Extended Data Fig. 8: Manipulations of CRH neurons and glucocorticoid-sensing neurons in ABX-treated and SPF mice.

a, Diagram of AAV-hSyn-DIO-hM4Di-mCherry (hM4Di) or AAV-hSyn-DIO-mCherry (mCherry) virus injection into the PVN of Crh-ires-Cre mice. b, Non-social activity in ABX-treated mCherry and hM4Di mice, with and without CNO (subject). hM4Di effect P = 0.4909. n = 10 mCherry, 11 hM4Di mice. c, d, Neuronal activity was measured by c-Fos staining of brain sections. Representative images (from 10 mCherry and 11 hM4Di mice) of c-Fos, mCherry and DAPI staining in the adBNST (c) and DG (d) in hM4Di and mCherry mice upon CNO injection after social interaction. Scale bars, 100 μm. e, f, Quantification of c-Fos⁺ cells in various brain regions of ABX hM4Di and mCherry mice. There was no change in c-Fos in the adBNST (e, P = 0.301) or DG (f, P = 0.5745) of ABX hM4Di mice upon CNO injection. n = 10 mCherry, 11 hM4Di mice per group. g, Top, timeline scheme of stereotaxic surgery, ABX treatment, drug administration, social behaviour, and sample collection. Crh-ires-Cre mice were stereotaxically injected with viruses into the BNST one week before ABX treatment at the age of 7–8 weeks. After three weeks of ABX treatment, social behaviour was tested in VEH- or CNO-injected mice. Bottom, diagram of virus injection into the BNST of Crh-ires-Cre mice to deliver AAV-hSyn-DIO-hM4Di-mCherry (hM4Di) or AAV-hSyn-DIO-mCherry (mCherry). h, Social activity was tested using the RSI paradigm in ABX test mice (subject), in the context of SPF novel mice. With VEH and CNO injection, there was no difference in social activity between mice injected with hM4Di and mice injected with mCherry. mCherry vs hM4Di P = 0.4722. n = 10 mCherry, 9 hM4Di mice per group (subject only). i, Non-social activity was recorded using the RSI paradigm in ABX mice (subject), in the context of SPF novel mice. With VEH and CNO injection, there was no difference in non-social activity between mice injected with hM4Di and mice injected with mCherry at the BNST. mCherry vs hM4Di P = 0.1921. n = 10 mCherry, 9 hM4Di mice per group (subject only). j, Serum corticosterone concentrations show no change after social interaction in ABX hM4Di mice when injected with CNO. P = 0.2063. n = 8 mice per group. k, l, Quantification of c-Fos⁺ cells in various brain regions of ABX hM4Di and mCherry mice. No change in c-Fos⁺ cells was detected in the PVN (k, P = 0.4792) or adBNST (l, P = 0.3054) after social interaction in ABX hM4Di mice with CNO injection, compared to mCherry mice. n = 10 mCherry, 8 hM4Di mice per group. m, Neuronal activity was measured by c-Fos staining of brain sections. Representative images (from 10 mCherry, 8 hM4Di mice) of c-Fos, mCherry and DAPI staining in the PVN and adBNST of hM4Di and mCherry mice with CNO injection after social interaction. Scale bars, 100 μm. a–m, All mice received antibiotics. n, Diagram of AAV-hSyn-DIO-hM3Dq-mCherry (hM3Dq) or AAV-hSyn-DIO-mCherry (mCherry) injection into the PVN. o, Relevant to Fig. 3h–j. Non-social activity was recorded using the RSI paradigm in SPF mice (subject), in the context of SPF novel mice. With VEH injection, there was no difference in non-social activity between mice injected with AAV-hSyn-DIO-hM3Dq-mCherry (hM3Dq) and mice injected with AAV-hSyn-DIO-mCherry (mCherry). Injection of CNO increased non-social activity in hM3Dq mice but not mCherry mice. SPF mCherry CNO vs SPF hM3Dq CNO  ****P < 0.0001. n = 10 mCherry, 11 hM3Dq mice per group (subject only). p, Relevant to Fig. 3k–m. Diagram of guide cannula implantation into the PVN to deliver VEH or CRF. q, Non-social activity was recorded using the RSI paradigm in SPF mice (subject), in the context of SPF novel mice. Injection of CRF (low) increased non-social activity, whereas injection of CRF (high) did not alter non-social activity compared to VEH mice. VEH vs CRF(low) *P = 0.0427, CRF(low) vs CRF(high) *P = 0.0134. n = 16 VEH, 7 CRF (low), 9 CRF (high) mice per group (subject only). r, s, Relevant to Fig. 3n–p. Diagram of guide cannula implantation into the DG (r) or BNST (s) to deliver VEH, CORT, or DEX. t, Relevant to Fig. 3k–m. Correct guide cannula implantation into the PVN was validated by histological sectioning and DAPI staining for visualization of the PVN to confirm CRF injection. The customized guide cannula set includes guide cannula, injector, dummy, and cap (left). The guide cannula was implanted 0.5 mm above the PVN. The tip of the guide cannula track can be visualized in the thalamus region in each implanted mouse. The injector was designed to reach 0.5 mm below the tip of the guide cannula. As the injector was made of 33G fine needle, the needle track of the injector was not visible in the brain slice and is depicted in the image (middle). Enlarged image to show the guide cannula track and the depicted needle track above the PVN (right). Scale bars, 500 μm. Data shown as individual points with mean ± s.e.m. Data analysed by two-way ANOVA, repeated measures (b, h, i, o) with Bonferroni’s multiple comparison post hoc test; two-tailed unpaired (e, f, k, l) or paired t-test (j); one-way ANOVA with Bonferroni’s multiple comparison post hoc test (q). *P < 0.05, ****P < 0.0001, ND: no difference. For more statistical details, see Supplementary Information.

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