Fig. 2. Osteocytes are positioned close to cholinergic nerves in vivo.

Intravital z-stack image (40 μm thick) of metatarsal bone osteocytes in mice expressing GCaMP6f in ACh transferase–expressing cells taken using two-photon microscopy at about 20 μm below the periosteal surface (A). The red fluorescence signal is driven by a 15-μl volume of ultrasmall RGD-functionalized integrin targeting nanoparticles (5 to 7 nm) at 10 μM concentration. This static 3D image indicates the colocalization of osteocytes, labeled in red, with cholinergic nerve fibers, labeled in green. (B and C) Yellow and blue insets depict single 2D frames where the nerve can be seen directly interacting with osteocytes. (D) Representative images for nerve-osteocyte proximity analysis showing the input image with osteocytes in red and ChAT signal containing cholinergic nerve fibers in green. The masks were drawn over the nerve fibers of the input image, and proximity analysis of the shortest distance between each osteocyte and a nerve was calculated and represented in the output image. (E) Histogram with an overlaying Gaussian curve showing the distances of osteocyte distribution around the nerve in both sexes. (F) Both males and females fail the normality test (P value < 0.0001), and a significant difference (P value = 0.001) exists in the distribution of osteocytes between the sexes (fig. S3). Kolmogorov-Smirnov test was used for normality assessment. Mann-Whitney nonparametric test was used for comparing between sexes.