Effects of Nitric Oxide on the Activity of P2X and TRPV1 Receptors in Rat Meningeal Afferents of the Trigeminal Nerve.

Nitric oxide is one of the endogenous molecules that play a key role in migraine. However, the interaction between NO and the main players in the nociceptive activity of the meningeal trigeminal afferents-TRPV1 and P2X3 receptors-remains unstudied. In the current project, the effects of acute and chronic NO administration on the activity of TRPV1 and P2X3 receptors in the peripheral afferents were studied using electrophysiological recording of action potentials of the trigeminal nerve in the rat hemiskull preparations. The data obtained indicate that exogenous and endogenous NO increased the activity of the trigeminal nerve independent on the inhibition of the TRPV1 and P2X3 receptors. The activity of the trigeminal nerve triggered by ATP changed neither in acute incubation in the NO donor-sodium nitroprusside (SNP) nor in the chronic nitroglycerine (NG)-induced migraine model. Moreover, the chronic NG administration did not increase in the number of degranulated mast cells in the rat meninges. At the same time, the capsaicin-induced activity of the trigeminal nerve was higher with chronic NO administration or after acute NO application, and these effects were prevented by N-ethylmaleimide. In conclusion, we suggested that NO positively modulates the activity of TRPV1 receptors by S-nitrosylation, which may contribute to the pro-nociceptive action of NO and underlie the sensitization of meningeal afferents in chronic migraine.

The role of the meningeal lymphatic system in local meningeal inflammation and trigeminal nociception.

A system of lymphatic vessels has been recently characterized in the meninges, with a postulated role in 'cleaning' the brain via cerebral fluid drainage. As meninges are the origin site of migraine pain, we hypothesized that malfunctioning of the lymphatic system should affect the local trigeminal nociception. To test this hypothesis, we studied nociceptive and inflammatory mechanisms in the hemiskull preparations (containing the meninges) of K14-VEGFR3-Ig (K14) mice lacking the meningeal lymphatic system. We recorded the spiking activity of meningeal afferents and estimated the local mast cells population, calcitonin gene-related peptide (CGRP) and cytokine levels as well as the dural trigeminal innervation in freshly-isolated hemiskull preparations from K14-VEGFR3-Ig (K14) or wild type C57BL/6 mice (WT). Spiking activity data have been confirmed in an acquired model of meningeal lymphatic dysfunction (AAV-mVEGFR3(1-4)Ig induced lymphatic ablation). We found that levels of the pro-inflammatory cytokine IL12-p70 and CGRP, implicated in migraine, were reduced in the meninges of K14 mice, while the levels of the mast cell activator MCP-1 were increased. The other migraine-related pro-inflammatory cytokines (basal and stimulated), did not differ between the two genotypes. The patterns of trigeminal innervation in meninges remained unchanged and we did not observe alterations in basal or ATP-induced nociceptive firing in the meningeal afferents associated with meningeal lymphatic dysfunction. In summary, the lack of meningeal lymphatic system is associated with a new balance between pro- and anti-migraine mediators but does not directly trigger meningeal nociceptive state.

Testing the Role of Glutamate NMDA Receptors in Peripheral Trigeminal Nociception Implicated in Migraine Pain.

The pro-nociceptive role of glutamate in the CNS in migraine pathophysiology is well established. Glutamate, released from trigeminal afferents, activates second order nociceptive neurons in the brainstem. However, the function of peripheral glutamate receptors in the trigeminovascular system suggested as the origin site for migraine pain, is less known. In the current project, we used calcium imaging and patch clamp recordings from trigeminal ganglion (TG) neurons, immunolabelling, CGRP assay and direct electrophysiological recordings from rat meningeal afferents to investigate the role of glutamate in trigeminal nociception. Glutamate, aspartate, and, to a lesser extent, NMDA under free-magnesium conditions, evoked calcium transients in a fraction of isolated TG neurons, indicating functional expression of NMDA receptors. The fraction of NMDA sensitive neurons was increased by the migraine mediator CGRP. NMDA also activated slowly desensitizing currents in 37% of TG neurons. However, neither glutamate nor NMDA changed the level of extracellular CGRP. TG neurons expressed both GluN2A and GluN2B subunits of NMDA receptors. In addition, after removal of magnesium, NMDA activated persistent spiking activity in a fraction of trigeminal nerve fibers in meninges. Thus, glutamate activates NMDA receptors in somas of TG neurons and their meningeal nerve terminals in magnesium-dependent manner. These findings suggest that peripherally released glutamate can promote excitation of meningeal afferents implicated in generation of migraine pain in conditions of inherited or acquired reduced magnesium blockage of NMDA channels and support the usage of magnesium supplements in migraine.

