Leopard zebrafish displayed more bottom-dwelling in the novel tank test (Fig. 1a; F 3,36 = 8.028, P = 0.0003; Tukey's post-test: q = 4.97, P < 0.05). Fluoxetine (5 mg/kg) decreased bottom-dwelling in leo mutants, but not in WT zebrafish [Tukey's post-test: q = 5.052 (0 vs. 5 mg/kg, leo), q = 0.02857 (0 vs. 5 mg/kg, WT), q = 5.023 (5 mg/kg, leo vs. WT)]. Nonetheless, albeit no differences were found between WT and leo in habituation (Tukey's post-test: q = 3.193), fluoxetine promoted habituation in WT, but not leo zebrafish (Fig. 1b; F 3,39 = 11.87, P < 0.0001). No further differences between strains or drug effects were observed in this test (Fig. 1c–f).
Figure 1. Differential effects of fluoxetine on WT and leo tq270/tq270 zebrafish in the novel tank test. (a–f) Effects of fluoxetine (0 or 5 mg/kg) on (a) bottom-dwelling, (b) habituation, (c) total locomotion, (d) homebase formation, (e) erratic swimming, and (f) freezing in the novel tank test in leo (gray bars, gray boxplots) and WT (black bars, black boxplots) zebrafish. ***, P < 0.001; *, P < 0.05. In (a), (b), (d) and (f), bars represent mean ± SEM. In (c) and (e), boxes represent data from the 25th to 75th percentiles, the trace represents the median, the plus (+) sign the mean, and whiskers represent the 2.5th and 97.5th percentile.
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In the scototaxis test, leo mutants spent less time in the white compartment than their WT counterparts (F 3,39 = 7.647, P = 0.0004; q = 5.152, Tukey's post-test; Fig. 2a). Treatment with fluoxetine (5 mg/kg) reverted this phenotype in leo animals (q = 6.314, 0 vs. 5 mg/kg, leo), without effects on white avoidance in WT animals (q = 0.6424, Tukey's post-test). No differences between groups were observed in the latency to explore the white compartment (χ 2 [df = 3] = 4.154, P = 0.2453; Fig. 2b) nor in the habituation of white compartment exploration (F 3,39 = 1.218, P = 0.3173; Fig. 2c). The number of entries in the white compartment (Fig. 2d) and its habituation (Fig. 2f), as well as the number of squares crossed in the white compartment (Fig. 2e), were likewise not affected (F 3,39 = 1.09; H < 3.9, NS [nonsignificant]). Although thigmotaxis (Fig. 2g) did not differ between phenotypes (q = 2.648, Tukey's post-test), fluoxetine did decrease it in leo, but not WT zebrafish (F 3,39 = 4.699, P = 0.0072). As in the novel tank test, freezing (F 3,39 = 0.8265, NS; Fig. 2i) did not differ between groups, nor did erratic swimming (H [df = 4] = 6.656, NS; Fig. 2h). Risk assessment was greater in leo than WT, and fluoxetine decreased it in leo but not WT (H [df = 4] = 21.19, P < 0.001; Fig. 2i).
Figure 2. Differential effects of fluoxetine on WT and leotq270/tq270 zebrafish in the scototaxis test. Effects of fluoxetine (0 or 5 mg/kg) on (a) white avoidance, (b) white avoidance habituation, (c) latency to white, (d) total locomotion on white, (e) entries on the white compartment, (f) locomotion habituation, (g) thigmotaxis, (h) freezing, (i) erratic swimming and (j) risk assessment in the light/dark tank in leo (black bars) and WT (gray bars) zebrafish. ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, nonsignificant. In (a–c) and (f–h), bars represent mean ± SEM. In (d), (e), (i) and (j), boxes represent data from the 25th to the 75th percentiles, the trace represents the median, the plus (+) sign the mean, and whiskers represent the 2.5th and 97.5th percentile.
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Tissue 5-HT levels were smaller in leo than in WT zebrafish (t [df = 18] = 3.093, P = 0.0063; Fig. 3a), while 5-HIAA levels were similar (t [df = 18] = 0.8371, NS; Fig. 3b). The 5-HT turnover was higher in leo than WT zebrafish (t [df = 18] = 2.201, P = 0.041; Fig. 3c). Monoamine oxidase activity was increased in leopard strain (t [df = 4] = 3.437, P = 0.0264; Fig. 4).