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, 364 (1519), 1021-32

Determining the Function of Zebrafish Epithalamic Asymmetry

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Determining the Function of Zebrafish Epithalamic Asymmetry

Lucilla Facchin et al. Philos Trans R Soc Lond B Biol Sci.

Abstract

As in many fishes, amphibians and reptiles, the epithalamus of the zebrafish, Danio rerio, develops with pronounced left-right (L-R) asymmetry. For example, in more than 95 per cent of zebrafish larvae, the parapineal, an accessory to the pineal organ, forms on the left side of the brain and the adjacent left habenular nucleus is larger than the right. Disruption of Nodal signalling affects this bias, producing equal numbers of larvae with the parapineal on the left or the right side and corresponding habenular reversals. Pre-selection of live larvae using fluorescent transgenic reporters provides a useful substrate for studying the effects of neuroanatomical asymmetry on behaviour. Previous studies had suggested that epithalamic directionality is correlated with lateralized behaviours such as L-R eye preference. We find that the randomization of epithalamic asymmetry, through perturbation of the nodal-related gene southpaw, does not alter a variety of motor behaviours, including responses to lateralized stimuli. However, we discovered significant deficits in swimming initiation and in the total distance navigated by larvae with parapineal reversals. We discuss these findings with respect to previous studies and recent work linking the habenular region with control of the motivation/reward pathway of the vertebrate brain.

