Genetically Encoded Reporters of Integrated Neural Activity for Functional Mapping of Neural Circuitry-Administrative Supplement

 描述（由申请人提供）： 神经科学的主要挑战之一是将神经回路的结构与功能联系起来。为了实现这一目标，我们需要了解定义的神经元群体之间的连接以及这些神经元对生理过程，行为反应和疾病状态的贡献。成像技术的最新进展使我们能够以细胞分辨率可视化大脑结构。当前一代基因编码光学工具（如传感器和控制器）的应用，正在促进对清醒行为动物中分子定义的细胞群的神经元活动的测量和操纵。然而，探索神经回路的动力学基础行为，特别是解剖功能定义的电路超出分子定义的电路，不仅取决于现有工具的改进，而且还需要新的工程。因此，我们建议开发一种全新的传感器，通过生物化学试剂标记功能相关的神经元，这些生物化学试剂可以在研究人员定义的行为时期将神经活动整合到永久增加的荧光信号中。我们的技术取决于效应蛋白，离子通道，特别是钾通道Kv2.1，其激活状态与综合神经活性直接相关。Kv2.1的激活由其构象和翻译后状态决定，并且离子通道激活驱动电信号。最近，我们已经开发出适合于创建探针来监测离子通道激活的分子工具。使用一珠一化合物（OBOC）组合技术，我们已经确定了遗传编码的短肽（12-16聚体，GESI），在给定的生物条件下，特异性激活有机染料的荧光。使用现有的GESIs作为支架，我们建议设计和筛选新的肽-染料对，其相互作用由电压诱导的构象变化或Kv2.1的磷酸化控制，从而将这种丰富的神经元离子通道的激活状态转化为荧光信号。我们的具体目标将从设计和筛选电压敏感和去磷酸化GESIs开始，以我们在离子通道结构-功能，Rosetta计算蛋白质设计和高通量OBOC文库方面的专业知识为指导。我们将表征有前途的Kv2.1- GESI电压激活和去磷酸化探针在分离的神经元培养物和脑切片中的表达、细胞毒性、灵敏度和动力学。我们将最终证明这个新工具集识别活体动物中激活的神经元的能力。这一建议的成功结果将使神经活动的动态映射通过一个新的透镜：可视化的激活状态的离子通道，是在大脑中的电活动的中央效应器。由于该工具集独特地提供了有关功能连接的信息，因此它代表了一种全新的功能电路分析方法，而不是基于结构和遗传学的电路解剖。结合行为，将这些小的动态活动标签应用于脑成像开辟了神经元回路功能理解的新维度。

 DESCRIPTION (provided by applicant): One of the major challenges in neuroscience is to link the structure to the function of neural circuits. To achieve this goal, we need to understand the connectivity between defined neuronal populations and the contribution of these neurons to physiological processes, behavioral responses and disease states. Recent advances in imaging techniques allow us to visualize the brain structure with cellular resolution. Application of the current generation of genetically encoded optical tools, such as sensors and controllers, is facilitating measurement and manipulation of neuron activity from molecular-defined cell populations in awake, behaving animals. However, probing the dynamics of neural circuitry underlying behavior, specifically for dissecting functional-defined circuitry beyond molecular-defined circuitry, not only depends on the improvement of existing tools, but also requires novel engineering. We thus propose to develop a radically novel sensor to label functionally related neurons through biochemical reagents that can integrate neural activity into permanently increased fluorescent signals during a researcher-defined behavioral epoch. Our technology hinges on effector proteins, ion channels, in particular the potassium channel Kv2.1, whose activation status is directly correlated to the integrated neural activity. The activation of Kv2.1is determined by their conformational and post-translational status, and ion channel activation drives electrical signaling. Recently, we have developed molecular tools appropriate for creating probes to monitor the activation of ion channels. Using one-bead-one-compound (OBOC) combinatorial technology, we have identified genetically encoded short peptides (12-16 mers, GESIs) that specifically activate the fluorescence of organic dyes under a given biological condition. Using existing GESIs as scaffolds, we propose to design and screen novel peptide-dye pairs whose interaction is controlled by voltage-induced conformational changes or phosphorylation of Kv2.1, thus transforming the activation status of this abundant neuronal ion channel into fluorescent signals. Our specific aims will start by designing and screening voltage-sensing and dephosphorylation GESIs, guided by our expertise in ion channel structure-function, Rosetta computational protein design and high-throughput OBOC library. We will characterize the expression, cytotoxicity, sensitivity and kinetics of promising Kv2.1- GESI voltage activation and dephosphorylation probes in dissociated neuronal culture and in brain slices. We will finally demonstrate the capability of this novel toolset to identify activated neurons in living animals. A successful outcome of this proposal will enable dynamic mapping of neural activity through a new lens: visualizing the activation states of ion channels that are central effectors of electrical activity in the brain. As this toolset uniquely provides informatio regarding functional connectivity, it represents a completely novel approach for functional circuitry analysis, instead of circuitry dissection based on structure and genetics. Combined with behavior, application of these small dynamic activity tags to brain imaging opens up new dimensions of functional understanding of neuronal circuitry.