Expression of vesicular glutamate transporters in transient receptor potential melastatin 8 (TRPM8)-positive dental afferents in the mouse

Abstract

Transient receptor potential melastatin 8 (TRPM8) is activated by innocuous cool and noxious cold and plays a crucial role in cold-induced acute pain and pain hypersensitivity. To help understand the mechanism of TRPM8-mediated cold perception under normal and pathologic conditions, we used light microscopic immunohistochemistry and Western blot analysis in mice expressing a genetically encoded axonal tracer in TRPM8-positive (+) neurons. We investigated the coexpression of TRPM8 and vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 in the trigeminal ganglion (TG) and the dental pulp before and after inducing pulpal inflammation. Many TRPM8+ neurons in the TG and axons in the dental pulp expressed VGLUT2, while none expressed VGLUT1. TRPM8+ axons were dense in the pulp horn and peripheral pulp and also frequently observed in the dentinal tubules. Following pulpal inflammation, the proportion of VGLUT2+ and of VGLUT2+/TRPM8+ neurons increased significantly, whereas that of TRPM8+ neurons remained unchanged. Our findings suggest the existence of VGLUT2 (but not VGLUT1)-mediated glutamate signaling in TRPM8+ neurons possibly underlying the cold-induced acute pain and hypersensitivity to cold following pulpal inflammation.

Morphological foundations of pain processing in dental pulp

“…Considering that both VGLUT2 and TRPM8 in the primary sensory neurons have been implicated in the mediation of acute nociceptive and pathologic pain [68,69], it seems likely that VGLUT2-mediated glutamate signaling is involved in the transmission of TRPM8-mediated noxious cold. Furthermore, the expression of VGLUT2 in TG neurons is upregulated following pulpal inflammation, whereas the expression of TRPM8 is not significantly changed [7]. Since both TRPM8 and VGLUT2 are implicated in cold hypersensitivity following inflammation [69][70][71], it is reasonable to assume that cold hypersensitivity following pulpal inflammation is induced by upregulation of the expression of VGLUT2 in the TRPM8-expressing TG neurons, rather than by the direct effect of TRPM8 upregulation.…”

“…Recent immunohistochemical studies showed the distribution of coldsensing channels TRPA1 and TRPM8 in the axons of dental pulp [7,22]: thus, nerve fibers expressing TRPA1 and/or TRPM8 are scant in the radicular pulp and the core of the coronal pulp, but they branch extensively and form a network of fibers in the peripheral pulp: many TRPM8-positive axons were also found to penetrate the dentinal tubules toward the enamel. The latter observation, in particular, may have clinical implications since it indicates that the TRPA1-and TRPM8-mediated pain from noxious cold is primarily perceived at the level of the peripheral pulp and dentin.…”

“…VGLUT and soluble NSF attachment protein receptor (SNARE), which are two classes of proteins associated with synaptic vesicle exocytosis, are densely expressed in the varicosities of pulpal axons, suggesting that glutamate may be released from the pulpal axons at those varicosities (similar to the terminals of the central axons in the spinal cord, which are the sites for transmitter glutamate release) [4,6,7,[62][63][64]. Reportedly, many axons possessing varicosities expressed VGLUT in the peripheral pulp and many of those axons entered the dentinal tubules [4].…”

“…For example, it was shown that activation of TRPM8 in neurons induces release of glutamate from their central afferents, which plays a crucial role in the transmission of TRPM8-mediated signaling of cold [66,67]. In addition, many TRPM8-expressing TG neurons and pulpal axons express VGLUT2 (but not VGLUT1) [7]. Considering that both VGLUT2 and TRPM8 in the primary sensory neurons have been implicated in the mediation of acute nociceptive and pathologic pain [68,69], it seems likely that VGLUT2-mediated glutamate signaling is involved in the transmission of TRPM8-mediated noxious cold.…”

“…Elucidation of the types and properties of nerve fibers innervating dental pulp and of the sensory receptors and ion channels expressed in each type of pulpal nerve fiber is crucial for understanding the peripheral mechanisms of dental pain. Recently, many studies have reported expressions of glutamate receptors and various molecules involved in the release of glutamate in pulpal axons [4,5] and their upregulation in inflamed pulp [6,7]. This suggests that glutamate signaling, associated with the pulpal nerve may play a crucial role in acute and pathologic dental pain.…”