Extended Data Fig. 1: AB-2004 administration lowers 4EPS levels in gnotobiotic mice and ameliorates anxiety-like behavior.
a, Bacterial strain pairs were engineered to produce 4-ethylphenol (4EP+) or not (4EP-). See Methods and [see accompanying manuscript, accepted for publication] for details. 4EP is converted to 4EPS by the mouse. b, Timeline schematic for colonization of germ-free mice, AB-2004 administration, and metabolite and behavioral analysis. c, Separate groups of mice were each colonized with either the 4EP+ or 4EP- strain pair. 4EPS levels quantified in urine of mice two weeks after dietary administration of AB-2004 or control diets. Limit of detection is 10 ng/ml (n number left to right: n = 18, 20, 12,18). All adj. p-values, <0.0001. d, Weight gain of mice on diet containing 5% AB-2004 compared to controls (n = 10 each). e, Colonization of mice with 4EP producing strain pairs of Lactobacillus plantarum and Bacteroides ovatus. Both bacterial strains equally colonize 4EP+ and 4EP- groups of mice, independent of diet (n number left to right: n = 4, 4, 3, 4). f-j, Behavioral test results from mice administered AB-2004 or control diet. f, Visual representations of the behavioral assays open field, elevated plus maze, marble burying and grooming. g, Open field (OF) test results presented as, from left to right, a ratio of time spent in the center of the arena over time spent in thigmotaxis along the perimeter during the 10-minute testing period, total distance mice traveled, time in center, and time in thigmotaxis along the perimeter of the arena (n number left to right: n = 31, 33, 26, 26). Exact adj. p-values: center/thigmotaxis, 0.0003; center, <0.0001; thigmotaxis, 0.03. h, Elevated plus maze (EPM) results presented, from left to right, as a ratio of time spent in the open arms of the maze over time spent in the closed arms during the 5-minute testing period, time mice spent in the open arms, time spent in the closed arms, and total time at the terminus (outermost third of open arms)(n number left to right: n = 27, 29, 26, 28). Exact adj. p-values: open/closed, both 0.014; open, 0.03; closed, 0.045, 0.01; terminus, 0.01. i, Number of marbles buried during the 10-minute testing period (n number left to right: n = 31, 33, 25, 26). Exact adj. p-values: 0.0006; 0.04. j, Amount of time spent grooming during the 10-minute testing period (n number left to right: n = 26, 32, 22, 25). Abbreviations: 4EP, 4-ethylphenol; 4EPS, 4-ethylphenyl sulfate. Data represent mean ± SEM analyzed by ordinary two-way ANOVA test with FDR correction, with individual variances computed for each comparison (* p adj. ≤ 0.05; ** p adj. ≤ 0.01; *** p adj. ≤ 0.001; **** p adj. ≤ 0.0001).
