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  • Since the introduction of zebrafish

    2024-05-18

    Since the introduction of zebrafish into the laboratory, many milestones have been achieved that firmly establish this organism as a prominent genetic model for biology and medicine. Many features make this species an organism of easy maintenance in laboratory which provides advantages to understand the molecular and cellular mechanisms of behavior and behavioral disorders (Chen et al., 2016). Forward genetic studies in zebrafish represent an important complementary approach to uncover behavioral sensitivity to ethanol, beyond novel molecular mechanisms underlying (Linney et al., 2004, Shin and Fishman, 2002). While high-throughput behavioral assays for ethanol sensitivity have been established (Lockwood et al., 2004), it has been difficult to recover behavioral mutants or underlying molecular lesions. Several neurotransmitter systems was described in zebrafish dopamine beta hydroxylase (Mahabir et al., 2014, Rico et al., 2011a, Rico et al., 2011b). In relation to purinergic system, our group demonstrated the presence of NTPDase (Rico et al., 2003), 5′-nucleotidase (Senger et al., 2004), and adenosine deaminase (Rosemberg et al., 2008) activities in zebrafish brain. Besides, we have shown that exposure to ethanol changes NTPDase and ecto-5′-nucleotidase activities in the CNS of this animal model (Rico et al., 2008). Our group also reported the differential expression pattern of ADA-related genes in zebrafish tissues, including brain, confirming that these genes (ADA1, ADAL, ADA2-1, and ADA2-2) are present in this species (Rosemberg et al., 2007a). Recently, two zebrafish A2A and one A2B genes were identified in developing embryos, and their expression was demonstrated in the CNS (Boehmler et al., 2009).
    Material and methods
    Results The results obtained for ADA activity after chronic ethanol exposure in zebrafish brain are presented in Fig. 1. As can be observed, in soluble fractions, ADA activity of zebrafish exposed for 7, 14 and 28days did not significantly change as compared to the control group (Fig. 1A). However, in membrane fractions, after 28days of ethanol exposure it was possible to observe a decrease of ADA activity (44%), while 7 and 14days of ethanol exposure did not promote significant changes in ADA activity (Fig. 1B). In order to verify whether the ADA-related genes could be modulated when zebrafish were exposed to chronic ethanol, we have performed semi-quantitative RT-PCR experiments after 7, 14 and 28days. Ethanol exposure modified the gene expression pattern of ADA-related genes in zebrafish brain (Fig. 2). ADA1 transcripts were not altered in all times of ethanol exposure in zebrafish brain (Fig. 2A). However, ADAL presented an increase in the level of transcripts after 28days of ethanol exposure (34%), while that 7 and 14days did not induce significant changes (Fig. 2B). Considering the member related to ADA2-1, it was previously identified an ADA2-1 truncated alternative splice isoform (ADA2-1/T), which was expressed at different intensities (Rosemberg et al., 2007b). There were not alterations of ADA2-1T after ethanol exposure for 7 and 14days, while transcript levels demonstrated a reduction after 28days (20%). Interestingly, ADA2-1 showed a decrease (26%) of transcripts followed by an increase of transcripts (17%) after 14 and 28days of ethanol exposure, respectively (Fig. 2C). Similarly to ADA2-1, ADA2-2 demonstrated a decrease (22%) of transcripts followed by a strong increase of transcripts (109%) after 14 and 28days of ethanol exposure, respectively (Fig. 2D).
    Discussion The chronic ethanol consumption is associated with neurochemical changes in the CNS. Ethanol is able to interfere the function of adenosinergic system, and may mediate some effects of ethanol, such as intoxication, motor coordination and sedation (Dohrman et al., 1997). Furthermore, studies have shown that extracellular concentrations of adenosine may also be regulated by ecto-ADA activity (Franco et al., 1998, Romanowska et al., 2007). In this study, we found an inhibition of ADA activity in membrane fractions of zebrafish brain exposed to 28days to ethanol exposure. Differently, we did not observe significant alterations in soluble fractions. Ethanol has an aliphatic moiety and provides a lipophilic group that can interact with non-polar domains of macromolecules. This physico-chemical property governs the forces of interaction of ethanol with biological substrates (Fadda and Rossetti, 1998). Although ethanol can disturb the natural thermal balance that maintains membrane architecture and can alter membrane microdomains that determine protein-membrane and protein-ligand interactions (Wang et al., 1993). Furthermore, studies demonstrate points to a specificity of action of ethanol directly on membrane proteins producing on conformational changes that alter their function (Li et al., 1994, Lovinger, 1997).