Identifying New Antimigraine Targets: Lessons from Molecular Biology
Authors: Lars Edvinsson, Kristian Agmund Haanes
Journal: Trends in Pharmacological Sciences (2021)

Neuroimaging experiments propose that the migraine attack originates in the hypothalamus. A further mismatch in the communication or tuning of the trigeminovascular system, might lead to calcitonin gene-related peptide (CGRP) release, sensitization, and migraine pain.Potential targets for CGRP include the middle meningeal artery, satellite glial cells, and importantly Aδ neurons. The CGRP receptor is also expressed in the node of Ranvier, and the current hypothesis recognizes this novel site as a regulator of excitability.At the molecular level, new targets can be found among modulators of excitability, both on the C fibers (to prevent neuropeptide release) and on Aδ fibers (to reduce action potential generation and sensitization), with novel targets being GPCRs or ion channels localized at the node of Ranvier. Primary headaches are one of the most common conditions; migraine being most prevalent. Recent work on the pathophysiology of migraine suggests a mismatch in the communication or tuning of the trigeminovascular system, leading to sensitization and the release of calcitonin gene-related peptide (CGRP). In the current Opinion, we use the up-to-date molecular understanding of mechanisms behind migraine pain, to provide novel aspects on how to modify the system and for the development of future treatments; acute as well as prophylactic. We explore the distribution and the expression of neuropeptides themselves, as well as certain ion channels, and most importantly how they may act in concert as modulators of excitability of both the trigeminal C neurons and the Aδ neurons. Primary headaches are one of the most common conditions; migraine being most prevalent. Recent work on the pathophysiology of migraine suggests a mismatch in the communication or tuning of the trigeminovascular system, leading to sensitization and the release of calcitonin gene-related peptide (CGRP). In the current Opinion, we use the up-to-date molecular understanding of mechanisms behind migraine pain, to provide novel aspects on how to modify the system and for the development of future treatments; acute as well as prophylactic. We explore the distribution and the expression of neuropeptides themselves, as well as certain ion channels, and most importantly how they may act in concert as modulators of excitability of both the trigeminal C neurons and the Aδ neurons. Primary headaches represent up to 300 separate diagnoses, with migraine being the most prevalent of these, with a prevalence of 15% in the global population. It is predominant in women (two to three times more than in men) and most frequent in the most active ages (18–50 years) [1.Headache Classification Committee of the International Headache Society The International Classification of Headache Disorders, 3rd edn.Cephalalgia. 2018; 38: 1-211Crossref PubMed Scopus (1535) Google Scholar,2.Feigin V.L. et al.The global burden of neurological disorders: translating evidence into policy.Lancet Neurol. 2020; 19: 255-265Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar]. Studies of the global burden of diseases, injuries, and risk factors have demonstrated that migraine is the leading cause of years of life lived with disabilities in under 50s in both sexes [3.Steiner T.J. et al.Migraine is first cause of disability in under 50s: will health politicians now take notice?.J. Headache Pain. 2018; 19: 17Crossref PubMed Scopus (236) Google Scholar]. Migraine is still characterized by symptoms [1.Headache Classification Committee of the International Headache Society The International Classification of Headache Disorders, 3rd edn.Cephalalgia. 2018; 38: 1-211Crossref PubMed Scopus (1535) Google Scholar] since no clear biomarker profile has emerged. A migraine attack can often be divided into four phases; (i) a premonitory phase on the day before the attack; (ii) early aura (in 20% of the patients); (iii) headache phase dominated by moderate–severe headache; and (iv) the postdromal phase [1.Headache Classification Committee of the International Headache Society The International Classification of Headache Disorders, 3rd edn.Cephalalgia. 