Abstract
Visualizing active neurons and circuits in vivo is critical for investigating the neural activity that underlies behavior. While several established methodologies are available to achieve this end in larval zebrafish, they are limited by the scale of tissue visualization, temporal resolution, need to restrain larvae, and/or accessibility of necessary instruments. Here, we establish a pipeline for the visualization and quantification of spatiotemporally precise whole-brain neural activity in larval zebrafish using CaMPARI2, a genetically-encoded calcium indicator. Using temporally specific photoconverting UV light exposures, we capture whole-brain "snapshots" of neural activity time-locked to stimuli during unrestrained larval behavior. We optimize experimental conditions for recording sub-second neuronal activity changes across acoustically-evoked behavioral paradigms spanning minutes to hours. We then leverage this system to pinpoint brain-wide neural activity changes during non-associative habituation learning, observing distinct activity signatures in the subpallium, preoptic area, and habenulae that are altered through pharmacological disruption of habituation learning. This approach effectively complements the temporal precision achievable through post-hoc activity detection methods and expands the accessibility of large-scale behavioral circuit dissection beyond highly specialized real-time volumetric imaging equipment.