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\begin{document}

\title{Biological and small molecule strategies in migraine therapy with
relation to the CGRP family of peptides.}



\author[1]{Lars Edvinsson (GUEST EDITOR)}%
\author[2]{Jacob Edvinsson}%
\author[3]{Kristian Agmund Haanes}%
\affil[1]{Institute of Clinical Science in Lund, Lund University}%
\affil[2]{University of Copenhagen Faculty of Health and Medical Sciences}%
\affil[3]{Copenhagen University Hospital, Glostrup}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Migraine is one of the most common of neurological disorders with a
global prevalence of up to 15\%. One in five migraineurs have frequent
episodic or chronic migraine requiring prophylactic treatment. In recent
years, specific pharmaceutical treatments targeting calcitonin
gene-related peptide (CGRP) signalling molecules have provided safe and
effective treatments; monoclonal antibodies for prophylaxis and gepants
for acute therapy. Albeit the beneficious impact of these new drugs, it
is important to understand the molecular mechanisms involved to better
understand migraine pathophysiology and improve the therapy. Here we
describe current views on the role of the CGRP family of peptides CGRP,
calcitonin (CT), adrenomedullin (AM), amylin (AMY) and their receptors
in the trigeminovascular system (TGV). All these molecules are present
within the TGV system but differ in expression and localization. It is
likely that they have different roles, which can be utilized in
providing additional drug targets.%
\end{abstract}%



\sloppy


\emph{Review Paper for Br. J. Pharmacol. 2021.}

\textbf{Biological and small molecule strategies in migraine therapy
with relation to the CGRP family of peptides.}

Lars Edvinsson\textsuperscript{1,2}, Jacob C. A.
Edvinsson\textsuperscript{2, 3}, Kristian A. Haanes\textsuperscript{1,2}

\textsuperscript{1}Departments of Internal Medicine, Lund University
Hospital, Lund, Sweden.

\textsuperscript{2}Department of Clinical Experimental Research,
Glostrup Research Institute, Glostrup Hospital, Rigshospitalet, Denmark.

\textsuperscript{3}Department of Drug Design and Pharmacology, Faculty
of Health and Medical Sciences, University of Copenhagen, Copenhagen,
Denmark.

Address for correspondence:

Lars Edvinsson, Professor MD

Department of Internal Medicine, Lund University Hospital, S-22185 Lund,
Sweden.

Phone: +46 703271484

e-mail: lars.edvinsson@med.lu.se

\section*{Abstract}

{\label{abstract}}

Migraine is one of the most common of neurological disorders with a
global prevalence of up to 15\%. One in five migraineurs have frequent
episodic or chronic migraine requiring prophylactic treatment. In recent
years, specific pharmaceutical treatments targeting calcitonin
gene-related peptide (CGRP) signalling molecules have provided safe and
effective treatments; monoclonal antibodies for prophylaxis and gepants
for acute therapy.

Albeit the beneficious impact of these new drugs, it is important to
understand the molecular mechanisms involved to better understand
migraine pathophysiology and improve the therapy. Here we describe
current views on the role of the CGRP family of peptides CGRP,
calcitonin (CT), adrenomedullin (AM), amylin (AMY) and their receptors
in the trigeminovascular system (TGV). All these molecules are present
within the TGV system but differ in expression and localization. It is
likely that they have different roles, which can be utilized in
providing additional drug targets.

\textbf{Key words} . CGRP, adrenomedullin, amylin, calcitonin, CLR,
RAMPs, receptors

\textbf{INTRODUCTION}

Migraine is a complex disorder, known for centuries but still not fully
understood. Affecting 1 billion people, migraine is one of the most
prevalent, disabling neurological disorders worldwide. Migraine has a
female predominance with debilitating impact in the most active years
(2018; GBD 2016 Disease and Injury Incidence and Prevalence
Collaborators, 2017). Currently, the migraine attack is proposed to
start in the central nervous system (CNS), mainly involving regions such
as hypothalamus and brainstem (Goadsby, Holland, Martins-Oliveira,
Hoffmann, Schankin \& Akerman, 2017). Evidence of migraine initiating in
the CNS has been demonstrated in longitudinal neuroimaging studies over
30 days in spontaneous migraine attacks (Schulte, Mehnert \& May, 2020;
Schulte, Menz, Haaker \& May, 2020). The studies revealed that
hypothalamic activation during the premonitory phase of a spontaneous
migraine attack, and ictally there was activity in brainstem regions
often discussed in migraine. However, the link with the
trigeminovascular (TGV) system and the CNS is still unclear; either the
CNS is the sole activator of the trigeminal nucleus caudalis (TNC) and
subsequently the TGV system or the incoming signals from the TGV system
can be modified by brainstem nuclei. Following activation of the
1\textsuperscript{st} order neurons in the TG, the sensory nerve fibres
transmit pain signals to the lower brainstem and spinal cord C1 - C3,
verified in tracing studies, and send signals to second order ascending
neurons to various CNS regions such as brainstem nuclei and thalamus
(Edvinsson, 2011).

Until 25 years ago, neurologists had few, and often insufficient,
options to treat patients with acute migraine attacks apart from general
analgesics alone or in combinations. Understanding the mechanisms of
ergotamine related molecules resulted in development of the triptan
group of acute medications; acting with high specificity on serotonin
(5-HT\textsubscript{1B} and 5-HT\textsubscript{1D}) receptors. The
triptans may act via at least 3 sites; (i) they are cranial
vasoconstrictors with possibility of cardiovascular side-effects (Saxena
\& Den Boer, 1991), (ii) they inhibit neuropeptide release (Amrutkar,
Ploug, Hay-Schmidt, Porreca, Olesen \& Jansen-Olesen, 2012; Goadsby \&
Edvinsson, 1994), and (iii) may inhibit second-order neurons of the TGV
pain pathway (Goadsby \& Knight, 1997).

Due to the high probability of cardiovascular adverse events, there was
a need for new drugs without vasoconstrictor effects. This work lead to
a new group of prophylactic agents, monoclonal antibodies (mAbs) towards
CGRP or selective binding to CGRP receptor components. These new mAbs
have since proven to be effective and have few side-effects (Edvinsson,
Haanes, Warfvinge \& Krause, 2018). Currently, upcoming additions to
acute anti-migraine pharmacotherapy includes the
5-HT\textsubscript{1F}receptor class of agonists, the ditans
(Labastida-Ramirez et al., 2020), and the CGRP receptor antagonists, the
gepants (Edvinsson, Haanes, Warfvinge \& Krause, 2018).

This review provides a brief description of mechanistic actions of the
molecules acting through CGRP related mechanisms in relation to migraine
therapy, and discusses the actions based on available molecular data.
Combined, we propose ways towards improving therapy of acute and
prophylactic treatments of migraine at sites involving other members of
the CGRP family of peptides.

