Anxiolytic and anti-stress effects of acute administration of acetyl-L-carnitine in zebrafish

Animals

A total of 240 adult zebrafish (Danio rerio, F. Hamilton 1822) wild-type short fin strain (6-month-old, 3–4 cm long) 50:50 male/female ratio were purchased from Delphis aquariums (Porto Alegre, Brazil). The fish were kept for 15 days in a closed acclimation tank system of 16 L (40 × 20 × 24 cm) identical to the experimental tanks. Housing conditions consisted only of a tank with water, heater, filter and aeration system, and were maintained as previously described in Marcon et al. (2016). The tanks contained non-chlorinated, aerated tap water (pH 7.0 ± 0.3; temperature 26 ± 1 °C; total ammonia at <0.01 mg/L; nitrite <0.01 mg/L; dissolved oxygen at 7.0 ± 0.4 mg/L; alkalinity at 22 mg/L CaCO₃ and total hardness at 5.8 mg/L), with a light/dark cycle of 14/10 h (lights on at 06:00 am). The fish were fed twice a day with a commercial flake fish food (Alcon BASIC^®; Alcon, São Paulo, Brazil). On the experimental days, all the fish were only fed early in the morning before behavioral testing began. The order of testing was counterbalanced so that fasting time was randomized across experimental groups. All experiments were approved by the Ethics Committee of Universidade Federal do Rio Grande do Sul (#30992/2015).

Drug and experimental design

O-Acetyl-L-carnitine hydrochloride (ALC, CAS number 5080-50-2) was acquired from Sigma-Aldrich (St Louis, Missouri, USA). In all experimental protocols (novel tank, light/dark, and acute chasing stress tests), the animals were treated or not with ALC (0.1, 1.0 and 10.0 mg/L) in a beaker for 10 min. In the first protocol, immediately after the treatment, the animals were placed in the novel tank test (NTT) for 6 min. In the second protocol, after the treatments, the animals were placed in the light/dark test (LDT) for 5 min. Finally, in the third protocol, the animals were treated as previously and then chased with a net for 2 min. Then, the animals were placed in the NTT. The biochemical analyses were performed in animals submitted to this last protocol. A control group was submitted to the same experimental conditions (stressed or not) but without treatment. Different sets of animals were used in each experimental protocol. The experimental design is shown in Fig. 1 and was based on the previously published study by Mocelin et al. (2015). All behavioral tests were performed between 09:00 am and 16:00 pm. The researchers who performed the behavioral tests and analyzed the data were unaware of the allocation of animals to the experimental groups. The concentrations were based on previous studies with another antioxidant compound (N-acetylcysteine) and pilot studies with a wider concentration range. The same concentrations were used in a chronic study with ALC (M Marcon, R Mocelin, A Araujo, A Herrmann & A Piato, 2018, unpublished data). We do not attempt to extrapolate the drug concentrations we used in a fish study to human dosage since there is not a straightforward calculation to be done. Since the half-life and other pharmacokinetic parameters of ALC in zebrafish are not known, it is difficult to precisely compare the concentration range that we observed here with the dose range for humans.

Novel tank test (NTT)

The novel tank test followed the protocol already described in Mocelin et al. (2015). Briefly, the animals were separately moved to the apparatus (2.7-L tank, 24 × 8 × 20 cm, virtually divided into three equal horizontal zones and filled with standard tank water up to 15 cm) and video recorded for 6 min to be later analyzed by the ANY-maze™ software (Stoelting Co., Wood Dale, IL, USA). To evaluate exploratory behavior and locomotion we measured the parameters: total distance moved (m), number of transitions between zones, time spent in the upper and bottom zones of the tank, and number of transitions to the upper zone. Total distance and crossings were used as an indicator of overall locomotor activity. The upper zone of the tank corresponds in rats and mice protocols to the periphery region of the open-field test. Alterations in time spent and number of crossings to this zone are frequently used as a parameter of anxiety in zebrafish (Mocelin et al., 2015; Giacomini et al., 2016; Marcon et al., 2016; Mocelin et al., 2017; Marcon et al., 2018).

Light/dark test (LDT)

The light/dark test followed the protocol already reported by Gebauer et al. (2011). Specifically, the apparatus consisted of a glass tank (18 × 9 × 7 cm) divided by a raised glass into a dark and a white compartment of equal sizes, with the water level set at 3 cm and the partition raised 1 cm above the tank floor. One fish at a time was positioned in the white zone of the apparatus immediately after treatment. We recorded the number of crossings and the time spent in the white compartment for 5 min. Zebrafish have a natural preference for dark environments and the white compartment is very anxiogenic for this species; anxiolytics increase the time spent in the white compartment (Maximino et al., 2010; Mocelin et al., 2015).

Acute chasing stress test (ACS)

The acute stress protocol was performed according to the previous study published by Mocelin et al. (2015). Briefly, the animals were treated for 12 min and then chased for the last 2 min with a net before being moved to the novel tank, where they were recorded for 6 min. The behavioral parameters were quantified as described above for the NTT.

Tissue preparation

Samples were collected and prepared as previously reported by Mocelin et al. (2018). Specifically, after the ACS fish were anesthetized by immersion in cold water and euthanized by decapitation. Each independent sample was then obtained by pooling four brains, which were homogenized on ice in 600 µL phosphate buffered saline (PBS, pH 7.4; Sigma-Aldrich, St. Louis, MO, USA). The homogenate was centrifuged at 10,000 g for 10 min at 4 °C in a cooling centrifuge, and the supernatant was packed in microtubes for further assays.

