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Revealing circuit control of neuronal excitation with next-generation voltage indicators

使用下一代电压指示器揭示神经元兴奋的电路控制

基本信息

批准号：
9380741
负责人：
Thomas Robert Clandinin
金额：
$ 289.39万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-08 至 2021-08-07
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9380741
关键词：
Action Potentials Acute Adoption Affect Axon Behavior Biological Assay Brain Computer Simulation Corpus striatum structure Data Decision Making Dendrites Detection Disease Electrophysiology (science)Feedback Fluorescence Generations Genetic Engineering Goals Huntington Disease Image Individual Kinetics Lighting Measurement Measures Membrane Membrane Potentials Methods Microscopy Mus Neurons Neurosciences Neurotransmitters Opsin Optical Methods Optics Output Parkinson Disease Pathogenesis Performance Phenotype Photons Physiological Population Process Protein Engineering Proteins Publishing Reporter Reporting Research Personnel Role Signal Transduction Slice Speed Synapses System Technology Testing Time Tissues Variant Visual system structure Work base cell type fluorescence imaging fly improved in vivo in vivo two-photon imaging information processing interest light emission method development mutant neurotransmitter release next generation optogenetics postsynaptic postsynaptic neurons presynaptic relating to nervous system response tool two-photon voltage

项目摘要

ABSTRACT Recording the electrical impulses of individual neurons in intact brain circuits in real time has been a longstanding goal in neuroscience. One potentially widely applicable use of voltage recording would be to test postsynaptic responses upon physiological or optogenetic activation of presynaptic partners. Recording a neuron while its inputs are controlled would enable a detailed understanding of how individual neurons process information. This understanding becomes important when circuity is altered in disease, e.g. in the striatum with Parkinson's or Huntington's diseases, as it would help explain the pathogenesis of the mutant phenotype and suggest possible therapies. However, the only way now to reliably measure membrane voltage in vivo is to perform electrophysiology, whose difficulty and low throughput hamper widespread adoption. We aim to develop a new paradigm for determining the input-output relationshps of neurons using genetically encoded voltage indicators (GEVIs) and two-photon imaging. GEVIs can provide information on subthreshold voltage changes, which form the basis of neuronal computation and modulate excitability, and on timing and order of neuronal action potentials. These constitute basic essential information required for understanding information processing in brain circuits. However, in vivo voltage imaging is currently limited. No published GEVIs respond with sufficient speed and amplitude for spike detection in single trials under two-photon excitation. In this project, we will develop methods to record electrical activity from individual neurons in the brain at depth using two- photon microscopy. Our approach combines engineering of GEVIs that can respond to two-photon illumination with the establishment of conditions for using GEVIs in brain slices and living brains. Specifically, we will carry out the following aims: (1) Generate brighter and more responsive variants of ASAP2s, the best performer under two-photon excitation, and of Ace-mNeonGreen, a leading performer under one-photon excitation; (2) validate GEVI variants for their ability to report contributions of specific inputs to subthreshold and action potential responses in a variety of neurons of the fly visual system, in single trials, in vivo; and (3) systematically test GEVI performance under two-photon excitation in mouse striatal spiny projection neurons in ex vivo acute brain slices and in living mice in vivo, using GEVIs to determine the role of cell type- specific inputs to a recently discovered phenomenon of long-lasting dendritic voltage plateaus. This project will integrate the expertise of three groups spanning protein engineering, optical method development, and systems neuroscience to improve two-photon imaging of GEVIs so they can be used to image voltage transients in single trials. If successful, this project will open up in vivo two-photon imaging of GEVIs to many interested researchers, potentially catalyzing a transformation in how we measure neuronal responses in living brains.

摘要在真实的时间内记录完整脑回路中单个神经元的电脉冲一直是一个长期的目标，神经科学电压记录的一个潜在的广泛适用的用途是测试突触后反应，突触前伴侣的生理或光遗传激活。在神经元的输入受到控制时记录它，使我们能够详细了解单个神经元如何处理信息。这种理解变得重要当疾病中的回路发生改变时，例如帕金森病或亨廷顿病的纹状体，这将有助于解释突变表型的发病机制，并提出可能的治疗方法。然而，现在唯一能可靠地测量膜电压在体内是进行电生理学，其难度和低通量阻碍了广泛的领养我们的目标是开发一种新的范式，用于确定神经元的输入输出关系，电压指示器（GEVI）和双光子成像。GEVI可以提供关于亚阈值电压变化的信息，它形成了神经元计算的基础，并调节兴奋性，以及神经元作用的时间和顺序潜力这些构成了理解大脑回路中的信息处理所需的基本信息。然而，体内电压成像目前是有限的。没有公布的GEVI响应足够的速度和幅度用于双光子激发下的单次试验中的尖峰检测。在这个项目中，我们将开发方法来记录从个别神经元在大脑深处的电活动，使用两个- 光子显微镜我们的方法结合了GEVI的工程设计，可以响应双光子照明，建立在脑切片和活体脑中使用GEVI的条件。具体来说，我们会进行以下工作目标：（1）产生更明亮，响应更灵敏的ASAP 2变体，在双光子激发下表现最好， Ace-mNeonGreen，在单光子激发下的领先性能;（2）验证GEVI变体的报告能力在果蝇视觉系统的各种神经元中，特定输入对阈下和动作电位反应的贡献，在单次试验中，在体内;和（3）在小鼠纹状体棘状突起中系统地测试在双光子激发下的GEVI性能投射神经元在离体急性脑切片和活的小鼠体内，使用GEVI来确定细胞类型的作用，对最近发现的持久树枝状电压平台现象的特定输入。该项目将整合三个小组的专业知识，涵盖蛋白质工程，光学方法开发，系统神经科学，以改善GEVI的双光子成像，使它们可以用于成像电压瞬变在单审判如果成功，该项目将为许多感兴趣的研究人员开放GEVI的体内双光子成像，可能会催化我们测量活体大脑神经元反应方式的转变。