Both P2X3 homomultimer and P2X2/3 heteromultimer receptors have been reported in a variety of tissues, including tooth pulp [9–13], stimulation of which causes a barrage of action potentials in tooth pulp afferents that are conveyed to the rat Vc and C1/C2, resulting in a purinergic-dependent central sensitization of Vc and C1/C2 nociceptive neurons [16, 38–40]. Recently it has been found that Vc central sensitization can be produced by specifically stimulating these P2X receptors in the pulp .
In the present study, we observed that GG muscle activity could be evoked by pulpal application of the P2X3,2/3 receptor agonist α,β-meATP 100 mM and was significantly larger in the rats when compared with PBS-administration to the pulp. The GG activity was significantly reduced in the rats receiving co-application of 100 mM α,β-meATP with the P2X3,2/3 receptor inhibitor TNP-ATP (1 mM). A large number of pERK-LI cells were also expressed in the Vc, Vi/Vc, C1/C2 and Pa5 5 min after pulpal application of 100 mM α,β-meATP. The pERK-LI cells and GG activity were significantly reduced after intrathecal injection of the MEK inhibitor PD98059. In addition, the number of pERK-LI cells was significantly reduced in rats receiving pulpal co-application of 100 mM α,β-meATP with 1 mM TNP-ATP compared with rats receiving 100 mM α,β-meATP alone.
In the present study, dental paper points soaked with PBS, α,β-meATP or TNP-ATP were inserted into the tooth pulp. The insertion of a paper point into the tooth pulp may itself cause strong mechanical stimulation of pulpal nerve fibers. We observed some GG activity and many pERK-LI cells were expressed in the Vc, C1/C2 and Pa5 following pulpal insertion of a PBS-dipped paper point. Thus, the GG activity and pERK-LI cells expression were likely a reflection of both mechanical stimulation of the tooth pulp as well as activation of purinergic receptors when α,β-meATP was applied. Nonetheless, the use of PBS as a vehicle control allowed for separation of the effects of α,β-meATP per se. Indeed, the GG activity was significantly larger following pulpal application of 100 mM (but not 10 mM) α,β-meATP when compared with PBS application. Likewise, pERK expression was significantly larger with 100 mM α,β-meATP than PBS. The dose of α,β-meATP required was higher than that reported in other parts of the body (e.g., reduction of hind paw withdrawal threshold, hind paw lifting and licking) [41–43]. This may be due to damage of ATP receptors in tooth pulp nerve fibers during the preparation of the pulp for subsequent drug administration since it has been reported that pulpal P2X3 receptors are highly concentrated in the odontoblastic layer [9, 12] and thus susceptible to damage by cavity preparation. This may explain why a high concentration of ATP was necessary to produce a reflex effect and ERK phosphorylation. Thus, the high concentration of ATP is needed for reflex and other effects relative to other body regions.
We could not observe significant activation of MA or DA muscle following α,β-meATP administration. Primary afferent activity is not sufficiently strong to activate MA motor neurons relative to GG muscle, following noxious mechanical stimulation of facial skin, based on our EMG recordings as described in the Results section. Furthermore, P2X receptor density is lower in the tooth pulp relative to other receptors . Therefore, the tooth pulp-MA and tooth pulp-DA pathways have a higher threshold for activation by tooth pulpal administration of ATP agonists compared with the tooth pulp-GG pathway.
Involvement of P2X receptor activation of tooth pulpal afferents
It is well known that peripheral inflammation or tissue injury causes peripheral ATP release from non-neuronal cells in the injured region [9, 10, 17]. The ATP released from the non-neuronal cells binds to purinergic receptors such as P2X3 and/or P2X2/3 receptors in C-fiber terminals, resulting in peripheral sensitization of the primary afferent neurons [1, 10, 11, 17, 44]. We observed strong activation of GG activity following pulpal application of α,β-meATP, indicating that activation of pulpal P2X3 and/or P2X2/3 receptors in the tooth pulp is sufficient for activation of the GG reflex. The increase in GG muscle activity with α,βme-ATP and its 1-2 sec latency are consistent with other studies of muscle (MA or DA muscle) responses evoked by tooth pulp stimulation  or other orofacial stimuli, eg. TMJ [36, 37]. Shigenaga et al. [45, 46] have reported that tooth pulp afferents project to the Vc and trigeminal spinal subnucleus interpolaris (Vi), and many Vi neurons send projection axons to GG muscle motor neurons . Furthermore, previous electrophysiological studies have also reported that GG or hypoglossal motor neuron activity is modulated by trigeminal nerve stimulation, suggesting that the trigeminal afferent is involved in modulation of GG muscle activity [48, 49]. Previous studies have also reported that the pulpal application of mustard oil or capsaicin induces EMG activity in the MA and DA muscles simultaneously, and these excitations could last more than 1 min [23, 24]. On the other hand, pulpal application of α,β-meATP in the present study caused a significant increase in EMG activity only in the GG muscle, and the duration of GG activity was less than 1 min. These differences between the studies might be explained by different experimental conditions in the studies or that the tooth pulp afferent-GG reflex pathway especially involves P2X3,2/3 receptors.
