Fig. 2. Two-photon calcium imaging during spontaneous eye movements.

a Brightfield image of a 7-day-old larval zebrafish along with a time-averaged calcium image showing all neurons within a single plane of the hindbrain. A, P, L, and R denote anterior, posterior, left, and right respectively. b Zoomed-in image of time-averaged nuclear-localized GCaMP6f expression. Red dots show the center-of-mass for automatically detected nuclei from time-averaged fluorescence intensity images (see “Methods”). Similar results were seen across 422 planes collected from 20 fish. Scale bar length = 5 µm. c–e Examples of simultaneously recorded eye movements and calcium activity along with saccade-triggered responses and averages. (Top panel) simultaneously recorded left and right eye position (gray and black), single-cell fluorescence (blue), and deconvolved fluorescence (black) traces versus time. Deconvolved fluorescence is scaled to fluorescence traces and units are arbitrary. The arrows labeled L/R indicate the direction of leftward/rightward movements. (Middle panel) heatmaps of saccade-triggered responses around leftward (top map) and rightward (bottom map) directed saccades. Each row displays fluorescence versus time before and after each saccade. Dashed vertical line shows time of saccade. Source data are provided in a Source data file. (Bottom panel) saccade-triggered averages around leftward (red) and rightward (purple) directed saccades. Data are shown as average fluorescence (solid line) along with 95% confidence intervals (shaded region) and average deconvolved fluorescence (dashed line). c Example of a cell with tonic post-saccadic activity (n = 16 (15) samples for leftward (rightward) saccades). d Example of a cell that displays a burst of activity triggered during the saccade (n = 7 (9) samples for leftward (rightward) saccades). e Example of a cell that displays pre-saccadic activity (n = 9 (9) samples for leftward (rightward) saccades).