Control of a specific motor program by a small brain area in zebrafish

Abstract

Complex motor behaviors are thought to be coordinated by networks of brain nuclei that may control different elementary motor programs. Transparent zebrafish larvae offer the opportunity to analyze the functional organization of motor control networks by optical manipulations of neuronal activity during behavior. We examined motor behavior in transgenic larvae expressing channelrhodopsin-2 throughout many neurons in the brain. Wide-field optical stimulation triggered backward and rotating movements caused by the repeated execution of J-turns, a specific motor program that normally occurs during prey capture. Although optically-evoked activity was widespread, behavioral responses were highly coordinated and lateralized. 3-D mapping of behavioral responses to local optical stimuli revealed that J-turns can be triggered specifically in the anterior-ventral optic tectum (avOT) and/or the adjacent pretectum. These results suggest that the execution of J-turns is controlled by a small group of neurons in the midbrain that may act as a command center. The identification of a brain area controlling a defined motor program involved in prey capture is a step toward a comprehensive analysis of neuronal circuits mediating sensorimotor behaviors of zebrafish.

Keywords: J-turn; motor control; optogenetics; prey capture; zebrafish.

Figure 1

Optical stimulation evokes J-turn in HuC:itTA/Ptet:ChR2YFP zebrafish larvae. (A) Pattern of ChR2YFP expression in the brain of a HuC:itTA/Ptet:ChR2YFP larva (17 dpf; z-projection of a confocal stack). OT-sp, Optic tectum-superficial layers; OT-PV, Optic tectum-periventricular layer. (B) Classification of behavioral responses to blue light stimulation in freely swimming zebrafish larvae. Backward movement was the dominant response in HuC:itTA/Ptet:ChR2YFP transgenics (ChR2⁺; n = 47 fish; one trial per fish) but never occurred in wt siblings (wt; n = 12). (C) Video sequence of a freely swimming HuC:itTA/Ptet:ChR2YFP larvae during an episode of backward movement. Note unilateral bends of the caudal tail, a characteristic of J-turns. Overlay illustrates net backward movement and rotation. (D) Examples of four different behaviors observed in head-fixed fish. Black traces show the curvature of the tail as a function of time. Red line represents the resting angle (straight tail). (E) Video sequence of a J-turn response in a head-fixed larva. Note unilateral bends of the caudal tail and symmetric movement of the pectoral fins (red arrow). (F) Classification of behavioral responses to blue light stimulation in head-fixed larvae. J-turns were frequently observed in HuC:itTA/Ptet:ChR2YFP (95 trials in 17 fish) but never in wt siblings (44 trials in 8 fish). (G) Latency (time from blue light onset to the initiation of motor response) and duration of J-turns evoked by blue light stimulation in freely swimming (n = 12) and head-fixed (n = 15) HuC:itTA/Ptet:ChR2YFP larvae (mean ± s.e.m.). ^*p = 0.019, Student's t-test.

Figure 2

Convergent eye movements during optically-evoked J-turns. (A) Eye position before (a) and during (b) a J-turn evoked by optical stimulation in a HuC:itTA/Ptet:ChR2YFP larva. White lines show orientation of anterior-posterior axis; colored lines show angle of the eye. Overlay shows convergence of eyes during J-turn. Traces show angular changes in eye position as a function of time. (B) Mean change in eye vergence angle (±s.e.m.) evoked by blue light stimulation in HuC:itTA/Ptet:ChR2YFP larvae (ChR2⁺) and wt siblings. Positive change indicates convergence of eyes. ^***p < 0.001, Student's t-test. (C) Classification of eye movements in HuC:itTA/Ptet:ChR2YFP larvae that did not respond to blue light (n = 49), in HuC:itTA/Ptet:ChR2YFP larvae responding with J-turns (n = 65), and in wt siblings, which never responded with J-turns (n = 32). An eye movement was defined as an angular change in eye position >2°. Convergent eye movements were closely associated with J-turns. ^***p < 0.001, Chi-square test for comparison of the frequency of convergent eye movements.

Figure 3

Quantitative comparison of J-turns and forward swimming. (A) Examples of tail curvature as a function of time during episodes of forward swimming and J-turning. Fish were stimulated with blue light starting at the onset of the trace. (B) Power spectral analysis of tail curvature during forward swimming and J-turns (average over all trials). (C–G) Asymmetry of tail movements, latency of motor response, duration of motor behavior, mean frequency of tail beats, and cumulative amplitude of tail beats for optically-evoked J-turns (n = 138 trials in 12 larvae; mean ± s.e.m.) and forward swims (n = 35 trials in 9 larvae). Mean frequency and cumulative amplitude were calculated over the total duration of the behavior, including short periods of inactivity. ^**p < 0.01; ^***p < 0.001; Student's t-test. (H,I) Latency and frequency of J-turns as a function of light intensity in four HuC:itTA/Ptet:ChR2YFP larvae. Because behavioral thresholds varied between individuals, data for each larvae were normalized to the maximum intensity used for each larva. Latencies were normalized to the maximum latency observed in each larva. (J) Dependence of behavioral response on intensity of blue light stimulation. Colored bars indicate the most frequently observed response as a function of light intensity in HuC:itTA/Ptet:ChR2YFP larvae (n = 10 fish) and wt siblings (n = 8). (K) Probability of J-turn responses as a function of age.

