Distinct retinal pathways drive spatial orientation behaviors in zebrafish navigation

Abstract

Navigation requires animals to adjust ongoing movements in response to pertinent features of the environment and select between competing target cues. The neurobiological basis of navigational behavior in vertebrates is hard to analyze, partly because underlying neural circuits are experience dependent. Phototaxis in zebrafish is a hardwired navigational behavior, performed at a stage when larvae swim by using a small repertoire of stereotyped movements. We established conditions to elicit robust phototaxis behavior and found that zebrafish larvae deploy directional orienting maneuvers and regulate forward swimming speed to navigate toward a target light. Using genetic analysis and targeted laser ablations, we show that retinal ON and OFF pathways play distinct roles during phototaxis. The retinal OFF pathway controls turn movements via retinotectal projections and establishes correct orientation by causing larvae to turn away from nontarget areas. In contrast, the retinal ON pathway activates the serotonergic system to trigger rapid forward swimming toward the target. Computational simulation of phototaxis with an OFF-turn, ON-approach algorithm verifies that our model accounts for key features of phototaxis and provides a simple and robust mechanism for behavioral choice between competing targets.

Figure 1

Zebrafish larvae perform positive and negative phototaxis. a Schematic showing the phototaxis conditions. Larvae were maintained under uniform illumination and tested for phototaxis by transitioning to a dark field with a single light spot. b Swim tracks for nine larvae during phototaxis superimposed over an image of the target arena showing the position of the target light (U/RTI=100/-1). Circles indicate starting positions. Scale bar 5 mm. c Phototaxis is associated with larvae rapidly orienting toward the target (red trace), reflected in a decreased Orientation to Target (OT) score (inset gives example of larvae oriented 45°). Low OT scores are maintained during target approach. Once larvae reach the target their orientation randomizes and mean OT returns to 90°. Mean distance from target is minimal after 60s of phototaxis (blue trace). n=6 groups (total 180 larvae). d The rate of phototaxis depends on the relative intensity of the target light and the light source to which larvae are pre-adapted. Larvae were pre-adapted to light intensities of 0.65 μW/cm² (grey), 6.5 μW/cm² (blue) or 65 μW/cm² (orange) and maintained in uniform illumination of the same intensity in the testing arena. Relative target intensity = log₁₀ ( (intensity in the center of the target spot light) / (pre-adapted intensity) ). We measured the mean larval distance to the target at 0.5 s intervals over the first 5 seconds of phototaxis, performed a linear fit to the data and calculated the rate of phototaxis by taking the gradient of the fitted line. n=4 groups per adaptation/target intensity pair. At the highest relative target light intensity, larvae showed a slow but significant rate of movement away from the target. * P < 0.05 for one-sample t-test versus 0, color indicates the pre-adaptation intensity for the point subject to statistical test. See also Figure S1 and Movie S1.

Figure 2

Motor control during phototaxis. a Larvae oriented away from the target during positive phototaxis show high rates of turn movements. Graph shows the frequency of turn initiations during uniform illumination (black) and for the first 5 seconds of positive phototaxis (blue, U/RTI=100/-1) for subsets of larvae in the schematized orientations (n=4 groups). In all figures, the x-axis shows orientation groups relative to a target light to the left. * P < 0.05 versus corresponding orientation during uniform illumination. b Turn movements are directed toward weak target lights and away from intense target lights. Bars show the percentage of turns initiated in a rightward direction: 50% represents no directional bias. During positive phototaxis (blue bars, n = 5 groups, U/RTI=100/-1) and negative phototaxis (white bars, n = 9 groups, U/RTI=20/+2) larvae oriented toward or away from the target show no bias, but larvae oriented perpendicularly to the target show significant directionality of turn movements toward or away from the target respectively. * P < 0.01 for one sample t-test against 50%. c During negative phototaxis, larvae facing an intense target light increase the frequency of turn movements. Turn frequency during uniform illumination (black) and during the first 5 seconds of negative phototaxis (open circles, U/RTI=15/+2) for larvae in the schematized orientations (n=6 groups). * P < 0.05 versus corresponding orientation during uniform illumination. d Larvae oriented toward the target produce high levels of scoot movements during positive phototaxis. The initiation frequency of scoot movements is shown during uniform illumination (black) and for subsets of larvae in the schematized orientations (blue, n=4 groups, U/RTI=100/-1). * P < 0.05 versus corresponding orientation during uniform illumination. e. During negative phototaxis (U/RTI=15/+2), larvae (n=6 groups) show a small reduction in the initiation frequency of scoot movements (‘Pht’, white bar) compared to uniform illumination (‘Uni’, black bar). Burst movements show a small increase above baseline (two-way ANOVA, main effect of test condition F(1,50)=11.2, P=0.002), but rates remain very low and are not modulated by orientation (no main effect of orientation F(4,50)=1.67, P=0.17 or interaction between test condition and orientation F(4,50)=0.59, P=0.67). * P < 0.05. See also Figure S2.

