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Whole-population brain stem V2a neurons recruitment during spontaneous swimming using high-speed, single-objective light sheet microscopy. a, Schematic illustration of the experiment investigating hindbrain V2a neurons recruitment during spontaneous locomotion in enucleated 6-dpf Tg(vsx2:GAL4;UAS:GCaMP6s) larvae. b, Pipeline workflow developed for the SCAPE microscopy adapted from refs. 45 and 46. b1, Example of deconvolved image using the system’s experimental PSF (Methods). c, Top left: typical tail angle trace exhibited by intact (red, top) and enucleated (violet, bottom) larvae during recordings (intact: 5 larvae, 512 swim bouts; enucleated: 12 larvae, 3,946 swim bouts). Bottom left: violin plots depicting the distribution of bout duration (intact: 432 ± 340 ms; enucleated: 532 ± 340 ms; enucleated > intact with P < 0.001, Wilcoxon rank-sum test), number of oscillations (intact: 7.51 ± 6.3; enucleated: 8.3 ± 6.3; enucleated > intact with P < 0.001, Wilcoxon rank-sum test), mean tail angle (intact: 2.14 ± 6.6°; enucleated: 0.45 ± 6.6°; enucleated = intact with P < 0.001, Wilcoxon rank-sum test) and maximum absolute tail angle (intact: 36.43 ± 23.48°; enucleated: 27.15 ± 23.48°; enucleated < intact with P < 0.001, two-sided Wilcoxon rank-sum test) of each episode, in each condition. The box plots depict the mean (center), first and third quartiles (lower and upper box limits), and minima and maxima (bottom and top whiskers). Right: density distribution of TBF across the two conditions computed as either the median iTBF or mean TBF. Mean iTBF was greater in intact larvae (intact: 17.12 ± 2.35 Hz; enucleated: 15.11 ± 2.35 Hz; P < 0.001, Wilcoxon rank-sum test), as well as median iTBF (intact: 19.68 ± 1.75 Hz; enucleated: 17.60 ± 1.75 Hz; P < 0.001, Wilcoxon rank-sum test; three asterisks correspond to P < 0.001). d, Top: tail angle traces of swimming episodes classified as forward (orange), left turns (magenta) and right turns (green) in one example experiment (1 fish, one recording, n = 311 bouts classified as 117 forward, 127 left turns, and 29 right turns; note that 38 bouts did not fall in any category and were discarded from further analysis). Bottom: tail curvature plots for example bouts of the three types. The curvature value of each segment (from rostral to caudal, in y) is represented in the color scale as a function of time (in x). e, Schematics of bout type decomposition into kinetic components. We assumed that a turn was composed of a directionality bend (steer component) executed in tandem with symmetric oscillating bends (forward component). f, Left: example volume image (as the average of multiple time steps) of a 6-dpf larval zebrafish after pipeline processing. Middle: position of example neurons (colored dots) among the population of all recorded V2a neurons (unfilled dots), in dorsal and sagittal views. Right: ΔF/F (colored trace) and inferred spike rate traces (gray) of the example neurons during several swim bouts. The periods during which the larva swam forward are shown as gray rectangles. ΔF/F and spike rate traces are represented as their z-score. g, Distribution of V2a neurons in a representative fish active during both forward bouts, left and right turns, corresponding to the forward component neurons (orange circles, 74 of 844 neurons, 8.9%). h, Distribution of V2a neurons for a representative fish active during left (pink, 327 of 844, 38.7%) or right turns (green, 223 out of 844, 26.4%) but not forward swim bouts. i, Density distribution of V2a neurons recruited for the forward or steering component (3 fish, median ± s.d.; forward component = 8.9 ± 3.2%; left steering component = 36.5 ± 1.5%; right steering component = 29.5 ± 4.5%). Source data
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