Many neuropsychiatric disorders are associated with both dopaminergic (DAergic) and developmental hypotheses. Since DAergic receptors are expressed in the developing brain, it is possible that alterations in dopamine (DA) signaling may impair brain development and consequent behavior. In our previous study, using a zebrafish model, we showed that an increase of DA during the 3 to 5 days postfertilization (dpf) developmental window (an important window for GABAergic neuronal differentiation) affects the motor behavior of 5 dpf larvae. In this study, we set out to determine whether these behavioral alterations were sustained in larvae at older stages (7 and 14 dpf). To test this hypothesis, we chronically treated zebrafish larvae from 3 to 5 dpf with DA. After washing the drug, we recorded and analyzed the first 5 and 30 min of the motor behavior of 5, 7, and 14 dpf subjects. We analyzed mobile episodes, distance traveled, time mobile, distance traveled per mobile episode, time in movement per mobile episode, and distance traveled per time mobile. We showed, once again, that an increase of DA during the 3 to 5 dpf developmental window reduces the number of movement episodes initiated by 5 dpf larvae. We also detected a decrease of other motor behavior parameters in 5 dpf DA‐treated larvae. We observed that these alterations are sustained in the 7 dpf larvae. However, we did not see these general locomotor alterations in the 14 dpf larvae. Moreover, we detected a decrease of distance traveled and an increase of time of locomotion per episode in the first 5 min of behavioral analyses in 14 dpf DA‐treated larvae. To test if the alterations in the first 5 min were due to anxiety‐like behavior, we used a light/dark preference paradigm. We recorded 5dpf, 7dpf, and 14dpf larvae for 5 min and analyzed time of freezing, preference for light or dark, number of entries to the dark, percentage of time in the light. We observed that 5dpf larvae treated with DA showed more freezing, less passages to the dark, and more time spent in the light as compared to their control counterparts. But 7dpf and 14dpf larvae did not show these alterations. Taken overall, therefore, our results suggest that DA does play a role in the development of zebrafish motor behavior, and, furthermore, that some behaviors are more sensitive than others to the effects of DAergic imbalances during development.

Study design and experimental tools are crucial for good quality science, and an important part of it is the choice of control groups to best test the hypothesis. Two of the most common control groups in research are needle prickling without substance injection (Sham) and/or vehicle injection (Saline). Very few studies investigated the behavioral effects of a single intraperitoneal (i.p) Sham and/or single i.p. Saline administration in mice, mostly using adult males. In this study, we investigated if a single i.p. Sham and/or i.p. Saline administration affects female and male prepubertal (4-weeks-old) mice behavior. After Sham or Saline injection, we examined exploratory/motor behavior (open field test; OFT), anxiety-like behavior (elevated plus-maze; EPM), and behavioral despair/depressive-like behavior (forced swimming test; FST). We observed that both Sham prepubertal females and males showed minor behavioral alterations in OFT and EPM, and Saline males showed behavioral alterations in OFT and FST. On the other hand, prepubertal Saline females showed an increase in exploratory behavior, risk assessment/anxiety-like behavior, and behavioral despair/depressive-like behavior. Thus, we observed that the experimental procedures used as control have mild (but not insignificant) consequences in prepubescent mice behavior. However, the consequences of control protocols in prepubescent females are stronger than in males. To the best of our knowledge, this is the first study investigating the consequences of Sham and Saline protocols in prepubertal mice. This study highlights how important is to include Naive animals (Negative control) together with Sham and/or Vehicle for a better and more honest interpretation of the data.