Over the past two decades, a convergence of basic and clinical evidence has established the neuropeptide calcitonin-gene-related peptide (CGRP) as a key player in migraine. Although CGRP is a recognised neuromodulator of nociception, its mechanism of action in migraine remains elusive. In this review, we present evidence that led us to propose that CGRP is well poised to enhance neurotransmission in migraine by both peripheral and central mechanisms. In the periphery, it is thought that local release of CGRP from the nerve endings of meningeal nociceptors following their initial activation by cortical spreading depression is critical for the induction of vasodilation, plasma protein extravasation, neurogenic inflammation and the consequential sensitisation of meningeal nociceptors. Mechanistically, we propose that CGRP release can give rise to a positive-feedback loop involved in localised increased synthesis and release of CGRP from neurons and a CGRP-like peptide called procalcitonin from trigeminal ganglion glia. Within the brain, the wide distribution of CGRP and CGRP receptors provides numerous possible targets for CGRP to act as a neuromodulator.

Background Calcitonin gene-related peptide (CGRP) is a neuropeptide that acts in the trigeminovascular system and is believed to play an important role in migraine. CGRP activates two receptors that are both present in the trigeminovascular system; the CGRP receptor and the amylin 1 (AMY) receptor. CGRP receptor antagonists, including olcegepant (BIBN4096BS) and telcagepant (MK-0974), can treat migraine. This study aimed to determine the effectiveness of these antagonists at blocking CGRP receptor signalling in trigeminal ganglia (TG) neurons and transfected CGRP and AMY receptors in Cos7 cells, to better understand their mechanism of action. Methods CGRP stimulation of four intracellular signalling molecules relevant to pain (cAMP, CREB, p38 and ERK) were examined in rat TG neurons and compared to transfected CGRP and AMY receptors in Cos7 cells. Results In TG neurons, olcegepant displayed signal-specific differences in antagonism of CGRP responses. This effect was also evident in transfected Cos7 cells, where olcegepant blocked CREB phosphorylation more potently than expected at the AMY receptor, suggesting that the affinity of this antagonist can be dependent on the signalling pathway activated. Conclusions CGRP receptor antagonist activity appears to be assay-dependent. Thus, these molecules may not be as selective for the CGRP receptor as commonly reported.

Background The neuropeptide calcitonin gene–related peptide (CGRP) plays a key role in migraine. CGRP gene expression involves an enhancer that is active in neurons, yet inactive in glia. In this report, we analyze epigenetic modifications that allow enhancer activation in glia. Methods DNA methylation and histone acetylation states were measured in rat and human-model cell lines and primary cultures of rat trigeminal ganglia glia. The functional consequence of altering the chromatin state was determined by quantitative measurements of both calcitonin (CT) and CGRP mRNAs. Results A hypermethylated CpG island flanking the enhancer was identified in glia and non-expressing cell lines. In addition, the chromatin was hypoacetylated. Treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine induced CT mRNA ~30-fold in glial cultures. Treatment with a histone deacetylase inhibitor alone had little effect; however, the combination of inhibitors yielded a synergistic ~80-fold increase in CT and ~threefold increase in CGRP mRNA. Treated glia contained CT precursor (pro-CT) immunoreactivity. Conclusions Epigenetic modulation is sufficient to induce the CGRP gene in glia. Because the CGRP gene is systemically activated by inflammatory conditions, this suggests that glial pro-CT may be an unexplored biomarker during migraine.

Objective To examine calcitonin gene-related peptide (CGRP) gene expression under inflammatory conditions using trigeminal ganglia organ cultures as an experimental system. These cultures have increased proinflammatory signaling that may mimic neurogenic inflammation in the migraine state. Background The trigeminal nerve sends peripheral pain signals to the central nervous system during migraine. Understanding the dynamic processes that occur within the trigeminal nerve and ganglion may provide insights into events that contribute to migraine pain. A neuropeptide of particular interest is CGRP, which can be elevated and play a causal role in migraine. However, most studies have overlooked a second splice product of the CALCA gene, which encodes calcitonin (CT), a peptide hormone involved in calcium homeostasis. Importantly, a precursor form of calcitonin called procalcitonin (proCT) can act as a partial agonist at the CGRP receptor and elevated proCT has recently been reported during migraine. Methods We used a trigeminal ganglion whole organ explant model, which has previously been demonstrated to induce pro-inflammatory agents in vitro. Quantitative PCR and immunohistochemistry were used to evaluate changes in mRNA and protein levels of CGRP and proCT. Results Whole mouse trigeminal ganglia cultured for 24 h showed a 10-fold increase in CT mRNA, with no change in CGRP mRNA. A similar effect was observed in ganglia from adult rats. ProCT immunoreactivity was localized in glial cells. Cutting the tissue blunted the increase in CT, suggesting that induction required the close environment of the intact ganglia. Consistent with this prediction, there were increased reactive oxygen species in the ganglia and the elevated CT mRNA was reduced by antioxidant treatment. Surprisingly, reactive oxygen species were increased in neurons, not glia. Conclusions These results demonstrate that reactive oxygen species can activate proCT expression from the CGRP gene in trigeminal glia by a paracrine regulatory mechanism. We propose that this glial recruitment pathway may occur following cortical spreading depression and neurogenic inflammation to increase CGRP nociceptive actions in migraine.

Objective The neuropeptide calcitonin gene-related peptide (CGRP) has now been established as a key player in migraine. However, the mechanisms underlying the reported elevation of CGRP in the serum and cerebrospinal fluid of some migraineurs are not known. A candidate mechanism is cortical spreading depression (CSD), which is associated with migraine with aura and traumatic brain injury. The aim of this study was to investigate whether CGRP gene expression may be induced by experimental CSD in the rat cerebral cortex. Methods CSD was induced by topical application of KCl and monitored using electrophysiological methods. Quantitative PCR and ELISA were used to measure CGRP mRNA and peptide levels in discrete ipsilateral and contralateral cortical regions of the rat brain 24 hours following CSD events and compared with sham treatments. Results The data show that multiple, but not single, CSD events significantly increase CGRP mRNA levels at 24 hours post-CSD in the ipsilateral rat cerebral cortex. Increased CGRP was observed in the ipsilateral frontal, motor, somatosensory, and visual cortices, but not the cingulate cortex, or contralateral cortices. CSD also induced CGRP peptide expression in the ipsilateral, but not contralateral, cortex. Conclusions Repeated CSD provides a mechanism for prolonged elevation of CGRP in the cerebral cortex, which may contribute to migraine and post-traumatic headache.