Exposure to water flow induces a state of increased locomotor activity. A, Frequency of swim initiation after movement from a holding area to the testing arena (diagrammed in the inset). Epochs of activity patterns are indicated. Black line and shaded area show mean and SEM (n = 6 × 25). *p < 0.05 compared with baseline. B, Change from baseline activity after exposure to sensory cues. Larvae were left on the testing arena for 30 min before testing. Baseline is the mean swim initiation frequency in the 5 min before testing. Responsiveness during the presentation of the stimulus was not recorded, so these graphs do not show acute responses to the stimuli tested. Vibration, 500 ms duration, 10 Hz vertical acceleration, 62 m/s2 peak (n = 5 × 25). Loom, A shadow (contrast ratio of 18:1) moves across arena at 20 mm/s (n = 11 × 25). Optomotor (Opto), 30 s duration, 10 mm grating width, 2 Hz frequency, 18:1 contrast ratio (n = 7 × 20). Light Flash, 30 s dark pulse from baseline illumination at 150 μW/cm2 (n = 7 × 20). Flow, 45 s water flow, 40 ml/min (n = 8 × 20). C, D, Activity is proportional to the duration (C) but not to the velocity (D) of the flow stimulus. Trials were 20 min apart, with measurements taken of the locomotor activity during the 3 min before (black circles) and after (gray circles) each stimulus and activity for each before/after pair normalized so that the average before-flow value was set to 100. To test duration, flow velocity was held at 40 ml/min (n = 6 × 20). To test velocity, flow duration was 30 s (n = 7 × 20). #p < 0.05, *p < 0.01 for paired t tests. E, Kinematic analysis of swimming. The gray bar marks 15 s flow stimulus at 30 min. Pairwise comparisons are to baseline movement at 25 min (circles, n = 8 × 20). *p < 0.05. Graphs show mean and SEM. F, Total displacement generated by swim bouts with pairwise comparisons as for E.
Visual sensitivity is selectively modulated by exposure to water flow. A, Flow chamber for testing activity and sensory thresholds. Optomotor stimuli are projected onto the diffuser using a mirror. Leads attached to grids enable electric stimuli. LED mounted above the chamber for visible illumination and flash responses. To test responsiveness, larvae were left in the chamber for 25 min before recording baselines, exposed to a 60 s flow stimulus at 30 min, and retested 3 min after flow. B, C, Responsiveness was measured to an electric stimulus (B) (n = 7 × 20) and a light flash stimulus (C) (n = 9 × 20). D, To test touch responsiveness, larvae expressing ChR2 in the trigeminal ganglion were exposed to five brief blue light pulses before and after water flow (open circles; n = 10 larvae) or before and after an equivalent delay without water flow (“no flow”) to control for habituation (filled circles; n = 11 larvae). E, Average orientation during the first 10 s of water flow. Orientation is the offset (0–180°) from the direction opposite to flow. Larvae orient more quickly to a second exposure to water flow, 3 min after the termination of the first exposure (open circles) than to the initial exposure (filled circles). F, Proportion of larvae in orientation bins (30 ° each) relative to flow direction, 5 s after flow stimulus starts, for first exposure (top) and second exposure (bottom). *p < 0.05 compared with the same orientation at first flow exposure. G, Rate of orienting during the OMR. The proportion of larvae (n = 19 × 20) oriented in the quadrant matching the direction of visual flow was measured throughout stimulus presentation and rate of increase calculated in baseline state (filled circles) or after exposure to flow (open circles). *p < 0.001. H, Turn bias during the OMR. Larvae (n = 23 × 20) were tested in baseline state (filled circles) and then after flow (open circles). *p < 0.05. Graphs show mean and SEM.
