Bone’s ability to adapt to mechanical loads is attributed to the osteocyte. Osteocytes reside embedded in the calcified bone matrix, where they function as the resident tissue mechanotransducer. Calcium (Ca2+) signaling is a key second messenger in osteocyte mechanotransduction, however the details regarding Ca2+ signaling regulation in osteocytes in vivo are not well stratified. The nervous system is known to regulate bone. Osteocytes express components for acetylcholine (ACh) receptors that are known for rapid firing at the neuromuscular junction. Although ACh is known to impact bone mass and fracture risk, the details regarding ACh signaling in bone mechanotransduction remain largely unexplored. This talk identifies osteocytes as a functional target for cholinergic signaling with impacts on bone mechanoadaptation and Ca2+ signaling. We generated osteocyte-targeted conditional knockout mice to remove key components used by ACh receptors. We assessed tissue material make-up, strength, and formations rates to anabolic loading. We also used intravital imaging via two-photon fluorescent microscopy to visualize the Ca2+ dynamics in osteocytes during active mechanical loading of the bone in living mice. Finally, we explore the ability of light-sheet fluorescent microscopy to create high resolution 3D reconstructions of nerve bodies in whole-mount and intact bone specimens. Our results establish a new signaling axis between the brain and bone via the osteocyte with far reaching implications in bone biology which may be vital to treat bone diseases.