A high-content platform for physiological profiling and unbiased classification of individual neurons

High-throughput physiological assays often lose single cell resolution, precluding subtype-specific analyses of neuronal activation mechanism and drug effects. Here, we demonstrate APPOINT, Automated Physiological Phenotyping Of Individual Neuronal Types. This physiological assay platform combines calcium imaging, robotic liquid handling, and automated analysis to generate physiological activation profiles of single neurons at a large scale. Using unbiased techniques, we quantify responses to multiple sequential stimuli, enabling subgroup identification by physiology and probing of distinct mechanisms of neuronal activation within subgroups. Using APPOINT, we quantify primary sensory neuron activation by metabotropic receptor agonists and identify potential contributors to pain signaling. Furthermore, we expand the role of neuroimmune interactions by showing that human serum can directly activate sensory neurons, elucidating a new potential pain mechanism. Finally, we apply APPOINT to develop a high-throughput, all-optical approach for quantification of activation threshold and pharmacologically separate the contributions of distinct ion channel subsets to optical activation.