rsCaMPARI: an erasable marker of neuronal activity

Identifying and comparing active neuron ensembles underlying complex behaviors is a key challenge in neuroscience. Recent tools such as CaMPARI have enabled the optical marking and selection of active neuron populations. However, CaMPARI photoconversion is permanent and irreversible, limiting its utility when multiple activity snapshots must be compared within the same sample. We sought to overcome these limitations by developing an erasable neuronal activity marker based on a reversibly switchable fluorescent protein. Here we introduce rsCaMPARI, a reversibly switchable calcium marker that enables spatiotemporally precise marking, erasing, and remarking of active neuron populations under brief, user-defined time windows of light exposure. rsCaMPARI photoswitching kinetics are modulated by calcium concentration when illuminating with blue light, and the fluorescence can be reset with violet light. We demonstrate the utility of rsCaMPARI for marking and remarking active neuron populations in freely-swimming zebrafish.