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结构与神经回路的功能联系起来。为了实现这一目标，我们需要了解特定神经元群体之间的连通性，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。当代遗传编码光学工具的应用，如传感器和控制器，正在促进对清醒、行为动物的分子定义细胞群中神经元活动的测量和操作。然而，探索神经电路潜在行为的动力学，特别是为了解剖分子定义的电路之外的功能定义的电路，不仅依赖于现有工具的改进，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，通过生化试剂标记功能相关的神经元，这种试剂可以在研究人员定义的行为时代将神经活动整合到永久增加的荧光信号中。我们的技术依赖于效应器蛋白、离子通道，特别是钾通道Kv2.1，它的激活状态与整体神经活动直接相关。Kv2.1的激活由其构象和翻译后状态决定，离子通道激活驱动电信号转导。最近，我们开发了适合于创建探针来监控离子通道激活的分子工具。利用一珠一化合物(OBOC)组合技术，我们已经鉴定出在给定生物条件下特异激活有机染料荧光的遗传编码短肽(12-16 MERS，Gesis)。我们建议利用现有的Gesis作为支架，设计和筛选新的多肽-染料对，其相互作用受电压诱导的Kv2.1构象变化或磷酸化控制，从而将这一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GEIS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。我们将研究Kv2.1-GeSI电压激活和去磷酸化探针在分离的神经元培养和脑片中的表达、细胞毒性、敏感性和动力学。我们将最终展示这一新的工具集识别活动物中激活的神经元的能力。这一提议的成功结果将使通过一种新的透镜动态绘制神经活动图成为可能：可视化作为大脑电活动中心效应器的离子通道的激活状态。由于该工具集独一无二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到大脑成像中，打开了对神经元电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.