2018; 38: 1-211Crossref PubMed Scopus (1535) Google Scholar,4.Dodick D.W. A phase-by-phase review of migraine pathophysiology.Headache. 2018; 58: 4-16Crossref PubMed Scopus (132) Google Scholar]. The underlying pathophysiology is still unclear, and consequently, over the years several hypotheses have occurred to explain the complexity of the biology of migraine. (i) The vascular theory of migraine. The attack starts in the cranial vasculature and has dominated the past few decades and still pertains in intravenous infusion studies [5.Ashina M. et al.Human models of migraine - short-term pain for long-term gain.Nat. Rev. Neurol. 2017; 13: 713-724Crossref PubMed Scopus (80) Google Scholar]. (ii) More recent work with detailed functional neuroimaging proposes that the migraine attack originates in the hypothalamus, connects to central nervous system (CNS) regions such as the thalamus and brainstem, and involves typical neurological aspects [6.Tfelt-Hansen P.C. Koehler P.J. One hundred years of migraine research: major clinical and scientific observations from 1910 to 2010.Headache. 2011; 51: 752-778Crossref PubMed Scopus (64) Google Scholar]. A study by Schulte and May showed, upon daily imaging of a single migraine patient, that the hypothalamus was active during 24 h preceding the migraine attack [7.Schulte L.H. May A. The migraine generator revisited: continuous scanning of the migraine cycle over 30 days and three spontaneous attacks.Brain. 2016; 139: 1987-1993Crossref PubMed Scopus (275) Google Scholar]. The activity was coupled/connected to the brainstem (e.g., the trigeminal nucleus caudalis, TNC) during the ictal stage (see Glossary), and similar data on hypothalamic connectivity has been reported to occur prior to a migraine attack [8.Meylakh N. et al.Altered regional cerebral blood flow and hypothalamic connectivity immediately prior to a migraine headache.Cephalalgia. 2020; 40: 448-460Crossref PubMed Scopus (8) Google Scholar]. We speculate that changes in the TNC causes further activation, potentiation, or modification of the function in the trigeminal ganglion (TG), either by direct activation or by modulation of incoming sensory information. This proposal is likely since the TNC is out of reach of current antimigraine medications since it is protected by the blood–brain barrier (BBB) [9.Eftekhari S. et al.Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.Brain Res. 2015; 1600: 93-109Crossref PubMed Scopus (138) Google Scholar, 10.Lundblad C. et al.Experimental inflammation following dural application of complete Freund's adjuvant or inflammatory soup does not alter brain and trigeminal microvascular passage.J. Headache Pain. 2015; 16: 91Crossref PubMed Scopus (35) Google Scholar, 11.Noseda R. et al.Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.Cephalalgia. 2020; 40: 229-240Crossref PubMed Scopus (21) Google Scholar]. A further mismatch in the communication or tuning of the trigeminovascular system (TVS), might lead to sensitization and possibly triggering the release of CGRP and the migraine attack is no longer only central. Here, we describe how current views on mechanisms behind migraine and migraine pain, particularly on a molecular level, can be used to provide novel aspects about how to modify the system and aid in development of novel treatments. By applying this approach, we propose ways for potential new antimigraine therapeutic targets. Although we do not consider that finding a cure for migraine lies within the TVS, this is still the area where novel reachable targets reside. Curing migraine would probably involve targeting CNS structures or genetic engineering, but that is outside of the scope of this paper. Within the trigeminal system there are many cellular targets that in part can explain the migraine pathophysiology that so far mostly involves the CGRP signaling system [12.Edvinsson J.C.A. et al.The fifth cranial nerve in headaches.J. Headache Pain. 2020; 21: 65Crossref PubMed Scopus (27) Google Scholar]. The original link between CGRP and migraine has its basis in the neurogenic inflammation theory [13.Waeber C. Moskowitz M.A. Migraine as an inflammatory disorder.Neurology. 