Figure 3

The retinal ON pathway triggers scoots a Initiation frequencies of scoots when larvae are tested with a single target (1, blue circles, U/RTI=100/-1, n=6 groups) or two equally intense lights on opposite sides of the testing arena (1+2, green circles, n=6 groups). Dotted line shows the rate of scoot initiations under uniform illumination (n=6 groups). As orientation relative to the target differs for the two lights, absolute orientation of the larvae was used and recordings restricted to a 15×15 mm patch in the center of the testing arena. * P < 0.05 verses uniform illumination. b Wildtype nrc siblings show increased scoot movements when oriented toward the target and reduced levels of scoot movements when facing away from the target. * P < 0.05 for schematized orientation during phototaxis (grey bars) compared to uniform illumination (black bars). In contrast, c nrc mutants show similar levels of scoot movements during uniform illumination (black bar) and when facing the target light during phototaxis (grey bars). d The SSRI fluoxetine (30 μM, green) increases the rate of scoot initiations compared to larvae treated with vehicle DMSO alone (black circles). This increase was specific to larvae facing the target light during phototaxis (U/RTI=100/-2, n=3 groups each treatment), and not apparent during baseline movement under uniform illumination (inset). Drug was added 15 minutes before testing. * P < 0.05 compared to same orientation in DMSO treated groups. e Methysergide (80 μM, red) a non-selective serotonin receptor antagonist, suppresses the increase in scoot initiations seen for vehicle larvae (black) facing the target during phototaxis (U/RTI=100/-1, n=5 groups per condition). Methysergide does not impair locomotor activity under uniform illumination (inset). Drug was added 120 minutes before testing. * P < 0.01 compared to same orientation in DMSO groups.

Figure 4

The retinotectal OFF pathway triggers turns allowing target selection a Percentage of turns initiated in a rightward direction after Mock operation (‘Mock’, n=9 groups), unilateral section of right optic nerve (‘R Opn’, n=5 groups) or unilateral section of left optic nerve (‘L OpN’, n=6 groups). Turn direction was separately assessed for larvae facing the target with left or right eye as indicated. * P < 0.01 against corresponding orientation in mock. U/RTI=100/-1 for all groups. b Percentage of turns initiated in a rightward direction after laser ablation of right arborization field 7 (‘R A7’, n=11 larvae), right optic tectum (‘R Tec’, n=14 larvae) or sham operation (‘Mock’, n=10 larvae). Turns show significant directional bias for all orientations in all groups (P < 0.05 for one sample t-test versus 50%). * P < 0.001. c Modulation of turn frequency during phototaxis in the nrc retinal ON pathway mutant. Larvae (n=20) oriented either perpendicular to or away from the target show a significant increase in the frequency of turn initiations during phototaxis (grey bars, U/RTI=300/-1 ) relative to uniform illumination (black bar). * P < 0.05. Moreover, d turns initiated by mutants show a robust directional bias toward the target light. One sample t-test, * P < 0.01 versus 50%. Mutants showed the same overall preference for turning toward the target light as sibling larvae (target bias in siblings 77.4 % ± 8.6%, mutants 83.3 % ± 16.7 %, t-test P=0.76). e Schematic illustrating information flow through retinotectal ON and OFF pathways when the target light is in the blind visual field. The OFF pathway model enables larvae to orient toward the target light by turning away from the eye perceiving a light decrement. f Simulation of zebrafish-like agents whose movement is controlled by an OFF-turn, ON-approach algorithm (blue traces) demonstrates that this is a plausible navigational strategy to implement phototaxis. g When a second target was simultaneously presented in the model, zebrafish-like agents efficiently selected a single navigational target, swimming directly toward it. h Zebrafish larvae show choice behavior in a two target test, swimming toward one target and ignoring the second. Traces show the swim paths of individual larvae starting in the center of the testing arena and followed for 15s of phototaxis (or until they reached a target spot). i Quantification behavior in two choice test. The graph shows the distribution of trajectories taken by larvae presented with a single target light at the top of the testing arena (n=27 larvae), on the right side of the arena (n=21 larvae) or in both positions simultaneously (n=16 larvae). The area of each circle indicates the proportion of larvae following the trajectory indicated on the Y-Axis. There were no instances were larvae swam in trajectories outside the range illustrated. Trajectories were measured over the first 5s of phototaxis with recordings made at 25 frames per second. See also Figure S3.