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
基本信息
- 批准号:9269378
- 负责人:
- 金额:$ 11.66万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:Action PotentialsAdministrative SupplementAnimalsBehaviorBehavior ControlBehavioralBiochemicalBiologicalBrainBrain imagingCalciumCellsChimeric ProteinsComplexDimensionsDiseaseDissectionDyesElectrophysiology (science)EngineeringFingerprintFluorescenceGenerationsGenetic StructuresGoalsHealthImageImageryImaging TechniquesImmediate-Early GenesIon ChannelIon Channel ProteinKineticsKnowledgeKv2.1 channelLabelLibrariesLifeLinkManuscriptsMapsMeasurementMethodsModelingMolecularMonitorMotor CortexNeurogliaNeuronsNeurosciencesNeurotransmittersOpticsOutcomePathway interactionsPatternPeptidesPerformancePhosphorylationPhysiological ProcessesPopulationPotassium ChannelProbabilityPropertyProtein DephosphorylationProtein EngineeringProteinsReagentReporterReportingResearch PersonnelResolutionRunningSignal TransductionSiteSliceSpecific qualifier valueStructureSurfaceSystemTechniquesTechnologyTestingTimeTissuesTrainingValidationawakebasebehavioral responsecombinatorialcytotoxicitydesignexperiencein vivoinnovationlensnervous system disorderneural circuitneural patterningneuronal cell bodyneuronal circuitrynew technologynovelnovel strategiesprotein structurerelating to nervous systemresponsescaffoldscreeningsensorspatiotemporaltemporal measurementtoolvoltage
项目摘要
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(应用程序提供):神经科学的主要挑战之一是将结构与神经元电路的功能联系起来。为了实现这一目标,我们需要了解定义的神经元种群与这些神经元对物理过程,行为反应和疾病状态的贡献之间的连通性。成像技术的最新进展使我们能够通过细胞分辨率可视化大脑结构。当前一代一般编码的光学工具(例如传感器和控制器)的应用正在支持醒着的分子定义的细胞群体对神经元活性的测量和操纵。但是,探测神经元电路的动力学,特别是用于剖析超出分子定义电路的功能定义的电路,不仅取决于现有工具的改进,而且还需要新颖的工程。因此,我们建议通过生物化学试剂在功能相关的神经元中开发一个新颖的传感器,这些试剂可以通过在研究人员定义的行为时期内永久增加荧光信号来整合神经元。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元直接相关。 Kv2.1is的激活由它们的构象和翻译后状态确定,离子通道激活驱动电信号传导。最近,我们开发了用于创建问题以监视离子通道激活的问题的分子工具。我们使用一珠一个化合物(OBOC)组合技术,我们已经确定了一般编码的短胡椒粉(12-16 Mers,Gesis),这些辣椒(12-16 Mers,Gesis)专门激活了给定生物条件下有机染料的荧光。我们建议使用现有的gesis作为脚手架,设计和筛选新型肽型染色对,其相互作用受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我们在离子通道结构功能,Rosetta计算蛋白设计和高通量OBOC库的指导下设计和筛选电压传感和去磷酸化gesis。我们将表征有望Kv2.1- GESI电压激活和脱磷酸化问题的表达,细胞毒性,灵敏度和动力学,并在解离神经元培养和脑切片中的去磷酸化问题。我们最终将证明这种新型工具集可以鉴定活动物中活化的神经元的能力。该提案的成功结果将通过新的晶状体进行动态映射:可视化离子通道的激活状态,这是大脑中电活动的核心效应。由于该工具集唯一地提供了有关功能连接性的信息,因此它代表了一种完全新颖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,将这些小型动态活性标签应用于大脑成像,可以开发出对神经元电路的功能理解的新维度。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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James S Trimmer其他文献
James S Trimmer的其他文献
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{{ truncateString('James S Trimmer', 18)}}的其他基金
Investigating the contributions of voltage gated sodium channels to oxaliplatin induced neuropathy
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10621059 - 财政年份:2022
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Defining the Proteomic Composition of ER:Plasma Membrane Junctions in Brain Neurons
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10454277 - 财政年份:2018
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UC Davis/NIH NeuroMab Facility-Administrative Supplement
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9138371 - 财政年份:2015
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Phosphorylation as a Determinant of BK Channel Expression and Localization
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