Structure guided design of photoselectable channelrhodopsins

项目摘要： 该提案概述了一种全新的光遗传学技术的发展， 灵活地操纵数千个神经元的活动， 分布式神经回路的单神经元分辨率。 目前没有任何方法， 远程满足了对分布在不同区域的数千个神经元进行灵活、选择性控制的需求。 大量的大脑。填补这一方法上的空白是本报告的一个中心研究目标。 大脑倡议，因为这样做将改变我们的能力，调查如何紧张， 系统编码、处理、利用、存储和检索信息。 本申请的总体目标是获得光敏化合物的关键结构知识， 状态的红移通道视紫红质，并使用这些工程师的光选择性通道 原型，展示了我们的方法在未来发展中的潜力， 动物这将允许视蛋白表达神经元被灵活地选择，激活， 用灯光取消选择。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， 有针对性的控制和进入一个时代的功能有针对性的，灵活的控制任何神经合奏。 我们的研究目的是获得红移通道视紫红质的第一级原子结构 在三种通道状态的突变体中，工程化具有高开放性的三态ReaChR突变体， 电导和优化的行动光谱，并证明可逆的光选择性控制 神经元与PReaChR原型。 我们预计，完成这些目标将产生以下预期成果。一是 将产生新的知识， 通道视紫红质光态，这将使光可选择的高效计算设计成为可能。 光遗传学工具。其次，它将产生第一个光选择性的例子， 用于神经兴奋的通道视紫红质。第三，它将评估这些新的 用于灵活控制分布式神经元集合的视蛋白。总的来说，这些将提供一个 路线图，以扩大变革性的新性状的光选择性，以广泛的 用于神经活动的激发、抑制和调节的现有光遗传学工具。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的 在行为过程中，头部固定和自由移动的动物中神经元的分布，这是研究 进一步加深我们对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， 以及神经系统疾病如何破坏电路动力学。

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.