*2.1. Thermo-Sensing Ion Channels in the Trigeminal Nerve*

TRPV1 is a polymodal receptor activated by high temperatures over 43 ◦C or irritant chemicals including capsaicin and proton. TRPV1 is believed to play a central role in nociception because it is primarily expressed in small- to medium-peptidergic nociceptive neurons, and its activation is modulated by various inflammatory and nerve-damage-inducing mediators. Immunohistochemical investigation demonstrated TRPV1 expression in 20% of rat trigeminal ganglion (TG) cells, mostly in small- to medium-sized, as expected [39]. Interestingly, a retrograde labeling study revealed that only 8% of tooth pulpal neurons were TRPV1-positive, whereas 26% of TG neurons innervating facial skin were TRPV1-positive, which was contrary to the previous speculation that most nerves innervating the tooth pulp are nociceptive [40–42]. Conversely, the functional analysis of retrograde-labeled dental primary afferent neurons showed the opposite results; the neuron response to capsaicin application

was more abundant for dental primary afferents than for TG neurons in calcium imaging studies [43] and in whole-cell patch clamp experiments [44]. Single-cell RT-PCR analysis revealed that most dental primary afferents are TRPV1-positive, whereas two other immunohistochemical analyses reported that only 17–34% of pulp-innervating neurons were TRPV1-positive [45,46]. The reason for this discrepancy is not clear. Of note, lipopolysaccharides (LPS) from Gram (−) bacteria upregulated TRPV1 expression in TG [47], and Complete Freund's Adjuvant (CFA) upregulated TRPV1 in TG neurons innervating adjacent teeth [48], suggesting the potential contribution of TRPV1 to tooth pain under the pulpitis condition. Interestingly, estrogen is also upregulated TRPV1 and anoctamin-1 (ANO1)—a potential heat-sensing ion channel—in female rat TG neurons and induced an increased pain response to TRPV1 agonists [49]. The physiological meaning of estrogen-induced upregulation for heat sensing ion channels is not clear and needs to be considered when designing pain studies.

TRPV2 is an ion channel homolog to TRPV1 with a higher threshold (>52 ◦C). TRPV2 is different from TRPV1 in that it does not respond to capsaicin nor acid and is preferentially expressed in mediumto large-sized myelinated neurons [50]. The immunohistochemical analysis of retrograde-labeled TG neurons revealed a TRPV2 expression pattern quite the opposite to that of TRPV1 [51]. While 14% of the TG cells showed immunoreactivity to TRPV2 mostly in medium- to large-sized, 37% of neurons innervating tooth pulp was TRPV2-positive, whereas only 9% of neurons to the facial skin were positive. Another immunohistochemical analysis using a double-labeling technique confirmed the mutually exclusive expression of TRPV1 and TRPV2 in pulpal neurons, with 32–51% TRPV2 positive cells [45,46]. These findings are consistent with previous reports that pulpal neurons are mostly medium- to large-sized myelinated neurons that lose their myelination upon entering tooth pulp [2,4,52–56], suggesting that teeth might have a distinct nociceptive system.

In addition, the expression of TRPV4 and TRPM3 was observed in retrogradely labeled dental afferent neurons [57–59]. Because TRPV4 activates at innocuously warm temperature between 27 and 35 ◦C, it is believed to play a role in the maintenance of body temperature, rather than in nociception [60]. On the other hand, TRPM3, or long TRPC3 as previously known, was recently discovered to have an activation threshold of 40 ◦C and became a prominent candidate of noxious heat detector [61].

Since cold stimuli induce tooth pain more frequently than hot, cold-sensitive TRP channels might play a role in the transduction of tooth pain. TRPA1 and TRPM8 are cold-sensitive TRP channel subtypes activated at temperatures below 17 and 25 ◦C, respectively [62,63]. Calcium imaging experiments with cold stimuli under 18 ◦C revealed that cold-sensitive neurons are more abundant in the TG than in the DRG (15% and 7%, respectively) [43]. TRPA1 upregulation in a tooth injury rat model proposes the importance of TRPA1 in tooth pain [64]. A subsequent study combining electrophysiological recording with single-cell RT-PCR and immunocytochemistry revealed the functional expression of TRPA1 and TRPM8 in rodent dental primary afferent neurons [43]. Interestingly, the expression of TRPA1 and TRPM8 channels was lower than that of TRPV1 in dental primary afferent (DPA) neurons. TRPA1 and TRPM8 were, moreover, co-expressed in some of the TRPV1-positive DPA neurons, suggesting an ambiguity between cold and hot stimuli-induced tooth pain. A recent study suggested that acute heat sensation requires any of functional TRPV1, TRPA1, and TRPM3 ion channels, and only triple knock-out mice showed a lack of acute withdrawal response to noxious heat compared to the intact normal response to cold stimuli, which suggests a redundant mechanism for heat detection [65]. Whether dental sensory systems utilize a similar mechanism is unclear.