2005; 64: S9-15Crossref PubMed Google Scholar] and laid the approach for the early approaches. This theory was based on the release of tachykinins and CGRP, leading to vasodilation, mast cell degranulation, and plasma protein extravasation, and finally causing pain [13.Waeber C. Moskowitz M.A. Migraine as an inflammatory disorder.Neurology. 2005; 64: S9-15Crossref PubMed Google Scholar,14.Levy D. Migraine pain, meningeal inflammation, and mast cells.Curr. Pain Headache Rep. 2009; 13: 237-240Crossref PubMed Scopus (117) Google Scholar]. This view is now abandoned because selective neurokinin receptor blockers are ineffective as antimigraine therapy [15.Edvinsson L. et al.Does inflammation have a role in migraine?.Nat. Rev. Neurol. 2019; 15: 483-490Crossref PubMed Scopus (79) Google Scholar]. In addition, recent work has clearly shown that CGRP-directed antibodies cannot prevent cortical-spreading-depression-induced dilatation, and plasma protein extravasation is unaffected by the anti-CGRP monoclonal antibody (mAb) fremanezumab [16.Schain A.J. et al.CSD-induced arterial dilatation and plasma protein extravasation are unaffected by fremanezumab: implications for CGRP's role in migraine with aura.J. Neurosci. 2019; 39: 6001-6011Crossref PubMed Scopus (19) Google Scholar]. Furthermore, human (unlike rodent) mast cells, do not express a functional CGRP receptor or show a CGRP response [17.Eftekhari S. et al.Differentiation of nerve fibers storing CGRP and CGRP receptors in the peripheral trigeminovascular system.J. Pain. 2013; 14: 1289-1303Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar]. Hence, the neurogenic inflammation theory most likely describes some epiphenomena but does not explain the link between CGRP and the genuine migraine attack. There is an increasing amount of data on neuropeptides being modulators of excitability, and we propose that understanding trigeminal excitability is the missing link to understanding how CGRP contributes to sensation of pain (Figure 1, Key Figure). We explore the effect of neuropeptides themselves, but most importantly as modulators of excitability, both on the C fibers (to prevent neuropeptide release) and on Aδ fibers (to reduce action potential generation and sensitization). In 1985, CGRP, stored in the TG and present in cranial nerve fibers, was suggested to be a key molecule involved in migraine [18.Edvinsson L. Functional-role of perivascular peptides in the control of cerebral-circulation.Trends Neurosci. 1985; 8: 126-131Abstract Full Text PDF Scopus (173) Google Scholar], and a few years later shown to be a neuronal messenger selectively released in migraine and cluster headache attacks [19.Goadsby P.J. Edvinsson L. The trigeminovascular system and migraine: studies characterizing cerebrovascular and neuropeptide changes seen in humans and cats.Ann. Neurol. 1993; 33: 48-56Crossref PubMed Scopus (909) Google Scholar,20.Goadsby P.J. Edvinsson L. Human in vivo evidence for trigeminovascular activation in cluster headache. Neuropeptide changes and effects of acute attacks therapies.Brain. 1994; 117: 427-434Crossref PubMed Scopus (538) Google Scholar]. The CGRP finding was robust and supported by studies from several research groups, and resulted in the development of a new class of antimigraine drugs for acute treatment, the gepants, and for preventive drugs consisting of mAbs against CGRP or the CGRP receptor, which took almost 25 years [21.Edvinsson L. et al.CGRP as the target of new migraine therapies - successful translation from bench to clinic.Nat. Rev. Neurol. 2018; 14: 338-350Crossref PubMed Scopus (329) Google Scholar]. The TG consists of neurons storing CGRP (C fibers) and others expressing CGRP receptors (Aδ fibers), as well as numerous satellite glial cells [22.Eftekhari S. et al.Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion.Neuroscience. 2010; 169: 683-696Crossref PubMed Scopus (202) Google Scholar]. The new drugs directed at the CGRP system likely act at these sites as they do not pass the BBB [11.Noseda R. et al.Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.Cephalalgia. 