An improved FLARE system for recording and manipulating neuronal activity.

Recording and manipulating neuronal ensembles that underlie cognition and behavior in vivo is challenging. FLARE is a light- and calcium-gated transcriptional reporting system for labeling activated neurons on the order of minutes. However, FLARE is limited by its sensitivity to prolonged neuronal activities. Here, we present an improved version of FLARE, termed cytoFLARE. cytoFLARE incorporates cytosolic expression of the transcription factor and a more sensitive pair of calcium sensing domains. We showed that cytoFLARE provides more calcium- and light- dependent signals in HEK293T cells and higher signal-to-background ratios in neuronal cultures. We further established cytoFLARE transgenic Drosophila models and applied cytoFLARE to label activated neurons upon sensory or optogenetic stimulation within a defined time window. Notably, through cytoFLARE-driven expression of an optogenetic actuator, we successfully reactivated neurons involved in the larval nociceptive system. Our findings demonstrate the first characterization and application of time-gated calcium integrators in Drosophila.