FIGURE 3

FIGURE 3

Comparison of the effects of the three Kampo-hozai on anxiety-like behavior using cold stress test in NPY-KO zebrafish. (A–J) The NPY-KO zebrafish were fed control, NYT, HET, or JTT twice daily for 4 days (3% concentration of NYT, HET, and JTT in diet). Controls were fish fed feed without Kampo-hozai. Anxiety-like behaviors, such as freezing and swimming in the wall area, were estimated after the cold stress test. (A) Experiment scheme for behavioral evaluation. (B) Test method and apparatus. (C) Tracking of control, NYT, HET, and JTT swimming behavior in 0–10 min (3% concentration of NYT, HET, JTT in diet). (D) Total freezing time by control and the three Kampo-hozai-fed NPY-KO zebrafish. (E) Total distance traveled by control and the three Kampo-hozai-fed NPY-KO zebrafish in 0–3 min. Control n = 10, NYT n = 10, HET n = 5, JTT n = 5. (F) Tracking of control, NYT, HET, and JTT swimming behavior in 15–17 min. (G) Total distance traveled by control and the three Kampo-hozai-fed NPY-KO zebrafish in 15–17 min. (H) Time spent in the wall area of the control and three Kampo-hozai-fed NPY-KO zebrafish. (I) Tracking of control, NYT, HET, and JTT swimming behavior in 0–10 min (0.3% concentration of NYT or HET in diet). (J) Total freezing time by control and the three Kampo-hozai-fed NPY-KO zebrafish. Control n = 9, NYT n = 8, HET n = 9. Results are shown as mean ± standard deviation. n.s., not significant. (a,b,c) 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 (E), and p < 0.01 in (D,H,J)].