Exploring CNS Effects of American Traditional Medicines using Zebrafish Models.

Although American traditional medicine (ATM) has been practiced for millennia, its complex multi-target mechanisms of therapeutic action remain poorly understood. Animal models are widely used to elucidate the therapeutic effects of various ATMs, including their modulation of brain and behavior. Complementing rodent models, the zebrafish (Danio rerio) is a promising novel organism in translational neuroscience and neuropharmacology research. Here, we emphasize the growing value of zebrafish for testing neurotropic effects of ATMs and outline future directions of research in this field. We also demonstrate the developing utility of zebrafish as complementary models for probing CNS mechanisms of ATM action and their potential to treat brain disorders.

Neurochemical mechanisms underlying acute and chronic ethanol-mediated responses in zebrafish: The role of mitochondrial bioenergetics.

Ethanol (EtOH) is a socially-accepted drug, whose consumption is a risk factor for non-intentional injuries, development of pathologies, and addiction. In the brain, EtOH affects redox signaling and increases reactive oxygen species (ROS) production after acute and chronic exposures. Here, using a high-resolution respirometry assay, we investigated whether changes in mitochondrial bioenergetics play a role in both acute and chronic EtOH-mediated neurochemical responses in zebrafish. For the first time, we showed that acute and chronic EtOH exposures differently affect brain mitochondrial function. Acutely, EtOH stimulated mitochondrial respiration through increased baseline state, CI-mediated OXPHOS, OXPHOS capacity, OXPHOS coupling efficiency, bioenergetic efficiency, and ROX/ETS ratio. Conversely, EtOH chronically decreased baseline respiration, complex I- and II-mediated ETS, as well as increased ROX state and ROX/ETS ratio, which are associated with ROS formation. Overall, we observed that changes in mitochondrial bioenergetics play a role, at least partially, in both acute and chronic effects of EtOH in the zebrafish brain. Moreover, our findings reinforce the face, predictive, and construct validities of zebrafish models to explore the neurochemical bases involved in alcohol abuse and alcoholism.

Protective role of jaboticaba Plinia peruviana peel extract in copper-induced cytotoxicity in Allium cepa.

Jaboticaba Plinia peruviana (Poir.) Govaerts is a Brazilian berry that presents high levels of polyphenols, which may play a key role in preventing cytotoxic and genotoxic effects of harmful agents. Although copper is an essential micronutrient that plays an important role in organisms, high copper concentrations may trigger toxicity to animals and plants. Here, we investigated whether Plinia peruviana hydroalcoholic extract prevents copper-induced cytotoxicity in Allium cepa root cells. Five different anthocyanins and phenolic compounds were identified in Plinia peruviana extract. Importantly, the exposure to 1.53 mg/L copper for 24 h impaired mitotic index, as well as increased mitosis disturbances and triggered DNA damage. Pre-incubation with Plinia peruviana extract (0.25 g/L and 0.75 g/L) for 3 h prevented copper-induced changes in the mitotic index and reduced the number of abnormal cells. In conclusion, we suggest that Plinia peruviana peel extract has protective effects against cellular and genetic disturbances induced by copper.

Hyperglycemia elicits anxiety-like behaviors in zebrafish: Protective role of dietary diphenyl diselenide.

Diabetes mellitus (DM) is a chronic metabolic disease that may comorbid with various psychiatric disorders, such as anxiety and depression. The search for effective therapeutics to alleviate hyperglycemia and complications resulting from DM is continuous. Here we investigate the effects of diphenyl diselenide (DD), an organoselenium compound with several pharmacological properties, in a zebrafish model of hyperglycemia. Fish were fed for 74 days with a diet containing 3 mg/Kg DD, a concentration chosen after experiments based in a dose-response curve (DD 1, 2 and 3 mg/Kg) that did not cause overt toxicity (mortality, weight loss and neurobehavioral deficits). In the last 14 days of the experimental period, fish were concomitantly exposed to a glucose solution (111 mM). Afterwards, blood glucose levels, brain selenium (Se) content, and behavioral analysis aiming to assess anxiety-like behaviors and locomotor/exploratory activities were performed. In the novel tank diving test, glucose decreased vertical exploration and fish spent less time in the lit area when tested in the light-dark test, suggesting increased anxiety-like behavior. Moreover, DD decreased blood glucose levels in hyperglycemic fish as well as prevented the development of anxiety-related symptoms. DD diet alone did not change glycemia and behavioral parameters, but increased Se levels in the brain without affecting the cellular viability. Collectively, our findings highlight the growing utility of this zebrafish hyperglycemia model as a valuable strategy for further research in DM field and neuroprotective approaches.

Protective effect of Uncaria tomentosa extract against oxidative stress and genotoxicity induced by glyphosate-Roundup® using zebrafish (Danio rerio) as a model.

