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.
Technologies to study the relative timing and patterns of action potentials (spikes) across large numbers of neurons are currently lacking. However, these spiking patterns are crucial to understanding how the brain functions. The use of multicolor voltage indicators is a new technology that could provide these much needed capabilities.