2020; 40: 229-240Crossref PubMed Scopus (21) Google Scholar]. Debate has been heated in discussions whether the gepants and mAbs have sites of action in the CNS. Our understanding is that they do not pass the BBB to any significant degree, exemplified by experiments with different tracers that have refuted their effects in the CNS [9.Eftekhari S. et al.Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.Brain Res. 2015; 1600: 93-109Crossref PubMed Scopus (138) Google Scholar, 10.Lundblad C. et al.Experimental inflammation following dural application of complete Freund's adjuvant or inflammatory soup does not alter brain and trigeminal microvascular passage.J. Headache Pain. 2015; 16: 91Crossref PubMed Scopus (35) Google Scholar, 11.Noseda R. et al.Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.Cephalalgia. 2020; 40: 229-240Crossref PubMed Scopus (21) Google Scholar,23.Edvinsson L. CGRP receptor antagonists and antibodies against CGRP and its receptor in migraine treatment.Br. J. Clin. Pharmacol. 2015; 80: 193-199Crossref PubMed Scopus (119) Google Scholar]. Neurons containing CGRP give rise to unmyelinated C fibers that are distributed both intra- and extracranially [24.Edvinsson L. Tracing neural connections to pain pathways with relevance to primary headaches.Cephalalgia. 2011; 31: 737-747Crossref PubMed Scopus (49) Google Scholar]. With specific antibodies, it has been revealed that about half of the neurons contain CGRP, while the others store CGRP receptors [9.Eftekhari S. et al.Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.Brain Res. 2015; 1600: 93-109Crossref PubMed Scopus (138) Google Scholar,22.Eftekhari S. et al.Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion.Neuroscience. 2010; 169: 683-696Crossref PubMed Scopus (202) Google Scholar,25.Eftekhari S. et al.Localization of CGRP receptor components and receptor binding sites in rhesus monkey brainstem: A detailed study using in situ hybridization, immunofluorescence, and autoradiography.J. Comp. Neurol. 2016; 524: 90-118Crossref PubMed Scopus (44) Google Scholar]. CGRP binds to CLR/RAMP1 (the CGRP receptor), which are both widely distributed in the TVS [17.Eftekhari S. et al.Differentiation of nerve fibers storing CGRP and CGRP receptors in the peripheral trigeminovascular system.J. Pain. 2013; 14: 1289-1303Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar], but could also activate the amylin receptor (CTR/RAMP1) although with lower potency [26.Hay D.L. Amylin.Headache. 2017; 57: 89-96Crossref PubMed Scopus (21) Google Scholar]. Amylin signaling is not covered in depth here, but has been reported to be a part of the TVS [27.Walker C.S. Hay D.L. CGRP in the trigeminovascular system: a role for CGRP, adrenomedullin and amylin receptors?.Br. J. Pharmacol. 2013; 170: 1293-1307Crossref PubMed Scopus (57) Google Scholar]. Amylin receptors could therefore play a part as a CGRP target, and indeed some antimigraine drugs, such as rimegepant, also bind the amylin receptor [28.Pan K.S. et al.Antagonism of CGRP signaling by rimegepant at two receptors.Front. Pharmacol. 2020; 11: 1240Crossref PubMed Scopus (11) Google Scholar]. Here, we focus further on the potential cellular targets for CGRP, which include the middle meningeal artery [29.Gupta S. et al.Characterisation of CGRP receptors in the human isolated middle meningeal artery.Life Sci. 2006; 79: 265-271Crossref PubMed Scopus (41) Google Scholar], satellite glial cells [30.Thalakoti S. et al.Neuron-glia signaling in trigeminal ganglion: implications for migraine pathology.Headache. 2007; 47: 1008-1023Crossref PubMed Scopus (210) Google Scholar], and importantly, Aδ neurons [31.Melo-Carrillo A. et al.Fremanezumab-A humanized monoclonal anti-CGRP antibody-inhibits thinly myelinated (Adelta) but not unmyelinated (C) meningeal nociceptors.J. Neurosci. 2017; 37: 10587-10596Crossref PubMed Scopus (68) Google Scholar]. There are some data showing that pituitary adenylate cyclase-activating polypeptide (PACAP) is increased during a migraine attack, but notably this is from a significantly lower baseline [32.Tuka B. et al.Alterations in PACAP-38-like immunoreactivity in the plasma during ictal and interictal periods of migraine patients.Cephalalgia. 