\textbf{2. THE CGRP SYSTEM and MIGRAINE.}

CGRP and its receptor are extensively expressed in two separate neuron
populations in the TGV system (Eftekhari, Salvatore, Calamari, Kane,
Tajti \& Edvinsson, 2010). CGRP is released from the TGV system in
conjunction of acute attacks of migraine and cluster headache (Ho,
Edvinsson \& Goadsby, 2010). The trigeminal C-fibres and ganglia are the
main source of stimulus-induced CGRP found in the jugular vein as shown
in man and cat (Goadsby, Edvinsson \& Ekman, 1988) and rat (Hoffmann,
Wecker, Neeb, Dirnagl \& Reuter, 2012). The C-fibres have both afferent
and efferent aspects; perivascular fibres release CGRP onto the vascular
smooth muscle cells to induce dilatation and on dural mast cells to
participate in induction of neurogenic inflammation (Pietrobon \&
Moskowitz, 2013). Many outstanding questions remain, particularly
regarding the other members of the CGRP family. This group of signalling
peptides include calcitonin (CT), adrenomedullin (AM) and amylin (AMY)
and their receptors (Table 1). A fair question is often asked: Can other
peptides be released in the TGV and will CGRP acting drugs have effects
that include other related receptor targets?

\textbf{CGRP family; peptides and receptors.}

The CGRP family of peptides (\selectlanguage{greek}α \selectlanguage{english}and \selectlanguage{greek}β \selectlanguage{english}forms) are ligands for a closely
related family of G protein-coupled receptors (GPCRs) with shared
structural homology (Hay et al., 2014). The CGRP family of peptides and
receptors show a varying degree of structural homology with CGRP and
have a widespread distribution throughout the body (Hendrikse, Bower,
Hay \& Walker, 2018).

The receptors in this group are formed from either of two
7-transmembrane (TM) G-protein coupled receptors (GPCRs), the calcitonin
receptor-like receptor (CLR) or the calcitonin receptor (CTR), which
interact with receptor activity-modifying protein (RAMPs) to form
heterodimers (Gingell, Hendrikse \& Hay, 2019). The RAMPs are a small
family of three proteins (RAMP 1-3) that are single TM-spanning proteins
that can modify binding characteristics, pharmacology, functionality and
cell trafficking of the specific GPCRs (Hay \& Pioszak, 2016). The
currently most central is the 7-TM complex -- CLR -- which is a required
element of receptors for CGRP and adrenomedullin (AM\textsubscript{1}and
AM\textsubscript{2}). Early studies showed that transfecting cells with
only CLR revealed no response to CGRP (McLatchie et al., 1998). It was
only after the demonstration that the dimerization of RAMP1 and CLR
resulted in formation of a functional receptor to CGRP (Hay, Garelja,
Poyner \& Walker, 2018). When CLR couples to RAMP2 or RAMP3 it forms
AM\textsubscript{1} receptor (CLR/RAMP2) or AM\textsubscript{2} receptor
(CLR/RAMP3). The AMY receptors are formed by association of the
Calcitonin (CT) receptor (CTR) with a RAMP: AMY\textsubscript{1}receptor
(CTR/RAMP1), AMY\textsubscript{3} receptor (CTR/RAMP3). The CTR can also
signal on its own, as a calcitonin receptor (without any RAMP) (Poyner
et al., 2002). Due to the complexity of this peptide-receptor system,
their expression in the trigeminal system is unclear and their
functional roles yet to be resolved (Edvinsson, Grell \& Warfvinge,
2020).

\textbf{Localization of the CGRP family of peptides in relation to the
TGV system.}

CGRP is expressed in a granular pattern in small (\textless{}30 \selectlanguage{greek}µ\selectlanguage{english}m) to
medium (30-60 \selectlanguage{greek}µ\selectlanguage{english}m) sized TG neurons (Eftekhari, Salvatore, Calamari,
Kane, Tajti \& Edvinsson, 2010; Lennerz et al., 2008) where CGRP is
packed in vesicles that are surrounded by the Golgi apparatus. In
addition, C-type of sensory unmyelinated nerves appear as pearl-like
CGRP immunoreactivity (ir) in boutons (also known as varicosities)
(Figure 1). The myelinated fibres do not contain CGRP as sometimes has
been proposed (Eftekhari, Warfvinge, Blixt \& Edvinsson, 2013; Lennerz
et al., 2008). The CGRP receptor components, CLR and RAMP1, are
co-expressed in medium to large (\textgreater{}60 \selectlanguage{greek}µ\selectlanguage{english}m) diameter neurons
(Eftekhari et al., 2016; Eftekhari, Salvatore, Calamari, Kane, Tajti \&
Edvinsson, 2010). In addition, CLR and RAMP1 have been observed in
satellite glial cells (SGC) and in thinly myelinated fibres, typical for
A\selectlanguage{greek}δ-\selectlanguage{english}fibres in various parts of the TGV system such as the TG, dura mater,
and root entry zone (Figure 1) (Eftekhari, Salvatore, Calamari, Kane,
Tajti \& Edvinsson, 2010; Lennerz et al., 2008; Miller et al., 2016).
Early findings, which have today been confirmed by many researchers, has
stood the test of time, and served a solid base for the successful
development of mAbs and small molecule gepants (Edvinsson, Haanes,
Warfvinge \& Krause, 2018).

\emph{CT} is a hormone produced mainly by C cells in the thyroid gland
with a role to reduce plasma calcium and to promote bone formation
(Findlay \& Sexton, 2004). In the clinic, CT is used in treatment of
bone disorders characterized by increased bone resorption, osteoporosis
and hypercalcemia due to malignancy, with some pain relief (Findlay \&
Sexton, 2004). Thus, CT and its receptor CTR are seen in different cell
types and tissues which suggests multiple physiological roles, including
bone metabolism (Findlay \& Sexton, 2004). CT has not been shown to be
expressed in the nervous system, however binding sites for CT are found
in many brain structures (Hendrikse, Bower, Hay \& Walker, 2018). In
addition, a recent study has revealed the presence of the CT receptor in
the human brainstem (Bower et al., 2016).

Recently, CT was found to be expressed in the trigeminal system; CT the
peptide was found in vesicles that are surrounded by the endoplasmic
reticulum/Golgi apparatus and occur in addition as pearl-like
CT-immunoreactivity (ir) in the C-type of sensory fibres (Figure 1B)
(Edvinsson, Grell \& Warfvinge, 2020).

. The CT-ir was expressed in a pattern like that of CGRP; granular
staining of small to medium sized neurons and pearl-like staining of
fibres in the TG. In addition, many SGCs were CT positive (Edvinsson,
Grell \& Warfvinge, 2020). In a subpopulation of TG neurons there is
granular and cytosolic expression of CT-ir (Figure 1).

