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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：
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
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：
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

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.