2013; 33: 1085-1095Crossref PubMed Scopus (111) Google Scholar,33.Zagami A.S. et al.Pituitary adenylate cyclase activating polypeptide and migraine.Ann. Clin. Transl. Neurol. 2014; 1: 1036-1040Crossref PubMed Scopus (76) Google Scholar], and migraine-like attacks can be induced in humans by both PACAP-38 and PACAP-27 [34.Amin F.M. et al.Investigation of the pathophysiological mechanisms of migraine attacks induced by pituitary adenylate cyclase-activating polypeptide-38.Brain. 2014; 137: 779-794Crossref PubMed Scopus (119) Google Scholar,35.Ghanizada H. et al.PACAP27 induces migraine-like attacks in migraine patients.Cephalalgia. 2019; 40: 57-67Crossref PubMed Scopus (28) Google Scholar]. In contrast, vasoactive intestinal peptide (VIP) is not released in migraine and does not cause migraine-like attacks [19.Goadsby P.J. Edvinsson L. The trigeminovascular system and migraine: studies characterizing cerebrovascular and neuropeptide changes seen in humans and cats.Ann. Neurol. 1993; 33: 48-56Crossref PubMed Scopus (909) Google Scholar,36.Rahmann A. et al.Vasoactive intestinal peptide causes marked cephalic vasodilation, but does not induce migraine.Cephalalgia. 2008; 28: 226-236Crossref PubMed Scopus (175) Google Scholar,37.Goadsby P.J. et al.Vasoactive peptide release in the extracerebral circulation of humans during migraine headache.Ann. Neurol. 1990; 28: 183-187Crossref PubMed Scopus (1131) Google Scholar]. Recently, a long-term infusion of VIP was shown to cause headache in healthy volunteers [38.Pellesi L. et al.Two-hour infusion of vasoactive intestinal polypeptide induces delayed headache and extracranial vasodilation in healthy volunteers.Cephalalgia. 2020; 40: 1212-1223Crossref PubMed Scopus (11) Google Scholar], and this protocol needs testing in migraine patients. Furthermore, PACAP does not appear to be present in the rodent dura mater, and its release from the TG is lower than that observed for CGRP [39.Edvinsson J.C.A. et al.Differences in pituitary adenylate cyclase-activating peptide and calcitonin gene-related peptide release in the trigeminovascular system.Cephalalgia. 2020; 40: 1296-1309Crossref PubMed Scopus (6) Google Scholar]. The results from the human anti-PAC1 antibody trial were negative when it came to migraine prophylaxis [40.Ashina M. et al.A phase 2, randomized, double-blind, placebo-controlled trial of AMG 301, a pituitary adenylate cyclase-activating polypeptide PAC1 receptor monoclonal antibody for migraine prevention.Cephalalgia. 2020; 41: 33-44Crossref PubMed Scopus (10) Google Scholar]. Alder (acquired by Lundbeck) has an anti-PACAP antibody (ALD1910) under development [41.Moldovan L.C. et al.Pharmacologic characterization of ALD1910, a potent humanized monoclonal antibody against the pituitary adenylate cyclase activating peptide.J. Pharmacol. Exp. Ther. 2019; 369: 26-36Crossref PubMed Scopus (25) Google Scholar]. This approach does not require activity at the PAC1 receptor, but could in addition prevent activation of both of VIP receptors VPAC1 and VPAC2. It has been suggested that an antimigraine effect could be mediated by other receptors, such as MrgX2, which is the human equivalent of the MrgB3 receptor [42.Pedersen S.H. et al.PACAP-38 and PACAP(6-38) degranulate rat meningeal mast cells via the orphan MrgB3-receptor.Front. Cell. Neurosci. 2019; 13: 114Crossref PubMed Scopus (13) Google Scholar]. The results from the clinical trial of ALD1910 will provide the final conclusion of the involvement of PACAP in migraine pathophysiology. Since an increase in CGRP release is associated with migraine, reducing its release is an obvious medical target. Indeed, the current view on triptans, the currently most used specific acute medication, is that they exert their effects through presynaptic inhibition of CGRP release from trigeminal sensory neurons [43.Goadsby P.J. Edvinsson L. Joint 1994 Wolff Award Presentation. Peripheral and central trigeminovascular activation in cat is blocked by the serotonin (5HT)-1D receptor agonist 311C90.Headache. 