AMY is currently in focus of much migraine research, due to its affinity
for the CGRP receptor that pharmacologically is almost equal to that of
CGRP and because CGRP has strong affinity for the AMY\textsubscript{1}
receptor (Hendrikse, Bower, Hay \& Walker, 2018). Comparing the binding
affinity of olcegepant and CGRP\textsubscript{8-37} to the
AMY\textsubscript{1} and CGRP receptors revealed similarities in binding
characteristics as well as in cAMP production (Walker et al., 2015).
Human AMY was first isolated in 1987 (Cooper, Willis, Clark, Turner, Sim
\& Reid, 1987). It is an endocrine hormone that signals to the brain and
acts as a satiety factor; its presence in plasma is about 10 times
higher than that of CGRP (Hay, 2017). Research on AMY deposition in
brain neurons has been discussed for a role in Alzheimer's disease
(Mietlicki-Baase et al., 2017) and AMY has been found to alter viability
of human brain pericytes (Schultz, Byman, Fex \& Wennstrom, 2017).

Previously, we reported that AMY is expressed in feline TG neurons, and
that AMY relax \emph{in vitro} and \emph{in vivo} cerebral vessels
(Edvinsson, Goadsby \& Uddman, 2001). A detailed report showed AMY
expression in a few small to medium sized TG neurons which co-express
CGRP and in C-fibres (Edvinsson, Grell \& Warfvinge, 2020).

Receptors for CGRP and AMY are related and share components, CLR/RAMP1
and CTR/RAMP1, respectively. Given the close relationship between AMY
and CGRP, and since release of the peptides may act on either or both
receptors, it is understandable that experiments with AMY aimed to
trigger migraine and to block migraine with mAbs towards AMY are on the
way. However, early result from a trial on migraine patients did not
reveal any prophylactic effect of the mAbs towards AMY (Ashina, 2019).

AM is most often considered an endothelial peptide and was first
isolated in 1993 (Kitamura et al., 1993). AM is generally expressed and
participates in a variety of physiological functions including
vasodilation, bronchodilatation, growth and hormone regulation (Ferrero
et al., 2018). Furthermore, AM is involved in pathophysiological
processes such as hypertension, retinopathy and tumour genesis (Ferrero,
Larrayoz, Gil-Bea, Martinez \& Ramirez, 2018). In mammals, endothelial
AM-ir is present in low concentrations in the vascular endothelium
(Satoh et al., 1996). AM is found in neurons and glial cells (Serrano et
al., 2000). We have reported the presence of AM-ir in the thin cytoplasm
of the glial cells, SGCs and cells enveloping neuronal processes,
probably myelinating cells. In addition, AM-ir has been noted to occur
in cranial vascular endothelium (Edvinsson, Grell \& Warfvinge, 2020).
Its molecular relation to CGRP has resulted in suggestion that AM may
have a role in migraine pathophysiology (Juhl, Petersen, Larsen,
Jansen-Olesen \& Olesen, 2006). However, intravenous AM infusion did not
cause migraine-like attacks in man (Ashina, Hansen, BO \& Olesen, 2017).
This view is in line with our demonstration that there is no AM in TG
neurons but presence in glial cells and vascular endothelium (Edvinsson,
Grell \& Warfvinge, 2020).

\textbf{Relation between expression of the peptides and their
receptors.}

CGRP and CT were found to be co-expressed in small to medium sized
neurons, and in some SGCs, suggesting that CT might have a role in
trigeminal function (Edvinsson, Grell \& Warfvinge, 2020). CGRP and AMY
are co-expressed in some of the small to medium sized neurons and in
thin C-fibres. The number of CGRP positive cells were in abundance
compared to those storing AMY (Edvinsson, Grell \& Warfvinge, 2020). AMY
is present in high concentrations in plasma, in migraine attacks higher
levels of CGRP is released than AMY. Since the CGRP receptor is more
sensitive to CGRP than to AMY, AMY is unlikely to have a central role in
migraine.

The CGRP receptor (CLR/RAMP1) is mainly expressed in large neurons and
SGCs (Edvinsson, Grell \& Warfvinge, 2020). The
AM\textsubscript{2}receptor (CLR/RAMP3) occurs in some SGCs, the
AMY\textsubscript{1}receptor (CTR/RAMP1) in large neurons and SGCs,
while the AMY\textsubscript{3} receptor (CTR/RAMP3) has been observed in
some SGCs.

\textbf{3. SITE OF ACTION OF CGRP AND THE CGRP RECEPTOR ACTIVE DRUGS.}

CGRP antagonistic molecules have by now a 2-decade history with
olcegepant and telcagepant as the first small molecules that
competitively antagonize the vasodilator effect of CGRP in human
arteries (Edvinsson et al., 2002). They were studied in several clinical
trials, but the program was halted because of hepatotoxicity. Following
molecular modifications three other gepants have now passed phase III
and are approved by the FDA; these are atogepant, ubrogepant
(Rubio-Beltran, Chan, Danser, MaassenVanDenBrink \& Edvinsson, 2020) and
rimegepant (Mulder et al., 2020). The drugs were initially designed for
acute therapy, but they are now also studied for prophylaxis. The other
class of molecules are humanized monoclonal antibodies directed towards
different parts of the CGRP molecule \emph{per se} (eptinezumab,
fremanezumab, galcanezumab) or a human antibody which binds to the
N-terminals of CLR and RAMP1 (erenumab). They have all passed extensive
clinical studies with significant effects and minor adverse events,
subsequently they are now approved for migraine prophylaxis. While these
molecules are effective in prophylaxis of chronic migraine their
respective sites of action remains unclear.

(i). \emph{Action on intracranial blood vessels.}

Dilatation of the middle meningeal arteries and neurogenic inflammation,
in the dura mater, have been suggested as a triggering mechanism
regarding migraine pathophysiology. The direct activation of trigeminal
afferents in the dura causes a painful headache in humans; this
observation was first made in 1940 via stimulation of these vascular
nerves during surgery and extended more recently to include pia mater
and small pial vessels (Fontaine et al., 2018). Similarities of the
occurring headache with a migraine headache led to the vascular theory
of migraine, which postulates that headaches is a disorder triggered by
dilation of intra- or extra-cranial blood vessels. One aspect worth
considering is that nearly all vasodilators given in a peripheral vein
results in ``migraine-like headache or migraine'' (Ashina, Hansen, BO \&
Olesen, 2017). However, this view is disputed since the same group of
researchers did not observe that cerebral and meningeal arteries but
primarily the extra-cranial arteries were dilated during a genuine
migraine attack (Amin et al., 2013).

