*4.3. TRPM8 as a Familial Migraine Instigator*

TRPM8 is found on both Aδ and C fiber afferents, and is important for the activation of peripheral sensory neurons by cold temperature. It is activated at non-noxious cold temperatures (< 26 ◦C) and by compounds that produce a cooling sensation such as menthol or eucalyptol [158,159]. While its role as a cold sensor has been firmly established, it is not the case regarding its role in pain sensation. It is still under debate whether TRPM8 reduces or exacerbates pain sensation, and the most recent view on the matter is that TRPM8-expressing afferent fibers have the ability to both produce and alleviate pain, and the outcome will be determined by context (see for review [133,160]). As such, TRPM8 has begun to gather attention in the migraine field. A genetic predisposition to migraine is well-recognized: migraineurs presenting a hereditary component account for 42% of patients with migraine, as shown in studies on families and twins [161,162]. Migraine is genetically complex because many genetic variants with small effects and environmental factors can confer migraine susceptibility [163]. However, several genome-wide association studies from different cohorts identified single nucleotide polymorphisms (SNPs) in the gene encoding TRPM8, suggesting an important role for this TRP channel in migraine pathophysiology [128–132]. Several of these variants are located in regions involved in transcriptional regulation and may therefore impact upon TRPM8 expression levels. Moreover, in calcium imaging experiments, some TRPM8 SNP variants heterologously expressed in HEK293 cells showed alterations of channel functionality [164]. Based on these results, TRPM8 variants identified in migraine patients likely contribute to migraine pathology. In adult mice, TRPM8 is also expressed in dural trigeminal nerve endings, albeit rather sparsely [136,165]. Age-dependent decreases in TRPM8 expression in TG neurons appears to play a role in pathways that are differentially regulated with age, in that both the density and number of branches of TRPM8-expressing fibers are comparable to CGRP-expressing fibers in postnatal mouse dura. Specifically, both are reduced by half in adult mouse dura [165]. However, the functional consequence of this reduction remains unclear.

Although TRPM8 is a well-established cold transducer, limited temperature fluctuations in the skull suggest that this activity is less important in dural tissue. Thus, endogenous TRPM8 activators within the dura are unknown. Similarly, whether TRPM8 activation within the dura has a proor anti-nociception effect is unclear. The most recent studies yielded opposite results. Ren and colleagues observed that dural application of menthol resulted in inhibition of nocifensive behavior in a mouse migraine model induced by inflammatory mediators, suggesting an anti-nociceptive effect of TRPM8 [165]. In contrast, dural application of icilin produced migraine-like behavior in mice, such as cutaneous facial and hind paw allodynia. Pretreatment with the TRPM8 antagonist, AMG1161,

attenuated these behaviors [133]. The contrary results obtained in these two studies may be due to the model used: when activated alone, dural TRPM8 appears to have a pro-nociceptive effect, but when activated together with inflammatory mediators, TRPM8 has an anti-nociceptive effect. Ultimately, TRPM8 activation may act as a migraine initiator in the first instance, but have another role during the neurogenic inflammation phase. Moreover, as suggested by Dussor and Cao, these different outcomes might also reflect how TRPM8-expressing fibers project to central neurons as well as the context dependence of TRPM8 activation [166]. More studies are needed to fully understand the role of TRPM8 in dural afferents and migraine pathophysiology.

#### *4.4. TRPV4 as an Indirect Migraine Modulator*

TRP Vanilloid 4 (TRPV4) is a widely distributed cationic channel that participates in the transduction of both physical (osmotic, mechanical, and heat) and chemical (endogenous, plant-derived, and synthetic ligands) stimuli (see for review [126]). As a mechanosensitive channel, TRPV4 has attracted increasing interest in the context of migraine. Indeed, headaches can be influenced by changes in intracranial pressure. Recently, TRPV4 was shown to be expressed in dural afferents, and its activation in the dura of freely-moving rats could produce migraine-like behavior such as cutaneous allodynia [125]. Although dural afferents are known to be mechanically sensitive, whether TRPV4 activation that contributes to migraine is due to mechanical stimulation or another endogenous mechanism remains to be elucidated. Another study showed that both TRPV4 and TRPA1 can be activated by the irritant formalin in the TGVS, and result in downstream MEK–ERK pathway activation and pain behavior in mice [127]. However, whether formalin directly or indirectly activates TRPV4 is unknown. Nevertheless, taken together, these findings suggest that TRPV4 could also be a promising target for agents that provide relief from pain that originates in the trigeminal system.