A tph2 promoter fragment labels DR serotonergic neurons. A, Genomic organization of the tph2 gene upstream region from which the 3.4 kb promoter was taken (brown bar) and schematic of the construct used to create the stable transgenic Tg(tph2:Gal4ff)y228 line. B, C, Whole-body lateral (B) and oblique (C) head views of 6 dpf Tg(tph2:Gal4ff)y228; Tg(UAS:Kaede)s1999t double-transgenic larva showing Kaede expression in the ventral midline of the brainstem (arrowheads) and spinal cord (asterisks). Dotted line in C outlines the brain. D, Immunohistochemistry against serotonin (red) confirms that Kaede-expressing cells in the double-transgenic line are serotonergic. Anterior is to the left. The posterior extension of serotonergic neurons that are not labeled in Tg(tph2:Gal4ff)y228 is the inferior raphe. A, Anterior; D, dorsal; L, left; P, posterior; R, right; V, ventral. Scale bar, 50 μm.
Calcium imaging of DR neurons during flow-induced arousal. A, Schematic of microscope mountable flow chamber. Larvae are head embedded and stimulated with water ejected from a solenoid water pump. Both input and output tube openings are set inside water in a 3 cm Petri dish. B, Dorsal view of DR neurons in Tg(tph2:Gal4ff)y228 transgenic fish injected with UAS:GCaMP3–v2a–mCherry. Anterior is to the top. GCaMP3 and mCherry are expressed mosaically but in the same DR cells. C, Time series for a single neuron. Flow exposure is at time 0. GCaMP3 but not mCherry fluorescence transiently increases after flow. D, Cumulative distribution of ΔR/Rm from 93 individual neurons for 1 min before (pre-flow) and after (post-flow) water-flow stimulus. E, Average ΔR/Rm for neurons responding to the flow stimulus (top trace; n = 28) and not responding (bottom trace; n = 65). Thick and thin lines indicate mean and SEM. Flow stimulus was applied for 10 s (dotted rectangle). *p < 0.05 compared with baseline activity.
Ablation of the DR blocks enhanced visual sensitivity during arousal. A, Timeline for experiments using metronidazole to ablate the DR in Tg(tph2:nfsB-mCherry)y226 fish. B, Detection of activated caspase-3 during ablation using PhiPhiLux G1D2 (green) reveals ongoing apoptosis selectively in DR mCherry-positive cells in metronidazole-treated (right) but not vehicle-treated (left) larvae. Scale bar, 25 μm. C, Confocal stacks in control (left) and metronidazole-ablated (right) fish. Immunohistochemistry against mCherry (red) and serotonin (green). Scale bar, 50 μm. D, Locomotor activity of control (gray) and ablated (red) fish (n = 9 × 20). A 60 s flow stimulus was applied at 30 min (dotted line). E, F, Turn bias in response to optomotor stimuli presented 5 min before (E) and 3 min after (F) the flow stimulus in control (black) and ablated (red) larvae (n = 14 × 20). *p < 0.05. G, Turn bias of the OMR during transfer arousal in control (black) and DR-ablated (red) larvae (n = 9 × 25). *p < 0.05, **p < 0.001. Graphs show mean and SEM.
The optic tectum receives DR input and is required for accurate responding to a weak visual stimulus. Optic tectum in double-transgenic Tg(tph2:Gal4ff); Tg(ath5:GFP) larvae injected with UAS:lyn–TagRFPT. A–C, Dorsal confocal stack through the tectal neuropil. D–F, Single sagittal confocal section. G, Schematic showing the location of the image planes. Green represents retinotectal projections. IR, Inferior raphe; TeO, optic tectum; RGC, retinal ganglion cells. A, D, TagRFPT labels projections from the DR (anti-TagRFP, red). B, E, GFP labels retinal ganglion cell axons in the tectum (anti-GFP, green). C, Overlap from dorsal view with TeO outlined. Scale bar, 100 μm. F, DR projections are found throughout the tectal neuropil layers, including in the stratum fibrosum et griseum superficiale (SFGS), stratum opticum (SO), and stratum album centrale (SAC). Scale bar, 20 μm. H, I, Turn bias during the OMR under baseline conditions (H) and during transfer arousal (I) for control (black) and optic tectum-ablated (red) larvae (n = 6). Graphs show mean and SEM. *p < 0.05.