Extended Data Fig. 4: Central motor circuits control neutrophil egress from the bone marrow during acute stress.

a, Quantification of circulating blood neutrophils under non-stressed conditions and after 1 h of restraint stress in sympathectomized (6-OHDA) vs. vehicle-treated mice (2 experiments combined, n = 10 non-stressed vehicle-treated mice, n = 10 non-stressed 6-OHDA-treated mice, n = 8 stressed vehicle-treated mice, n = 8 stressed 6-OHDA-treated mice). Two-way ANOVA. b, Quantification of circulating blood neutrophils under non-stressed conditions and 40 min after a 20 min restraint stress episode in high-dose alpha- (phentolamine, 30mg/Kg) and beta-adrenergic receptor blocker (propranolol, 30mg/Kg) vs. vehicle-treated mice (n = 6 per group). The non-stressed baseline was collected 75 min after the i.p. injection of vehicle or alpha-/ and beta-blocker. Two-way ANOVA. c, Quantification of circulating blood neutrophils under non-stressed conditions and after 1 h of restraint stress in WT mice, beta 2/beta 3 adrenergic receptor double knockout mice (B2/B3 KO), and beta 1/beta 2 adrenergic receptor double knockout mice (B1/B2 KO) (n = 11 WT mice, n = 10 B2/B3 KO mice, n = 8 B1/B2 KO mice). The non-stressed baseline was collected from the same mice one week apart from the actual stress experiment, at the same time of the day. Data from the B1/B2 KO mice were generated in an independent experiment. Two-way ANOVA. d, Quantification of circulating blood neutrophils in WT mice 1 h after injection of vehicle or the indicated doses of noradrenaline (n = 5 in the 2000µg/Kg group, n = 4 in all other groups). One-way ANOVA. e, Change in circulating blood neutrophil numbers induced by 1 h of restraint stress in adrenalectomized (ADX) vs. sham-operated WT mice (calculated as neutrophil number after 1h h of stress - leukocyte number at baseline; n = 4 mice per group). Unpaired t-test. f, Quantification of circulating blood neutrophils under non-stressed conditions and after 1 h of restraint stress in WT mice vs. interleukin-6 (IL-6) knockout mice (n = 4 per group). Two-way ANOVA. g, Schematic of optogenetic approach used to stimulate hChR2-expressing neurons in the rostroventrolateral medulla (RVLM) of DBH Cre mice. Quantification of circulating blood neutrophils 55 min after a 5 min LED stimulation period compared to hChR2-injected DBH^Cre mice without light stimulation (cross-over design with each mouse analysed with and without LED stimulation 1d apart, total of n = 8 per group). Two-tailed unpaired t-test. h, Quantification of circulating blood neutrophils in WT mice 1 h after injection of vehicle or the indicated doses of CXCL1 (n = 3 per group). i, Cxcl1 transcript expression in the gluteal muscle measured after the indicated time of recovery from a 3 min restraint stress episode (expressed as fold change from non-stressed control mice, n = 3 mice per time point). One-way ANOVA. j, Cxcl1 transcript expression in the gluteal muscle measured immediately after 1 h of restraint stress vs voluntary running on a running wheel compared to non-stressed control mice without a running wheel (n = 4 per group). One-way ANOVA. k, Wireless EMG telemetry recordings from the gluteal and upper leg musculature in WT mice under conditions of restraint stress vs voluntary running on a running wheel compared to non-stressed controls without a running wheel (each mouse was recorded under each condition on subsequent days during their active period, data expressed as number of amplitude peaks above a mouse-specific threshold of 0.2 mV (2 mice) or 0.4 mV (1 mouse) during the first 10min of restraint) (n = 3 mice total, each recorded under each condition as outlined above). One-way ANOVA. l, Cxcl1 transcript expression in the gluteal muscle and CXCL1 protein levels in the serum measured in WT mice immediately after 1 h of exposure to the indicated stressor compared to non-stressed controls (Cxcl1 transcript expression expressed as fold change from non-stressed control mice (n = 5 in the aggressive intruder group, n = 6 in all other groups). One-way ANOVA. m, Muscle damage parameters in blood serum at different time points after the onset of a 30 min restraint stress episode, compared to non-stressed controls. Serum myoglobin measured by ELISA. Serum creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) activities in units/liter (U/L) were determined by IDEXX BioAnalytics (n = 5 non-stressed mice, n = 5 mice 30min post stress onset, n = 5 mice 1h post stress onset, n = 5 mice 4h post stress onset, n = 4 mice 12h post stress onset, n = 4 mice 24h post stress onset). One-way ANOVA. n, Representative injection site of the channelrhodopsin encoding virus pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA with optic fibre position in the medulla of a vGlut2^Cre mouse stained with anti-mCherry primary antibody and AF594-labelled secondary antibody. Optic cannula positions indicated in the inset. Scale bar indicates 500 µm. o, CXCL1 protein levels in the serum and circulating blood leukocyte numbers measured immediately after 30 min of LED stimulation (LED on) compared no-LED stimulation (LED off) in vGlut2^Cre mice that had previously received an mCherry control virus injection with cannula implantation into the medulla (two experiments with changed groups combined, total of n = 6 mice per group). Two-tailed unpaired t-test. p, Schematic of the bilateral motor cortex ablation approach with coordinates indicated as distance from Bregma. Representative Cresyl Violet stained brain sections showing the extend of ablation. q, Representative bilateral injection sites of the inhibitory DREADD encoding virus pAAV-hSyn-DIO-hM4D(Gi)-mCherry in the medulla of a vGlut2^Cre mouse stained with anti-mCherry primary antibody and AF594-labelled secondary antibody. Scale bar indicates 1000 µm. Data are mean ±s.e.m.; ^★P < 0.05, ^★★P < 0.01, ^★★★P < 0.001, statistical tests used as indicated.