Constant light exposure causes stress and constipation in zebrafish larvae. (A) Schematic illustration showing experimental design. (B) Comparison of cortisol concentration between the control and light-stress groups (n = 13). (C,D) Lateral images of the fluorescent tracer in the intestinal tracts of zebrafish larvae. Scale bar: 100 μM. (E) Comparison of the number (#) of intestinal contractions in zebrafish larvae (n = 8). (F) Fluorescence spectrophotometer of fecal accumulation over time (n = 3). Each replicate had 45 zebrafish embryos. * p < 0.05; **** p < 0.0001.

Probiotic intakes reduced stress response and constipation in zebrafish larvae. (A) Schematic illustration showing experimental design. Larvae were fed Bifidobacterium longum subsp. longum probiotic-based food on 6 and 8 dpf during constant light exposure. (B) Comparison of cortisol concentration between light-stress group and probiotic-feeding light-stress group (n = 12). (C) Relative fold change of crhb mRNA expression (n = 3). Each replicate had 20 zebrafish embryos. (D–F) Lateral images of the fluorescent tracer in the intestinal tracts of zebrafish larvae. Scale bar: 100 μM. (F) Comparison of the number (#) of intestinal contractions in zebrafish larvae. n = 12 for light-stress group, n = 17 for light-stress/B. longum group. * p < 0.05; *** p < 0.001.

Probiotics reduced inflammatory cell recruitment by constant light exposure. (A,C,E) In vivo intestinal images of 10 dpf Tg (mpeg1:gal4vp16;uas:egfp) show macrophage infiltration. Scale bar: 100 μM. (B,D,F) In vivo intestinal images of 10 dpf Tg (mpx:mcherry) show neutrophil infiltration. Arrows indicate neutrophils in the intestine. Scale bar: 100 μM. (A,B) Control group. (C,D) Stress-induced group by constant light exposure. (E,F) B. longum-feeding stress-induced group. (G) Comparison of the number (#) of intestine-infiltrated macrophages among all groups: n = 16 for control group; n = 21 for light-stress group; n = 13 for light-stress/B. longum group. (H) Comparison of the number (#) of intestine-infiltrated neutrophils among all groups: n = 18 for control group; n = 21 for light-stress group; n = 21 for light-stress/B. longum group. (I) Relative fold changes of inflammatory genes, including tnfα, nfkb, il1b, and il6 (n = 4). Each replicate had 20 zebrafish embryos. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Inflammation induced by stress affects intestinal folds. (A–C) Cross-sectional images of 10 dpf larvae labeled with phalloidin (red) and DAPI (blue). Scale bar: 100 μM. (D) Comparison of the number (#) of intestinal folds of mid-intestines among all groups: n = 13 for control group; n = 10 for light-stress group; n = 8 for light-stress/B. longum group. ns, not significant; *** p < 0.001.

Inflammation-induced activity of enteric neurons. (A–C) Intestinal images of 10 dpf larvae labeled with anti-Hu (green) and anti-5-HT (red) antibodies. Arrows indicate 5-HT+/Hu+ neurons in the intestine. Scale bar: 50 μM. (D–F) Intestinal images of 10 dpf larvae labeled with anti-Hu (green) and anti-pERK (red) antibodies. Arrows indicate pERK+/Hu+ neurons in the intestine. Scale bar: 50 μM. (G) Comparison of the number (#) of Hu+ enteric neurons among all groups: n = 25 for control group; n = 30 for light-stress group; n = 38 for light-stress/B. longum group. (H) Comparison of the number (#) of 5-HT+/Hu+ serotonin enteric neurons among all groups: n = 12 for control group; n = 17 for light-stress group; n = 15 for light-stress/B. longum group. (I) Comparison of the number (#) of pERK+/Hu+ enteric neurons among all groups: n = 13 for control group; n = 20 for light-stress group; n = 21 for light-stress/B. longum group. ns, not significant; * p < 0.05; ** p < 0.01.

Probiotics relieve stress-induced constipation by affecting intestinal water metabolism. Relative fold changes in aqp3a (A), aqp4 (B), aqp8a (C), and vip (D) mRNA expressions (n = 3). Each replicate had 20 zebrafish embryos. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.