A viral system for light-dependent trapping of activated neurons

 描述（由申请人提供）：神经科学的一个中心目标是识别支持精神和行为状态的细胞系综，但这些系综无法先验定义。例如，齿状回（DG）包含超过1 M的颗粒细胞，这些细胞本质上彼此难以区分，但这些看似相同的神经元中只有不到5%在任何一个行为事件期间活跃，这表明相关的精神状态是由一小部分神经元介导的。我们建议开发一种新的方法，用于识别和获得这种短暂的，行为相关的神经元在清醒的动物大会的遗传访问。我们的方法的关键独特之处在于：（1）它的时间精度是前所未有的，因为它是第一个与自然行为的时间尺度相匹配的神经元标记技术;（2）它能够标记中小动物中的多个细胞群，从而能够比较特定状态的细胞系。我们的新分子遗传技术首先根据细胞内钙离子的升高来识别激活的神经元，然后用光标记它们。光的应用特别有吸引力，因为它在时间上是精确的：就像其他光遗传学方法通过近似细胞活动的时间尺度来辅助神经元电路分析一样，光依赖性标记技术也将照亮功能性细胞组装。这项技术将完全基于病毒，因此它可以在不依赖转基因动物的情况下跨物种使用。根据该奖项，我们将通过开发和测试两项关键创新来建立该技术：（1）合成双向启动子系统，以及（2）用于启动子功能的多波长可见光调控的笼化化学。最终，这项技术将用于阐明恐惧、饥饿、抑郁、焦虑和成瘾等不同心理状态的神经元基底，从而推进对关键大脑网络的探索。这个高风险、高回报的项目包含多个创新功能。这里描述的新生报告系统的元素，如启动子强度，调节基因表达的机制，活化配体的选择，笼化化学，以及体内配体和光传递代表了将受益于广泛优化的起点。一旦现有的报告成分已经足够完善，我们设想用重组酶取代荧光报告，这样致动器就可以在鉴定的细胞中表达，用于测试神经元功能。其他功能，包括新的笼状配体的发展，以及其他方法的全脑活动报告也将解决以下实现我们的目标。尽管存在固有的风险，但我们相信，我们提出的系统代表了对现有技术的根本性和迫切需要的偏离。我们相信，在BRAIN倡议的支持下，我们的方法将从目前作为一项有前途的奋进发展成为一种广泛使用的工具。

 DESCRIPTION (provided by applicant): A central goal in neuroscience is to identify cellular ensembles supporting mental and behavioral states, but these ensembles cannot be defined a priori. The dentate gyrus (DG), for example, contains more than 1M granule cells, which are essentially indistinguishable from each other, but less than 5% of these seemingly identical neurons are active during any one behavioral event, suggesting that the associated mental states are each mediated by a small subset of neurons. We propose to develop a novel method for identifying and gaining genetic access to such transient, behaviorally-relevant assemblies of neurons in awake animals. The key unique features of our approach are (1) its temporal precision is unprecedented because it is the first neuronal tagging technique that matches the timescale of naturalistic behavior; and (2) its ability to label multiple cell populations in the sme animal enables the comparison of state-specific cell ensembles. Our novel molecular-genetic technique first identifies activated neurons on the basis of elevated intracellular calcium and then tags them using light. Light application is especially attractive because it is temporally precise: just as other optogenetic methods have aided neuronal circuit analysis by approximating the timescale of cell activity, so too will a light-dependent labeling technique illuminate functional cell assemblies. The technique will be entirely virus-based, so it is usable across species without relying on transgenic animals. Under this award we will establish the technique by developing and testing two critical innovations: (1) a synthetic bidirectional promoter system, and (2) caging chemistry for multi-wavelength visible light regulation of promoter function. Ultimately this technique will be used to elucidate the neuronal substrates of diverse mental states, such as fear, hunger, depression, anxiety, and addiction, thereby advancing the exploration of critical brain networks. This high-risk, high-reward project comprises multiple innovative features. Elements of the nascent reporter system described here, such as promoter strength, mechanism for regulating gene expression, choice of activating ligand, caging chemistry, and in vivo ligand and light delivery represent starting point that will benefit from extensive optimization. Once existing reporter components have been sufficiently refined, we envision replacing fluorescent reporters with recombinases, so that actuators can be expressed in identified cells for testing neuronal function. Other features, including the development of novel caged ligands, as well as additional methods for brain-wide activity reporting will also be addressed following achievement of our Aims. Despite the inherent risks, we are confident that our proposed system represents a fundamental and much-needed departure from existing techniques. We believe that our approach will evolve from its present status as a promising endeavor into a widely-used tool with the support of the BRAIN Initiative.