Cholinergic neural signals regulate HSPC and leukocyte traffic by modulating sympathetic noradrenergic tone centrally. (A-B) Representative immunofluorescence of CD31⁺ endothelial cells (blue), tyrosine hydroxylase–positive sympathetic nerve fibers (TH; red), and nestin-GFP⁺ cells (green) in the skull BM of Nes-gfp;Gfra2^+/− and Nes-gfp;Gfra2^−/− compound mice. Scale bars, 100 μm. (C) Quantification of the skull BM area covered by TH⁺ sympathetic noradrenergic nerve fibers from Gfra2^+/− mice and Gfra2^−/− mice. (D) Nocturnal (black) and diurnal (yellow) norepinephrine concentration in the urine of Gfra2^+/− and Gfra2^−/− compound mice. (E) Scheme illustrating the different types of cholinergic antagonists used and their capacity to cross the BBB. Mecamylamine and scopolamine are BBB-permeable antagonists, whereas hexamethonium and methylatropine are BBB-nonpermeable antagonists. Blood-circulating HSPCs, measured as CFU-Cs (F) and WBCs (G) at ZT13 in WT mice treated with acetylcholine antagonists (i.p.) at ZT5. (C-D,F-G) Data are mean ± standard error of the mean; n (inside bars) and P values (multivariate analysis for >2 groups) are indicated. *P < .05, **P < .01, unpaired 2-tailed t test (C), 1-way analysis of variance with Bonferroni comparisons (D,F-G). a.u, arbitrary units; ns, not significant.