Diffuse, spectrally-resolved optical strategies for detecting activity of individual neurons from in vivo mammalian brain with GEVIs

测量和理解单个神经元的活动对于理解神经元回路如何运作和导致行为是至关重要的。钙敏感指标的双光子显微镜已经产生了深刻的数据与群体单个神经元的作用。随着头部固定或小型化版本的使用，这种光学技术已经导致神经动力学和行为之间的联系。然而，这些方法还没有转化为电压指示器，因此对细胞群中的尖峰如何影响电路和行为的理解是缺乏的。许多困难与双光子显微镜需要扫描整个体积的小激光焦点有关，这导致有限的时间分辨率。来自记者的信号与神经元有关，因为当激光聚焦在该神经元的位置时产生荧光。本文提出了一种基于使用多种颜色的指标对神经输出进行颜色编码的替代策略。该策略完全依赖于光谱信息，因此不需要位置信息或图像生成。这使高速数据采集成为可能。该策略利用了基因编码电压指示器的多种颜色的可用性，并将单个神经元与独特的颜色组合联系起来。一种腺相关病毒载体的混合物，每一种都携带一种特定颜色指示物的DNA，被注射到大脑中。因为感染过程是随机的，神经元被几个粒子感染，所以神经元被随机组合的颜色标记。在这个建议中，颜色的数量，传递方法和信号的分析被优化，以识别群体中的单个神经元。为了对这项技术进行基准测试，将这些新信号与多光子显微镜和神经活动分析中的电生理学性能进行比较。

Measuring and understanding the activity of individual neurons is critical for understanding how neuronal circuits function and lead to behavior. Two-photon microscopy of calcium-sensitive indicators has produced insightful data on the role of individual neurons with populations. With the use of head-fixed or miniaturized versions, such optical techniques have lead to links between neural dynamics and behavior. However, these methods have not translated to voltage indicators, so that the understanding of how spikes across populations of cells affects circuits and behavior is lacking. Much of the difficulty is related to the need in two-photon microscopy to scan a small laser focus throughout a volume which results in limited time resolution. Signals from reporters are associated with a neuron because the fluorescence was generated when the laser focus was at the location of that neuron. An alternate strategy based on using multiple colors of indicators to color code neural output is proposed here. This strategy relies entirely on spectral information, so no location information or image formation is required. This enables high-speed data acquisition. This strategy takes advantage of the availability of multiple colors of genetically encoded voltage indicators and associates individual neurons with unique color combinations. A mix of adeno-associated virus vectors, each carrying DNA for a indicator of a particular color, is injected into the brain. Because the infection process is stochastic and neurons are infected by several particles, neurons are labeled by a random combination of colors. In this proposal, the number of colors, delivery methods and analysis of signals is optimized for the identification of individual neurons within a population. For the benchmarking this technology, these novel signals are compared to the performance of multiphoton microscopy and electrophysiology in assays of neural activity.