Protein voltage sensors: kilohertz imaging of neural dynamics in behaving animals

蛋白质电压传感器：行为动物神经动力学的千赫兹成像

• 批准号: 8827201
• 负责人: Michael Z. Lin
• 金额: $ 93.24万
• 依托单位: STANFORD UNIVERSITY
• 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 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8827201
• 关键词: Action Potentials; Animals; Benchmarking; Brain; Cells; Chronic; Code; Color; Communities; Custom; Data; Detection; Disease; Drosophila genus; Event; Fluorescence; Frequencies; Genetic; Goals; Grant; Head; Hybrids; Image; In Vitro; Individual; Interneurons; Kinetics; Knowledge; Label; Lead; Learning; Left; Libraries; Life; Lighting; Mammals; Membrane; Membrane Potentials; Methods; Metric; Microscope; Monitor; Mus; Nature; Nematoda; Neurons; Neurosciences; Neurosciences Research; Optics; Paper; Performance; Proteins; Reporting; Research; Resolution; Resource Sharing; Rodent; Science; Signal Detection Analysis; Signal Transduction; Slice; Speed; System; Testing; Time; Tissues; Training; Variant; Viral; Work; awake; brain research; design; experience; fluorescence imaging; fly; imaging modality; improved; information processing; instrument; instrumentation; millisecond; miniaturize; mutant; neural circuit; new technology; novel; public health relevance; relating to nervous system; response; screening; sensor; small molecule; theories; tool; voltage

 DESCRIPTION (provided by applicant): Attaining effective genetically encoded optical voltage-indicators has been a longstanding goal in neuroscience research and is a key near-term aim of the BRAIN Initiative. Unlike small molecule sensors or hybrids of fluorescent proteins with organic molecules, optical voltage-indicators that can be fully encoded genetically are readily combined with genetic tools and viral delivery methods that enable long-term expression and chronic imaging studies without addition of exogenous agents. Genetically encoded Ca2+-sensors offer similar targeting advantages, but Ca2+-imaging fails to reveal individual spikes in many neuron types, poorly captures sub- threshold membrane dynamics, and has insufficient temporal resolution to capture spike timing to better than ~50-100 ms. Voltage-indicators directly sense the membrane potential and promise faithful reporting of spike waveforms, spike bursts and sub-threshold dynamics, in cells targeted by their genetic class or connectivity. An ideal voltage-indicator would produce large fluorescence responses, to facilitate spike detection, and have millisecond-scale kinetics, to study synchrony and spike-timing aspects of neural coding. However, prior protein voltage-indicators have generally suffered performance-limiting tradeoffs between modest brightness, sluggish kinetics, and limited signaling dynamic range in response to action potentials. To date, no protein voltage-indicator combines the attributes needed for accurate reporting of voltage activity in behaving animals. However, if such a sensor emerged, this would likely have even greater impact on brain science than the surge in research enabled by recent advanced versions of the GCaMP Ca2+-indicator. This proposal seeks to create broad voltage-imaging capabilities and involves two Co-PDs who are highly experienced in fluorescence imaging of neural activity. Working collaboratively, we recently created two new classes of voltage-indicators, of distinct colors and voltage-sensing mechanisms, each of which has substantially superior signaling fidelity than earlier protein voltage-indicators while offering faster kinetics and higher brightness. Using thes two sensor types, we have imaged fast spike trains in cultured neurons and brain slices. Calculations using signal detection theory show our indicators are now on the brink of transitioning into a mainstay approach to monitor large numbers of individual neurons in behaving animals. To enact this, we will use novel massively parallel methods to screen variants of our protein indicators at 100-1000¿ greater throughput than screening methods used previously in the field. We will validate and iteratively optimize the resulting indicators in cultred neurons, mammalian brain slices, and behaving flies, nematodes and mice, by using signal detection theory to benchmark indicator performance. To accompany these voltage-indicators, we will also create imaging instrumentation custom-designed for high-speed (~1 kHz) voltage-imaging in awake head-restrained and freely behaving mice. If our work succeeds, it will be a game-changer for brain research, propelling studies of how cells and circuits function normally and go awry in disease.

 描述（由申请人提供）：获得有效的基因编码光学电压指示剂一直是神经科学研究的长期目标，也是 BRAIN Initiative 的近期关键目标，与小分子传感器或荧光蛋白与有机分子的混合物不同，光学。可以完全基因编码的电压指示剂很容易与遗传工具和病毒递送方法相结合，从而无需添加外源性试剂即可进行长期表达和慢性成像研究。 Ca2+ 传感器具有类似的定位优势，但 Ca2+ 成像无法揭示许多神经元类型中的单个尖峰，很难捕获亚阈值膜动力学，并且时间分辨率不足以捕获优于约 50-100 毫秒的尖峰时间。指示器直接感测膜电位，并保证在其遗传类别或连接性的目标细胞中忠实地报告尖峰波形、尖峰爆发和亚阈值动态。理想的电压指示器将产生大的荧光响应，促进尖峰检测，并具有毫秒级动力学，以研究神经编码的同步性和尖峰时序方面。然而，先前的蛋白质电压指示剂通常在适度的亮度、缓慢的动力学和有限的信号动态范围之间进行性能限制。迄今为止，还没有蛋白质电压指示器能够结合准确报告行为动物电压活动所需的属性，但是，如果出现这样的传感器，这可能会对大脑产生更大的影响。这项提案旨在创造广泛的电压成像功能，并涉及两位在神经活动荧光成像方面经验丰富的 Co-PD 的合作。创建了两种新的电压指示器，具有不同的颜色和电压感应机制，每种电压指示器都比早期的蛋白质电压指示器具有更高的信号保真度，同时提供更快的动力学和更高的亮度，我们使用这两种传感器类型。使用信号检测理论对培养神经元和大脑切片中的成像快速尖峰序列进行计算表明，我们的指标现在正处于转变为监测行为动物中大量个体神经元的主要方法的边缘。并行方法以 100-1000 筛选我们的蛋白质指示剂变体我们将通过使用信号检测理论来衡量指标性能，在培养的神经元、哺乳动物脑切片以及行为果蝇、线虫和小鼠中验证和迭代优化所得指标。 -指标，我们还将创建专门设计的成像仪器，用于对清醒的头部受限且自由行为的小鼠进行高速（~1 kHz）电压成像。大脑研究的游戏规则改变者，推动对细胞和电路如何正常运作以及在疾病中出错的研究。