Animals and housing conditions
Adult wild-type Zebrafish (Danio rerio), of both sexes, obtained from the department of Biochemistry at Federal University of Rio Grande do Sul (UFRGS). Zebrafish were conditioned at the Translational Psychiatry laboratory of UNESC and maintained according to standard husbandry procedures. The water was kept at 26 ± 2°C under 14h/10h light dark cycle photoperiod and fed twice a day with artemia and commercial flake fish food. All procedures presented in this study were approved by the Ethics Committee of University of Southern Santa Catarina (UNESC) protocol number 030/2019-1.
Melatonin exposure and seizure induction
The molecule was administered directly into the aquariums half an hour before the lights went off in the zebrafish facility at a final concentration of 100nM, according previous described [22]. Fish were exposed to melatonin in two different time periods, being: melatonin added for 3 nights, and 7 nights treatment in which seizures were induced the morning after each period (Fig. 1A). To ensure the same concentration of melatonin daily, the water in the tanks was completely exchanged every day. After melatonin treatment, fish were immersed in 160 µg ⁄ mL of tricaine and then inoculated (10µL) intraperitoneally (i.p.) with KA at 5mg/kg [21]. The fish groups that were not induced seizures were similarly anaesthetized and injected with PBS. After seizure induction, fish were observed individually for a period of 60 min. The seizures presents classic behaviors and abnormal brain activity, which can be observed through a numerical system of scores, varying from 1–7, being: 1) immobility and hyperventilation; 2) whirlpool-like swimming; 3) rapid movements from right to left; 4) abnormal and spasmodic muscular contractions, 5) rapid whole-body clonus-like convulsions; 6) sinking to the bottom of the tank and spasms for several minutes; 7) death [16]. Seizure intensity, latency, and SE as duration of 5 and 6 score time was evaluated.
After the preliminary test verifying the effect of different times of melatonin exposure, we established 7 days of pretreatment for biochemical analyzes (Fig. 2A). For this, fish were divided into four groups according to each treatment: CTL + PBS, Mel + PBS, CTL + KA and Mel + KA. Control groups underwent the same procedures without the administration of any molecule into the water. Seizure- induced by KA generate neurochemical consequences after different periods, including glutamate transport and microglial markers [21]. In order to verify whether melatonin is able to influence the changes caused by KA-induced glutamatergic hyperstimulation, glutamate uptake and oxidative stress parameters were evaluated. Thus, the end of 12 hours post seizure induction the animals were anesthetized by immersing them in 160 mg/ml of tricaine (4 oC), suffered euthanasia and their brain contents were dissected for neurochemical analysis.
Evaluation oxidative stress parameters
Tissue preparation
Five zebrafish brains were pooled and used to prepare each homogenate fraction (n = 6). Tissues homogenized in 1 mL of 20 mM sodium phosphate buffer, pH 7.4, containing 140 mM KCl. Homogenates were centrifuged at 750 × g for 10 min at 4°C to discard nuclei and cell debris [23]. The pellet was discarded, and the supernatant was collected and used for the evaluation of oxidative stress parameters.
Determination of oxidation of DCFH
The production of reactive oxygen species (EROS) was measured from the oxidation of 2 ′,7′-dihydrodichlorofluorescein diacetate forming DCF, where the intensity of fluorescence is in accordance with the formation of EROS [24]. A calibration curve was performed with standard DCF (0.25–10 µM) and the levels of reactive species were expressed as nmol DCF formed.mg of protein− 1.
Determination of TBA-RS levels
The levels of substances reactive to thiobarbituric acid (TBA-RS) evaluate the lipid peroxidation produced by reactive oxygen species. This technique is based on the principle of species reactive to thiobarbituric acid forming a pink color in the samples [25]. A calibration curve was established using 1,1,3,3-tetramethoxypropane and each curve point was subjected to the same treatment as supernatants. TBA-RS values were measured and expressed as nmol of TBA-RS.mg protein− 1.
Antioxidant enzyme activities determination
Determination of SOD and CAT activities
Superoxide Dismutase (SOD) was carried out using methods already established, based on the oxidation of adrenaline by the enzyme SOD, disabling the dismutation of the radical O2− in H2O2 [26]. The reaction medium consisted of 50 mM glycine buffer, pH 10.2, 0.1 mM catalase and 1 mM epinephrine. Absorbance was measured at 480 nm. SOD specific activity is expressed as nmol.min− 1.mg protein− 1. Catalase (CAT) activity assay was performed through the measuring of absorbance decrease at 240 nm in a reaction medium containing 20 mM H2O2, 0.1 % Triton X-100, 10 mM potassium phosphate buffer, pH 7.0, and the supernatants containing 0.1–0.3 mg protein.mL− 1 [27]. The specific activity was represented as nmol.min− 1.mg protein− 1. The set of two enzymatic activities were expressed using the SOD/CAT ratio.
Determination of GR and GPx activities.
Glutathione reductase (GR) is responsible for catalyzing the GSSG reduction reaction in NADPH-dependent GSH [28]. The enzyme activity was assessed in a solution containing 50mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA, 0.2 mM NADPH and the supernatant containing 0.3–0.5 mg protein.ml− 1. The reaction was initiated by the addition of 1 mM oxidized glutathione and a change in absorbance was measured at 340 nm. GR activity was expressed as nmol NADPH oxidized.min− 1 .mg protein− 1. The enzymes Glutathione peroxidases (GPx), are responsible for the consumption of NADPH to generate reduced glutathione (GSH) from oxidized glutathione (GSSG), acting on the metabolism of H2O2 [29]. This method was performed using Tert-butylhydroperoxide as a substrate. The enzyme activity was determined by monitoring the NADPH disappearance at 340 nm in 50 mM potassium phosphate buffer, pH 7.0, containing 1mM EDTA, 1 mM glutathione, 0.2 U.ml− 1 glutathione reductase, 1 mM azide, 0.2 mM tert-butyl-hydroperoxide, 0.2 mM NADPH and the supernatant containing 0.2–0.3 mg protein.ml-1. GPx activity was expressed as nmol NADPH oxidized.min− 1 .mg protein− 1.
Glutamate uptake assay
Glutamate uptake assay was performed as previously described [20, 29]. The brains were set in microplates of 24 wells and total glutamate uptake was measured with the addition of 0.33 µCi mL− 1 L-[3H] glutamate (PerkinElmer, Madrid, Spain) to the incubation medium (HBSS-HEPES buffer) pH 7.2 at 37°C for 7 minutes. After this period, the uptake was stopped with two subsequent washes with 1 mL ice-cold HBSS-HEPES buffer). Na+-independent glutamate uptake was measured using the same conditions as described above, except that N-methyl-D-glucamine was used instead of sodium. Na+-dependent glutamate uptake was measured as the difference of incorporated radioactivity between the total glutamate uptake and the Na+-independent glutamate uptake. Radioactivity was measured by liquid scintillation and uptake was expressed as nmol [3H]-Glu.min− 1.mg of protein− 1).
Protein determination
Total protein quantification in the samples was performed by Lowry et al. (1951)[31] method using bovine serum albumin as standard.
Statistical Analysis
Statistical analyzes were performed using the statistical program SPSS Statistics (Armonk, New York, USA). The construction of all the graphs present in this study was performed using the Graph Pad Prism software version 8.4. The results were found as mean ± standard error of the mean. The results were evaluated by one- or two-way analysis of variance (ANOVA) in which the degree of significance was evaluated, where p < 0.05. As a result, where there was a difference, the Tukey test was performed as a post hoc test when necessary.