The first study of CGRP on intracranial arteries revealed very potent
vasodilatation, independent of endothelial function and associated with
activation of adenylyl cyclase in the vascular smooth cells (Edvinsson,
Fredholm, Hamel, Jansen \& Verrecchia, 1985) and with a parallel
reduction in intracellular calcium ion concentration (Erdling,
Sheykhzade \& Edvinsson, 2017). Subsequent studies revealed that other
members of the CGRP family of peptides also were vasodilators but had
lower potency. While the perivascular sensory nerves had a rich supply
of CGRP containing fibres, there is less expression of AMY and no ADM in
nerve fibres on cerebral vessels. A subpopulation of CGRP positive TG
neurons co-localized AMY, while no ADM-ir was seen (Edvinsson, Goadsby
\& Uddman, 2001). The receptor components CLR and RAMP1-3 were
demonstrated with qPCR, suggesting that AMY receptors might occur. In
agreement human cerebral pial arteries, middle cerebral, superficial
temporal and middle meningeal arteries have CLR, RAMP 1, RAMP2 and RAMP3
in the smooth muscle layers (Oliver, Wainwright, Edvinsson, Pickard \&
Hill, 2002). The vascular endothelium contained CLR and RAMP2, but no
RAMP1 or RAMP3. Functional study of cerebral arteries revealed strong
vasodilator effects by CGRP and AMY (with or without endothelium) while
only at very high concentration did ADM show a relaxant effect;
CGRP\textsubscript{8-37} blocked the relaxant response. Intracortical
CGRP, AMY and ADM caused increased local cerebral blood flow by 42\%,
32\% and 15\%, respectively (Edvinsson, Goadsby \& Uddman, 2001). The
responses to CGRP and AMY were blocked by CGRP\textsubscript{8-37},
suggesting that the vasodilatation is mediated via CGRP receptors (both
mRNA for CLR and RAMP1 were seen in the MCA) (Edvinsson, Goadsby \&
Uddman, 2001). Studies of CT early revealed very low relaxant effect of
brain vessels (Edvinsson, Ekman, Jansen, McCulloch \& Uddman, 1987);
recent preliminary studies have not shown CT receptor-ir in the vessel
walls, while the CGRP receptor components CLR and RAMP1 are present in
human cerebral and middle meningeal arteries (Edvinsson et al., 2010).
While gepants showed competitive inhibition (Edvinsson et al., 2010;
Rubio-Beltran, Chan, Danser, MaassenVanDenBrink \& Edvinsson, 2020) the
mAbs showed competitive antagonism with no depression of maximum CGRP
induced relaxation in human middle meningeal and cerebral arteries
(Ohlsson, Haanes, Kronvall, Xu, Snellman \& Edvinsson, 2019; Ohlsson,
Kronvall, Stratton \& Edvinsson, 2018). The data supports a strong role
for CGRP but the lack of AMY receptor component CTR, and few AM
receptors are present their role is unlikely if induction of a migraine
attack if vasodilatation is the key mechanism (Ashina, Hansen, BO \&
Olesen, 2017; Walker et al., 2015). This view is in support by others
who stated that the TGV system does not require a peripheral sensory
input to be activated (Goadsby \& Akerman, 2012).

\emph{(ii). Relation to the blood-brain barrier.}

In light of a vascular mechanisms of anti-migraine drugs, the possible
relation of CGRP or its antagonists and the effect on dilation of
cerebral arteries, must be considered. Importantly, the endothelium in
these vessels restricts passage of molecules from the vessel lumen to
the smooth muscle layers of the vascular wall containing CGRP nerve
endings. The hypothesis was tested in isolated rat middle cerebral
arteries that were cannulated and luminally perfused, enabling
application of drugs to either the endothelium exposed in the lumen or
the smooth muscle layers on the abluminal side of the artery (Edvinsson,
Nilsson \& Jansen-Olesen, 2007). In these experiments, CGRP as well as
AM, AMY and CT were relaxing the artery only when applied to the
abluminal surface of the cerebral artery (Edvinsson, Nilsson \&
Jansen-Olesen, 2007). Moreover, neither CGRP receptor antagonists
(olcegepant and telcagepant) nor anti-CGRP antibodies blocked
CGRP-mediated dilation when applied to the lumen; they were effective
only when applied to the abluminal side. On this basis, circulating CGRP
receptor antagonists and mAbs against CGRP and the CGRP receptor, all of
which are effective in migraine, do not seem to be able to cross the
endothelial barrier to access targets in the brain vasculature.

A neurogenic inflammation occurring in the dura has also been proposed
as trigger of migraine attacks (Pietrobon \& Moskowitz, 2013). However,
numerous drugs that block the plasma protein extravasation component of
neurogenic inflammation in the dura of animals have been tested in
clinical trials but have not exhibited anti-migraine efficacy. Moreover,
CGRP does not induce neurogenic inflammation in humans or rodents but
mediates only the vasodilatory aspect of inflammation (Levy, Burstein \&
Strassman, 2005; Schain, Melo-Carrillo, Stratton, Strassman \& Burstein,
2019). Although the idea of neurogenic inflammation has been discussed
at length over the years the role in migraine is still not clear
(Edvinsson, Haanes \& Warfvinge, 2019). Aspects for and against this
hypothesis are still being discussed (Hadjikhani et al., 2020; Khan et
al., 2019).

Considering the BBB, the question remains ``where do the gepants and
mAbs act'' to relieve migraine headache. Looking at the dura mater, the
cerebral circulation, the brain and the TG after administration of
tracers like Evans Blue or radioactive compounds to calculate the
permeability surface (PS product) passage showed that the dura and the
TG were freely accessible (Eftekhari, Salvatore, Johansson, Chen, Zeng
\& Edvinsson, 2015; Lundblad, Haanes, Grande \& Edvinsson, 2015).
Induction of neurogenic inflammation using the Freud's Adjuvant or the
``inflammation soup'' revealed activation within the TG but no
quantitative increase in PS product into the CNS. Subsequent
measurements revealed minor passage by gepants (2\%) or none of the mAbs
(\textless{} 0, 01\%) (Johnson, Morin, Wroblewski \& Johnson, 2019).
This is in support of a more recent study by Noseda showing that
labelled fremanezumab was distributed to cranial sensory and autonomic
ganglia and to the dura mater but not to the CNS in rats with
uncompromised blood-brain barrier (Noseda et al., 2020). Since the TG
and its peripheral ramifications (TGV system) are without the protection
of the blood-brain barrier it is likely that the antimigraine site of
action of the CGRP group of anti-migraine drugs reside here. This is
also the reason why there is few CNS related side-effects.

\emph{(iii). Action on neuron cell bodies and nociceptive fibres.}

The trigeminal system provides the link between the peripheral primary
afferents and the central terminals of the trigeminal nucleus caudalis
(TNC). Activation of this pathway may result in sensitization within
second-order neurons and drive the CNS aspects of the migraine attacks
(May \& Burstein, 2019). Secondly, it has been demonstrated that
activation of hypothalamus is early (prodromal) site where the migraine
attack starts (May \& Burstein, 2019; Schulte, Mehnert \& May, 2020;
Schulte, Menz, Haaker \& May, 2020). Connectivity studies have revealed
that other CNS regions are subsequently activated including the
brainstem from with links are available to activate or modulate the
trigeminal system function (May \& Burstein, 2019). In both hypothesis
the trigeminal system plays a key role and currently available mAbs
towards CGRP and the CGRP receptor are effective (Edvinsson, Haanes,
Warfvinge \& Krause, 2018) despite their inability to penetrate the
blood-brain barrier (Lundblad, Haanes, Grande \& Edvinsson, 2015; Noseda
et al., 2020).

Each of the peptides of the CGRP family exhibits a distinct selection of
biological actions (Poyner et al., 2002). CGRP and AMY are the most
closely related peptides in terms of amino acid sequence, which may
cause an overlap in their ability to activate their receptors. CGRP and
AMY are reported to have effects related to pain, though there are still
limited data and it is unclear how much overlap there is because the
peptides are usually not studied simultaneously. The relative potency of
CGRP, CT, AMY and AM at the different receptor complexes is complicated
and challenging because of cross-reactivity (Hay, Garelja, Poyner \&
Walker, 2018; Hendrikse, Bower, Hay \& Walker, 2018). In addition,
release of a peptide will result in a very high concentration just at
the receptor site, while circulating levels vary considerably. The study
of the nodes of Ranvier in the trigeminal system demonstrated CGRP
containing boutons with CGRP containing vesicles in C-fibres that may
directly release CGRP to reach the adjacent A\selectlanguage{greek}δ-\selectlanguage{english}fibres with CGRP
receptors (Edvinsson et al., 2019).

The possibility of intercellular cross-talk in the TG was recently
proposed to result in a feedback loop sensitizing neurons (Messlinger,
Balcziak \& Russo, 2020). Apart from direct gap-junction communication
between SGC\selectlanguage{ngerman}´s and neurons, paracrine signalling may also occur. CGRP
locally released by neurons or C-fibres could potentially activate
receptors located on SGC´s, other neurons or auto receptors. This
activation may in turn affect gene expression, neurotropic factors,
neuropeptides or receptor regulations.

There exists few direct functional studies on the various cells in the
TG. Hypothetically, a range of plausible targets, available to
circulating drugs with low BBB permeability, can be noted: (i)
Peripheral terminals of nociceptive A\selectlanguage{greek}δ-\selectlanguage{english}fibers and C-fibres innervating
various cranial structures, (ii) Receptors expressed at or proximal to
the nodes of Ranvier on A\selectlanguage{greek}δ-\selectlanguage{english}fibers (Edvinsson et al., 2019), (iii) Neuron
cell bodies (notably, in the TG these are vigorously enveloped by SGCs
which may act as a gate-keeper to systemically introduced drugs) and
(iv) the SGCs and Schwann cells in the TG (likely a complex signalling
relationship between these cells and proximal neurons/axons (Messlinger,
Balcziak \& Russo, 2020).

A recent study demonstrated that the CGRP receptor antagonist Erenumab
was internalized by both the CGRP receptor and AMY1 receptor in cultured
TG neurons (Bhakta, Vuong, Taura, Wilson, Stratton \& Mackenzie, 2021).
Thus, there is a possibility of paracrine CGRP signalling occurring with
SGC\selectlanguage{ngerman}´s expressing either the CGRP receptor or the AMY1 receptor. Both
receptor activation pathways lead to an upregulation of cAMP which in
turn could further sensitize neurons and axons to noxious stimuli.

Non-myelinating Schwann cells form Remak bundles around parts of C-fibre
axons. They are vital in providing trophic factors and regenerating
damaged C-fibres (Murinson \& Griffin, 2004). Disruption of
non-myelinating Schwann cell ErbB signalling in mice resulted in a
progressive sensory loss. This suggests intercellular signalling between
non-myelinating Schwann cells and unmyelinated sensory fibres and is
critical for C-fibre and Schwann cell survival. The implications of SGC
and Schwann cell interactions with neurons is a field of research not
yet fully understood. In part due to difficulties in studying the
structures \emph{in vitro} without disrupting the neuron-glia
architecture.

\emph{(iv). Action in the nodes of Ranvier -- axon-axon interaction.}

The exploration of CGRP and its receptor have so far yielded successful
scientific breakthroughs and therapies. RAMP1 and CLR are co-expressed
mainly in larger neurons and in the thinly myelinated fibres, typically
identified as A\selectlanguage{greek}δ-\selectlanguage{english}fibres (Edvinsson et al., 2019; Eftekhari, Salvatore,
Calamari, Kane, Tajti \& Edvinsson, 2010). CGRP receptors are found
distally in the dura mater, associated both with the vessels and at
avascular sites. In larger arteries, these fibres run in the adventitia,
close to the C-fibres. Interestingly, the C-fibres storing CGRP could
align with adjacent nodes of Ranvier in the A\selectlanguage{greek}δ-\selectlanguage{english}fibres (Edvinsson et al.,
2019). This led to the suggestion that at the nodes of Ranvier local
release of CGRP may act on CGRP receptors on the A\selectlanguage{greek}δ-\selectlanguage{english}fibres to modify the
sensory signalling. In this region, CLR/RAMP1 as well as a complete CGRP
receptor antibody immunoreactivity were observed on the A\selectlanguage{greek}δ-\selectlanguage{english}fibres and
associated with protein kinase A, which provides a link towards the
possible phosphorylation of ion channels in A\selectlanguage{greek}δ-\selectlanguage{english}fibres (Edvinsson et al.,
2019). This mechanism may alter ion channel activity at the A\selectlanguage{greek}δ-\selectlanguage{english}fibres
and contribute to the understanding of pain signalling and perhaps also
the sensitization processes. The nodes are defined by their lack of the
many layers of myelinated Schwann cells, which is why they may be a
target for therapeutic drugs such as gepants and mAbs to interact within
the trigeminal system and the pain perception.

\textbf{4. FUTURE PERSPECTIVES.}

Future research should aim to compare quantitatively the expression of
the discussed peptides and receptors, both in relation to sex and age.
Preliminary expression studies suggest that (i) CGRP and CT are the most
expressed of the studied peptides, (ii) that CGRP receptor and CTR are
the most frequently expressed receptor types and (iii) that
AM\textsubscript{2,} AMY\textsubscript{1} and
AMY\textsubscript{3}receptors occur in rat TG, but are mainly localized
to the SGCs. The demonstration of the specific ligands and receptor
sites in TG neurons highlights the importance of CGRP and the CGRP
receptor as viable contributors in primary headache disorders. Future
investigations of the expression of the CGRP-family of peptides near the
node of Ranvier is warranted. Understanding the location and
distribution is important in deciding on how to identify future targets
for anti-migraine medications and this will facilitate future drug
development and ultimately enrich the therapeutic options for the
patients.

\textbf{Figure Legends.}

\textbf{Figure 1.} Immunohistochemical demonstration of localization of
CGRP, CT, Amylin and Adrenomedullin in the trigeminal
ganglion.\textbf{A.} CGRP is expressed in a granular pattern in many
neurons, mainly in small- to medium-sized neurons (arrow heads). The
cellular CGRP is expressed in vesicles (insert). In addition, pearl-like
CGRP immunoreactivity was detected in fibers that are of the C-type of
sensory unmyelinated nerves (long arrows). The myelinated fibers do not
contain CGRP. Short arrow points at a large negative neuron. \textbf{B}
. CT immunoreactivity displayed a similar pattern as for CGRP; granular
staining of small- to medium-sized neurons (arrow heads, insert) and
pearl-like staining of fibers. Also, SGCs were CT immunoreactive
(asterisks). Arrows point at a large negative neuron. \textbf{C} . AMY
was exclusively expressed in the neurons, mainly small to medium sized
(arrow heads). In some of the cells, the expression was granular, but in
others, a general cytoplasmic immunoreactivity. Arrows point at a large
negative neuron. Insert shows three different cells: one negative and
two amylin immunoreactive. \textbf{D} . AM was expressed in the glial
cells, both the SGCs (arrow heads) and cells enveloping the neuronal
processes (arrows), probably myelinating cells.

\textbf{Figure 2.} Schematic illustration of the differential
distribution of the CGRP family of peptides and receptors. The CGRP
receptor (CLR/RAMP1) is mainly expressed in the large neurons and SGCs,
the Adrenomedullin\textsubscript{2} receptor (CLR/RAMP3) in some SGCs,
the Amylin\textsubscript{1} receptor (CTR/RAMP1) in~large neurons and
SGCs, and the Amylin\textsubscript{3} receptor (CTR/RAMP3) in some SGCs
Modified from Edvinsson, Grell \& Warfvinge, 2020.

\textbf{REFERENCES}

(2018). Global, regional, and national incidence, prevalence, and years
lived with disability for 354 diseases and injuries for 195 countries
and territories, 1990-2017: a systematic analysis for the Global Burden
of Disease Study 2017. Lancet (London, England) 392\textbf{:} 1789-1858.

Amin FM, Asghar MS, Hougaard A, Hansen AE, Larsen VA, de Koning
PJ\emph{, et al.} (2013). Magnetic resonance angiography of intracranial
and extracranial arteries in patients with spontaneous migraine without
aura: a cross-sectional study. Lancet Neurol 12\textbf{:} 454-461.

Amrutkar DV, Ploug KB, Hay-Schmidt A, Porreca F, Olesen J, \&
Jansen-Olesen I (2012). mRNA expression of 5-hydroxytryptamine 1B, 1D,
and 1F receptors and their role in controlling the release of calcitonin
gene-related peptide in the rat trigeminovascular system. Pain
153\textbf{:} 830-838.

Ashina M, Hansen JM, BO AD, \& Olesen J (2017). Human models of migraine
- short-term pain for long-term gain. Nat Rev Neurol
13\textbf{:}713-724.

Ashina MD, D. Bonner, JH et al (2019) \emph{A phase 2, randomized,
double-blind, placebo-controlled study to evaluate the efficacy and
safety of AMG 301 in migraine prevention.} . vol. Athens.

Bhakta M, Vuong T, Taura T, Wilson DS, Stratton JR, \& Mackenzie KD
(2021). Migraine therapeutics differentially modulate the CGRP pathway.
Cephalalgia\textbf{:} 0333102420983282.

Bower RL, Eftekhari S, Waldvogel HJ, Faull RL, Tajti J, Edvinsson
L\emph{, et al.} (2016). Mapping the calcitonin receptor in human brain
stem. Am J Physiol Regul Integr Comp Physiol 310\textbf{:} R788-793.

Charles A, \& Pozo-Rosich P (2019). Targeting calcitonin gene-related
peptide: a new era in migraine therapy. Lancet (London, England)
394\textbf{:} 1765-1774.

Cooper GJ, Willis AC, Clark A, Turner RC, Sim RB, \& Reid KB (1987).
Purification and characterization of a peptide from amyloid-rich
pancreases of type 2 diabetic patients. Proc Natl Acad Sci U S A
84\textbf{:} 8628-8632.

Edvinsson JCA, Warfvinge K, Krause DN, Blixt FW, Sheykhzade M, Edvinsson
L\emph{, et al.} (2019). C-fibers may modulate adjacent Adelta-fibers
through axon-axon CGRP signaling at nodes of Ranvier in the trigeminal
system. J Headache Pain 20\textbf{:} 105.

Edvinsson L (2011). Tracing neural connections to pain pathways with
relevance to primary headaches. Cephalalgia 31\textbf{:} 737-747.

Edvinsson L, Alm R, Shaw D, Rutledge RZ, Koblan KS, Longmore J\emph{, et
al.} (2002). Effect of the CGRP receptor antagonist BIBN4096BS in human
cerebral, coronary and omental arteries and in SK-N-MC cells. Eur J
Pharmacol 434\textbf{:} 49-53.

Edvinsson L, Chan KY, Eftekhari S, Nilsson E, de Vries R, Saveland
H\emph{, et al.} (2010). Effect of the calcitonin gene-related peptide
(CGRP) receptor antagonist telcagepant in human cranial arteries.
Cephalalgia 30\textbf{:} 1233-1240.

Edvinsson L, Ekman R, Jansen I, McCulloch J, \& Uddman R (1987).
Calcitonin gene-related peptide and cerebral blood vessels: distribution
and vasomotor effects. J Cereb Blood Flow Metab 7\textbf{:} 720-728.

Edvinsson L, Fredholm BB, Hamel E, Jansen I, \& Verrecchia C (1985).
Perivascular peptides relax cerebral arteries concomitant with
stimulation of cyclic adenosine monophosphate accumulation or release of
an endothelium-derived relaxing factor in the cat. Neurosci Lett
58\textbf{:} 213-217.

Edvinsson L, Goadsby PJ, \& Uddman R (2001). Amylin: localization,
effects on cerebral arteries and on local cerebral blood flow in the
cat. ScientificWorldJournal 1\textbf{:} 168-180.

Edvinsson L, Grell AS, \& Warfvinge K (2020). Expression of the CGRP
Family of Neuropeptides and their Receptors in the Trigeminal Ganglion.
J Mol Neurosci 70\textbf{:} 930-944.

Edvinsson L, Haanes KA, \& Warfvinge K (2019). Does inflammation have a
role in migraine? Nat Rev Neurol 15\textbf{:} 483-490.

Edvinsson L, Haanes KA, Warfvinge K, \& Krause DN (2018). CGRP as the
target of new migraine therapies - successful translation from bench to
clinic. Nat Rev Neurol 14\textbf{:} 338-350.

Edvinsson L, Nilsson E, \& Jansen-Olesen I (2007). Inhibitory effect of
BIBN4096BS, CGRP(8-37), a CGRP antibody and an RNA-Spiegelmer on CGRP
induced vasodilatation in the perfused and non-perfused rat middle
cerebral artery. Br J Pharmacol 150\textbf{:} 633-640.

Eftekhari S, Gaspar RC, Roberts R, Chen TB, Zeng Z, Villarreal S\emph{,
et al.} (2016). 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
524\textbf{:} 90-118.

Eftekhari S, Salvatore CA, Calamari A, Kane SA, Tajti J, \& Edvinsson L
(2010). Differential distribution of calcitonin gene-related peptide and
its receptor components in the human trigeminal ganglion. Neuroscience
169\textbf{:} 683-696.

Eftekhari S, Salvatore CA, Johansson S, Chen TB, Zeng Z, \& Edvinsson L
(2015). Localization of CGRP, CGRP receptor, PACAP and glutamate in
trigeminal ganglion. Relation to the blood-brain barrier. Brain Res
1600\textbf{:} 93-109.

Eftekhari S, Warfvinge K, Blixt FW, \& Edvinsson L (2013).
Differentiation of nerve fibers storing CGRP and CGRP receptors in the
peripheral trigeminovascular system. J Pain 14\textbf{:} 1289-1303.

Erdling A, Sheykhzade M, \& Edvinsson L (2017). Differential inhibitory
response to telcagepant on alphaCGRP induced vasorelaxation and
intracellular Ca2+ levels in the perfused and non-perfused isolated rat
middle cerebral artery. J Headache Pain 18\textbf{:} 61.

Ferrero H, Larrayoz IM, Gil-Bea FJ, Martinez A, \& Ramirez MJ (2018).
Adrenomedullin, a Novel Target for Neurodegenerative Diseases. Mol
Neurobiol.

Ferrero H, Larrayoz IM, Martisova E, Solas M, Howlett DR, Francis
PT\emph{, et al.} (2018). Increased Levels of Brain Adrenomedullin in
the Neuropathology of Alzheimer's Disease. Mol Neurobiol
55\textbf{:}5177-5183.

Findlay DM, \& Sexton PM (2004). Calcitonin. Growth Factors
22\textbf{:}217-224.

Fontaine D, Almairac F, Santucci S, Fernandez C, Dallel R, Pallud
J\emph{, et al.} (2018). Dural and pial pain-sensitive structures in
humans: new inputs from awake craniotomies. Brain
141\textbf{:}1040-1048.

GBD 2016 Disease and Injury Incidence and Prevalence Collaborators
(2017). Global, regional, and national incidence, prevalence, and years
lived with disability for 328 diseases and injuries for 195 countries,
1990-2016: a systematic analysis for the Global Burden of Disease Study
2016. Lancet (London, England) 390\textbf{:} 1211-1259.

Gingell JJ, Hendrikse ER, \& Hay DL (2019). New Insights into the
Regulation of CGRP-Family Receptors. Trends Pharmacol Sci
40\textbf{:}71-83.

Goadsby PJ, \& Akerman S (2012). The trigeminovascular system does not
require a peripheral sensory input to be activated--migraine is a
central disorder. Focus on 'Effect of cortical spreading depression on
basal and evoked traffic in the trigeminovascular sensory system'.
Cephalalgia 32\textbf{:} 3-5.

Goadsby PJ, \& Edvinsson L (1994). Joint 1994 Wolff Award Presentation.
Peripheral and central trigeminovascular activation in cat is blocked by
the serotonin (5HT)-1D receptor agonist 311C90. Headache
34\textbf{:}394-399.

Goadsby PJ, Edvinsson L, \& Ekman R (1988). Release of vasoactive
peptides in the extracerebral circulation of humans and the cat during
activation of the trigeminovascular system. Ann Neurol
23\textbf{:}193-196.

Goadsby PJ, Holland PR, Martins-Oliveira M, Hoffmann J, Schankin C, \&
Akerman S (2017). Pathophysiology of Migraine: A Disorder of Sensory
Processing. Physiol Rev 97\textbf{:} 553-622.

Goadsby PJ, \& Knight YE (1997). Direct evidence for central sites of
action of zolmitriptan (311C90): an autoradiographic study in cat.
Cephalalgia 17\textbf{:} 153-158.

Hadjikhani N, Albrecht DS, Mainero C, Ichijo E, Ward N, Granziera
C\emph{, et al.} (2020). Extra-Axial Inflammatory Signal in Parameninges
in Migraine with Visual Aura. Ann Neurol 87\textbf{:} 939-949.

Hay DL (2017). Amylin. Headache 57 Suppl 2\textbf{:} 89-96.

Hay DL, Garelja ML, Poyner DR, \& Walker CS (2018). Update on the
pharmacology of calcitonin/CGRP family of peptides: IUPHAR Review 25. Br
J Pharmacol 175\textbf{:} 3-17.

Hay DL, Harris PW, Kowalczyk R, Brimble MA, Rathbone DL, Barwell
J\emph{, et al.} (2014). Structure-activity relationships of the
N-terminus of calcitonin gene-related peptide: key roles of alanine-5
and threonine-6 in receptor activation. Br J Pharmacol
171\textbf{:}415-426.

Hay DL, \& Pioszak AA (2016). Receptor Activity-Modifying Proteins
(RAMPs): New Insights and Roles. Annu Rev Pharmacol Toxicol
56\textbf{:}469-487.

Hendrikse ER, Bower RL, Hay DL, \& Walker CS (2018). Molecular studies
of CGRP and the CGRP family of peptides in the central nervous system.
Cephalalgia\textbf{:} 333102418765787.

Ho TW, Edvinsson L, \& Goadsby PJ (2010). CGRP and its receptors provide
new insights into migraine pathophysiology. Nat Rev Neurol
6\textbf{:}573-582.

Hoffmann J, Wecker S, Neeb L, Dirnagl U, \& Reuter U (2012). Primary
trigeminal afferents are the main source for stimulus-induced CGRP
release into jugular vein blood and CSF. Cephalalgia
32\textbf{:}659-667.

Irimia P, Mart\selectlanguage{ngerman}ínez-Valbuena I, Mínguez-Olaondo A, Domínguez-Vivero C,
Sánchez-Arias J-A, Martínez-Vila E\emph{, et al.} Interictal amylin
levels in chronic migraine patients: A case-control study. Cephalalgia
0\textbf{:} 0333102420977106.

Johnson KW, Morin SM, Wroblewski VJ, \& Johnson MP (2019). Peripheral
and central nervous system distribution of the CGRP neutralizing
antibody {[}(125)I{]} galcanezumab in male rats. Cephalalgia
39\textbf{:} 1241-1248.

Juhl L, Petersen KA, Larsen EH, Jansen-Olesen I, \& Olesen J (2006). The
in vivo effect of adrenomedullin on rat dural and pial arteries. Eur J
Pharmacol 538\textbf{:} 101-107.

Khan S, Amin FM, Fliedner FP, Christensen CE, Tolnai D, Younis S\emph{,
et al.} (2019). Investigating macrophage-mediated inflammation in
migraine using ultrasmall superparamagnetic iron oxide-enhanced 3T
magnetic resonance imaging. Cephalalgia 39\textbf{:} 1407-1420.

Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H\emph{,
et al.} (1993). Adrenomedullin: a novel hypotensive peptide isolated
from human pheochromocytoma. Biochem Biophys Res Commun
192\textbf{:}553-560.

Labastida-Ramirez A, Rubio-Beltran E, Haanes KA, Chan KY, Garrelds IM,
Johnson KW\emph{, et al.} (2020). Lasmiditan inhibits calcitonin
gene-related peptide release in the rodent trigeminovascular system.
Pain 161\textbf{:} 1092-1099.

Lennerz JK, Ruhle V, Ceppa EP, Neuhuber WL, Bunnett NW, Grady EF\emph{,
et al.} (2008). Calcitonin receptor-like receptor (CLR), receptor
activity-modifying protein 1 (RAMP1), and calcitonin gene-related
peptide (CGRP) immunoreactivity in the rat trigeminovascular system:
differences between peripheral and central CGRP receptor distribution. J
Comp Neurol 507\textbf{:} 1277-1299.

Levy D, Burstein R, \& Strassman AM (2005). Calcitonin gene-related
peptide does not excite or sensitize meningeal nociceptors: implications
for the pathophysiology of migraine. Ann Neurol 58\textbf{:} 698-705.

Lundblad C, Haanes KA, Grande G, \& Edvinsson L (2015). Experimental
inflammation following dural application of complete Freund's adjuvant
or inflammatory soup does not alter brain and trigeminal microvascular
passage. J Headache Pain 16\textbf{:} 91.

May A (2017). Understanding migraine as a cycling brain syndrome:
reviewing the evidence from functional imaging. Neurol Sci
38\textbf{:}125-130.

May A, \& Burstein R (2019). Hypothalamic regulation of headache and
migraine. Cephalalgia\textbf{:} 333102419867280.

McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N\emph{, et
al.} (1998). RAMPs regulate the transport and ligand specificity of the
calcitonin-receptor-like receptor. Nature 393\textbf{:} 333-339.

Messlinger K, Balcziak LK, \& Russo AF (2020). Cross-talk signaling in
the trigeminal ganglion: role of neuropeptides and other mediators.
Journal of Neural Transmission 127\textbf{:} 431-444.

Messlinger K, \& Russo AF (2019). Current understanding of trigeminal
ganglion structure and function in headache. Cephalalgia
39\textbf{:}1661-1674.

Mietlicki-Baase EG, McGrath LE, Koch-Laskowski K, Krawczyk J, Reiner DJ,
Pham T\emph{, et al.} (2017). Amylin receptor activation in the ventral
tegmental area reduces motivated ingestive behavior. Neuropharmacology
123\textbf{:} 67-79.

Miller S, Liu H, Warfvinge K, Shi L, Dovlatyan M, Xu C\emph{, et
al.}(2016). Immunohistochemical localization of the calcitonin
gene-related peptide binding site in the primate trigeminovascular
system using functional antagonist antibodies. Neuroscience
328\textbf{:} 165-183.

Mulder IA, Li M, de Vries T, Qin T, Yanagisawa T, Sugimoto K\emph{, et
al.} (2020). Anti-migraine Calcitonin Gene-Related Peptide Receptor
Antagonists Worsen Cerebral Ischemic Outcome in Mice. Ann Neurol
88\textbf{:} 771-784.

Murinson BB, \& Griffin JW (2004). C-fiber structure varies with
location in peripheral nerve. Journal of Neuropathology \& Experimental
Neurology 63\textbf{:} 246-254.

Noseda R, Schain AJ, Melo-Carrillo A, Tien J, Stratton J, Mai F\emph{,
et al.} (2020). 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 40\textbf{:}229-240.

Ohlsson L, Haanes KA, Kronvall E, Xu C, Snellman J, \& Edvinsson L
(2019). Erenumab (AMG 334), a monoclonal antagonist antibody against the
canonical CGRP receptor, does not impair vasodilatory or contractile
responses to other vasoactive agents in human isolated cranial arteries.
Cephalalgia 39\textbf{:} 1745-1752.

Ohlsson L, Kronvall E, Stratton J, \& Edvinsson L (2018). Fremanezumab
blocks CGRP induced dilatation in human cerebral, middle meningeal and
abdominal arteries. J Headache Pain 19\textbf{:} 66.

Oliver KR, Wainwright A, Edvinsson L, Pickard JD, \& Hill RG (2002).
Immunohistochemical localization of calcitonin receptor-like receptor
and receptor activity-modifying proteins in the human cerebral
vasculature. J Cereb Blood Flow Metab 22\textbf{:} 620-629.

Pietrobon D, \& Moskowitz MA (2013). Pathophysiology of migraine. Annu
Rev Physiol 75\textbf{:} 365-391.

Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W\emph{, et
al.} (2002). International Union of Pharmacology. XXXII. The mammalian
calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin
receptors. Pharmacol Rev 54\textbf{:} 233-246.

Rubio-Beltran E, Chan KY, Danser AJ, MaassenVanDenBrink A, \& Edvinsson
L (2020). Characterisation of the calcitonin gene-related peptide
receptor antagonists ubrogepant and atogepant in human isolated
coronary, cerebral and middle meningeal arteries. Cephalalgia
40\textbf{:} 357-366.

Satoh F, Takahashi K, Murakami O, Totsune K, Sone M, Ohneda M\emph{, et
al.} (1996). Immunocytochemical localization of adrenomedullin-like
immunoreactivity in the human hypothalamus and the adrenal gland.
Neurosci Lett 203\textbf{:} 207-210.

Saxena PR, \& Den Boer MO (1991). Pharmacology of antimigraine drugs. J
Neurol 238 Suppl 1\textbf{:} S28-35.

Schain AJ, Melo-Carrillo A, Stratton J, Strassman AM, \& Burstein R
(2019). CSD-Induced Arterial Dilatation and Plasma Protein Extravasation
Are Unaffected by Fremanezumab: Implications for CGRP's Role in Migraine
with Aura. J Neurosci 39\textbf{:} 6001-6011.

Schulte LH, \& May A (2017). Of generators, networks and migraine
attacks. Curr Opin Neurol 30\textbf{:} 241-245.

Schulte LH, Mehnert J, \& May A (2020). Longitudinal Neuroimaging over
30 Days: Temporal Characteristics of Migraine. Ann Neurol
87\textbf{:}646-651.

Schulte LH, Menz MM, Haaker J, \& May A (2020). The migraineur's brain
networks: Continuous resting state fMRI over 30 days. Cephalalgia
40\textbf{:} 1614-1621.

Schultz N, Byman E, Fex M, \& Wennstrom M (2017). Amylin alters human
brain pericyte viability and NG2 expression. J Cereb Blood Flow Metab
37\textbf{:} 1470-1482.

Serrano J, Uttenthal LO, Martinez A, Fernandez AP, Martinez de Velasco
J, Alonso D\emph{, et al.} (2000). Distribution of adrenomedullin-like
immunoreactivity in the rat central nervous system by light and electron
microscopy. Brain Res 853\textbf{:} 245-268.

Walker CS, Eftekhari S, Bower RL, Wilderman A, Insel PA, Edvinsson
L\emph{, et al.} (2015). A second trigeminal CGRP receptor: function and
expression of the AMY1 receptor. Ann Clin Transl Neurol
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