1994; 34: 394-399Crossref PubMed Scopus (166) Google Scholar, 44.Humphrey P.P. The discovery of a new drug class for the acute treatment of migraine.Headache. 2007; 47: S10-S19Crossref PubMed Scopus (56) Google Scholar, 45.Haanes K.A. Edvinsson L. Pathophysiological mechanisms in migraine and the identification of new therapeutic targets.CNS Drugs. 2019; 33: 525-537Crossref PubMed Scopus (42) Google Scholar]. Triptans lead to activation of Gi-coupled 5-HT1B/1D receptors, reducing the intracellular cAMP [46.Adham N. et al.The rat 5-hydroxytryptamine1B receptor is the species homologue of the human 5-hydroxytryptamine1D beta receptor.Mol. Pharmacol. 1992; 41: 1-7PubMed Google Scholar]. This leads to reduced excitability of the C fibers and reduced CGRP release. Recently, we investigated whether similar mechanisms also applied to PACAP release. The induced PACAP release only occurred in the TG and not from its peripheral ramifications in the dura mater. The PACAP release was lower than that of CGRP; it was a challenge to demonstrate a significant inhibition by sumatriptan on CGRP release with capsaicin stimulation and the results were inconclusive, with only a tendency toward inhibition [39.Edvinsson J.C.A. et al.Differences in pituitary adenylate cyclase-activating peptide and calcitonin gene-related peptide release in the trigeminovascular system.Cephalalgia. 2020; 40: 1296-1309Crossref PubMed Scopus (6) Google Scholar]. Although the role of substance P in migraine was abandoned as the NK1 inhibitors showed no effect, there are still some older data suggesting that triptans in some animal models inhibit substance P release [47.Arvieu L. et al.Sumatriptan inhibits the release of CGRP and substance P from the rat spinal cord.Neuroreport. 1996; 7: 1973-1976Crossref PubMed Scopus (61) Google Scholar]. The combined data show that targeting Gi-coupled receptors and lowering cAMP reduce neuropeptide release, with the reduction in CGRP release giving the most likely clinical effect for migraine patients. There are currently two good models to screen for compounds targeting CGRP release. The in vitro/ex vivo approach uses a rat hemi skull model and TG setup, where CGRP release is stimulated either with depolarization through increases in the extracellular K+ [39.Edvinsson J.C.A. et al.Differences in pituitary adenylate cyclase-activating peptide and calcitonin gene-related peptide release in the trigeminovascular system.Cephalalgia. 2020; 40: 1296-1309Crossref PubMed Scopus (6) Google Scholar], or capsaicin can be used to trigger TRPV1 channels [48.Meents J.E. et al.Two TRPV1 receptor antagonists are effective in two different experimental models of migraine.J. Headache Pain. 2015; 16: 57Crossref PubMed Scopus (21) Google Scholar]. A model that is closer to the real world CGRP release is the cranial window model, where the nerves surrounding the middle meningeal artery are stimulated to release CGRP [49.Haanes K.A. et al.Exploration of purinergic receptors as potential anti-migraine targets using established pre-clinical migraine models.Cephalalgia. 2019; 39: 1421-1434Crossref PubMed Scopus (11) Google Scholar]. These models have revealed that sumatriptan has inhibitory effects [50.Gupta S. et al.Intravital microscopy on a closed cranial window in mice: a model to study trigeminovascular mechanisms involved in migraine.Cephalalgia. 2006; 26: 1294-1303Crossref PubMed Scopus (37) Google Scholar], and are useful in studying future drug targets. Some of the currently used triptans additionally bind to the 5-HT1F receptor [51.Rubio-Beltran E. et al.Is selective 5-HT1F receptor agonism an entity apart from that of the triptans in antimigraine therapy?.Pharmacol. Ther. 2018; 186: 88-97Crossref PubMed Scopus (51) Google Scholar]. The further developments in targeting the 5-HT1F receptor have led to the development of LY344864 and LY334370. These drugs belong to a new class called neutrally acting antimigraine agents (NAAMAs) or ditans for the acute treatment of migraine with low affinity for the 5-HT1B/1D receptors. These drugs were developed as specific 5-HT1F agonists that prevent dural protein extravasation and are without significant vasoconstrictor effects [52.Rubio-Beltran E. et al.Characterization of binding, functional activity, and contractile responses of the selective 5-HT1F receptor agonist lasmiditan.Br. J. Pharmacol. 