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

 描述（由申请人提供）： 获得有效的遗传编码光学电压指标一直是神经科学研究的长期目标，也是BRAIN Initiative的关键近期目标。与小分子传感器或荧光蛋白与有机分子的杂交体不同，可以完全遗传编码的光学电压指示剂很容易与遗传工具和病毒递送方法相结合，从而能够在不添加外源性试剂的情况下进行长期表达和慢性成像研究。遗传编码的Ca 2+传感器提供类似的靶向优势，但Ca 2+成像未能揭示许多神经元类型中的个体尖峰，捕获阈下膜动力学不佳，并且时间分辨率不足以捕获优于~50-100 ms的尖峰定时。电压指示剂直接感测膜电位并承诺尖峰波形、尖峰爆发和阈下动力学的忠实报告，在细胞中的遗传类别或连接性。 理想的电压指示器将产生大的荧光响应，以促进尖峰检测，并具有毫秒级动力学，以研究神经编码的同步性和尖峰定时方面。然而，现有的蛋白质电压指示剂通常在适度的亮度、缓慢的动力学和响应于动作电位的有限的信号传导动态范围之间遭受性能限制的权衡。迄今为止，没有蛋白质电压指示剂结合了准确报告行为动物中电压活动所需的属性。然而，如果这种传感器出现，这可能会对脑科学产生更大的影响，而不是最近GCaMP Ca 2+指标的高级版本所带来的研究激增。 该提案旨在创建广泛的电压成像能力，并涉及两名在神经活动荧光成像方面经验丰富的Co-PD。通过合作，我们最近创建了两种新的电压指示剂，具有不同的颜色和电压感应机制，每种都具有比早期蛋白质电压指示剂更上级的信号保真度，同时提供更快的动力学和更高的亮度。使用这两种传感器类型，我们已经在培养的神经元和脑切片中成像了快速尖峰序列。使用信号检测理论的计算表明，我们的指标现在正处于过渡到一种主流方法的边缘，以监测行为动物中大量的单个神经元。为了实现这一点，我们将使用新的大规模并行方法来筛选我们的蛋白质指示剂的变体，其通量比该领域以前使用的筛选方法高100-1000倍。我们将使用信号检测理论对指标性能进行基准测试，从而验证并迭代优化培养神经元、哺乳动物脑切片和行为果蝇、线虫和小鼠中的指标。伴随着这些电压指标，我们还将创建成像仪器定制设计的高速（~1 kHz）电压成像清醒的头部限制和自由行为的小鼠。如果我们的工作成功，它将改变大脑研究的游戏规则，推动对细胞和电路如何正常工作以及在疾病中出错的研究。

 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.