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示器一直是神经科学研究的长期目标，也是大脑倡议的一个关键近期目标。与小分子传感器或荧光蛋白与有机分子的杂交不同，可在基因上完全编码的光学电压指示器很容易与遗传工具和病毒传递方法相结合，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。基因编码的钙传感器提供了类似的靶向优势，但钙成像无法显示许多神经元类型中的单个棘波，捕捉亚阈值下的膜动力学很差，并且没有足够的时间分辨率来捕捉好于~50-100ms的棘波计时。电压指示器直接感知膜电位，并承诺忠实地报告细胞中的尖峰波形、尖峰爆发和亚阈值动态，这些都是根据它们的遗传类别或连接性来确定的。理想的电压指示器应该产生大的荧光响应，以便于检测尖峰，并具有毫秒级的动力学，以研究神经编码的同步性和尖峰计时方面。然而，以前的蛋白质电压指示器通常在适度的亮度、迟缓的动力学和有限的信号动态范围之间进行性能限制的权衡，以响应动作电位。到目前为止，还没有蛋白质电压指示器结合了准确报告行为动物电压活动所需的属性。然而，如果出现这样的传感器，这可能会对脑科学产生比最近先进版本的GCaMP钙指示器带来的研究激增更大的影响。这项提议寻求创建广泛的电压成像能力，并涉及两个在神经活动的荧光成像方面经验丰富的Co-PD。通过合作，我们最近创造了两种新的电压指示器，具有不同的颜色和电压感应机制，每一种都具有比以前的蛋白质电压指示器更高的信号保真度，同时提供更快的动力学和更高的亮度。使用这两种类型的传感器，我们已经在培养的神经元和脑片中成像了快速的棘波序列。使用信号检测理论进行的计算表明，我们的指示器现在即将过渡到监测行为动物中大量单个神经元的主要方法。为了实现这一点，我们将使用新颖的大规模并行方法来筛选我们的蛋白质指示剂的变体，其吞吐量比以前在该领域使用的筛选方法高出100-1000。我们将通过使用信号检测理论对指标性能进行基准测试，验证并迭代优化培养的神经元、哺乳动物脑片以及表现良好的苍蝇、线虫和小鼠的指标。为了配合这些电压指示器，我们还将创建定制的成像仪器，用于在清醒、头枕和行为自由的小鼠中进行高速(~1 kHz)电压成像。如果我们的工作成功，它将改变大脑研究的游戏规则，推动细胞和电路如何正常运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.