Genetically Encoded Light-Production and Light-Sensing for Neuronal Manipulation

描述(申请人提供)：在活体实验动物中，迫切需要在完整的神经回路中操纵确定的神经元群体，以研究大脑障碍，特别是那些可能由异常回路引起的障碍，如许多经典的精神障碍。长期目标是开发和改进更复杂的在体神经元操作方法。这一特殊应用的总体目标是创造一套新的光驱动技术，用于研究完整大脑中的神经电路动力学。这些技术是基于光遗传学和生物发光的结合，即通过荧光素酶将光感应分子(Opsins)与生物产生的光结合起来。具有光产生和光感知各自基因编码的概念允许a)扩大来自标准光遗传应用的视蛋白的使用，以包括化学遗传激活；b)通过分别针对产生光和感光的蛋白质的突触前和突触后靶向，对突触信息流进行功能测试；以及c)通过钙敏感的荧光素酶将神经活动转化为光，后者将信号转给光感蛋白。这项拟议研究的基本原理是，对电路进行双峰询问，从功能上绘制远程和局部连接的图，并使神经元能够进行基因靶向的非侵入性自我调节，将增加我们对神经元信息流的理解，可能导致治疗神经精神疾病的新的和改进的方法。基于强大的初步数据，这些目标将通过追求三个具体目标来实现：1)确定激活和抑制神经元活动的最佳荧光素酶-视蛋白组合；2)跨突触伙伴在细胞中表达产生光的荧光素酶和光敏视蛋白；以及3)将神经元激活时产生光的荧光素酶与感光质子泵相结合。在第一个目标下，已经在工作的神经元激活剂版本将被优化并扩展到神经元沉默。在第二和第三个目标下，将探索两个新的概念，特别是跨突触光激活和神经元活动控制的光激活。这种方法是创新的，因为它 使用基因编码的光源来激活基因编码的光传导分子。这项拟议的研究意义重大，因为它使人们能够提出目前无法用现有技术解决的长期问题，从而有望促进和扩大对神经元回路功能和功能障碍的理解。归根结底，这种加深的理解有可能为新的疗法提供信息，有助于减少美国日益严重的精神健康问题。

DESCRIPTION (provided by applicant): The need to manipulate defined neuronal populations in intact neural circuits in the living experimental animal is urgent for the study of brain disorders, specifically those which are likely caused by abnormal circuitry, such as many of the classical psychiatric disorders. The long-term goal is to develop and refine ever more sophisticated methods for in vivo neuronal manipulation. The overall objective of this particular application is to create a set of new light-driven technologies for studying neural circuit dynamics in the intact brain. These technologies are based on combining optogenetics with bioluminescence, i.e. combining light-sensing molecules (opsins) with biologically produced light through luciferases. The concept of having light-production and light-sensing each genetically encoded permits a) to expand the use of opsins from standard optogenetic applications to also include chemical genetic activation; b) functional testing of synaptic information flow through pre- and postsynaptic targeting of the light-producing and the light-sensing proteins, respectively; and c) translating neural activity via calcium-sensitive luciferases into light whichin turn signals to a light-sensing protein. The rationale for the proposed research is that bimodal interrogation of circuits, functionally mapping long-range and local connectivity, and enabling genetically targeted non-invasive self-regulation of neurons will increase our understanding of neuronal information flow, potentially resulting in new and improved approaches to treatment of neuropsychiatric disorders. Based on strong preliminary data, the objectives will be achieved by pursuing three specific aims: 1) Identify the optimal luciferase-opsin combinations for activating and inhibiting neuronal activity; 2) Express the light-producing luciferase and the light-sensing opsin in cells across synaptic partners; and 3) Combine a luciferase which produces light upon neuronal activation with a light-sensing proton pump. Under the first aim, already working versions of neuronal activators will be optimized and extended to neuronal silencing. Under the second and third aims two novel concepts will be explored, specifically transsynaptic light activation and neuronal activity-controlled light activation. The approach is innovative in that it uses a genetically encoded light source to activate a genetically encoded light transducing molecule. The proposed research is significant, because it enables to ask longstanding questions which currently cannot be addressed with available technology and thus is expected to advance and expand understanding of neuronal circuit function and dysfunction. Ultimately, such increased understanding has the potential to inform new therapeutics that will help reduce the growing mental health problems in the United States.