Transient knockdown of tyrosine hydroxylase during development has persistent effects on behaviour in adult zebrafish (Danio rerio)

Abstract

Abnormal dopamine (DA) signaling is often suggested as causative in schizophrenia. The other prominent hypothesis for this disorder, largely driven by epidemiological data, is that certain adverse events during the early stages of brain development increase an individual's risk of developing schizophrenia later in life. However, the clinical and preclinical literature consistently implicates behavioural, cognitive, and pharmacological abnormalities, implying that DA signaling is abnormal in the adult brain. How can we reconcile these two major hypotheses underlying much of the clinical and basic research into schizophrenia? In this study we have transiently knocked down tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis) gene expression in the early stages of brain development in zebrafish using morpholinos. We show that by adulthood, TH and DA levels have returned to normal and basic DA-mediated behaviours, such as locomotion, are also normal. However, when they were exposed to a novel environment the levels of freezing and immediate positioning in deeper zones were significantly reduced in these adult fish. The neurochemistry underlying these behaviours is complex, and the exact mechanisms for these abnormal behaviours remains unknown. This study demonstrates that early transient alterations in DA ontogeny can produce persistent alterations in adult brain function and suggests that the zebrafish may be a promising model animal for future studies directed at clarifying the basic neurodevelopmental mechanisms behind complex psychiatric disease.

Figure 1. The open field, place preference and the novel diving tank.

Illustration of the open-field tank (A), place preference (B), and the novel diving test tank (C) with specific dimensions. Virtual divisions in the open-field tank and in four zones of the diving tank (Z1/top – Z4/bottom) were used to evaluate fish positioning.

Figure 2. 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.

Figure 3. 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.

Figure 4. 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 α-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.

Figure 5. 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.

Figure 6. 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.

Figure 7. 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.