Protein determination

Protein was determined by the Coomassie blue method described in detail by Bradford (1976). Specifically, we used bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) as standard and the absorbance of samples was measured at 595 nm.

Lipid peroxidation (TBARS)

Lipid peroxidation was measured by the quantification of thiobarbituric acid reactive species (TBARS) production according to the method reported by (Draper & Hadley, 1990). More specifically, we followed the protocol described by Mocelin et al. (2018), in which 50 µL of the sample (80–100 µg protein) was mixed with 75 µL of trichloroacetic acid (TCA 10%; Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 6,000 rpm for 5 min at 4 °C in a cooling centrifuge. In the supernatants were added to 75 µL thiobarbituric acid (TBA 0.67%; Sigma-Aldrich, St. Louis, MO, USA), then homogenized in a vortex for 5s and heated at 100 °C for 30 min. TBARS levels were measured by absorbance (532 nm) in a microplate reader, using malondialdehyde (MDA; Sigma-Aldrich, St. Louis, MO, USA) as a standard, and results were expressed as nmol MDA/mg protein.

Reduced thiol (SH) and Non-protein thiols levels (NPSH)

SH and NPSH levels were determined and measured at 412 nm in a microplate reader according to the method described by Ellman (1959). More specifically, we followed the steps described by Mocelin et al. (2018). Briefly, for SH the samples (60–80 µg protein) were added to 10 mM 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) dissolved in ethanol, developing yellow color after 1 h. The NPSH were similarly assessed, except that the sample was mixed with equal volumes of the 10% trichloroacetic acid (TCA) and centrifuged (6,000 rpm, 5 min). The supernatant was used for the biochemical assay. Results were expressed as µmol SH/mg protein.

Superoxide dismutase (SOD) and catalase (CAT) activities

SOD and CAT activities were determined according to the method reported by Misra & Fridovich (1972) and Aebi (1984), respectively. The protocol followed the more specific details described by Dal Santo et al. (2014). Specifically, SOD activity was quantified in a microplate reader (480 nm) by testing the inhibition of radical superoxide reaction of the sample (20–30 µg protein) in the presence of adrenalin, monitoring adrenochrome formation in a medium containing a glycine-NaOH buffer (pH 10) and adrenaline (1 mM). CAT activity was assessed by measuring the decrease in H₂O₂ absorbance in a microplate reader (240 nm). The assay mixture consisted of sample (20–30 µg protein), phosphate buffered saline (pH 7.4), and 5 µL H₂O₂(0.3 M). Results were expressed as units/mg protein.

Statistics

Normality and homogeneity of variance of the data were checked by D’Agostino-Pearson and Levene tests, respectively. Results were analyzed by one- or two-way ANOVA followed by Tukey’s post hoc test. Two-way ANOVA was used to identify the main effects of stress and treatment, as well as their interactions. Data are expressed as a mean + standard error of the mean (S.E.M.). The level of significance was set at p < 0.05.

Behavioral parameters

Figure 2 shows the effects of ALC (0.1, 1.0 and 10.0 mg/L) on the novel tank test in zebrafish. ALC significantly increased the time spent in the top (0.1 and 1.0 mg/L, Fig. 2D) and decreased the time spent the bottom (0.1 mg/L, Fig. 2E) zone of the tank (F_3,77 = 8.0, p = 0.0001 and F_3,77 = 5.6, p = 0.0016, respectively). Locomotor parameters of groups treated with ALC (0.1, 1.0, and 10.0 mg/L) did not differ from control (Figs. 2A and 2B).

In the light/dark test, ALC (0.1 and 10.0 mg/L) significantly increased the time spent in the lit side of the tank when compared to control (F_3,92 = 3.6, p = 0.0161, Fig. 3B). The number of crossings between the light and dark compartments was not altered by any of the concentrations (F_3,92 = 0.9, p = 0.4284, Fig. 3A).

Figure 4 shows the effects of ALC in the acute chasing stress (ACS) in zebrafish and Table 1 summarizes the two-way ANOVA analysis. As expected, ACS decreased the distance total traveled, crossings, entries and time in the top area (Figs. 4A–4D, respectively) and increased the time in the bottom area (Fig. 4E). ALC (0.1, 1.0 and 10.0 mg/L) prevented the effects of ACS on the time in the top and bottom areas in the novel tank test (Figs. 4D and 4E ). Also, ALC (0.1 mg/L) prevented the effects of ACS on the total distance traveled.

Biochemical parameters

Figure 5 shows the effects of ALC (0.1, 1.0 and 10.0 mg/L) on oxidative status. ACS significantly increased lipid peroxidation (TBARS), non-protein sulfhydryl (NPSH) and superoxide dismutase (SOD) activity (Figs. 5A, 5C and 5D, respectively), but did not alter sulfhydryl (SH) content and catalase (CAT) activity (Figs. 5B and 5E, respectively). Treatment with ALC (0.1, 1.0 and 10.0 mg/L) prevented oxidative damage as measured by TBARS. ALC also prevented the increase of antioxidant defenses as measured by NPSH (0.1 mg/L) and SOD (0.1, 1.0 and 10.0 mg/L). Two-way ANOVA analyses were summarized in Table 2.