Novel fluorescent sensors based on GPCRs for imaging neuromodulation

神经调质是调节许多神经过程的重要信号分子，包括 认知，情绪，记忆和睡眠，通过它们对大脑回路的影响。监测释放 神经调质在行为动物中的分布对于理解 这些分子的功能。发展这种理解的一个主要障碍是缺乏工具 可以在时间、空间和浓度尺度上监测这些化合物， 大脑活动填补这一技术空白是神经科学最迫切的需求之一 research.我们的建议通过开发一个新工具平台，直接弥合了这一差距， 以毫秒、亚细胞和纳摩尔分辨率对神经调质进行侵入性监测。 钙和谷氨酸的遗传编码荧光指示剂改变了对 主要模型系统中的动态大脑过程，包括蠕虫，苍蝇，啮齿动物，以及越来越多的 灵长类动物根据我们以前开发这些工具的经验，我们现在建议建立一个新的 一套基于GPCR激活（GRAB）基因编码荧光指示剂， 神经调质我们的初步数据显示，我们可以生成具有>500%荧光的GRAB 变化和纳摩尔亲和力。我们建议进一步开发和验证这些 原型在培养的神经元，苍蝇，啮齿动物脑切片，麻醉和行为小鼠，以最大限度地提高 他们的效用。 在目标1中，我们将开发乙酰胆碱、5-羟色胺和去甲肾上腺素的GRAB指标， 迭代筛选在插入位点、接头、cpGFP序列方面系统变化的文库， FP-GPCR蛋白表面界面。优化的维度将是dF/F，膜表面 表达、亲和力和内源性信号传导的不中断。我们的目标性能水平是 > 10 x dF/F，纳摩尔范围亲和力和<10毫秒体外结合速率。 在目标2中，将验证体外筛选的首选GRAB指示剂的性能 在果蝇嗅觉系统、脑切片、麻醉和 行为小鼠皮层。来自这些实验的反馈将指导目标1中的迭代优化。 成功完成我们的目标将产生一套强大的分子结构，细胞类型特异性 病毒工具和技术方法，将广泛传播到神经科学界。 GRAB指标可以很容易地与现有的人类精神障碍小鼠模型相结合。 由于这些用于神经调质的探针非常适合于各种各样的制备， 研究人员的数量，他们将对我们的神经回路的理解产生倍增的影响 功能和功能障碍时，结合其他进步支持的大脑倡议。

Neuromodulators are essential signaling molecules that regulate many neural processes, including cognition, mood, memory, and sleep, through their influence on brain circuits. Monitoring the release and distribution of neuromodulators in behaving animals is critical for understanding the diverse functions of these molecules. A major impediment to developing this understanding is the lack of tools that can monitor these compounds at the temporal, spatial and concentration scales relevant to these brain processes. Filling this technological gap is one of the most pressing needs in neuroscience research. Our proposal directly bridges this gap by developing a platform of new tools for chronic, non- invasive monitoring of neuromodulators at millisecond, subcellular, and nanomolar resolution. Genetically-encoded fluorescent indicators for calcium and glutamate have transformed investigation of dynamic brain processes in the major model systems, including worms, flies, rodents, and increasingly primates. Building on our prior experience in developing these tools, we now propose to build a new suite of GPCR-activation-based (GRAB) genetically-encoded fluorescent indicators for neuromodulators. Our preliminary data shows we can generate GRABs with >500% fluorescence change and nanomolar affinity in mammalian cells. We propose to further develop and validate these prototypes in cultured neurons, flies, rodent brain slices, anesthetized and behaving mice to maximize their utility. In Aim 1, we will develop GRAB indicators for acetylcholine, serotonin, and norepinephrine by iteratively screening libraries that systematically vary in insertion site, linkers, cpGFP sequence, and FP-GPCR protein surface interface. The dimensions of optimization will be dF/F, membrane surface expression, affinity, and non-disruption of endogenous signaling. Our targeted performance levels are >10x dF/F, nanomolar range affinity and <10 millisecond on-rates in vitro. In Aim 2, performance of top candidate GRAB indicators from the in vitro screen will be validated following long-term expression in drosophila olfactory system, in brain slice, in anesthetized and behaving mouse cortex. Feedback from these experiments will guide iterative optimization in Aim 1. Successful completion of our Aims will yield a suite of powerful molecular constructs, cell-type specific viral tools and technical approaches that will be broadly disseminated to the neuroscience community. The GRAB indicators can be easily integrated with existing mouse models of human mental disorders. Since these probes for neuromodulators are well-suited for a wide range of preparations, and a large number of investigators, they will have a multiplicative impact on our understanding of neural circuit function and dysfunction when combined with other advances supported by the BRAIN Initiative.