Figures

Figure 1
Figure 1
L–R reversal of anatomical asymmetry in larval zebrafish. (a,b) Dorsal views of the pineal and asymmetrically positioned parapineal (arrowhead) at 3 dpf, following injection of the southpaw MO into the Tg(foxd3:GFP)fkg17 (Gilmour et al. 2002) line. (c,d) Labelling of GFP in the pancreas and dsRed in the liver in 5 dpf Tg(ela3l:GFP)gz2;Tg(fabp10:dsRed)gz4 (Wan et al. 2006; Dong et al. 2007) larvae viewed ventrally ((c) right pancreas and (d) left pancreas). (e) Frequencies of the four asymmetric configurations in spaw MO-injected, mock-injected and uninjected larvae.
Figure 2
Figure 2
Equivalent startle responses in L–R reversed larvae. (a) Initiation frequencies for the short latency C-start (SLC) and (b) long latency C-start (LLC) responses. Movement initiation frequencies correspond to the percentage of trials in which SLC and LLC responses were observed. Larvae were tested in a 9-well grid and scored individually (n=18 per group). (c) Percentage of SLC and (d) LLC responses initiated in a rightward direction. A few larvae produced either no SLC (n=7/72) or LLC (n=1/72) responses and these were excluded from the analysis of directionality. Startle stimuli were generated and responses were recorded as previously described (Burgess & Granato 2007a) using a 1000 Hz horizontal vibrational stimulus of 3 ms duration and maximum acceleration 150 ms. Each set of larvae was tested with a series of 40 stimuli, presented at 15 s intervals. For these and all other assays, larvae were raised at a standard density of 30 larvae per 6 cm plastic Petri dish in E3 embryo media (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 and 0.33 mM MgSO4; Nüsslein-Volhard & Dahm 2002) and maintained at 27–28°C under uniform lighting in a 14 L : 10 D cycle.
Figure 3
Figure 3
Directional behaviours are unaffected by epithalamic reversal. The (a) initiation frequency and (b) directionality of O-bend responses to dark flash stimuli were measured in Lpp and Rpp larvae (7 or 8 dpf). Dark flashes were generated as previously described (Burgess & Granato 2007b), by extinguishing an array of LEDs (800 μW cm−2) positioned at one end of the dish. Each group (8–10 larvae) was tested with a series of 24 such stimuli, presented at 60 s intervals. Only larvae oriented within 45° of perpendicular to the light source were scored. Bias measures the directionality of responses, where a score of +100 means all O-bends are in the direction of the recently extinguished light source (bias=(% O-bends towards target)×2–100). Lpp (n=9 plates) and Rpp larvae (n=8 plates) show very similar levels of dark flash responsiveness and directional bias (see text for statistics). The (c) initiation frequency and (d) directionality of turning manoeuvres in response to a looming shadow were measured in Lpp and Rpp (7 dpf) larvae. A projector was used to illuminate the testing arena (200 μW cm−2) and to cast an area of darkness (4 μW cm−2) expanding at 70 mm s−1 across the plate. Groups of 8–10 larvae were tested with eight repetitions of the looming stimulus, which was presented at 60 s intervals in alternating directions. Five groups of Lpp and four groups of Rpp larvae were tested. Only larvae oriented perpendicular to the direction of movement of the shadow were scored. Turn bias is calculated as for (b), but values are negative because larvae turn away from the approaching shadow. For both assays, 1000 ms recording windows were used to measure responses.
Figure 4
Figure 4
Larval populations do not show consistent eye preference in the mirror test. (a) The mirror test is conducted in a rectangular tank (10×4 cm) with two mirrors as the longer walls and two white screens as the shorter walls. The tank contains 28°C water at a depth of 3 cm, is evenly illuminated by overhanging 15 W fluorescent lamps and can be monitored in its entirety by a video camera suspended above the apparatus. Measurements of L–R eye use are confined to the lateral monocular visual field and scored by a larva's body position with respect to the closest mirror at 1 s intervals. Larvae in the 10 mm wide central area of the testing chamber (shaded in light grey) or at angles of either 0° or more than 90° with respect to the mirror are not scored. The frequency of right-eye use was calculated as (frequency of right-eye use)/(frequency of right-eye use+frequency of left–eye use)×100. Analysis of variance was carried out using SPSS v. 16.0 (SPSS Inc., Chicago, IL) to detect significant differences between anatomical classes. Mean and standard deviation of right eye use in (b) spaw MO-injected, (c) mock-injected and spontaneous anatomical larval groups. LppLpa larvae were not found spontaneously from transgenic intercross progeny (refer to figure 1). (d) Percentage of spaw MO-injected larvae showing a statistically significant bias (left or right) or no bias in eye use for each anatomical group. For every individual, the statistical significance of eye use was determined by a chi-squared test at a level of 5%. (e) Percentage of larvae showing a statistically significant bias (left or right) or no bias in eye use for mock-injected and uninjected spontaneous anatomical larval groups, calculated as in (d) (white bars, left bias; grey bars, right bias; black bars, no bias). (f) Mean and standard error of eye use during each minute of viewing by spaw MO-injected larvae with a left (n=65) or right (n=85) positioned parapineal (grey squares, left parapineal; black squares, right parapineal).
Figure 5
Figure 5
Larvae with reversed epithalamic asymmetry show altered navigational behaviour. (a) Mean and standard error of the elapsed time (in seconds) before a larva moves a distance equivalent to twice its body length in spaw MO-injected larvae. Differences between the four classes were calculated using the Kruskal–Wallis test (**p<0.001). (b) Mean and standard error of the onset of navigation behaviour in mock-injected or uninjected LppRpa and uninjected RppRpa and RppLpa larvae. Spontaneous LppLpa larvae were not recovered. Differences between the three classes were calculated using the Kruskal–Wallis test (**p<0.001). (c) Representative swim paths of two spaw MO-injected larvae over 5 min. Swimming behaviour was recorded to videotape (30 frames s−1) and was subsequently digitized. Video processing and analysis were performed using Matlab (The MathWorks, Natick, MA). Larval position is indicated by an open circle at the start, and a black square at the end of recording ((i) left parapineal and (ii) right parapineal). (d) Mean and standard deviation of the total distance covered (in mm) over a 5 min period starting from the first movement of individual spaw MO-injected larvae. Differences between the four classes were calculated using the ANOVA test (**p<0.001). (e) Mean and standard deviation of the average speed (mm s−1) for the total swimming episodes of spaw MO-injected larvae. Differences between the four classes were calculated using the ANOVA test (**p<0.001).

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