Protective Effects of Hydrogen Sulfide Against the ATP-Induced Meningeal Nociception.

We previously showed that extracellular ATP and hydrogen sulfide (H2S), a recently discovered gasotransmitter, are both triggering the nociceptive firing in trigeminal nociceptors implicated in migraine pain. ATP contributes to meningeal nociception by activating the P2X3 subunit-containing receptors whereas H2S operates mainly via TRP receptors. However, H2S was also proposed as a neuroprotective and anti-nociceptive agent. This study aimed to test the effect of H2S on ATP-mediated nociceptive responses in rat meningeal afferents and trigeminal neurons and on ATP-induced degranulation of dural mast cells. Electrophysiological recording of trigeminal nerve activity in meninges was supplemented by patch-clamp and calcium imaging studies of isolated trigeminal neurons. The H2S donor NaHS induced a mild activation of afferents and fully suppressed the subsequent ATP-induced firing of meningeal trigeminal nerve fibers. This anti-nociceptive effect of H2S was specific as an even stronger effect of capsaicin did not abolish the action of ATP. In isolated trigeminal neurons, NaHS decreased the inward currents and calcium transients evoked by activation of ATP-gated P2X3 receptors. Moreover, NaHS prevented ATP-induced P2X7 receptor-mediated degranulation of meningeal mast cells which emerged as triggers of migraine pain. Finally, NaHS decreased the concentration of extracellular ATP in the meningeal preparation. Thus, H2S exerted the multiple protective actions against the nociceptive effects of ATP. These data highlight the novel pathways to reduce purinergic mechanisms of migraine with pharmacological donors or by stimulation production of endogenous H2S.

Antidromic Spike Propagation and Dissimilar Expression of P2X, 5-HT, and TRPV1 Channels in Peripheral vs. Central Sensory Axons in Meninges.

Background: The terminal branches of the trigeminal nerve in meninges are supposed to be the origin site of migraine pain. The main function of these peripheral sensory axons is the initiation and propagation of spikes in the orthodromic direction to the second order neurons in the brainstem. The stimulation of the trigeminal ganglion induces the release of the neuropeptide CGRP in meninges suggesting the antidromic propagation of excitation in these fibers. However, the direct evidence on antidromic spike traveling in meningeal afferents is missing. Methods: By recording of spikes from peripheral or central parts of the trigeminal nerve in rat meninges, we explored their functional activity and tested the expression of ATP-, serotonin-, and capsaicin-gated receptors in the distal vs. proximal parts of these nerves. Results: We show the significant antidromic propagation of spontaneous spikes in meningeal nerves which was, however, less intense than the orthodromic nociceptive traffic due to higher number of active fibers in the latter. Application of ATP, serotonin and capsaicin induced a high frequency nociceptive firing in peripheral processes while, in central parts, only ATP and capsaicin were effective. Disconnection of nerve from trigeminal ganglion dramatically reduced the tonic antidromic activity and attenuated the excitatory action of ATP. Conclusion: Our data indicate the bidirectional nociceptive traffic and dissimilar expression of P2X, 5-HT and TRPV1 receptors in proximal vs. distal parts of meningeal afferents, which is important for understanding the peripheral mechanisms of migraine pain.