Dramatic decrease of innervation density in bone after ovariectomy.

Recent studies have demonstrated that bone is highly innervated and contains neuromediators that have functional receptors on bone cells. However, no data exist concerning the quantitative changes of innervation during bone loss associated with estrogen withdrawal. To study the involvement of nerve fibers in the regulation of bone remodeling, we have evaluated the modifications of innervation in a classical in vivo model of osteopenia in rats, ovariectomy (OVX). Skeletal innervation was studied by immunocytochemistry using antibodies directed against specific neuronal markers, neurofilament 200 and synaptophysin, and the neuromediator glutamate. Sciatic neurectomy, another model of bone loss due to limb denervation and paralysis, was used to validate our quantitative image analysis technique of immunostaining for nerve markers. Female Wistar rats at 12 wk of age were sham-operated (SHAM) or ovariectomized (OVX). Bone mineral density measurement and bone histomorphometry analysis of tibiae 14 d after surgery demonstrated a significant bone loss in OVX compared with SHAM. We observed an important reduction of nerve profile density in tibiae of OVX animals compared with SHAM animals, whereas innervation density in skin and muscles was similar for OVX and control rats. Quantitative image analysis of immunostainings demonstrated a significant decrease of the percentage of immunolabeling per total bone volume of neurofilament 200, synaptophysin, and glutamate in both the primary and secondary spongiosa of OVX rats compared with SHAM. These data indicate for the first time that OVX-induced bone loss in rat tibiae is associated with a reduction in nerve profile density, suggesting a functional link between the nervous system and the bone loss after ovariectomy.

Molecular identification of NMDA glutamate receptors expressed in bone cells.

The N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor has recently been identified in bone, but the molecular composition of this receptor expressed by bone cells is unknown. NMDA receptor (NMDAR) is a hetero-oligomeric protein composed of two classes of subunits, the essential subunit NR1 and NR2A to D subunits that do not by themselves produce functional channels but potentiate NR1 activity and confer functional variability to the receptor. These subunits coassemble in different combinations to form functionally distinct NMDAR. In this study, we have investigated the molecular composition of NMDAR expressed by osteoblasts and osteoclasts in culture, using RT-PCR analysis, in situ hybridization and immunocytochemistry. Specific probes were designed for the different subunits of the NMDAR, and we showed by RT-PCR analysis that mammalian osteoclasts expressed NR2B and NR2D subunits mRNAs but not NR2A and NR2C mRNAs. Rat calvaria and MG63 osteoblastic cells also expressed several NR2 subunits mRNAs, namely NR2A, NR2B, and NR2D. In situ hybridization on isolated rabbit osteoclasts and MG63 cells has confirmed the localization of NR1, NR2B, and NR2D transcripts in osteoclasts and NR1, NR2A, NR2B, and NR2D transcripts in MG63 cells. The expression of NR2D protein by bone cells was shown by immunofluorescence. These results demonstrate for the first time that osteoblasts and osteoclasts express several NR2 subunits, suggesting a molecular diversity of NMDAR channels similar to what was shown for brain. The presence of distinct functional NMDAR on bone cells may be associated with various states of bone cell differentiation and function.

Specific antagonists of NMDA receptors prevent osteoclast sealing zone formation required for bone resorption.

N-Methyl-d-aspartate (NMDA) glutamate receptors, widely distributed in the nervous system, have recently been identified in bone. They are expressed and are functional in osteoclasts. In the present work, we have studied the effects of specific antagonists of NMDA receptors on osteoclast activation and bone resorption. Using an in vitro assay of bone resorption, we showed that several antagonists of NMDA receptors binding to different sites of the receptor inhibit bone resorption. Osteoclast activation requires adhesion to the bone surface, cytoskeletal reorganization and survival. We demonstrated by autoradiography that the specific NMDA receptor channel blocker, MK 801, binds to osteoclasts. This antagonist had no effect on osteoclast attachment to bone and did not induce osteoclast apoptosis. In contrast, MK 801 rapidly decreased the percentage of osteoclasts with actin ring structures that are associated with actively resorbing osteoclasts. These results suggest that NMDA receptors expressed by osteoclasts may be involved in adhesion-induced formation of the sealing zone required for bone resorption.

Active NMDA glutamate receptors are expressed by mammalian osteoclasts.

1. The N-methyl-D-aspartate (NMDA) glutamate receptor, widely distributed in the mammalian nervous system, has recently been identified in bone. In this study, we have investigated whether NMDA receptors expressed by osteoclasts have an electrophysiological activity. 2. Using the patch clamp technique two agonists of the NMDA receptor, L-glutamate (Glu) and NMDA, were shown to activate whole-cell currents recorded in isolated rabbit osteoclasts. 3. The current-voltage (I-V ) relationships of the currents induced by Glu (IGlu) and NMDA (INMDA) were studied using Mg2+-free solutions. The agonist-induced currents had a linear I-V relationship with a reversal potential near 0 mV, as expected for a voltage independent and non-selective cationic current. 4. IGlu and INMDA were sensitive to specific blockers of NMDA subtype glutamate receptors, such as magnesium ions, (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten -5,10-imine (MK-801) and 1-(1,2-diphenylethyl) piperidine (DEP). The block of IGlu and INMDA by these specific antagonists was voltage dependent, strong for negative potentials (inward current) and absent for positive potentials (outward current). 5. These results demonstrate that NMDA receptors are functional in rabbit osteoclasts, and that their electrophysiological and pharmacological properties in these cells are similar to those documented for neuronal cells. Active NMDA receptors expressed by osteoclasts may represent a new target for regulating bone resorption.