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

描述（由申请人提供）：需要在活体实验动物的完整神经回路中操作限定的神经元群体，这对于研究脑疾病是迫切的，特别是那些可能由异常回路引起的脑疾病，例如许多经典的精神疾病。长期目标是开发和完善更复杂的体内神经元操作方法。这项特殊应用的总体目标是创建一套新的光驱动技术，用于研究完整大脑中的神经回路动力学。这些技术基于光遗传学与生物发光的结合，即通过生物发光酶将光敏分子（视蛋白）与生物产生的光结合。使光产生和光感测各自遗传编码的概念允许a）将视蛋白的使用从标准光遗传学应用扩展到还包括化学遗传激活; B）分别通过光产生和光感测蛋白的突触前和突触后靶向对突触信息流进行功能测试;和c）通过钙敏感性磷酸酶将神经活动翻译成光，光又向光敏蛋白发出信号。拟议研究的基本原理是，电路的双峰询问，功能性映射远程和局部连接，并使神经元的遗传靶向非侵入性自我调节将增加我们对神经元信息流的理解，可能导致新的和改进的方法来治疗神经精神疾病。基于强有力的初步数据，这些目标将通过追求三个具体目标来实现：1）鉴定用于激活和抑制神经元活性的最佳的荧光素酶-视蛋白组合; 2）在突触配偶体之间的细胞中表达光产生荧光素酶和光感测视蛋白;以及3）将在神经元激活时产生光的荧光素酶与光感测质子泵联合收割机组合。在第一个目标下，已经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性控制的光激活。该方法的创新之处在于， 使用遗传编码光源来激活遗传编码光转换分子。这项研究意义重大，因为它能够提出目前无法用现有技术解决的长期存在的问题，因此有望推进和扩大对神经元回路功能和功能障碍的理解。最终，这种增加的理解有可能为新的治疗方法提供信息，这将有助于减少美国日益严重的心理健康问题。

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.