FIGURE 5

FIGURE 5

Comparison of the effects of the three Kampo-hozai on anxiety-like behavior using low water level stress test in wild zebrafish. (A–H) Wild zebrafish were fed control, NYT, HET, or JTT twice daily for 4 days (3% concentration of NYT, HET, or JTT in the diet). Controls were fish fed feed without Kampo-hozai. Anxiety-like behaviors, such as freezing and swimming in the wall area, were estimated after low water level stress. (A) Experimental scheme for behavioral evaluation. (B) Test methods and equipment (C) Tracking of control, NYT, HET), and JTT swimming behavior at 0–10 min (3% concentration of NYT, HET, and JTT in diet). (D) Total freezing time in the control and three Kampo-hozai-fed wild zebrafish. n = 8. (E) Total distance traveled by the control and three Kampo-hozai-fed wild zebrafish at 0–5 min. n = 9. (F) Tracking of control, NYT, HET, and JTT swimming behavior at 15–18 min. (G) Total distance traveled by the control and three Kampo-hozai-fed wild zebrafish. (H) Time spent in the wall area by the control and three Kampo-hozai-fed wild zebrafish. n = 6. Results are shown as mean ± standard deviation. n.s., not significant. (a,b) Groups with different letters in the same row are significantly different by one-way ANOVA-test with Tukey’s multiple comparison test [p < 0.05 in (H), and p < 0.01 in (D,E)].