Age-related changes in afferent responses in sensory neurons to mechanical stimulation of osteoblasts in coculture system

Abstract

Asada K, Obata K, Horiguchi K, Takaki M. Age-related changes in afferent responses in sensory neurons to mechanical stimulation of osteoblasts in coculture system. Am J Physiol Cell Physiol 302: C757-C765, 2012. First published November 16, 2011 doi:10.1152/ajpcell.00362.2011.-Bone homeostasis is regulated by mechanical stimulation (MS). The sensory mechanism of bone tissue for MS remains unknown in the maintenance of bone homeostasis. We aimed to investigate the sensory mechanism from osteoblasts to sensory neurons in a coculture system by MS of osteoblasts. Primary sensory neurons isolated from dorsal root ganglia (DRG) of neonatal, juvenile, and adult mice and osteoblasts isolated from calvaria of neonatal mice were cocultured for 24 h. The responses in DRG neurons elicited by MS of osteoblasts with a glass micropipette were detected by increases in intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) with fluo 3-AM. In all developmental stages mice, [Ca 2ϩ ]i-increasing responses in osteoblasts were promptly elicited by MS. After a short delay, [Ca 2ϩ ]i-increasing responses were observed in neurites of DRG neurons. The osteoblastic response to second MS was largely attenuated by a stretch-activated Ca 2ϩ channel blocker, gadolinium. The increases of [Ca 2ϩ ]i in DRG neurons were abolished by a P2 receptor antagonist; suramin, a P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2=,4=-disulfonate; and an ATP-hydrolyzing enzyme, apyrase. Satellite cells were found around DRG neurons in cocultured cells of only neonatal and juvenile mice. After satellite cells were removed, excessive abnormal responses to MS of osteoblasts were observed in neonatal neurites with unchanged osteoblast responses. The present study indicated that MS of bone tissue elicited afferent P2X receptor-mediated purinergic transmission to sensory neurons in all stages mice. This transmission is modulated by satellite cells, which may have protective actions on sensory neurons. dorsal root ganglia; calcium imaging; satellite cell; 5=-triphosphate; bone formation BONE HAS IMPORTANT ROLES IN supporting the body to maintain motor functions against the gravity and in storing minerals such as calcium and phosphate, resulting in an essential role player maintaining life. In the maintenance of bone homeostasis, mechanical stimulation (MS) to the bone plays critical roles. Mechanically unloading by prolonged bed rest, immobilization, or microgravity in space causes a marked loss of bone (28, 34). The atrophic changes in bone or heterotopic bone formations are observed in patients with neurological disorder (7, 10). It would be beneficial in treatments for the patients with neurological disorder and for aged patients with bone loss (24) to reveal the transduction mechanism of MS into bone homeostasis.Such studies (17) have been initiated from the molecular mechanism in bone underlying transduction of MS into a cellular response, although the benefits of MS have been well demonstrated in clinical settings. Activation of a variety of factors s...

Bidirectional communication between sensory neurons and osteoblasts in an in vitro coculture system

“…2A,B). Preceding studies have demonstrated close membrane contact and synaptic structure in cocultures comprising DRG-derived sensory neurons and osteoblasts, by electron microscopy analysis [32,40]. Immunoreactivity to synapsin, a synaptic-related protein, has also been observed in nerve fibers attached to osteoblasts [40].…”

Innervation of bones: Why it should not be neglected?

“…Some of these nerve fibers clearly showed "local dilatations in contact with medullary cells and bone cells that were immunolabeled for synaptophysin, a nerve terminal marker" (11). A more detailed assessment was possible in cell co-culture of sensory neurons and osteoblasts, showing that they established a close synapse-like contact (38)(39)(40). Moreover, cell culture experiments showed that osteoblasts and sensory neurons communicate bidirectionally: peripheral neurite terminals release glutamate and substance P by exocytosis (efferent signal to osteoblasts) and osteoblasts release adenosine triphosphate -ATP (afferent signal to neurites) (39,40).…”

“…A more detailed assessment was possible in cell co-culture of sensory neurons and osteoblasts, showing that they established a close synapse-like contact (38)(39)(40). Moreover, cell culture experiments showed that osteoblasts and sensory neurons communicate bidirectionally: peripheral neurite terminals release glutamate and substance P by exocytosis (efferent signal to osteoblasts) and osteoblasts release adenosine triphosphate -ATP (afferent signal to neurites) (39,40). It is interesting that mechanical stimulation of osteoblasts in cell culture was able to activate the neurite of the co-cultured sensory neurons, which was based on the release of ATP from osteoblasts and its binding on the purinergic receptors on the neurite (40).…”

Purinergic signalling and development of the autonomic nervous system

“…Expression of the vanilloid receptor subtype 1 was not altered by desamethasone treatment, suggesting a potential role of P2X 3 receptors in the unique profile of pain perception in the newborn (Wang et al, 2008). In a recent study carried out in co-cultures of osteoblasts and DRG neurons isolated from neonatal (0-5 days old), juvenile (14-18 days old) and adult (2-3 months old) BALB/c mice, mechanical stimulation of bone cells induced increases of intracellular Ca ++ concentration [Ca ++ ] i in neurites of DRG neurons which were abolished by the antagonists suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS), suggesting the involvement of a P2X receptor, as well as by an ATP hydrolyzing enzyme, apyrase (Asada et al, 2012). P2X receptor-mediated responses were similar at all developmental stages, however, when satellite cells were removed from DRG culture, excessive responses to mechanical stimulation were recorded only in neonatal and juvenile preparations, suggesting a protective role for glial cells on development of sensory neurons.…”