Extended Data Fig. 4: IEG expression in the brains of GF mice and stress-related gene expression in the hippocampus and hypothalamus of ABX-treated mice.
From: Microbiota regulate social behaviour via stress response neurons in the brain
a–d, IEG expression was measured in GF mice taken from the isolator and temporarily mixed with other mice from different cages. SPF mice were sampled and handled following the same procedure. Mice were immediately killed and the brains were collected and analysed. Brains were dissected into hippocampus (a), hypothalamus (b), midbrain (c), and brainstem (d). IEG expression in each region was analysed by qRT–PCR. n = 6 mice per group. a, In the hippocampus, Arc (*P = 0.024), Fos (**P = 0.0036), cJun (*P = 0.0152), JunB (**P = 0.0077), Egr1 (P = 0.0532), Egr2 (*P = 0.0328), Gadd45b (***P = 0.0005), Gadd45g (*P = 0.0435), and Bdnf (*P = 0.0142) were upregulated in GF mice. Map2 P = 0.3874. n = 6 mice per group. b, In the hypothalamus, Arc (*P = 0.0108), Fos (***P = 0.0001), and Egr1 (**P = 0.0022) were upregulated in GF mice, with no change in Map2 gene expression. cJun P = 0.3859, JunB P = 0.106, Egr2 P = 0.0864, Gadd45b P = 0.8544, Gadd45g P = 0.4398, Bdnf P = 0.1517, Map2 P = 0.8255. n = 6 mice per group. c, No changes in IEG expression were found in the midbrains of GF mice. Arc P = 0.4733, Fos P = 0.455, cJun P = 0.6153, JunB P = 0.6154, Egr1 P = 0.4102, Egr2 P = 0.283, Gadd45b P = 0.424, Gadd45g P = 0.0852, Bdnf P = 0.9779, Map2 P = 0.4018. n = 3 mice per group. d, In the brainstem, cJun (*P = 0.0479), JunB (*P = 0.0244), Egr1 (P = 0.0502), Gadd45b (**P = 0.0022), Gadd45g (**P = 0.0075), and Bdnf (*P = 0.0127) were downregulated in GF mice. No change in Map2 was detected in all four brain regions in GF mice. Arc P = 0.8297, Fos P = 0.5208, Egr2 P = 0.5391, Map2 P = 0.1266. n = 6 mice per group. e, Timeline schematic of ABX treatment, single housing, behavioural manipulations, and tissue sampling. Tissues collected include hippocampal dentate gyrus (DG; red), hippocampal Ammon’s horn, and hypothalamus. f, Gene expression analysis in Ammon’s horn and DG was performed by qRT–PCR. Mgr1b is enriched in Ammon’s horn. Dsp and Tdo2 are specific to the DG. Analysis confirmed the specific dissection of the hippocampus into Ammon’s horn and DG. All ****P < 0.0001. n = 24 mice per group. g, h, Gene expression of glucocorticoid receptor (Nr3c1) and mineralocorticoid receptor (Nr3c2) in the hippocampus analysed by qRT–PCR. There was no difference between vehicle and ABX mice in DG (g, Nr3c1 P = 0.1016, Nr3c2 P = 0.8947) or Ammon’s horn (h, Nr3c1 P = 0.1379, Nr3c2 P = 0.9764) after RSI. n = 6 mice per group. i, Expression of stress-related genes (Crhr1 P = 0.4032, Crhr2 P = 0.6778, Ucn P = 0.2477, Ucn2 P = 0.0636, and Ucn3 P = 0.0797) and neuropeptide genes (Avp P = 0.9861 and Oxt P = 0.1445) were unchanged after reciprocal social interaction in ABX mice. n = 5 vehicle, 6 ABX mice per group. j, k, Expression of Nr3c1 and Nr3c2 does not differ between vehicle and ABX mice in DG (j, Nr3c1 P = 0.6206, Nr3c2 P = 0.5075) or Ammon’s horn (k, Nr3c1 P = 0.4873, Nr3c2 P = 0.0931) after novel cage exposure. n = 6 mice per group. l, Ucn gene expression is upregulated after novel cage exposure in ABX mice (*P = 0.013). Other stress-related genes (Crh1 P = 0.649, Crhr2 P = 0.3875, Ucn2 P = 0.1409, and Ucn3 P = 0.511) and neuropeptide genes (Avp P = 0.5809 and Oxt P = 0.8216) were unchanged. n = 6 mice per group. Data shown as individual points with mean ± s.e.m. Data analysed by two-tailed unpaired t-test (a–d, f–l). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ND: no difference. For more statistical details, see Supplementary Information.
