Resumo

An imbalance in dopamine-mediated neurotransmission and neurodevelopmental abnormalities are features of schizophrenia (SCZ). The main target of antipsychotics, the dopamine D2 receptor, modulates the activity of Akt, which has an important role in the regulation of cellular processes that are critical for brain development, and it is known to be downregulated in the brain of SCZ patients. Thus, it is possible that altered D2-dependent Akt signalling leads to abnormal neurodevelopment in SCZ. To address this hypothesis we used zebrafish as a model, because of three main advantages: (1) fundamental molecular genetic processes underlying brain development are conserved between fish and humans; (2) the patterns of neural circuit development are simplified, easily recognizable and occur in a stereotypical time window of just a few days; (3) pharmacological experiments are relatively easy to perform. Exogenous dopamine was applied to the rearing media of 5 day post fertilization (dpf) larvae and we used ELISA to verify that increased levels of brain dopamine were achieved. Using this approach, we observed an increase in brain dopamine levels after a 30 min exposure. To evaluate if dopamine modulates Akt signalling in the developing brain, we treated 3dpf and 5dpf larvae with dopamine and used Western blot to examine the phosphorylation status of Akt. Our results indicate that dopamine causes dephosphorylation at threonine 308 (T308), but not at serine 473 (S473), in a time- and concentration-dependent manner. We treated 5dpf larvae with dopaminergic agonists and antagonists and demonstrated that dopamine regulates Akt activity by D2 receptors, but not by D1 receptors. These patterns in dopamine regulation of Akt activity have also been observed from similar experiments in mice. Confocal imaging revealed that the decrease in pAkt(T308) levels occurs in forebrain regions that are innervated by dopaminergic neurons. We examined dlx6:gfp transgenic 3dpf larvae, which express GFP in several forebrain GABAergic neuronal populations, chronically exposed to dopamine. This treatment resulted in an increase the number of neurons expressing GFP, but not the total number of cells or the proportion of glial cells, suggesting a specific effect on neuronal differentiation. Furthermore, we observed that dopamine affects motor behaviour in 3-5dpf larvae. These results for motor activity were especially intriguing, as the effects of the dopamine were actually opposite depending on whether the larvae were in a group (increase in movement initiation) or isolated (decrease in movement initiation). Together, our data suggest that increased dopamine signalling represses Akt signalling and leads to defects in GABAergic neuronal differentiation in the zebrafish larval brain. Furthermore, the increase in forebrain GABAergic neurons is correlated with altered context-dependent motor behaviour. Thus, with this model system, we could holistically assay the biochemical, morphological, and behavioural consequences of altered dopamine signalling during a 3-5 day developmental window. These results will help shape our understanding of the role of dopamine in brain development and provide new mechanistic insight for further assessing the neurodevelopmental origin model of SCZ.