P2X7 Signaling Contributes to Meningeal Sensitization

By Fred Schwaller | October 31, 2023 | Posted in

Researchers provide a new explanation for how cortical spreading depression affects afferent nociceptive neurons in the meninges.

Cortical spreading depression (CSD) – a wave of electrical activity across the surface of the brain – is thought to underlie migraine aura, a phenomenon characterized by sensory, visual, and motor disturbances that precede the onset of headache. CSD also excites and increases the sensitivity of afferent nociceptive neurons in the meninges that are responsible for the headache, but the underlying mechanisms have remained unclear.

Now, a new study led by Dan Levy, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, US, provides evidence of a signaling pathway that may underlie this meningeal afferent sensitization.

Using a rat model of CSD, the investigators show that P2X7, a receptor involved in adenosine triphosphate (ATP) signaling, sensitizes nociceptive neurons in the meninges, as measured by the cells’ response to mechanical stimulation. ATP is an organic compound that had previously been shown to act directly on nociceptive afferent neurons via other receptors in the P2X family.

Interestingly, P2X7 played a role only in the sensitization of the afferent neurons, but not in their ongoing activation that takes place after CSD. That finding suggests that researchers need to more carefully consider afferent sensitization and activation as distinct mechanisms during migraine.

“This study provides another key piece to the puzzle of how the brain event, cortical spreading depression, that underlies the migraine aura acts on the sensory neurons in the meninges that are thought to initiate the migraine headache,” according to Andrew Strassman, a pain and headache researcher also at Beth Israel and Harvard but who was not involved with the new work.

The study appeared in the Journal of Neuroscience on August 16, 2023.

P2X7 receptors are to blame
Levy’s group, including first author Jun Zhao and co-author Samantha Harrison, began by modeling migraine aura using a rat CSD model. Here, the investigators induced CSD in anesthetized rats by briefly inserting a glass micropipette into the frontal cortex; this stimulus triggers brain activity akin to migraine aura in a human brain. Then they recorded the electrical activity of individual meningeal afferent neurons to see how CSD might affect the electrical excitability of the neurons.

The team’s initial experiments replicated previous findings that CSD increases the sensitivity of meningeal nociceptors to mechanical stimuli applied to the dura. (In people, this increased mechanical sensitivity is thought to make a headache worse from head movements and other normally harmless activities that can increase pressure inside the skull and mechanically deform the sensitized cells.)

Previous evidence showed that after CSD, a number of nociceptive chemical mediators are released in the brain that can reach the meninges and sensitize the neurons. But which mediators, specifically?

“We know ATP is locally released during CSD, so we thought it might be sensitizing nociceptors in the meninges – either directly or somehow indirectly,” Levy told Migraine Science Collaborative.

To investigate a possible role for ATP signaling, Levy and colleagues applied drugs directly to the meninges. They started by using drugs that block ATP receptors called P2X receptors.

“We focused here on one class of ATP receptors, P2X. There’s old work on P2X2/3 receptors, which are expressed primarily on nociceptive afferents, and if you apply ATP it excites neurons via this receptor. We also looked at another receptor, P2X7, which is more inflammatory related and expressed on some immune cells,” said Levy.

The group began by using a broad-spectrum antagonist to block all P2X receptors. Then they would selectively block P2X7 receptors, and then P2X2/3 receptors. They assessed how these agents affected the meningeal afferents by recording the electrical activity of individual neurons. The investigators recorded from the cell bodies of the afferents, which are located in the trigeminal ganglion.

Applying the broad-spectrum P2X receptor antagonist reduced meningeal afferent sensitization evoked by CSD. This suggested that ATP released in the meninges after CSD sensitizes meningeal afferents via P2X receptors.

To understand which P2X receptor was involved, the team blocked the P2X2/3 receptor, but that had no effect on sensitization of meningeal afferents after CSD. Finally, a P2X7 receptor inhibitor did block CSD-induced sensitization of the afferents.

The investigators also found that inhibiting Panx1, a channel that forms a complex with P2X7, had an effect on afferent sensitization after CSD similar to that of blocking P2X7. Control experiments also showed that the P2X and Panx1 drugs were acting at the meninges and not in the brain.