2019; 176: 4681-4695Crossref PubMed Scopus (26) Google Scholar]. The 5-HT1F agonist developments have led to LY573144, which is now approved by the FDA and EMA for use in the clinic as lasmiditan for acute treatment of migraine attacks [53.Kuca B. et al.Lasmiditan is an effective acute treatment for migraine: A phase 3 randomized study.Neurology. 2018; 91: e2222-e2232Crossref PubMed Scopus (123) Google Scholar]. Mechanistically, this drug acts via the intracellular signaling pathway, the 5-HT1F receptor, which is a Gi protein coupled receptor, and prevents the activated release of CGRP from the TVS [54.Labastida-Ramirez A. et al.Lasmiditan inhibits calcitonin gene-related peptide release in the rodent trigeminovascular system.Pain. 2020; 161: 1092-1099Crossref PubMed Scopus (26) Google Scholar], showing the validity of this approach. A side-note to developing Gi-targeted receptors is the risk of hyperalgesic priming [55.Araldi D. et al.Gi-protein-coupled 5-HT1B/D receptor agonist sumatriptan induces type I hyperalgesic priming.Pain. 2016; 157: 1773-1782Crossref PubMed Scopus (26) Google Scholar], which is typically seen with several pain-preventing medications. Although the mechanisms are not fully known, Gi-coupled activation could be linked to medication overuse headache [56.Holland P.R. et al.Medication overuse headache: divergent effects of new acute antimigraine drugs.Cephalalgia. 2020; 40: 889-891Crossref PubMed Scopus (2) Google Scholar], and must be considered with this approach. With the current success of the novel migraine medications directly targeting CGRP/CGRP receptors, we propose that the migraine pain must originate from the Aδ fibers, as these pain-transmitting neurons are the only nerve fibers containing CGRP receptors in the periphery [9.Eftekhari S. et al.Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.Brain Res. 2015; 1600: 93-109Crossref PubMed Scopus (138) Google Scholar,17.Eftekhari S. et al.Differentiation of nerve fibers storing CGRP and CGRP receptors in the peripheral trigeminovascular system.J. Pain. 2013; 14: 1289-1303Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar,25.Eftekhari S. et al.Localization of CGRP receptor components and receptor binding sites in rhesus monkey brainstem: A detailed study using in situ hybridization, immunofluorescence, and autoradiography.J. Comp. Neurol. 2016; 524: 90-118Crossref PubMed Scopus (44) Google Scholar]. This view is supported by results from Burstein who showed that CGRP mAbs have antimigraine effects by preventing binding of CGRP to trigeminal Aδ neurons and thereby act to prevent the response to CGRP [31.Melo-Carrillo A. et al.Fremanezumab-A humanized monoclonal anti-CGRP antibody-inhibits thinly myelinated (Adelta) but not unmyelinated (C) meningeal nociceptors.J. Neurosci. 2017; 37: 10587-10596Crossref PubMed Scopus (68) Google Scholar]. When CGRP binds to the CGRP receptor (CLR/RAMP1) the Gαs subunit activates adenylyl cyclase, which in turn catalyzes the conversion of ATP into the second messenger cAMP. Increasing cytosolic cAMP concentration results in activation of protein kinase A (PKA) [57.Egea S.C. Dickerson I.M. Direct interactions between calcitonin-like receptor (CLR) and CGRP-receptor component protein (RCP) regulate CGRP receptor signaling.Endocrinology. 2012; 153: 1850-1860Crossref PubMed Scopus (35) Google Scholar]. By PKA activation, CGRP regulates ion channels (for sodium and potassium) essential for nerve signal propagation [58.Scheuer T. Regulation of sodium channel activity by phosphorylation.Semin. Cell Dev. Biol. 2011; 22: 160-165Crossref PubMed Scopus (57) Google Scholar] and contributes to mechanisms of neural sensitization and/or increased intensity of pain transmission in migraine. When CGRP increases cAMP in Aδ fibers these can become hyperexcitable for other stimuli [59.Hurley J.H. et al.Role of intraganglionic transmission in the trigeminovascular pathway.Mol. Pain. 2019; 151744806919836570Crossref Scopus (11) Google Scholar]. Aδ fibers are known to be high-threshold receptors; they are activated