Extended Data Fig. 3: Anxiety-like behaviour, locomotion, water intake, and olfactory investigative behaviour in GF and/or ABX-treated mice.
From: Microbiota regulate social behaviour via stress response neurons in the brain
a–h, Anxiety-like behaviour and locomotion were tested using the open-field test, light–dark box and elevated plus maze in SPF, GF, VEH-treated and ABX-treated mice. a, Locomotor activity measured in open-field test in GF mice showed no difference in distance travelled in open-field chamber. P = 0.8371. n = 9 SPF, 8 GF mice per group. b, Centre zone duration in open-field test in GF mice. GF mice spent longer in the centre of the open-field chamber than SPF mice. *P = 0.0108. n = 9 SPF, 8 GF mice per group. c, Light chamber duration measured in light–dark box for GF mice. GF mice spent longer in the light chamber than SPF mice. *P = 0.0442. n = 9 mice per group. d, Open arm duration measured in elevated plus maze for GF mice. GF mice spent longer in the open arm than SPF mice. *P = 0.0261. n = 9 mice per group. e, Locomotor activity measured in open-field test for ABX mice showed no difference in distance travelled in the open-field chamber. P = 0.3606. n = 10 mice per group. f, Duration in the centre zone measured in open-field test shows no difference in centre duration for ABX mice. P = 0.3372. n = 10 mice per group. g, Duration in the light chamber measured in light–dark box shows no difference in light chamber duration for ABX mice. P = 0.9381. n = 10 mice per group. h, Duration spent in open arm measured in elevated plus maze shows no difference in open arm duration for ABX mice. P = 0.8743. n = 10 mice per group. i, ABX mice show no change in the distance travelled in the open-field chamber. VEH vs ABX P = 0.6223. n = 15 mice per group. j, Water intake was monitored in GF mice housed in IVC cages for one week. GF mice showed no difference in water consumed. SPF vs GF P = 0.7591. n = 5 mice per group. k, The olfactory habituation/dishabituation test involves exposure to a series of odours: baseline (water), volatile non-social odours (almond and banana extracts), and social odours (C57BL/6 and BTBR cages). Time-course of sniffing time shows no difference for GF mice when exposed to water, almond extract, or banana extract. GF mice displayed increased sniffing time when exposed to the social odour from C57BL/6J mice, but not from BTBR mice. SPF vs GF: B6 Cage(1-1) *P = 0.0172, B6 Cage(1-2) *P = 0.0402. n = 10 SPF, 11 GF mice per group. l, Percentage investigation time in response to the first exposure to social odour was measured in the olfactory habituation/dishabituation test in SPF and GF mice. The percentage of investigation time decreased at the second and third exposures to the same social odour in both SPF and GF mice. The investigation time decreased when switched to BTBR odour in GF mice. SPF: B6 Cage 1-1 vs 1-2 **P = 0.0020, B6 Cage 1-1 vs 1-3 ***P = 0.0002, BTBR Cage 1-1 vs 1-2 ***P = 0.0002, BTBR Cage 1-1 vs 1-3 ****P < 0.0001; GF: B6 Cage 1-1 vs 1-2 *P = 0.0120, B6 Cage 1-1 vs 1-3 *P = 0.0397, BTBR Cage 1-1 vs 1-2 ****P < 0.0001, BTBR Cage 1-1 vs 1-3 ****P < 0.0001, B6 Cage 1-1 vs BTBR Cage 1-1 *P = 0.0143. n = 10 SPF, 11 GF mice per group. Data shown as individual points with mean ± s.e.m. Data analysed by two-tailed unpaired t-test (a–h) and two-way ANOVA, repeated measures (i–l) with Bonferroni’s multiple comparison post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ND: no difference. For more statistical details, see Supplementary Information.