Overall, the data indicated that P2X7 receptors, but not P2X2/3 receptors, sensitize afferent nociceptors in the meninges, in the CSD model.

Sensitization but not activation
So far, all of the experiments had been performed after CSD had already sensitized meningeal nociceptors. But the researchers hadn’t yet tested whether P2X receptors are also involved with nociceptor activation at the early stages of CSD.

“Activation of meningeal afferents produces headaches. But sensitization of these neurons is a different process; it produces the amplification of the headache, say, during head movements,” said Levy.

So the investigators next analyzed the effect of P2X inhibitors on the baseline ongoing activity of meningeal nociceptors. However, blocking all P2X receptors did not affect their activation post-CSD. When the group got more specific, blocking P2X2/3 receptors, or P2X7 receptors, once again there were no changes in activation of the neurons in response to CSD.

“We were expecting the P2X3 receptor to be involved at least with activation of the afferents, but it wasn’t. But these data do show that inhibiting P2X7 receptors blocked sensitization of nociceptors during CSD, but not their activation,” according to Levy.

How does ATP reach the meninges during aura?
Together, Levy’s findings indicate that ATP signaling in the meninges is important for driving the sensitization of meningeal nociceptive afferents that spurs migraine headache pain during normally harmless physical activities, at least in the study’s CSD model.

According to Strassman, the paper opens up many exciting questions, but the one that makes him most curious is how ATP reaches the meninges during aura to sensitize meningeal nociceptors.

“Could ATP somehow flow in the cerebrospinal fluid from the brain to get to the meninges? There’s a separate line of work that struck me that’s relevant for the authors’ findings, and that’s related to the glymphatic system,” said Strassman.

The glymphatic system is a system for waste clearance in the central nervous system, similar to the lymphatic system in the body. Waste is cleared in cerebrospinal fluid that flows in the perivascular space around cerebral arteries.

Glial cells, especially astrocytes, are non-neuronal cells critical for the function of the glymphatic system and draining out the waste. Strassman suspects that astrocytes, and their known role in ATP-P2X7 signaling, may be important for headache pain during CSD.

“Importantly for migraine, this glymphatic system goes through the meninges, meaning it could be a potential path for chemicals to reach the meninges during migraine aura,” he said.

However, Strassman noted it’s also possible that ATP might originate from cells in the meninges themselves in response to other mediators that are released from the brain during aura.

“This isn’t really the topic of the paper, but it’s an example of a fascinating question in front of us,” said Strassman.

There’s even more to the story
Levy thinks the immune system could be a crucial factor in sensitizing meningeal nociceptive neurons during migraine aura. His group has been building the case for some time that inflammatory signaling in the meninges plays a key role in migraine.

“Research, at least in animals, has shown that macrophages in the meninges get activated after CSD. What’s important for our study is that P2X7 activation affects macrophages, so maybe there’s a link between the macrophage immune response and migraine,” said Levy.

While Levy is aiming to pursue this question, he also suspects other cell types are involved. That line of inquiry focuses on what happens downstream of ATP-P2X7 signaling, but it could also illuminate upstream mechanisms of ATP signaling. A link here could be astrocytes.

“ATP could prime astrocytes, too. Our previous work has shown that if you block astrocytic function, you block the sensitization but not the activation of meningeal nociceptors,” said Levy.

In the end, the study identifies a culprit for driving headache after migraine aura, but also shows that the mechanism is far from simple. There could be many factors to blame.

Fred Schwaller, PhD, is a freelance science writer based in Germany. Follow him on Twitter @SchwallerFred

Image credit: zodyak53/123RF Stock Photo.

Meningeal P2X7 signaling mediates migraine-related intracranial mechanical hypersensitivity.
Zhao et al.
J Neurosci. 2023 Aug 16;43(33):5975-85.

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Fred Schwaller is a science writer and communicator based in Berlin, Germany. Fred spent a decade in pain research during his doctoral degree at University College London, UK, and his postdoc at the Max Delbrück Centre in Berlin, Germany. After transferring to science communication in 2020, he has been writing and podcasting about life sciences and medicine, specializing in somatosensation and pain. Follow him on Twitter @SchwallerFred.



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