Fig. 6

Changes in nerve subtype distribution and neurovascular interactions during postnatal development. a MIP images of the parietal bone region of 4 weeks and 12 weeks mice with NeuF⁺ nerves (blue) and vasculature stained with CD31 (green) and Emcn (red). Scale bar is 300 μm. b Nerve volume fraction calculation for 4 weeks and 12 weeks NeuF⁺ nerves. c Periosteal and dural nerve fraction calculations for NeuF⁺ nerves in 4 weeks and 12 weeks mice. d, e Periosteal and dural nerve volume density calculations for NeuF⁺ nerves in 4 weeks and 12 weeks samples in (d) Overall Calvaria Periosteum, e Overall Calvaria Dura Mater. f Maximum intensity projections of the parietal bone region of 4 weeks and 12 weeks mice with CGRP⁺ nerves (blue) and vasculature stained with CD31 (green) and Emcn (red). Scale bar is 300 μm. g Nerve volume fraction calculation for 4 weeks and 12 weeks CGRP⁺ nerves. h Periosteal and dural nerve fraction calculations for CGRP⁺ nerves in 4 weeks and 12 weeks mice. i, j Periosteal and dural nerve volume density calculations for CGRP⁺ nerves in 4 weeks and 12 weeks samples in (i) Overall Calvaria Periosteum, j Overall Calvaria Dura Mater. k Maximum intensity projections of the parietal bone region of 4 weeks and 12 weeks mice with TH⁺ nerves (blue) and vasculature stained with CD31 (green) and Emcn (red). Scale bar is 300 μm. l Nerve volume fraction calculation for 4 weeks and 12 weeks TH⁺ nerves. m Periosteal and dural nerve fraction calculations for TH⁺ nerves in 4 weeks and 12 weeks. n,o Periosteal and dural nerve volume density calculations for TH⁺ nerves in 4 weeks and 12 weeks samples in (n) Overall Calvaria Periosteum, o Overall Calvaria Dura Mater. p–r Spatial association calculations for nerves and vessels phenotypes for (p) NeuF⁺, (q) CGRP⁺, and (r) TH⁺ nerves. The fraction of nerves within 10 μm is displayed for each vessel phenotype. Data are mean ± SD. Statistics were performed with a two-way ANOVA with post-hoc Tukey HSD test and a two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1 where designated