Novel Platforms for Systematic Optical Control of Complex Neural Circuits In Vivo

这项拨款申请是对我们小组的关键NIH拨款的第二次更新，该拨款支持以下项目的开发： 光遗传学工具-微生物视蛋白，能够安全，时间精确和高幅度控制神经元 在清醒的哺乳动物和其他物种的神经元活动在神经科学的重要性。由于我们 该基金于2010年首次授予，它支持了光遗传学工具的开发，如Arch（第一个 光遗传学神经沉默子导致清醒行为小鼠中神经活动的~100%光遗传学沉默）， ArchT（Arch的3倍感光度），Chronos（超快光遗传激活剂，用于上下文） 其中速度是必不可少的），Chrimson（最红移的光遗传学激活剂，用于激活大的 大量的脑组织以及避免果蝇的行为伪影），大白鲨（最红移 光遗传学沉默子）、SoCoChR（其实现单细胞、单尖峰分辨率光遗传学）和 ChromeQ（一种钾和钠选择性光遗传激活剂），产生了50篇同行评审论文， 导致下一代光遗传学工具在整个神经科学中广泛分布。到目前为止， 主要使用基因组搜索发现新的视蛋白，挖掘公共和私人数据库，以确定新的 候选人在筛选了大量的基因组资源以鉴定这些分子之后， 然而，一个令人担忧的问题是，收益递减，一些目标将无法完全通过 基因组搜索，甚至是结构导向的定点突变。定向进化，通过筛选 亲本基因的大量突变体，以确定朝着某个目标改进的版本，提供了希望， 由于在哺乳动物中进行定向进化的困难， 细胞（必需的，因为在定向进化中常用的细胞中表达良好的光遗传学工具，如 E.大肠杆菌，在哺乳动物细胞中不能很好地表达，在这些细胞中进化光遗传学工具可能会使 优化它们在哺乳动物细胞中的表达），以及进行多维定向表达的困难。 进化（至关重要，因为我们需要优化光遗传学工具以实现多个目标-例如， 局部化、频谱和幅度-沿沿着一个轴优化过多将使工具沿沿着失去优化 其他轴）。我们在这里提出了一种定向进化方法，用于光遗传学工具工程（Aim 1），并将其应用于光遗传学中的几个长期开放的需求： 多光子优化沉默剂的产生，目标3;以及 动力学和离子选择性的优化（通过开发和应用自动膜片钳技术）， 旨在改善上述光遗传工具的光遗传工具动力学以及钾 光门控钾通道的电导（目的4）。我们的目标是向神经科学界提供一个 广泛实用的光遗传学控制器的强大工具箱，并在整个 研究世界。

This grant application is for a second renewal of our group’s key NIH grant that supports development of optogenetic tools -- microbial opsins that enable safe, temporally precise, and high-magnitude control of neural activity in neurons in awake behaving mammals and other species of importance in neuroscience. Since our grant was first awarded in 2010, it has supported the development of optogenetic tools such as Arch (the first optogenetic neural silencer to result in ~100% optogenetic silencing of neural activity in awake behaving mice), ArchT (a 3x more light-sensitive relative of Arch), Chronos (an ultrafast optogenetic activator, used in contexts where speed is essential), Chrimson (the most redshifted optogenetic activator, useful for activation of large volumes of brain tissue as well as avoiding behavioral artifacts in Drosophila), Jaws (the most redshifted optogenetic silencer), SoCoChR (which enables single-cell, single-spike resolution optogenetics) and ChromeQ (a potassium- and sodium-selective optogenetic activator), resulting in 50 peer reviewed papers, and resulting in wide distribution of next-generation optogenetic tools throughout neuroscience. To date, we have primarily used genomic search to discover novel opsins, mining public and private databases to identify new candidates. Having screened through a large number of genomic resources to identify these molecules, however, one concern is that there are diminishing returns, and that some goals will not be met purely through genomic search, or even structure-guided site-directed mutagenesis. Directed evolution, which sifts through a large number of mutants of a parent gene to identify versions improved towards some goal, offers hope, but has not been applied to optogenetic tools due to the difficulty of performing directed evolution in mammalian cells (essential, since optogenetic tools that express well in cells commonly used in directed evolution, such as E. coli, do not express well in mammalian cells, and evolving optogenetic tools in such cells would likely de- optimize them for expression in mammalian cells), and the difficulty of performing multidimensional directed evolution (essential, because we need to optimize optogenetic tools towards multiple goals – for example, localization, spectrum, and magnitude – and optimizing too much along one axis will de-optimize the tool along other axes). We here propose to develop a directed evolution approach for optogenetic tool engineering (Aim 1), and apply it to several longstanding open needs in optogenetics: the creation of redshifted and blue spectrum-trimmed optogenetic activators, Aim 2; the creation of multiphoton-optimized silencers, Aim 3; and the optimization (by developing and applying automated patch clamp technology) of kinetics and ion selectivity, aiming to improve optogenetic tool kinetics for the aforementioned optogenetic tools as well as potassium conductances of light-gated potassium channels (Aim 4). We aim to deliver to the neuroscience community a powerful toolbox of optogenetic controllers of widespread utility, and to disseminate them freely throughout the research world.