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

用于神经回路功能图谱的综合神经活动的基因编码报告-管理补充

• 批准号: 9269378
• 负责人: James S Trimmer
• 金额: $ 11.66万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9269378
• 关键词: Action Potentials; Administrative Supplement; Animals; Behavior; Behavior Control; Behavioral; Biochemical; Biological; Brain; Brain imaging; Calcium; Cells; Chimeric Proteins; Complex; Dimensions; Disease; Dissection; Dyes; Electrophysiology (science); Engineering; Fingerprint; Fluorescence; Generations; Genetic Structures; Goals; Health; Image; Imagery; Imaging Techniques; Immediate-Early Genes; Ion Channel; Ion Channel Protein; Kinetics; Knowledge; Kv2.1 channel; Label; Libraries; Life; Link; Manuscripts; Maps; Measurement; Methods; Modeling; Molecular; Monitor; Motor Cortex; Neuroglia; Neurons; Neurosciences; Neurotransmitters; Optics; Outcome; Pathway interactions; Pattern; Peptides; Performance; Phosphorylation; Physiological Processes; Population; Potassium Channel; Probability; Property; Protein Dephosphorylation; Protein Engineering; Proteins; Reagent; Reporter; Reporting; Research Personnel; Resolution; Running; Signal Transduction; Site; Slice; Specific qualifier value; Structure; Surface; System; Techniques; Technology; Testing; Time; Tissues; Training; Validation; awake; base; behavioral response; combinatorial; cytotoxicity; design; experience; in vivo; innovation; lens; nervous system disorder; neural circuit; neural patterning; neuronal cell body; neuronal circuitry; new technology; novel; novel strategies; protein structure; relating to nervous system; response; scaffold; screening; sensor; spatiotemporal; temporal measurement; tool; voltage

 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.

 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群体之间的连接性以及这些神经元对生理过程的贡献。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（例如传感器和控制器）的应用正在促进从分子角度测量和操纵神经元活动。 -定义的细胞群然而，探索神经回路潜在行为的动力学，特别是剖析分子定义电路之外的功能定义电路，不仅取决于现有工具的改进，而且还需要新的工程设计。一种全新的传感器，通过生化试剂标记功能相关的神经元，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，其激活状态与整合的神经活动直接相关，Kv2.1 的激活由其构象和翻译后状态决定，并且离子通道激活驱动电信号传导。最近，我们开发了适合创建探针来监测的分子工具。使用一珠一化合物 (OBOC) 组合技术，我们鉴定了可特异性激活有机染料荧光的基因编码短肽（12-16 mers，GESI）。在给定的生物条件下，我们建议使用现有的GESI作为支架，设计和筛选新型肽-染料对，其相互作用由电压诱导的构象变化或Kv2.1磷酸化控制，从而改变这种丰富的神经离子的激活状态。我们的具体目标将从设计和筛选电压传感和去磷酸化 GESI 开始，以我们在离子通道结构功能、Rosetta 计算蛋白设计和高通量 OBOC 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离解神经培养物和脑切片中的表达、细胞毒性、敏感性和动力学。我们最终将证明这一新型工具集识别活体神经元的能力。该提案的成功结果将通过新的镜头实现神经活动的动态映射：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，因为该工具集提供了独特的信息。关于功能连接，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，将这些小的动态活动标签应用于大脑成像开辟了神经电路功能理解的新维度。