Distance travelledEthics statementThis research was conducted in accordance with the Ethical Principles in Animal Research adopted by the National Council for the Control of Animal Experimentation – Brazil (CONCEA?Conselho Nacional de Controle de Experimentacao Animal?Stress-Induced Antinociception in FishFigure 1. Locomotor activity of L. macrocephalus subjected to Epigenetics restraint for 3 and 5 min, followed by subcutaneous injection of 3 formaldehyde. Data are presented as the difference (D) between post-stimulus and baseline. Autophagy Experimental groups: saline (SAL, n = 8), formaldehyde (FOR, n = 8), 3 min of restraint + saline (RES (3) + SAL, n = 8), 3 min of restraint + formaldehyde (RES (3) + FOR, n = 8), 5 min of restraint + saline (RES (5) + SAL, n = 8) and 5 min of restraint + formaldehyde (RES (5) + FOR, n = 8). Different letters indicate significant difference (Tukey test, P,0.05). doi:10.1371/journal.pone.0071175.gFigure 2. Time course of the effect of 3 min of restraint on the locomotor activity of L. macrocephalus subjected to subcutaneous injection of 3 formaldehyde. Data are presented as the difference (D) between post-stimulus and baseline. Experimental groups: subcutaneous injection of formaldehyde without restraint (FOR) and applied immediately (0 min, n = 8), 5 (n = 7), 10 (n = 7) or 15 (n = 7) min after the restraint. Different letters indicate significant difference (Tukey test, P,0.05). doi:10.1371/journal.pone.0071175.gand swimming speed were significantly lower than those presented by non-immobilized animals when formaldehyde was applied immediately after the restraint (0 min) and 5 min after the restraint (Tukey, P,0.001). The formaldehyde injections 10 and 15 min after the restraint promoted increases in locomotor activity, and the distance and swimming speed were similar to those presented by non-immobilized animals (Figure 3).Experiment 3: Influence of naloxone pre-treatment on the inhibition of the nociceptive response induced by 3 min of restraintA significant effect of the naloxone intraperitoneal injection (30 mg.kg21) on the inhibition of the nociceptive response induced by 3 min of restraint was observed (ANOVA, F3,28 = 14.65, P,0.001). The naloxone injection 30 min before 3 min of restraint blocked the restraint-induced inhibition of the locomotor response to formaldehyde. In the NAL + RES (3) + FOR group (naloxone-treated animals before the restraint followed by formaldehyde subcutaneous injection), the distance and speed values were significantly higher than those presented by SAL + RES (3) + SAL, SAL + RES (3) + FOR and NAL + RES (3) + SAL (Tukey, P,0.001) (Figure 4 A).Figure 3. Time course of the effect of 5 min of restraint on the locomotor activity of L. macrocephalus subjected to subcutaneous injection of 3 formaldehyde. Data are presented as the difference (D) between post-stimulus and baseline. Experimental groups: subcutaneous injection of formaldehyde without restraint (FOR) and applied immediately (0 min, n = 8), 5 (n = 8), 10 (n = 8) or 15 (n = 7) min after the restraint. Different letters indicate significant difference (Tukey test, P,0.05). doi:10.1371/journal.pone.0071175.gExperiment 4: Influence of naloxone pre-treatment on inhibition of the nociceptive response induced by 5 min of restraintNo effect of the naloxone intraperitoneal injection (30 mg.kg21) on the inhibition of the nociceptive response induced by 5 min of restraint was observed (ANOVA, F3,28 = 0.169, P = 0.916). In the NAL + RES (5.Distance travelledEthics statementThis research was conducted in accordance with the Ethical Principles in Animal Research adopted by the National Council for the Control of Animal Experimentation – Brazil (CONCEA?Conselho Nacional de Controle de Experimentacao Animal?Stress-Induced Antinociception in FishFigure 1. Locomotor activity of L. macrocephalus subjected to restraint for 3 and 5 min, followed by subcutaneous injection of 3 formaldehyde. Data are presented as the difference (D) between post-stimulus and baseline. Experimental groups: saline (SAL, n = 8), formaldehyde (FOR, n = 8), 3 min of restraint + saline (RES (3) + SAL, n = 8), 3 min of restraint + formaldehyde (RES (3) + FOR, n = 8), 5 min of restraint + saline (RES (5) + SAL, n = 8) and 5 min of restraint + formaldehyde (RES (5) + FOR, n = 8). Different letters indicate significant difference (Tukey test, P,0.05). doi:10.1371/journal.pone.0071175.gFigure 2. Time course of the effect of 3 min of restraint on the locomotor activity of L. macrocephalus subjected to subcutaneous injection of 3 formaldehyde. Data are presented as the difference (D) between post-stimulus and baseline. Experimental groups: subcutaneous injection of formaldehyde without restraint (FOR) and applied immediately (0 min, n = 8), 5 (n = 7), 10 (n = 7) or 15 (n = 7) min after the restraint. Different letters indicate significant difference (Tukey test, P,0.05). doi:10.1371/journal.pone.0071175.gand swimming speed were significantly lower than those presented by non-immobilized animals when formaldehyde was applied immediately after the restraint (0 min) and 5 min after the restraint (Tukey, P,0.001). The formaldehyde injections 10 and 15 min after the restraint promoted increases in locomotor activity, and the distance and swimming speed were similar to those presented by non-immobilized animals (Figure 3).Experiment 3: Influence of naloxone pre-treatment on the inhibition of the nociceptive response induced by 3 min of restraintA significant effect of the naloxone intraperitoneal injection (30 mg.kg21) on the inhibition of the nociceptive response induced by 3 min of restraint was observed (ANOVA, F3,28 = 14.65, P,0.001). The naloxone injection 30 min before 3 min of restraint blocked the restraint-induced inhibition of the locomotor response to formaldehyde. In the NAL + RES (3) + FOR group (naloxone-treated animals before the restraint followed by formaldehyde subcutaneous injection), the distance and speed values were significantly higher than those presented by SAL + RES (3) + SAL, SAL + RES (3) + FOR and NAL + RES (3) + SAL (Tukey, P,0.001) (Figure 4 A).Figure 3. Time course of the effect of 5 min of restraint on the locomotor activity of L. macrocephalus subjected to subcutaneous injection of 3 formaldehyde. Data are presented as the difference (D) between post-stimulus and baseline. Experimental groups: subcutaneous injection of formaldehyde without restraint (FOR) and applied immediately (0 min, n = 8), 5 (n = 8), 10 (n = 8) or 15 (n = 7) min after the restraint. Different letters indicate significant difference (Tukey test, P,0.05). doi:10.1371/journal.pone.0071175.gExperiment 4: Influence of naloxone pre-treatment on inhibition of the nociceptive response induced by 5 min of restraintNo effect of the naloxone intraperitoneal injection (30 mg.kg21) on the inhibition of the nociceptive response induced by 5 min of restraint was observed (ANOVA, F3,28 = 0.169, P = 0.916). In the NAL + RES (5.
