br Material and methods br Results
Material and methods
Discussion Neurotransmitters pathways within vertebrates, including fish, are well conserved (Kreke and Dietrich, 2008, Valenti et al., 2012). Therefore psycopharmaceuticals that are design to act on these pathways in mammals, will possibly interact with the same pathways in aquatic organism and affect the neurotransmission systems. Moreover, our previous study also demonstrated that FLU, at the same concentrations tested in the present study, impacts the development of zebrafish embryos (Cunha et al., 2016). Hence, given the reported mode of action of FLU in vertebrates, this study aimed at evaluating the impact of FLU in monoamine receptors and transporters at the transcription level. Serotonin (sert, 5-ht1a, 5-ht2c), as well as dopamine (dat, drd1b, drd2b) and adrenergic (net, adra2a, adra2b, adra2c) transporters and receptors and vmat and mao in early stages of zebrafish embryos were analyzed. In mammalian model species, FLU leads to a down regulation of Sert, 5-Ht1a and 5-Ht2c and this decrease has been attributed to the increase of serotonin accumulation in the synaptic clefts by repeated SERT inhibition by FLU (Oliva et al., 2005, Gomez et al., 2015, Lesemann et al., 2012, Barbon et al., 2011, Messripour and Clark, 1985) that promotes behavioural and emotional changes by serotonergic system modulation. In this study a down regulation was observed for sert and 5-ht2c, at the lowest and environmentally relevant concentration. Hence, FLU seems to affect the serotonergic neurotransmission system in zebrafish embryos in a similar manner as in mammals, leading to a clear down regulation of serotonin transporters and receptors transcription. This decrease in mRNA can possibly affect the neurotransmission and provoke behavioural changes as described by previous studies (Santos et al., 2010, Pelli and Connaughton, 2015). In this study behavioural changes associated with FLU exposure were also evaluated. A significant decrease in the sensorimotor reflexes, mainly in tail reflexes, was observed. Signals from head and tail touches are conveyed to hindbrain Mauthner neurons stimulating escape responses (Pinho et al., 2016). In vertebrates, Mauthner cell signalling is hormone responsive and requires coordination of multiple neural signalling systems (Iwata et al., 2000, Faber et al., 1991). Therefore, exposure to toxics that can modulate neurotransmitters or block postsynaptic receptors, can results in impairment of the neurotransmission (Little and Brewer, 2001). This can disrupt the communication between sensory receptors and efferent motor systems that results in reduce behavioural responses (Little and Brewer, 2001) as the ones observed in this study. Serotonin and the serotonergic system play a role in vertebrate akt inhibitor development (Buznikov et al., 2001, Levin, 2006), hence a down regulation of genes involved in this system can also affect fish embryo development. This hypothesis is further supported by the results of a previous study performed in our laboratory reporting that FLU affects zebrafish embryos development (Cunha et al., 2016). Given that the changes in transcription levels were observed at environmentally relevant concentration, it will be important to perform additional studies, such as pathways silencing to better address FLU interaction with the serotonergic system. This work also evaluated the effects of FLU in the transcription of receptors and transporters of dopaminergic and adrenergic pathways, since previous studies suggest an interaction between these systems and the serotonergic (Kreke and Dietrich, 2008, Boulay et al., 2011, Lesemann et al., 2012). Similarly to the serotonergic systems a tendency for mRNA down regulation was also observed for dopamine transporter (dat) and receptors (drd1b, drd2b) and adrenergic receptors (adra2b, adra2c) in embryos exposed mainly at higher concentrations of FLU. In agreement with our results, FLU has been reported to decrease dopaminergic D2 receptor transcription in mammals (Dziedzicka-Wasylewska et al., 2002, Lesemann et al., 2012). The decrease of mRNA levels of dopamine receptors and transporter may have a negative impact in key functions, known to be controlled by this system, such as cognition, locomotor activity, motivation and reward, mood, attention and learning (Beaulieu and Gainetdinov, 2011). In fish, dopamine inhibits basal and GnRH-stimulated gonadotropin release by binding to the Dopamine 2 receptors (DR2) on gonadotropes, and by decreasing pituitary GnRH receptor expression which can impair gonadal growth and spawning (Chang et al., 2000, Dufour et al., 2005, Crago and Schlenk, 2015). In another study, Sebastiscus marmoratus was exposed to Tributyltin (TBT) and the results revealed a decrease in the transcription levels of dopamine receptors affecting the predatory activities of the fish (Yu et al., 2013). Therefore, the down regulation of dopamine transporter and receptors by FLU may negatively influence the reproduction and the behaviour of fish. Adrenoceptors are present in fish melanophores, modulating the pigmentation, as reported in previous studies with other pharmaceutical classes (Fujii, 2000, Xu and Xie, 2011). In agreement, zebrafish embryos that were exposed to the same FLU concentrations showed a high percentage of pigmentation anomalies, mainly at the highest concentration (0.8 μM) (Cunha et al., 2016). The storage process of monoamines is performed by VMAT that is released at the synapse following neuronal membrane depolarization. When monoamines are not stored in presynaptic vesicles by VMAT, they are degraded by MAO enzyme (Maximino, 2012). Transcription levels of vmat were diminished in the presence of FLU, while no changes were observed for mao. In contrast to our results, a previous study with rats reported an increased the transcription levels of Vmat following FLU exposure (Rehavi et al., 2002). Our results demonstrated a down regulation tendency upon exposure to FLU. However, an up regulation was observed for some genes. This seems to point to non-monotonic responses to FLU exposure which should be study in future studies.