Fig. 4

The Retina Computes Direction-Selective Glider Responses (A) Geometry of the how retinal axons arborize in the central zebrafish brain. Here we show a projected view of a 3D reconstruction of the left hemisphere arborization fields (AFs) using retinal-labeled Tg:Islt2b:SyGCAMP6s zebrafish (7 dpf). Each color highlights a single AF. AF8 is hidden behind AF7 in this view. Because AF5 and AF6 were difficult to distinguish and functionally similar (Figure S3A), we merged them into AF5&6 for most figure panels. Auto-fluorescence artifacts from the eye were masked prior to other data analyses (STAR Methods). P indicates the anterior-posterior axis, V the ventral-dorsal axis, and M the medial-lateral axis. Scale bar represents 50 μm. (B) Example planes showing ROIs identified with our imaging and segmentation routines. We extracted many ROIs from most AFs, but we did not observe functional responses in AF7. A and L indicates the anterior-posterior and left-right axes. Scale bar represents 20 μm. (C) Individual retinal ROIs were consistently activated or suppressed by visual stimuli across the stimulus conditions. We display fluorescence responses from a randomly selected subpopulation of 2,500 ROIs (out of 270,596). The ROIs were sorted by the significance of their selectivity for stimulus-on versus stimulus-off periods across all stimuli (Wilcoxon test). Solid and dashed yellow lines indicate the start and end of stimulus presentation periods respectively. Blue lines mark the p value thresholds (p = 0.05) used to define the activated and suppressed ROI populations. (D) More retinal ROIs were activated and suppressed than expected by chance, and the fraction of ROIs activated or suppressed varied across the AFs. Error bars represent standard error of the mean, and confidence intervals were estimated by assuming Poisson variability in counting statistics (N = AF4: 62,173; AF5&6: 59,450; AF8: 5,676; and AF9: 73,722 ROIs). (E) The number of retinal ROIs that were direction selective for drifting gratings was only above chance levels in AF5&6, as assessed by a shuffle test (STAR Methods). In particular, error bars represent estimated 95% confidence intervals and excluded the chance level of 0 in AF5&6 only. (F) Top: Leftmost column represents two example planes showing a region encompassing AF5 and AF6. ROIs preferring leftward (rightward) gratings are colored cyan (red). Panels in the middle/rightmost columns are from left/right side of the brain. The green boxes in the leftmost panels indicate subregions rotated and shown at higher resolution to the right. A indicates the anterior-posterior axis, and L indicates the left-right axis. Scale bars, left column: 30 μm, all the other columns: 5 μm. Bottom: Z-scored fluorescence traces of the three example ROIs circled in yellow at top. Each ROI was identified by its direction selectivity to drifting grating stimuli, but they also showed direction-selective responses to two-point and three-point glider stimuli. (G) Mean z-scored fluorescence traces of all ROIs direction selective for leftward- or rightward-drifting gratings in the left or right AF5&6. The directional stimulus pairs marked with asterisks were significantly different at the p = 0.01 level (one tailed Wilcoxon test). All other directionally paired stimuli did not show statistically significant differences. N = 240 ROIs (top left), 330 (bottom left), 234 (top right), 449 (bottom right). See also Figure S3.