Figure 4

Effects of optical stimulation in different transgenic fish lines. (A) Swimming speed as a function of time (1 s bins) in four different zebrafish lines and two different age groups before, during and after a 20 s exposure to blue light (bar; LED). n, number of fish. (B–D) Mean change in swimming speed during a 2 s period after light onset (20–22 s), a 10 s period before light offset (30–40 s) and a 20 s period after light offset (40–60 s). (E) Percentage of J-turn responses evoked by blue light stimulation in different fish lines. Error bars show s.e.m. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ANOVA and Bonferroni post-hoc test.

Figure 5

Calcium signals evoked by optical stimulation. (A) Changes in fluorescence intensity of the calcium indicator rhod-2 in an ex-vivo preparation of the larval head after optical stimulation with blue light. Top: HuC:itTA/Ptet:ChR2YFP; bottom: wt. Four different brain areas are outlined (left optic tectum, right optic tectum, torus longitudinalis, cerebellum). (B) Mean changes in fluorescence intensity in these brain areas evoked by stimuli of two different intensities. Error bars show s.e.m. (C,D) Same experiments performed in preparations without eyes. Error bars show s.e.m. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; Student's t-test.

Figure 6

J-turn responses depend on efficiency of optical stimulation. (A) ChR2YFP expression in two HuC:itTA/Ptet:ChR2YFP larvae that responded to blue light stimulation with J-turns (left) and two siblings that did not respond (right). Arrowheads depict the optic tectum, the tecto-toral pathway, and a prominent group of hindbrain neurons. Images are z-projections of confocal stacks acquired using the same settings. (B) Sum of binarized and registered images of seven HuC:itTA/Ptet:ChR2YFP larvae that responded to blue light stimulation with J-turns and seven siblings that did not respond. Gray levels indicate the number of larvae in which each pixel was covered by pigment. Pigmentation was then quantified within the outlined area. (C) Distribution of pixel counts in the images in (B). The distribution is shifted to the left for images from larvae that responded with J-turns, indicating less pigmentation (Kolmogorov-Smirnov test, p < 0.001). Insert shows cumulative distributions of pixel counts. (D) Probability of J-turning in HUC:itTA/Ptet:ChR2YFP fish in the nacre background and in pigmented siblings. ^*p = 0.021, Chi-square test. (E) Normalized probability of J-turn responses in pigmented HuC:itTA/Ptet:ChR2YFP larvae (n = 20) as a function of light intensity (number of LEDs).

Figure 7

Mapping of J-turn responses by local optical stimulation. (A) Approximate regions illuminated with blue light from an epifluorescence lamp (circles) superimposed on an outline of the zebrafish head (dorsal view). Numbers within circles show the probability of J-turn responses to optical stimulation in each location in HuC:itTA/Ptet:ChR2YFP larvae (ChR2⁺; n = 8 fish, 3 trials at each position) and wt siblings (n = 5 fish, 3 trials at each position). (B) Same with more restricted blue light illumination (field aperture closed; n = 5 fish, 3 trials at each position for ChR2⁺ and wt siblings). (C) Mapping of J-turn responses to blue light stimulation through a vertical optic fiber (diameter, 200 μm; n = 8 fish for ChR2⁺ and n = 7 fish for wt; 3 trials at each position). (D) Mapping of J-turn responses to optical stimulation in the midbrain using a vertical optical fiber with 50 μm diameter. Numbers indicate the probability of J-turn responses at the corresponding positions (n = 4 fish, between 3 and 13 trials each position). Arrows indicate the direction of tail bends when the probability of J-turning was >0.25. (E) Images showing tail bends evoked by optical stimulation in the anterior tectum with a small optical fiber (50 μm) on different sides. Arrows show the side of the brain that was stimulated. (F,G) Mean angular movement and movement speed of the ipsilateral and contralateral eyes evoked by stimulation with an optical fiber (50 μm) above the anterior tectum on one side. (H) Mean latency of eye movements and tail movements. All error bars show s.e.m. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; Student's t-test.

Figure 8

Localization of the brain area triggering J-turns in 3-D. (A) Probabilities of evoking J-turns in the midbrain of HuC:itTA/Ptet: ChR2YFP larvae with a 50 μm optical fiber tilted by 45° to the left or to the right. Numbers indicate the probability of J-turn responses at the corresponding positions (n = 3 fish, 3 trials at each position). Arrows indicate the direction of evoked tail bends when the probability of J-turning was >0.25. (B) Schematic illustration of the localization of the brain area triggering J-turns by triangulation. Image shows a coronal section of the zebrafish brain stained with anti-GFP, approximately at the rostro-caudal position where J-turns were evoked with highest probability (level of anterior tectum). Dashed lines indicate the light paths of optical stimuli (vertical and 45°) that produced J-turns with maximal probability. Light paths intersect in a region containing the anterior-ventral optic tectum (avOT) and part of the pretectum. (C) Photoconversion of kaede after illumination with UV light through an optic fiber (50 μm) oriented vertically or at 45°. The fiber was positioned at sites that produced maximal probabilities of J-turn. Images (z-projections of small multiphoton stacks) are shown at two depths for two fish illuminated at different angles. Most photoconverted neurons (red) were found in the avOT. OT, optic tectum; pv, periventricular layer; sp, superficial layers; PT, pretectum.