Efficiency of th1 gene knock down at 27 hpf. RNA expression levels of zebrafish th1 transcripts are presented as a percentage relative to the expression of the zebrafish housekeeping gene elf1a. [F2,8 = 4.9 p<0.05] Values are mean±SEM. * p<0.05.

Altered patterning of TH-positive cells in the larval brain of th1 morphants.

Immunofluorescence of TH-containing cells in the brains of control MO (A), th1-MO1 (B) and th1-MO2 (C) injected embryos (6 dpf). Confocal z-projections of larval brains are shown from a dorsal perspective, anterior to the left. OB olfactory bulb, LC locus coeruleus, SP subpallium, vDC ventral diencephalon. Dotted lines indicate brain outline. Scale bar = 100 μm.

TH protein and dopamine content are reduced in th1 morphant larvae.

(A) Western blot reveals the expression of TH1 protein is significantly reduced in th1-MO1 and th1-MO2 injected embryos compared with control MO (6 dpf) [F2,8 = 20.4 p<0.01]. Protein expression levels are presented as a percentage relative to the expression of ±-tublin (B) Dopamine (DA) content is significantly reduced in th1-MO1 and th1-MO2 injected embryos compared with control MO (6 dpf) [F2,11 = 17.5 p<0.01]. Values are mean±SEM. **p<0.01, ***p<0.001.

TH protein expression and dopamine content return to normal by adulthood.

Western blot and HPLC show that zebrafish TH1 protein (A) and synthesis of DA (B) return to control levels in both th1-MO1 and th1-MO2 adult morphants. TH protein expression levels are presented as a percentage relative to the expression of α-tublin.

Adult morphants show normal locomotor and habituation but impaired freezing behaviour.

Locomotor activity was measured as (A) distance travelled in metres in the open field tank, (B) time spent in the centre of the open field tank, (C) time spent in the dark compartment of the place preference tank, and (D) Percent time fish spent freezing in the open field tank. Habituation within these paradigms was assessed by comparing swimming behaviour in the 1^st minute with the 10^th minute. Shaded areas represent percentage area of tank defined as centre (open field tank) or dark (place preference tank). Freezing behaviour was abolished in both th1-morphants. (repeated measures ANOVA followed by Bonferroni′s test) *p<0.05, **p<0.01, ***p<0.001.

Adult morphants have impaired diving behaviour in a novel environment.

Early disruptions of th1 expression have long-lasting effects on the vertical diving behaviour in the novel diving tank task. Diving activity was assessed as the percentage time that was spent in each vertical zone (1-4) during a 10-minute trial. (A) shows behaviour that was displayed during the 1^st minute, and (B) shows behaviour during the 10^th minute. Letters indicate statistically significant differences between groups (one-way ANOVA followed by Bonferroni′s test), p≤0.05. Corresponding heat maps are displayed to reflect fish location in each zone.