Cortical bone is an extraneuronal site of norepinephrine uptake in adult mice

Abstract

The sympathetic nervous system is a major efferent pathway through which the central nervous system controls the function of peripheral organs. Genetic and pharmacologic evidence in mice indicated that stimulation of the β2 adrenergic receptor (β2AR) in osteoblasts promotes bone loss, leading to the paradigm that high sympathetic nervous activity is deleterious to bone mass. However, considerably less data exist to understand the putative impact of endogenous norepinephrine (NE), released by sympathetic nerves, on bone homeostasis. In this study, we investigated the in vivo expression and activity of the norepinephrine transporter (NET), a membrane pump known to actively uptake NE from the extracellular space in presynaptic neurons. Consistent with previously published in vitro data showing NET uptake activity in differentiated osteoblasts, we were able to detect active NET-specific NE uptake in the mouse cortical bone compartment in vivo. This uptake was the highest in young mice and accordingly with an age-related reduction in NET uptake, NE bone content increased whereas Net RNA and protein expression decreased with age. Histologically, NET expression in adult mouse bones was detected in osteocytes via immunofluorescence. Lastly, taking advantage of tissue-specific fluorescent reporter mice, we used CLARITY imaging and light sheet microscopy to visualize the 3D distribution of sympathetic fibers in whole mount preparations of bone tissues. These analyses allowed us to detect tyrosine hydroxylase (TH)-positive sympathetic nerve fibers penetrating the cortical bone, where NET+ osteocytes reside. Together, these in vitro results support the existence of an age-dependent extraneuronal and osteocytic function of NET with potential to buffer the bone catabolic action of endogenous NE released by sympathetic nerves in vivo.

Nerves in Bone: Evolving Concepts in Pain and Anabolism

“…Although these studies are powerful for comparing relative distribution of skeletal innervation, the high degree of fiber branching and winding through the tissue and the limited working depth in standard histological sections remain obstacles to quantifying the absolute number of neurons that innervate different bone compartments. Advances in tissue clearing techniques and whole‐mount preparations are beginning to be applied to bone that will improve quantitative mapping of skeletal innervation and secondary architecture such as glial cells …”

“…In bone metastasis, sympathetic signaling via activation of β 2 ‐AR on osteoblasts leads directly to cancer cell recruitment and indirectly to vascular changes that promote colonization of metastatic cancer cells in the bone marrow . Last, NE uptake by bone and immune cells may also contribute to extraneuronal control of local NE homeostasis and actions on the skeleton …”

“…Advances in tissue clearing techniques and whole-mount preparations are beginning to be applied to bone that will improve quantitative mapping of skeletal innervation and secondary architecture such as glial cells. (54)(55)(56) Pathways to the Brain There have only been a few studies that have mapped skeletal sensory input to and within the CNS. Retrograde labeled sensory neurons innervating rat tibial periosteum, medullary cavity, and trabecular bone have been observed in ipsilateral DRG spanning lumbar regions L 1 to L 6 , but are concentrated in L 3 .…”

“…(121,123,124) Last, NE uptake by bone and immune cells may also contribute to extraneuronal control of local NE homeostasis and actions on the skeleton. (56,125,126) NPY NPY is the most abundant neuropeptide in the mammalian CNS and is also released from peripheral sympathetic nerves with NE. NPY contributes to neural control of a wide variety of physiological processes including stress and anxiety, appetite, circadian rhythm, cardiovascular activity, immune function, and pain.…”

Simultaneous Regeneration of Bone and Nerves Through Materials and Architectural Design: Are We There Yet?

“…[ 351,352 ] Much of bone's innervation is centripetally distributed, derived from periosteal fibers that branch within the compact bone and bone marrow. [ 19,353–355 ]…”