Fig. 1. Effects of 4 h of ammonia exposure in the open-field test in zebrafish. (A) Schematic illustration of the arena, which was divided into inner and outer regions for data analysis. (B) Trajectories of fish moving under exposure to 0, 90 and 180 mg/L (NH₄⁺) (C) The time spent in the inner zone of the arena increased in fish exposed to 180 mg/L (NH₄⁺). (D) The total moving distance in fish did not differ among the 0, 90 and 180 mg/L groups. Locomotor activity was divided into three states according to average individual velocity per trial as followed: (E) The percentage of cumulative duration of moderate mobility (MM), 20–80 %; (F) The percentage of cumulative duration of high mobility (HM), > 80 %; (G) The percentage of cumulative duration of low mobility (LM), < 20 %. (H) Effects of 4 h of ammonia exposure in the novel tank test in zebrafish. The time spent in upper zone (sec) was similar in all zebrafish. Data are shown as the mean ± SD. * Indicates a significant difference from the control (0 mg/L) by a one-way ANOVA with Tukey’s pair-wise comparison, p < 0.05. NS, not significant.

Fig. 2. Mirror-biting test and shoaling behavior of zebrafish after treatment with ammonia for 4 h. (A) Schematic illustration of the mirror-biting test. (B) Adult zebrafish were treated with 0, 90, and 180 mg/L (NH₄⁺), and the number of times a fish bit the mirror was counted. Fish treated with 180 mg/L (NH₄⁺) showed a lower mirror-biting number. (C) Schematic illustration of the shoaling behavior test. (D) Adult zebrafish were treated with 0, 90, and 180 mg/L (NH₄⁺), and the average inter-fish distance during shoaling was analyzed. Data are shown as the mean ± SD. * Indicates a significant difference from the control (0 mg/L) by Student’s t-test, p < 0.05. NS, not significant.

Fig. 3. Effect of ammonia exposure for 4 h on social preference and social recognition of adult zebrafish. (A) Schematic illustration of the social-preference test. (B) Trajectories of fish moving in the test tank. (C) All of the 0, 90, and 180 mg/L (NH₄⁺) groups spent more time in the conspecific than the empty zone of the tank. (D) Schematic illustration of the social-recognition test. (E) Trajectories of fish moving in the test tank. (F) Fish treated with 180 mg/L (NH₄⁺) did not show a preference for staying in the familiar or unfamiliar zones of the tank. Data are shown as the mean ± SD. * Indicates a significant difference from the control (0 mg/L) by a one-way ANOVA with Tukey’s pair-wise comparison, p < 0.05. NS, not significant.

Fig. 4. Cued-fear conditioning and learning and memory behaviors of zebrafish after treatment with ammonia for 4 h. (A) Schematic illustration of the cued-fear conditioning test. The number of times turning (B) and the freezing ratio during a retrieval session (C) of the 0, 90, and 180 mg/L (NH₄⁺) groups. The number of times turning increased after 90, and 180 mg/L (NH₄⁺) exposure. (D) Schematic illustration of the system of the active-avoidance test. (E) Ratio of fish which satisfied the success criteria (learner; 90 % avoidance in 10 sequential trials by the third training session in the active-avoidance task). There were no differences among the groups. (F) Ratio of fish that achieved the criterion in the short-term memory (STM) test. There were no differences among the 0, 90, and 180 mg/L (NH₄⁺) groups. (B and C) Data are shown as the mean ± SD. * Indicate a significant difference from 0 mg/L, according to a one-way ANOVA with Tukey’s pair-wise comparison, p < 0.05. (B and C). NS, not significant according to a Chi-squared test (E and F).

Fig. 5. Effects of ammonia exposure for 4 h on mRNA expressions in the brain of zebrafish. Schematic illustration of the glutamate-glutamine cycle. mRNA expressions of glutamate dehydrogenase 1a (glud1a) and glud1b (A); glutaminase 1a (gls1a) and gls1b (B); and glutamine synthase 1a (glula) and glulb (C); in the 0, 90, and 180 mg/L (NH₄⁺) groups. mRNA expression of glud1b increased in the brain of the 180 mg/L (NH₄⁺) group while gls1b increased the 90, and 180 mg/L (NH₄⁺) groups. mRNA levels of c-fos (D) in the forebrain, midbrain and hindbrain of zebrafish. c-fos expression was stimulated in the hindbrain of the 180 mg/L (NH₄⁺) groups, while it was not affected in the forebrain. Data are shown as the mean ± SD. * Indicates a significant differences from 0 mg/L, according to a one-way ANOVA with Tukey’s pair-wise comparison, p < 0.05.