Differentiation of Nerve Fibers Storing CGRP and CGRP Receptors in the Peripheral Trigeminovascular System

  • Sajedeh Eftekhari
    Address reprint requests to Sajedeh Eftekhari, MSc, Department of Clinical Sciences, Division of Experimental Vascular Research, BMC A13, Sölvegatan 17, SE-22184 Lund, Sweden.
    Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University, Lund, Sweden
    Search for articles by this author
  • Karin Warfvinge
    Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University, Lund, Sweden

    Department of Clinical Experimental Research, Glostrup Research Institute, University of Copenhagen, Glostrup Hospital, Copenhagen, Denmark
    Search for articles by this author
  • Frank W. Blixt
    Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University, Lund, Sweden
    Search for articles by this author
  • Lars Edvinsson
    Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University, Lund, Sweden

    Department of Clinical Experimental Research, Glostrup Research Institute, University of Copenhagen, Glostrup Hospital, Copenhagen, Denmark
    Search for articles by this author
Published:August 19, 2013DOI:


      Primary headaches such as migraine are postulated to involve the activation of sensory trigeminal pain neurons that innervate intracranial blood vessels and the dura mater. It is suggested that local activation of these sensory nerves may involve dural mast cells as one factor in local inflammation, causing sensitization of meningeal nociceptors. Immunofluorescence was used to study the detailed distribution of calcitonin gene–related peptide (CGRP) and its receptor components calcitonin receptor–like receptor (CLR) and receptor activity–modifying protein 1 (RAMP1) in whole-mount rat dura mater and in human dural vessels. The relative distributions of CGRP, CLR, and RAMP1 were evaluated with respect to each other and in relationship to mast cells, myelin, substance P, neuronal nitric oxide synthase, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal peptide. CGRP expression was found in thin unmyelinated fibers, whereas CLR and RAMP1 were expressed in thicker myelinated fibers coexpressed with an A-fiber marker. CLR and RAMP1 immunoreactivity colocalized with mast cell tryptase in rodent; however, expression of both receptor components was not observed in human mast cells. Immunoreactive substance P fibers coexpressed CGRP, although neuronal nitric oxide synthase and vasoactive intestinal peptide expression was very limited, and these fibers were distinct from the CGRP-positive fibers. Few pituitary adenylate cyclase-activating polypeptide immunoreactive fibers occurred and some colocalized with CGRP.


      This study demonstrates the detailed distribution of CGRP and its receptor in the dura mater. These data suggest that CGRP is expressed in C-fibers and may act on A-fibers, rodent mast cells, and vascular smooth muscle cells that express the CGRP receptor. These sites represent potential pathophysiological targets of novel antimigraine agents such as the newly developed CGRP receptor antagonists.

      Key words

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to The Journal of Pain
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Andres K.H.
        • von During M.
        • Muszynski K.
        • Schmidt R.F.
        Nerve fibres and their terminals of the dura mater encephali of the rat.
        Anat Embryol (Berl). 1987; 175: 289-301
        • Bahra A.
        • Matharu M.S.
        • Buchel C.
        • Frackowiak R.S.
        • Goadsby P.J.
        Brainstem activation specific to migraine headache.
        Lancet. 2001; 357: 1016-1017
        • Csati A.
        • Tajti J.
        • Tuka B.
        • Edvinsson L.
        • Warfvinge K.
        Calcitonin gene-related peptide and its receptor components in the human sphenopalatine ganglion—Interaction with the sensory system.
        Brain Res. 2012; 1435: 29-39
        • Dieterle A.
        • Fischer M.J.
        • Link A.S.
        • Neuhuber W.L.
        • Messlinger K.
        Increase in CGRP- and nNOS-immunoreactive neurons in the rat trigeminal ganglion after infusion of an NO donor.
        Cephalalgia. 2011; 31: 31-42
        • Edvinsson L.
        Innervation of the cerebral circulation.
        Ann N Y Acad Sci. 1987; 519: 334-348
        • Edvinsson L.
        • Cantera L.
        • Jansen-Olesen I.
        • Uddman R.
        Expression of calcitonin gene-related peptide1 receptor mRNA in human trigeminal ganglia and cerebral arteries.
        Neurosci Lett. 1997; 229: 209-211
        • Edvinsson L.
        • Cervos-Navarro J.
        • Larsson L.I.
        • Owman C.
        • Ronnberg A.L.
        Regional distribution of mast cells containing histamine, dopamine, or 5-hydroxytryptamine in the mammalian brain.
        Neurology. 1977; 27: 878-883
        • Edvinsson L.
        • Chan K.Y.
        • Eftekhari S.
        • Nilsson E.
        • de Vries R.
        • Saveland H.
        • Dirven C.M.
        • Danser A.H.
        • MaassenVanDenBrink A.
        Effect of the calcitonin gene-related peptide (CGRP) receptor antagonist telcagepant in human cranial arteries.
        Cephalalgia. 2010; 30: 1233-1240
        • Edvinsson L.
        • Elsas T.
        • Suzuki N.
        • Shimizu T.
        • