REVIEW

Neuropeptide Effects in the Trigeminal System: Pathophysiology
and Clinical Relevance in Migraine
Karl Messlinger1, Michael J. M. Fischer2 and Jochen K. Lennerz3
1Institute of Physiology and Pathophysiology, University of Erlangen-Nuernberg, Erlangen, Germany
2Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom

3Department of Pathology, Massachusetts General Hospital/Harvard Medical School, Massachusetts, USA(Received for publication on November 26, 2010)(Accepted for publication on January 20, 2011)

The neuropeptides substance P, calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) have been considered as important mediators in migraine and other primary headaches.
CGRP and VIP have been found at increased concentrations in jugular venous plasma during attacks
of migraine or cluster headache, and CGRP receptor antagonists have recently been shown to be effective in migraine therapy. Substance P and CGRP are produced from a subset of trigeminal afferents,
whereas VIP derives from parasympathetic efferents. Release of these neuropeptides in the meninges
can cause arterial vasodilatation, mast cell degranulation and plasma extravasation in animal experiments, but only CGRP seems to be relevant in migraine. Animal models have confirmed the importantrole of CGRP in meningeal nociception. The activity of spinal trigeminal neurons is a sensitive integrative measure of trigeminal activity and is partly under the control of CGRP, most likely via central
mechanisms. CGRP released from central terminals of trigeminal afferents in the spinal trigeminal
nucleus seems to facilitate nociceptive transmission via presynaptic mechanisms. The central effect of
CGRP is substantiated by suppression of nociceptive c-fos activation and neuronal activity in the spinal
trigeminal nucleus following CGRP receptor inhibition. These proposed functions are supported by the
localization of CGRP receptor components in the rat cranial dura mater, trigeminal ganglion and spinal
trigeminal nucleus. The currently available data indicate multiple sites of CGRP action in trigeminal
nociception and the pathogenesis of migraine; however, central CGRP receptors are likely to be theessential targets in the treatment of migraine using CGRP receptor antagonists. (Keio J Med 60 (3) :82–89, September 2011)

Keywords: CGRP, headache, dura mater, trigeminal ganglion, trigeminal nucleus

process termed neurogenic inflammation 2. The idea thatneurogenic inflammation of meningeal tissues underlies
the generation of migraine pain, in particular, has been
put forward by distinguished groups that have performed
a considerable number of animal experiments.3
The basic idea used to be that neurogenic inflammation, once it occurs in the cranial dura mater, may releaseor produce noxious substances. These lead to increasing activation of nociceptive primary afferents with theconsequence of activation and sensitization of central

Introduction
It has long been known that neuropeptides such as
substance P and calcitonin gene-related peptide (CGRP)
are expressed by afferent neurons which have been ascribed a nociceptive function in various tissues.1 They
have been found to be released in the innervated tissues
upon noxious stimulation and to exert efferent, or socalled effector, functions such as arterial vasodilatationand plasma extravasation from postcapillary venules, a

Presented at the 1780th Meeting of The Keio Medical Society in Tokyo, September 28, 2010.
Reprint requests to: Karl Messlinger, MD, Institute of Physiology and Pathophysiology, University of Erlangen-Nuernberg, Universitaetsstr. 17,
Erlangen 91054, Germany, E-mail: Messlinger@physiologie1.uni-erlangen.de
Copyright © 2011 by The Keio Journal of Medicine

82

Keio J Med 2011; 60 (3): 82–89

trigeminal neurons that are involved in headache generation. This theory was substantiated by the effect ofspecific 5-HT1B/D agonists, the triptans, which inhibit
neurogenic inflammation on one hand and on the other
hand are effective drugs for migraine therapy.4 However,blockade of the receptor for substance P, the NK1 receptor, though very effective in blocking plasma extravasation in the cranial dura mater in animal experiments,5was ineffective in the therapy of migraine pain.6 What
has survived of the neurogenic inflammation theory is
the importance of CGRP as a key mediator in migraine
generation.7 Here we propose a new interpretation of theneurogenic events following neuropeptide release in themeninges and how these events may interplay with newtherapeutic options that arise as a consequence of neuropeptide functions. We also discuss the locations of themost important receptor sites for CGRP in the trigeminovascular system, and where they may have relevance astargets for an effective migraine therapy.
Role for Neuropeptides in the Trigeminovascular
SystemSubstance P, neurokinin A and CGRP are released
by primary afferent neurons in the peripheral nervous
system (i.e., the cranial meninges), while other neuropeptides such as neuropeptide Y (NPY) and vasoactive
intestinal polypeptide (VIP) are typically produced bypostganglionic sympathetic and parasympathetic efferents, respectively.8,9 The latter two neuropeptides will
not be discussed further because they do not seem to playa significant role in migraine; however, increased jugular venous blood levels of VIP have been found in those
suffering attacks of cluster headache.10 Based on thefindings of historical intraoperative studies of the Wolff
group from 1930 to 1950, i.e., that noxious stimulationof cerebral arteries and blood vessels of the dura mater,
but not the pia mater, produce headache-like sensations,11
the cranial dura mater of animals became the preferred
tool for basic research on headache generation. With the
implicit understanding that the human trigeminovascularsystem is very similar to that of other mammals, majorparts of our knowledge about meningeal pathophysiology
and headache generation are now derived from animal
experiments. However, histochemical and pharmacological studies on isolated human intracerebral arterial bloodvessels have been performed,12 but functional data fromthe intact human dura mater is virtually lacking becauseof technical and ethical considerations.
The distribution of neuropeptides has been extensively
studied by immunohistochemistry in the cranial duramater of mammals.8,13 Ramifications of the middle meningeal artery as well as the meningeal sinuses are densely innervated by CGRP-immunoreactive nerve fibers,
whereas innervation with substance P-immunoreactive
fibers is comparably sparse.14 Although terminals of im-

83

munopositive nerve fibers are found everywhere in thedura, it is noticeable that single CGRP-immunopositive
nerve fibers run tightly along arterial and precapillary
vessels, suggesting a vascular function. Vigorous stimulation of the rat dura in vivo or in preparations in vitro (using trains of electrical stimulation or capsaicin) changes
the appearance of immunopositive nerve fibers and results in thickened varicosities with a peculiar “beads-ona-string” appearance.15,16 This may indicate redistribution of CGRP. In an established in vitro preparation of
the hemisected rat skull combined with enzyme-linked
immunoassays, electrical and noxious chemical stimuli
caused marked release of CGRP into the superfusate,
whereas the release of substance P remained at baseline.15,17 These release studies are relevant in terms ofpathological changes in migraine for the following reasons. During migraine attacks, increased jugular venous
plasma levels of CGRP, but not substance P, have beenreported18; this increase probably indicates massive activation of CGRP release from trigeminal afferents. Furthermore, the site of CGRP release in migraine patients is
not clear; however, sites other than peripheral trigeminal
fibers have to be considered.
The inflammatory response to neuropeptides has beenstudied based on principal functional components inanimal experiments. For example, the main function ofsubstance P has been described as plasma extravasation,
whereas CGRP has a pronounced role in arterial vasodilatation and increase in blood flow, as outlined above.
CGRP has the most potent vasodilator effect at intracranial arteries,19 and the vasodilatory mechanism of CGRP
(but not substance P) is linked to an intracellular increasein cyclic adenosine monophosphate (cAMP), which canbe fully antagonized by CGRP8-37, the classical competitive receptor antagonist.20 CGRP-dependent vasodilatory effects in the rodent dura can be observed through acranial window by video microscopy21 or by using bloodflow as a readout via laser Doppler flow recordings.22 For
example, administrations of the CGRP receptor antagonists CGRP8-37 or olcegepant (BIBN4096BS, Boehringer
Ingelheim) were able to inhibit increases in meningeal
blood flow induced by periodic electrical stimulation of
the dura in rats. Interestingly, inhibition was observed
when CGRP inhibitors were applied topically or administered systemically.22,23 These striking actions of theinhibitors raise questions about the localization of the
CGRP receptors.
Role for CGRP Receptors in the Cranial Meninges
The CGRP receptor expressed by vascular smoothmuscle cells and other tissues has a rather complicatedstructure. A large peptide with seven transmembrane domains, named the calcitonin receptor-like receptor (CLRor CRLR), is supplemented by a small single transmembrane peptide, named the receptor activity modifying

84

protein 1 (RAMP1), to form the ligand binding site thatis specific for CGRP.24 Exchange of RAMP1 with other
RAMP molecules changes the receptor specificity, e.g.,
the adrenomedullin receptor is formed with RAMP2.
In addition, a small intracellular protein component,
called receptor component protein (RCP), is necessary
for receptor protein trafficking and links the receptor to
the intracellular signal-transduction machinery mainly
through Gs proteins and adenylate cyclase. As a consequence, only the combination of these three components(CLR, RAMP1, RCP) reliably constitutes the functional
CGRP receptor. Given the documented role of cAMP in
sensitization, the hypothesis that CGRP may cause sensitization in meningeal afferents has been addressed via
recordings from trigeminal ganglion neurons in rat nociceptors.25 While cAMP analogues induced sensitization to mechanical stimuli, topical application of CGRP
caused no change in activity or mechanical sensitivityof meningeal afferents, although CGRP did induce increased blood flow.26 The results were surprising at thetime of publication; however, our studies elucidating the
CGRP receptor distribution within the dura disentangled
these puzzling findings.
The distribution of CGRP receptors in the rat cranial dura has been extensively studied by our group.27
Multiple-color immunofluorescence examinations demonstrated co-localization of CLR and RAMP1 immunoreactivity in meningeal nerves, indicating functional
CGRP receptors; however, CLR was neither co-localizedwith protein gene product 9.5 (a pan-neuronal axonal
marker) nor with CGRP. Instead, CLR was co-localizedwith glial fibrillary acidic protein (GFAP) as a marker
for glia, indicating that CGRP receptor components may
be expressed by Schwann cells.27 While the functionalsignificance of CGRP receptors on Schwann cells is currently unexplored, findings of CGRP receptors on arterial blood vessels are in line with CGRP functions (Fig.
1A). In addition, we detected CGRP receptors in large,
round to elongated mononuclear cells in juxtaposition tomeningeal vessels (Fig. 1B). The morphology and immunophenotype of these cells were compatible with mast
cells, which have been found to accompany dural arteries
in considerable numbers.28 In vivo treatment of rat durawith mast cell degranulator compound 48/80 causes lossof immunoreactivity for histamine in mast cells, indicating degranulation. The treatment also caused delayed andprolonged activation of meningeal afferents.29 Although
the exact mediators and mechanisms responsible for themast cell activation are unknown, these results indicate
mast cell factors and possibly histamine as key contributors. We have observed that histamine is able to excite
a subpopulation of primary meningeal afferents in a rat
hemisected skull preparation.30 Interestingly, histamine
was not only released by superfusion with compound48/80 but also to a minor extent by CGRP in the rat hemisected skull preparation.30 Thus, localization of CGRP

Messlinger K, et al: Neuropeptide Effects in the Trigeminal System

receptor components on mast cells may indicate that
CGRP can act as a mild mast cell degranulator.27 Together, the dural CGRP receptor distribution is in line withexisting functional data and suggests targets importantin our understanding of the pharmacologic effector sitesof CGRP inhibitors. This forms the starting point in our
understanding of the exact interplay of substructures inmeningeal nociception and the relation to CGRP signaling and headache generation.
Possible Role for CGRP Receptors in the Trigeminal
GanglionIn the rat trigeminal ganglion, immunoreactivity of the
CGRP receptor components CLR and RAMP1 was seenin partly overlapping populations of mostly small-diameter neurons.27 Subpopulations of CGRP-immunoreactive
neurons were also immunopositive for either CLR or
RAMP1, but the co-existence of CGRP with CLR and
RAMP1 was extremely rare (Fig. 1C, D). Taken together,
this pattern suggests the existence of separate CGRPreleasing neurons on one hand and CGRP-receptive
neurons on the other hand. Whether CGRP can directly
activate primary afferent neurons is disputed by calcium
imaging in which CGRP increases intracellular calcium
in a subpopulation of cultivated rat dorsal root ganglion
neurons.31 In cultivated rat trigeminal ganglion neurons,
treatment with CGRP has been found to induce CGRP
promoter activity and to increase CGRP release.32 In a
separated culture of rat trigeminal ganglion glia cells, theexpression of inducible nitric oxide synthase and the production of nitric oxide has been found to be dose-dependently increased by CGRP.33 Immunoreactivity for both
CGRP receptor components has indeed been localized onsatellite glia cells in the rat trigeminal ganglion.33
Thus, signaling between trigeminal ganglion neurons
using CGRP as a mediator seems possible, although
functional data should be handled with care because of
the limitations imposed by cell culture.
Possible Role for CGRP Receptors in the Trigeminal
Brainstem
Recordings from neurons that receive afferent input
from the cranial dura in the subnucleus caudalis of the
spinal trigeminal nucleus (SpV) are excellently suited toassess the activation state of the rat meningeal nociceptive system and to test conditions that may be relevant forheadache generation. We employed this model to examine the effect of the CGRP receptor antagonist olcegepant (BIBN4096BS) on the spontaneous and heat-evoked
activity of neurons with receptive fields in the exposedparietal dura.34 Olcegepant decreased both spontaneousand evoked activity in a dose-dependent manner whenapplied intravenously (Fig. 2A-C). However, topical application to the dura was ineffective. These findings in-Keio J Med 2011; 60 (3): 82–8985   






 







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Fig. 1 Distribution of CGRP receptor components CLR and RAMP1 in the cranial dura, trigeminal ganglion and medulla oblongata.
(A) Triple immunolabeling of CGRP (red), CLR (green) and smooth muscle marker SMA (blue). CLR immunoreactivity is associatedwith arterial vessels (turquoise) but not with veins (asterisks). CGRP fibers (arrows) frequently run close to CLR-positive arteries andmononuclear cells with no co-localization. (B) Larger mononuclear cells (arrow) exhibited granular cytoplasmic CLR staining shown
here in close proximity to a CGRP-positive branching fiber. (C) CLR (green) and RAMP1 (blue) staining in the trigeminal ganglion
showed homogeneous cytoplasmic staining; co-localization results in the mixed color turquoise (arrows). (D) CGRP (red) and RAMP1(blue) staining in the trigeminal ganglion showed granular to homogeneous staining with nuclear sparing in many small- and mediumsized neuronal cell bodies. Co-localization results in the mixed color magenta (arrow). (E) Dark-field photomicrograph of an unstained
horizontal section through the medulla oblongata. The spinal trigeminal nucleus (SpV) is outlined in yellow and the images F and G
were taken from the posterior-lateral zone (black square). (F) CLR (green) and CGRP (red) immunolabeling in the SpV demonstrates
irregular networks arranged around unstained regions (asterisks, “glomerular structures”). Laminae I and II demonstrate three staining patterns: CLR only (green), CGRP only (red) and CLR+CGRP (yellow). Note the incoming primary afferent CGRP-positive fibers(arrows). (G) Double labeling of RAMP1 (blue) with CGRP (red) in the SpV also shows irregular networks around unstained regions(asterisks, “glomerular structures”). Laminae I and II demonstrate three staining patterns: RAMP1 only (blue), CGRP only (red) and
CGRP+RAMP1 (magenta). Note incoming primary afferent fibers (arrows). Scale bars: 50 μm in A, C and D; 10 μm in B, F and G;
1 mm in E.

dicate that the site of CGRP receptor inhibition may belocated centrally to the primary afferents innervating thedura mater. As a complementary method, c-fos activation
confirmed our extracellular recordings.35 Specifically, a
massive noxious stimulus in the form of a systemic infusion of capsaicin caused a marked increase in the numberof c-fos-immunoreactive neurons in the superficial layers of SpV. The number of c-fos-activated neurons was
significantly reduced when olcegepant was infused prior

to the capsaicin infusion. Together, these experimental
results argue that CGRP is an important nociceptive neuromodulator in the trigeminal brainstem.
Accordingly, we observed CLR and RAMP1 immunoreactivity in the spinal trigeminal tract and in the trigeminal nucleus of the spinal medulla (Fig. 1E). We noted thehighest density in superficial laminae,27 and the distribution of CGRP receptors components (CLR and RAMP1)
overlapped with that of CGRP (Fig. 1F, G). Interestingly,

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Messlinger K, et al: Neuropeptide Effects in the Trigeminal System

Fig. 2 Effects of i.v. BIBN4096BS (olcegepant) on the activity of spinal trigeminal nucleus neurons with dural afferent input.
(A) Normalized activity of 17 separate extracellular single-unit recordings on a real-time scale. Each point represents the mean activity
during intervals of 150 s, normalized to the 150-s period before first drug application (open square). Hatched bars indicate injection ofolcegepant (BIBN4096BS). Note the longer time between some 150-s intervals that were used for thermal testing. (B) Representative
spinal trigeminal neuron with phasic and tonic coding of dural heat stimulation. The activity is an average of 4 subsequent heat step
stimulation trials. The temperature of the dura was stepwise increased as indicated, from 32 °C to 44 °C, each step lasting for 30 s.
Stepwise temperature increase caused a phasic response in neuronal activity (mean of first 10-s periods of temperature steps versus
reference activity). (C) Normalized neuronal activity of 13 neurons in the spinal trigeminal nucleus. Average and phasic responses tothermal stimulation protocols were reduced after administration of BIBN4096BS (olcegepant) compared to the control period. After acumulative dose of 1.2 mg/kg BIBN4096BS, no significant activation by heat stimuli was observed. * p < 0.05 versus control period.

Keio J Med 2011; 60 (3): 82–89

87

Fig. 3 Schematic representation of calcitonin gene-related peptide (CGRP) signaling in the cranial dura mater encephali, the trigeminal ganglion and the spinal trigeminal nucleus.
In the dura, CGRP causes dilatation of arterial vessels (AV) and secretion of substances such as histamine (HA) from mast cells (MC).
HA amplifies vasodilatation and can activate specific primary afferents. In the trigeminal ganglion, CGRP may signal to other trigeminal ganglion neurons (Aδ/C) that express CGRP receptors. In the trigeminal nucleus, CGRP facilitates nociceptive transmission
presumably via a presynaptic mechanism by increasing neurotransmitter (e.g., glutamate, Glu) release from adjacent primary afferent
terminals. Possible contributions of Schwann cells and satellite cells to CGRP signaling are not depicted.

there was no co-localization with the glial cell marker
GFAP. CGRP receptor immunoreactivity was exclusively
seen in structures resembling nerve fibers but never incell bodies. The functional interpretation of this data isthat CGRP-releasing terminals of primary afferents synapse at CGRP receptor-expressing axons, which most
likely include the terminals of primary trigeminal afferents. Therefore we postulate that CGRP actions within
the trigeminal nucleus follow a presynaptic mechanism
whereby distinct terminals of primary afferents control
the neurotransmitter release in other primary afferents(Fig. 3). The advantage of such a presynaptic mechanism
would be that distinct afferent inputs could be selectively
activated under the control of CGRP receptors (or inhibited in the case of CGRP receptor antagonists). CGRP
receptor inhibition in the C1/2 segments of the cervicaldorsal horn brought about by iontophoretic application of
CGRP receptor antagonists reduces spontaneous and glutamate-induced neuronal activity in neurons with facial
receptive fields.36 In rat brain stem slices, the excitabilityof neurons in the spinal trigeminal nucleus (SpV) is increased by CGRP superfusion and prevented by inhibition of CGRP receptors using CGRP8-37.37 Therefore, theevidence is convincing that CGRP is an activating neuromodulator in the spinal trigeminal nucleus.
Where Is the Effective Site of Action of CGRP
Receptor Antagonists?
Given the multiple receptor sites in the trigeminovascular system – arterial vessels, mononuclear cells and

Schwann cells in the dura; neurons and satellite glial
cells in the trigeminal ganglion; and central/afferent projections in the trigeminal nucleus – the question arises
as to where CGRP receptor antagonists most effectively
inhibit trigeminal activity (Fig. 3). The answer is of considerable importance, since CGRP receptor antagonists
such as olcegepant and telcagepant have been shown tobe effective in migraine therapy.38,39 Since CGRP does
not activate meningeal afferents26 and local applicationof CGRP antagonist onto the cranial dura is not effective in reducing spinal trigeminal activity,34 a peripheral
site of action, at least for acute effects of CGRP receptor inhibition, is not very likely. In contrast, all experiments are consistent with a central action of CGRP receptor antagonists. However, the trigeminal ganglion itself
has not been excluded as an important receptor site andmay function as a central component in the postulated
mechanism. In recent studies, our group has addressed
this question using functional animal experiments inwhich phosphorylation of extracellular receptor kinase(pERK) is a very quick step in the nociceptive cascadeof primary afferents. We assessed pERK in both trigeminal ganglia after unilateral injection of capsaicin into thefacial skin.35 As expected, significantly more neurons in
the trigeminal ganglion on the injected side showed thepERK signal compared to the control side. Intravenous
infusion of CGRP receptor inhibitor olcegepant before
the capsaicin injection showed no difference in pERK in
the ganglions compared to controls without olcegepant.
Most recently, we succeeded in injecting olcegepant vs.
vehicle through the infraorbital channel directly into the88Messlinger K, et al: Neuropeptide Effects in the Trigeminal System

rat trigeminal ganglion while monitoring ongoing activity in neurons that receive input from meningeal afferents
where we evoked short mechanical stimuli to the exposed
dura (unpublished data). The injection of olcegepant into
the ganglion did not significantly change ongoing or mechanically evoked activity compared to vehicle injections.
Therefore we suggest that there is reasonable evidence toexclude the trigeminal ganglion as an important site ofacute actions of CGRP receptor antagonist.

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Conclusion
Systemic administration of CGRP receptor antagonist
reduced ongoing and heat-evoked activity of spinal trigeminal neurons with meningeal afferent input. Likewise, pretreatment with CGRP receptor antagonist reduced the increase in c-fos activation of neurons in the
spinal trigeminal nucleus induced by capsaicin infusion.
In contrast, pretreatment with CGRP receptor antagonist
did not reduce the increase in ERK phosphorylation in
the trigeminal ganglion induced by facial capsaicin injection. Direct injection of CGRP receptor antagonist into
the trigeminal ganglion did not reduce ongoing and mechanically evoked activity of spinal trigeminal neurons
with meningeal afferent input. Therefore, CGRP receptor
antagonists may act centrally rather than peripherally andpromptly inhibit incoming activity from the trigeminal
system involved in meningeal nociception. Similarly, wehypothesize that the action of CGRP receptor antagonists
such as olcegepant in migraine therapy may, at least inpart, depend on central mechanisms.
Acknowledgments
Parts of this work were supported by the Federal Ministry of Education and Research of the Federal Republic of
Germany (German Headache Consortium). BIBN4096BS(olcegepant) was a gift from Boehringer Ingelheim.
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