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

摘要 为了了解健康时大脑的功能，以及疾病时感觉、运动和认知功能如何受到影响， 对于能够在真实的时间内记录大量单个神经元的活动至关重要。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定性视图， 在特定的神经元类型或在各种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征。然而，体细胞钙通常对 阈下活动，即对突触输入，其使膜电位降低而不引发动作电位，以及 缺乏确定电路内动作电位之间的时序关系的时间精度。 我们对大脑的理解将大大受益于了解神经元回路如何使用跨膜电压 to represent代表and process处理information信息.悬而未决的问题包括神经元类型在其对神经元的总和方面如何不同。 输入以启动动作电位输出，神经元回路如何提取显著特征或基于 输入活动的复杂模式，以及经验或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 在活体动物中记录大量神经元的阈下和尖峰活动。特别是，我们发现， 正调谐GEVI具有检测具有比GECI大许多倍的信噪比的尖峰的潜力 同时实现亚阈值电位的有用的可辨别性。 我们提出了一个紧张的努力，以发展这种积极调整GEVI对理想的性能规格确定， 定量建模目的包括：（1）全面筛选原型积极调整GEVI中的残留物， 鉴定调节电压调谐和荧光响应性的位置，然后进行深度组合诱变 （2）在苍蝇和小鼠中进行1-光子和2-光子成像的体内指标验证，以及（3） 开发超高通量单细胞筛选系统，以进一步加速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.