Direct Neurite-osteoclastic Cell Communication in Co-culture System

Objective: It has been well known that sympathetic nerve system implicate in bone metabolism. Recently, we have demonstrated that nerve-osteoblastic cells communication can directly occur using our in vitro co-culture models comprising mouse nerve and osteoblastic cells. Our previous studies also showed that osteoclastic cells equipped with adrenergic and peptidergic receptors and that β-adrenergic agonists directly stimulate bone-resorbing activity in matured osteoclasts. In the present study, we examined the functional interaction between nerve and osteoclastic cells. Methods: Primary cultures of superior cervical ganglia neurons were dissected from newborn mice. Osteoblastic cells were used RANKL-induced TRAP-positive multinucleated cells in murine macrophage-like cell line RAW264.7. For co-culture experiments, nerve cells were added to 72-h-old cultures of RAW264.7 with soluble RANKL. After co-culture for 3days, cells were treated with culture medium containing the calcium fluorophore Fluo-3, nerve-osteoclastic cell units were monitored intracellular Ca²⁺ mobilization by confocal laser scanning microscopy. Results: We found that the addition of scorpion venom (SV) elicited nerve cells activation via intracellular Ca²⁺ mobilization and, after a lag period, osteoclastic Ca²⁺ mobilization. SV did not have any direct effect on osteoclastic cells in absence of nerve cells. These results suggested that osteoclastic cells activation occurred as a direct response to neuronal activation. We also observed that treatment with an α1-adrenergic agonist, phenylephrine evoked a transient Ca²⁺ mobilization in Fluo-3-loaded osteoclastic cells. Conclusion: Using our in vitro co-culture models, we demonstrated that nerve-osteoclastic cell communication can occur without intervening cells and that this osteoclastic activation might be mediated by α1-adrenergic receptor. These results implicate the direct action of the peripheral sympathetic nerve system in bone metabolism.