The power of positivity: a novel class of voltage indicators for high-fidelity brain activity imaging

摘要 为了了解大脑在健康中的功能，以及在疾病中感觉、运动和认知功能是如何受到影响的，它 能够实时记录大量单个神经元的活动是至关重要的。在过去的二十年里， 神经元细胞体中的钙成像提供了神经元活动的方便的定性观察，使得 特定神经元类型或不同大脑区域的潜在放电与感觉输入、决策或 情绪或生理参数的内部表示。然而，体细胞钙通常对 阈值下活动，即突触输入使膜电位去极化而不引发动作电位，以及 缺乏时间精度来确定电路内动作电位之间的时序关系。 我们对大脑的了解将大大得益于了解神经元电路如何使用跨膜电压 来表示和处理信息。悬而未决的问题包括神经元类型在它们的总结中有何不同 启动动作电位输出的输入，神经元电路如何提取显著特征或根据 输入活动的复杂模式，以及经验、神经调节或疾病如何影响这些过程。 我们建议通过创建一类高性能的遗传编码电压指示器(GEVI)来解决这个问题 记录活体动物大量神经元的亚阈值和尖峰活动。特别是，我们发现， 正向调谐的GEVI具有检测尖峰的潜力，其信噪比是GECI的数倍 同时实现了亚阈值电位的有用可区分性。 我们建议加紧努力，开发这种积极调整的GEVI，以实现以下理想的性能规格 定量建模。目标包括(1)全面筛选正向调整的GEVI原型中的残留物以 识别调节电压调谐和荧光响应性的位置，然后进行深度组合突变 (2)在苍蝇和小鼠的单光子和双光子成像中体内指示剂的验证，以及(3) 开发超高通量单细胞筛选系统，以进一步加快GEVI的改进。

ABSTRACT To understand how the brain functions in health, and how sensory, motor, and cognitive functions are affected in disease, it is crucial to be able to record the activities of large numbers of individual neurons in real time. In the past two decades, calcium imaging in neuronal cell bodies has provided a conveniently qualitative view of neuronal activity, allowing action potential firing in specific neuron types or in various brain regions to be correlated with sensory input, decision-making, or internal representations of emotional or physiological parameters. However, somatic calcium is generally insensitive to subthreshold activity, i.e. to synaptic inputs that depolarize the membrane potential without eliciting action potentials, and lacks the temporal precision to determine the timing relationship between action potentials within a circuit. Our understanding of the brain would beneift greatly from understanding how neuronal circuits use transmembrane voltage to represent and process information. Outstanding questions include how neuronal types differ in their summatation of inputs to initiate an action potential output, how neuronal circuits extract salient features or makes a decision based on complex patterns of input activity, and how experience or neuromodulation or disease affects these processes. We propose to address this problem by creating a class of high-performance genetically encoded voltage indicators (GEVIs) to record both subthreshold and spiking activity in large numbers of neurons in living animals. In particular, we find that positively tuned GEVIs have the potential for detecting spikes with many times greater signal-to-noise ratio than GECIs while achieving useful discriminability of subthreshold potentials. We propose an intense effort to develop such positively tuned GEVIs toward ideal performance specifications identified by quantitative modeling. Aims include (1) comprehensive screening of residues in a prototype positively tuned GEVI to identify positions modulating voltage tuning and fluorescence responsiveness, followed by deep combinatorial mutagenesis of identified sites, (2) validation of indicators in vivo in 1-photon and 2-photon imaging in flies and mice, and (3) development of an ultra-high-throughput single-cell screening system to further accelerate GEVI improvement.