Oxidative stress and DNA damage are involved in the glyphosate-based herbicide toxicity. Uncaria tomentosa (UT; Rubiaceae) is a plant species from South America containing bioactive compounds with known beneficial properties. The objective of this work was to evaluate the antioxidant and antigenotoxic potential of UT extract in a model of acute exposure to glyphosate-Roundup® (GR) in zebrafish (Danio rerio). We showed that UT (1.0 mg/mL) prevented the decrease of brain total thiols, the increase of lipid peroxidation in both brain and liver, and the decrease of liver GPx activity caused after 96 h of GR (5.0 mg/L) exposure. In addition, UT partially protected against the increase of micronucleus frequency induced by GR exposure in fish brain. Overall, our results indicate that UT protects against damage induced by a glyphosate-based herbicide by providing antioxidant and antigenotoxic effects, which may be related to the phenolic compounds identified in the extract.

Neuroprotection of luteolin against methylmercury-induced toxicity in lobster cockroach Nauphoeta cinerea.

Luteolin (3', 4', 5, 7-tetrahydroxyflavone) is a polyphenolic compound found in foods of plant origin and has been reported to possess antioxidant and neuroprotective properties. However, there is dearth of information on the beneficial effects of luteolin on methylmercury (MeHg), a long-established neurotoxic compound in animals and humans. This study evaluated the effect of luteolin on MeHg-induced behavioral and biochemical deficits, using lobster cockroach Nauphoeta cinerea as an alternative and complementary animal model. The insects were exposed for 35 consecutive days to either MeHg alone (0.05 mg/g feed) or in combination with luteolin at 0.25, 0.5 and 1.0 mg/g feed. Locomotor behavior was assessed using video-tracking software during a 10-min trial in a novel arena and subsequently, biochemical analyses were carried out using the cockroaches' heads. Luteolin supplementation dose-dependently reversed the MeHg-induced locomotor deficits and enhanced the exploratory profiles of MeHg-exposed cockroaches as confirmed by track and occupancy plot analyses. Luteolin reversed the MeHg-induced acetylcholinesterase activity inhibition, decreased dichlorofluorescein oxidation and lipid peroxidation levels, but increased total thiol level and catalase and glutathione S-transferase activities in the treated cockroaches. In conclusion, luteolin prevented oxidative stress indices and neurobehavioral deficits in a Nauphoeta cinerea model of MeHg toxicity.

Long-term proline exposure alters nucleotide catabolism and ectonucleotidase gene expression in zebrafish brain.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures cognitive dysfunctions, and psychotic disorders. However, the underlying mechanisms of these symptoms are still unclear. Since adenine nucleotides play crucial roles in neurotransmission and neuromodulation, we evaluated the in vivo and in vitro effects of proline on ectonucleotidase activities and gene expression in zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0 mM) during 1 h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 µM) were tested. Short-term proline exposure did not promote significant changes on the ectonucleotidase activities and gene expression. Long-term proline exposure significantly increased ATP catabolism in both concentrations tested (14 % and 22 %, respectively), whereas ADP and AMP hydrolysis were increased only at 3.0 mM proline (21 % and 17 %, respectively) when compared to control. Moreover, the relative gene expression of enpd3 increased in both treated groups after long-term proline, whereas enptd1 increased only at 3.0 mM proline. Proline in vitro did not promote significant changes on ectonucleotidase activities. Altogether, these data indicate that the enzymes responsible for the control of extracellular nucleotides levels might be altered after proline exposure in zebrafish, contributing to better understand the pathophysiology of this disease. Moreover, such findings might facilitate the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

Behavioral effects of taurine pretreatment in zebrafish acutely exposed to ethanol.

Taurine (TAU) is an amino sulfonic acid that plays protective roles against neurochemical impairments induced by ethanol (EtOH). Mounting evidence shows the applicability of zebrafish for evaluating locomotor parameters and anxiety-like behavioral phenotypes after EtOH exposure in a large scale manner. In this study, we assess the effects of TAU pretreatment on the behavior of zebrafish in the open tank after acute 1% EtOH (v/v) exposure (20 and 60 min of duration) and on brain alcohol contents. The exposure for 20 min exerted significant anxiolytic effects, which were prevented by 42, 150, and 400 mg/L TAU. Conversely, the 60-min condition induced depressant/sedative effects, in which the changes on vertical activity were associated to modifications on the exploratory profile. Although all TAU concentrations kept locomotor parameters at basal levels, 150 mg/L TAU, did not prevent the impairment on vertical activity of EtOH[60]. Despite the higher brain EtOH content detected in the 60-min exposure, 42, 150, and 400 mg/L TAU attenuated the increase of alcohol content in EtOH[60] group. In conclusion, our data suggest that both protocols of acute EtOH exposure induce significant changes in the spatio-temporal behavior of zebrafish and that TAU may exert a preventive role by antagonizing the effects induced by EtOH possibly due to its neuromodulatory role and also by decreasing brain EtOH levels. The hormetic dose-response of TAU on vertical exploration suggests a complex interaction between TAU and EtOH in the central nervous system.