Together with previous results our findings suggested that tooth pulp nerve fibers were sensitized by ATP released from the non-neuronal pulpal cells following tooth pulp inflammation or injury, resulting in the barrage of action potentials in the tooth pulp nerve fibers which were conveyed to Vc and C1/C2 neurons.
Sensitization of Vc, C1/C2 and Pa5 neurons
It has been documented that noxious inputs from the orofacial region are somatotopically organized in the Vc complex and C1/C2 regions, and nociceptive neurons in these areas are involved in the localization of orofacial pain . Anterograde tracing studies have revealed that the rat's tooth pulp afferents are distributed in the ipsilateral Vc, C1/C2 and Pa5 [45, 46, 51–55]. In particular, the maxillary first molar pulp afferent projects to the ipsilateral Vc, Vi/Vc, Pa5 and C1/C2 regions . Shimizu et al. have also reported that pulpal application of capsaicin produces pERK-LI cell expression in these regions suggesting that neurons in Pa5, Vc, Vi/Vc and Vc/C2 regions are involved in tooth pulp nociceptive processing . Consistent with these previous studies, the present study documented expression of pERK-LI cells following α,β-meATP pulpal application in the dorsal portion of the ipsilateral Vc, Vi/Vc, C1/C2 and bilaterally in the Pa5 region. It is well established that Vc, Vi/Vc and C1/C2 nociceptive neurons manifest marked central sensitization following orofacial inflammation or trigeminal nerve injury [40, 56, 57]. It is also known that a barrage of action potentials is elicited in primary afferent fibers following tooth pulp inflammation . Peripheral inflammation is thought to be involved in enhancement of a variety of receptor activities, including purinergic receptor in peripheral nerve terminals , which results in the peripheral and central sensitization of the trigeminal nociceptive system [16, 22]. Together, these data and the present results suggest that purinergic receptors in pulpal nerve terminals are involved in peripheral sensitization of the trigeminal nociceptive system. It has also been reported that Pa5 neurons are involved in autonomic regulation as well as in nociceptive processes .
ERK is one of the MAPK families, and has been documented in the spinal DH as well as dorsal root ganglion neurons that are phosphorylated by noxious peripheral stimulation in an intensity-related manner [60–64]. ERK has also be shown to be phosphorylated in Vc and C1/C2 neurons within 5 min after noxious stimulation of the orofacial region , strongly suggesting that ERK phosphorylation is involved in the activation of nociceptive neurons in the Vc and C1/C2 soon after orofacial noxious stimulation. Consistent with these findings, we observed that many pERK-LI cells were expressed in Vc, Vi/Vc, C1/C2 and Pa5 regions 5 min after pulpal application of α,β-meATP, and showed that the pERK-LI cells in Vc showed NeuN immunoreactivity, indicating that the phosphorylation of ERK occurred in neurons. Furthermore, following intrathecal administration of the MEK inhibitor PD 98059 [27, 30, 31, 65], the pERK-LI cells were significantly reduced in Vc, Vi/Vc, C1/C2 and Pa5 in rats receiving pulpal administration of α,β-meATP, suggesting that the intracellular ERK cascade is involved in the activation of Vc, Vi/Vc, C1/C2 and Pa5 nociceptive neurons. We also observed that the GG activity evoked by pulpal application α,β-meATP was significantly suppressed by intrathecal administration of PD 98059. Since it has been reported that Vc neurons are involved in masticatory muscle activity evoked by noxious stimulation of orofacial tissues [66, 67], the ERK phosphorylation in Vc neurons was likely involved in our documented enhancement of GG activity following pulpal administration of α,β-meATP.