Comparison of the effects of the three Kampo-hozai on anxiety-like behavior using novel tank test in NPY-KO zebrafish. (A–I) The NPY-KO 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 upper area, were assessed after the novel environmental stress. (A) Experimental scheme for behavioral evaluation. (B) Test apparatus. (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 by control and the three Kampo-hozai-fed NPY-KO zebrafish. (E) Total distance traveled by the control and three Kampo-hozai-fed NPY-KO zebrafish at 0–3 min n = 8. (F) Tracking of control (left), NYT (middle), HET (middle), and JTT (right) swimming behavior at 10–13 min. (G) Total distance traveled by the control and three Kampo-hozai-fed NPY-KO zebrafish. (H) Time spent in the upper area by the control and three Kampo-hozai-fed NPY-KO zebrafish. (I) Frequency of invasion into the upper area in the control and three Kampo-hozai-fed NPY-KO zebrafish. n = 7. 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).
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