Molecular tools for labeling and manipulating functional brain circuits

项目摘要 标记和操纵功能性脑回路的分子工具 神经科学的一个基本目标是发现与特定神经元相关的神经元亚群。 行为。例如，当我们感到恐惧或口渴时，我们会利用大脑中的哪些神经元？什么是神经元 当我们学习将特定的背景或刺激与恐惧感联系起来时，我们会使用什么？该领域经历了 革命与工具的出现，如通道视紫红质，DREADDS，实时钙指标， 多光子显微镜，使监测和操纵神经元活动清醒，行为的动物。 然而，当实验者已经有了一个特定的假设， 亚群可能与感兴趣的行为相关。缺乏的是一种技术来指导研究人员 特定的大脑区域和神经元亚群，当研究行为的先验信息不存在时， 不完整 在这里，我们建议开发一系列分子工具，统称为“FLARE”，用于快速光- 活动调节表达，这可能是非常有用的行为的神经回路基础的研究。耀斑是 光和升高的细胞溶质钙（神经元活动的代表）的符合检测器，其给出任何 选择报告基因作为其输出。如果FLARE成分在整个大脑区域表达，并且光被传递 与感兴趣的刺激一致，那么转基因表达应该是 选择性地只在光传递过程中激发的神经元亚群中打开，这可能是 短至几分钟到几秒。通过使用转基因如GFP通道视紫红质， 成像感兴趣的神经元亚群，并驱动其活动，从而检查与行为的因果关系。 我们的初步结果表明，第二代FLARE 2标记激活的神经元在培养与标签 时间窗口只有60秒。在这个项目中，我们建议充分表征和验证FLARE 2及其变体 （包括单链FLARE、scFLARE），同时应用蛋白质工程 技术和定向进化（我们的实验室拥有丰富的经验），以迭代地改进和 优化FLARE工具系列。 为了允许神经回路的全脑映射，我们还提出了一种FLARE变体，称为“nanoFLARE”， 可以通过光或小分子释放，所述小分子可以通过IP注射递送到整个大脑中， 那个禽兽NanoFLARE的特点是荧光素酶部分融合到工具的蛋白酶组分，我们发现它可以 通过邻近依赖性BRET释放光敏LOV结构域。因此，nanoFLARE可以通过以下方式解除锁定： 直接蓝光照射，或通过将小分子荧光素酶底物阿曲马嗪递送至脑。 这一提议的成功结果将创建一个工具包，使神经科学家能够发现神经 亚群以前所未有的速度和准确性，成为广泛行为和认知过程的基础， 并提供一种方法来询问这些电路编码功能的机制。

Project Summary Molecular tools for labeling and manipulating functional brain circuits A fundamental goal of neuroscience is to discover the subpopulations of neurons that are associated with specific behaviors. For example, what neurons in the brain do we utilize when we experience fear, or thirst? What neurons are utilized when we learn to associate a specific context or stimulus with the feeling of fear? The field has experienced a revolution with the emergence of tools such as channelrhodopsins, DREADDS, real-time calcium indicators, and multiphoton microscopes that enable monitoring and manipulation of neuronal activity in awake, behaving animals. However, these tools are most useful when the experimentalist already has a specific hypothesis for which neuronal subpopulations may be relevant to the behavior of interest. What is lacking is a technology to guide the researcher to specific brain regions and neuronal subpopulations, when prior information about the behavior under study is absent or incomplete. Here we propose to develop a family of molecular tools, collectively called “FLARE”, for Fast Light- and Activity-Regulated Expression, that may be highly useful for the study of the neural circuit basis of behaviors. FLARE is a coincidence detector of light and elevated cytosolic calcium (a proxy for neuronal activity) that gives expression of any reporter gene of choice as its output. If FLARE components are expressed throughout a brain region, and light is delivered to that region via an implanted fiber, coincident with a stimulus of interest, then transgene expression should be selectively turned on only in the subpopulation of neurons that fired during the moment of light delivery, which could be as short as a few minutes to seconds. By using a transgene such as GFP-channelrhodopsin, it would be possible to both image the neuronal subpopulation of interest, and drive its activity, thereby examining the causal relationship to behavior. Our preliminary results show that second-generation FLARE2 marks activated neurons in culture with a tagging time window of only 60 seconds. In this project, we propose to fully characterize and validate FLARE2 and its variants (including single chain FLARE, scFLARE) in vivo (mouse and fly) while simultaneously applying protein engineering techniques and directed evolution (with which our laboratory has extensive experience), to iteratively improve and optimize the family of FLARE tools. To permit brain-wide mapping of neural circuits, we also propose a FLARE variant, called “nanoFLARE”, that can be uncaged by either light or a small-molecule that can be delivered throughout the entire brain via IP injection into the animal. NanoFLARE features a luciferase moiety fused to the protease component of the tool, that we discovered can uncage the light-sensitive LOV domain via proximity-dependent BRET. Hence, nanoFLARE can be uncaged either by direct blue light illumination, or by delivery of the small-molecule luciferase substrate furimazine to the brain. A successful outcome of this proposal would create a toolkit that will empower neuroscientists to discover neural subpopulations that underlie a wide range of behaviors and cognitive processes, with unprecedented speed and accuracy, and provide a means to interrogate the mechanisms by which these circuits encode function.