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

这项赠款申请是为了第二次续签我们集团的关键NIH赠款，该赠款支持 光遗传工具--微生物视蛋白，能够安全、及时、精确和高幅度地控制神经 清醒的哺乳动物和其他在神经科学中具有重要意义的物种的神经元活动。因为我们的 Grant于2010年首次获奖，它支持了Arch(第一个)等光遗传工具的开发 光遗传神经消音器导致清醒行为小鼠神经活动的~100%光遗传沉默)， ARCHT(对光的敏感度比Arch高3倍)，Chronos(一种超快光遗传激活剂，用于上下文 在速度至关重要的地方)，Chrimson(红移最大的光遗传激活剂，适用于激活大型 脑组织的体积以及避免果蝇的行为伪影)，Jaws(红移最严重的 光遗传消音器)、SoCoChR(实现单细胞、单峰分辨率光遗传学)和 ChromeQ(一种选择性钾和钠的光遗传激活剂)，导致了50篇同行评审的论文，以及 导致下一代光遗传工具在神经科学中广泛分布。到目前为止，我们有 主要使用基因组搜索来发现新的Opsins，挖掘公共和私人数据库以识别新的 候选人。在筛选了大量基因组资源以鉴定这些分子后， 然而，一个令人担忧的问题是，回报正在递减，一些目标将不会仅仅通过 基因组搜索，甚至是结构导向的定点突变。定向进化，它筛选出一个 大量的亲本基因突变，以确定朝着某个目标改进的版本，提供了希望，但 由于在哺乳动物中执行定向进化的困难，尚未应用于光遗传工具 细胞(必不可少，因为光遗传工具在定向进化中通常使用的细胞中表达良好，例如 大肠杆菌在哺乳动物细胞中不能很好地表达，在这种细胞中进化的光遗传工具可能会破坏 优化它们以在哺乳动物细胞中表达)，以及执行多维定向的难度 进化(至关重要，因为我们需要朝着多个目标优化光遗传工具-例如， 局部化、光谱和震级-沿一个轴进行过多优化将使工具沿一个轴取消优化 其他轴)。我们提出了一种用于光遗传工具工程(AIM)的定向进化方法 1)，并将其应用于光遗传学中几个长期开放的需求：创造红移和蓝移 光谱修整的光遗传激活剂，AIM 2；创造多光子优化消音器，AIM 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)， 旨在改善前述光遗传工具以及钾的光遗传工具动力学 光门控钾通道的电导(目标4)。我们的目标是向神经科学界提供一个 强大的光遗传控制器工具箱，具有广泛的实用价值，并能在全球范围内自由传播 研究领域。

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.