Microbiota regulate social behaviour via stress response neurons in the brain

a–c, Social activity in SPF and GF test mice (subject), in the context of SPF (a) or GF (b) novel mice. a, Anogenital sniff  ****P < 0.0001, nose–nose sniff  ****P < 0.0001, active approach ***P = 0.0003, push-crawl **P = 0.0055; b, anogenital sniff  ****P < 0.0001, nose–nose sniff  **P = 0.003, active approach  **P = 0.0023; c, novel mouse effect P = 0.1133 (data from Fig. 1b, c). n = 20 SPF, 19 GF (vs SPF); 8 SPF, 8 GF (vs GF) mice. d, e, Social activity in vehicle (VEH)- and ABX-treated test mice (subject), in the context of SPF (d) or ABX-treated (e) novel mice. d, Anogenital sniff  ***P = 0.0005, nose–nose sniff **P = 0.0028, active approach ***P = 0.0004, push-crawl P = 0.4601. n = 26 mice per group. e, P = 0.7039, n = 10 VEH, 9 ABX mice. f, Non-social activity in the reciprocal social interaction (RSI) paradigm in SPF, GF and ABX-treated mice (subject), in the context of SPF or GF novel mice. GF vs SPF novel mouse: P = 0.8086, n = 20 SPF, 19 GF mice. GF vs GF novel mouse: P = 0.1205, n = 8 mice per group. ABX vs SPF novel mouse: P = 0.5044, n = 26 mice per group. g, Non-social behaviour in a novel cage without the presence of a novel mouse. Grooming P = 0.482, digging P = 0.8689, rearing P = 0.4608 or total P = 0.1743. n = 8 SPF, 10 GF mice. h, Timeline schematic of guide cannula stereotaxic surgery, intracerebroventricular (ICV) injection of vehicle, ampicillin (A) and metronidazole (M), social behaviour, and sample collection. Social activity was tested in SPF mice injected ICV with antibiotics (subject), in the context of SPF novel mice. Social activity P = 0.5294, nose-to-nose P = 0.1784, nose-to-tail P = 0.4477. n = 12 mice per group. i, j, Timeline schematic of intraperitoneal (i.p.) injection of vehicle, ampicillin and metronidazole, open-field (OF) test, social behaviour, and sample collection. RSI (i) and open-field (j) test were tested in antibiotic-injected SPF mice (subject), in the context of SPF novel mice. Social activity P = 0.5583 (i); locomotion P = 0.3705 (j). n = 6 VEH, 4 ampicillin, 6 metronidazole mice. k, Social activity was tested in SPF, GF, VEH-treated and ABX-treated female mice (subject), in the context of SPF female novel mice. GF **P = 0.0053 and ABX *P = 0.0191. n = 10 SPF, 9 GF, 5 VEH, 5 ABX mice. l, The length of isolation shown in Fig. 1b did not affect social activity in GF or SPF controls. Microbiota effect ****P < 0.0001. n = 4 SPF (4–5 h), 7 SPF (5–6 h), 5 SPF (6–7 h), 2 SPF (7–8 h), 5 SPF (8–9 h), 10 GF (4–5 h), 3 GF (5–6 h), 4 GF (6–7 h), 6 GF (7–8 h), 4 GF (8–9 h) mice (data from Fig. 1b, c). m, Age of GF mice had no effect on social activity. P = 0.379. n = 6 (11–12 w), 5 (13 w), 8 (14–15 w) GF mice (data from Fig. 1b,d). n–q, The distance travelled and social activity in the three-chamber social test for SPF and GF male mice (n, o) and VEH- and ABX-treated mice (p, q), in the context of SPF novel mice. n = 9 SPF, 10 GF, 10 VEH, 10 ABX mice. Distance moved in the sociability phase (n, GF **P = 0.0047; p, ABX P = 0.1246), time in chamber (o, left, SPF mouse (M) vs object (O) ****P < 0.0001, GF M vs O ***P = 0.0005; q, left, VEH M vs O P = 0.1220, ABX M vs O **P = 0.0063), and frequency entering chamber (o, right, SPF M vs O P = 0.1420, GF M vs O P = 0.0872; q, right, VEH M vs O P = 0.2194, ABX M vs O ***P = 0.0008). n = 9 SPF, 10 GF, 10 VEH, 10 ABX mice. r, Left, schematic of brain regions with high c-Fos expression after RSI (relevant to Fig. 1e–l). Right, representative images (from 6 SPF, 6 GF, 5 VEH, 5 ABX mice) of c-Fos staining in BLA in SPF, GF, VEH, and ABX after RSI. Scale bars, 200 μm. s, Quantification of c-Fos⁺ cells in BLA of GF and ABX mice. SPF vs GF P = 0.1014, VEH vs ABX P = 0.1095. n = 6 SPF, 6 GF, 5 VEH, 5 ABX mice. t, Quantification of c-Fos⁺ cells in various brain regions of SPF and GF mice. PVN P = 0.4239, adBNST P = 0.2571, DG P = 0.0818, BLA P = 0.2552. n = 5 SPF, 6 GF mice. u, Serum corticosterone levels in ABX-treated mice at isolation (iso), 0 h and 1 h post-RSI. VEH: Iso vs 0 h ^#P = 0.0129, Iso vs 1 h ^##P = 0.0027, 0 h vs 1hr ^##P = 0.0023; ABX: Iso vs 0 h P = 0.0624, Iso vs 1 h ^##P = 0.0016, 0 h vs 1 h ^##P = 0.0062. n = 5 mice per group. v, Serum corticosterone levels after novel cage exposure in GF and ABX-treated mice. ABX vs GF **P = 0.0085, SPF vs GF *P = 0.0125. n = 11 VEH, 11 ABX, 5 SPF, 4 GF mice. w, x, Serum corticosterone levels at different times of death in SPF and GF (w) and VEH and ABX mice (x) tested in Fig. 1m–o. GF ***P = 0.0005 (w), ABX **P = 0.0011 (x). w, n = 2 SPF (0–1 h), 5 SPF (1–2 h), 4 SPF (2–3 h), 4 SPF (3–4 h), 3 SPF (4–5 h), 9 GF (0–1 h), 3 GF (1–2 h), 4 GF (2–3 h), 6 GF (3–4 h), 4 GF (4–5 h) mice (data from Fig. 1m, n). x, n = 9 VEH (0–100 m), 17 VEH (100–200 m), 22 ABX (0–100 m), 4 ABX (100–200 m) mice (data from Fig. 1o). y, Social activity in SPF, GF and exGF test mice (subject), in the context of SPF novel mice. SPF vs GF  ****P < 0.0001, SPF vs exGF  ***P = 0.0001, GF vs exGF  ****P < 0.0001. n = 20 SPF, 19 GF, 8 exGF mice (SPF and GF data from Fig. 1b, c). z, Serum corticosterone levels after RSI in exGF mice. SPF vs GF **P = 0.0012, GF vs exGF *P = 0.0467. n = 13 SPF, 18 GF, 8 exGF mice (SPF and GF data from Fig. 1b, c). Data shown as individual points with mean ± s.e.m. Data analysed by two-tailed unpaired t-test (a, b, d–g, k, n, p, s, t); one-tailed paired t-test (o, q); one-way ANOVA (i, j, m, y, z), repeated measures (h) with Bonferroni’s multiple comparison post hoc test; two-way ANOVA (c, l, v–x), repeated measures (u) 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.

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