutant with high open conductance and optimized action spectra, and demonstrate reversible photoselective control of neurons in vivo with PReaChR prototypes. We anticipate that completion of these aims will yield the following expected outcomes. First, it will produce new knowledge of the underlying structural transformations between channelrhodopsin photostates that will enable efficient computational design of photoselectable optogenetic tools. Second, it will produce the first examples of photoselective channelrhodopsins useful for neural excitation. Third, it will assess the utility of these new opsins for flexible control of distributed sets of neurons. Collectively, these will provide a roadmap to extending the transformative new trait of photoselectabilty to a wide range of existing optogenetic tools for excitation, inhibition and modulation of neural activity. Further research in this direction should ultimately enable flexible control of spatially complex distributions of neurons in head-fixed and freely moving animals during behavior, a key to furthering our understanding of the intricate neural dynamics that underlie our thoughts, feeling, and actions and how circuit dynamics are disrupted by neurological disorders.