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Figure 1 Volumetric two-photon Ca2+ imaging of dark flash (DF) habituation. (A) In response to a DF, larval zebrafish execute a high-amplitude turn called an O-bend response. (B) Habituation results in a progressive decrease in response probability to DFs repeated at 1 min intervals, delivered in four blocks of 60 stimuli, separated by 1 hr of rest (from 0:00 to 7:00), and after a 5 hr retention period (12:00-). Inset (i) shows an expanded view of the first training block. (C) Tg(elavl3:H2B-GCaMP7f) larvae were imaged across 12 z-planes at 10 µm steps. Regions of Interest (ROIs) are overlaid in random colours. (D) Density of detected ROIs registered and plotted in the Z-Brain coordinate space. n = 1,050,273 ROIs across 34 larvae. (E) Cropped field of view used for plotting and analysing Ca2+ imaging data and approximate anatomical localizations of major brain areas: dien, diencephalon; mid-b, midbrain; cb, cerebellum; hind-b, hindbrain; io, inferior olive; ret, retina; tec, tectum. (F) Functional responses of neurons to 60 DFs at 1 min intervals, plotted as a clustered heatmap (‘rastermap’; Pachitariu et al., 2017, https://github.com/MouseLand/rastermap, copy archived at MouseLand, 2023) where rows represent individual neurons ordered by the similarities in their activity. Darker shades reflect increased activity. This clustering reveals neurons that are tuned to the DF stimuli (pink box) or motor events (green box). Dashed trace above the heatmap depicts the DF stimulus convolved with a kernel approximating H2B-GCaMP7f kinetics. (G) ROIs in an individual fish plotted based on their correlation and tuning to regressors defining either motor or DF stimulus events, highlighting the spatial distributions of these tunings across the imaged population. Plotted as a maximum intensity projection. (H) Same analysis as (G), but across the entire population of 34 larvae. (I) ROIs in an individual fish plotted based on their correlation and tuning to regressors defining either the first or last three DF stimuli. (J) Same analysis as (I), but across the entire population of 34 larvae. tl, torus longitudinalis.