Efficient mesoscale phenotypic screening in cultured primary neuron culture

A longstanding question in neuroscience is how the activity of ion channels shapes neuronal activity and, as a result, computation in circuits and networks. Optogenetic reagents are tools to answer this question by enabling precise and dynamic perturbation of cellular states. However, development of these reagents can be hampered by low-throughput assays in non-physiological contexts. Here, we develop an all optical phenotypic screen in cultured primary hippocampal neurons that enables the functional assessment of large libraries of genetically encoded optogenetic actuators. Combining real-time analysis and data reduction methods allows for continuous observation of several thousand neurons for several days without onerous data storage overhead. This screening system may be useful in a diversity of research questions that can be coupled to optical perturbation and sensing.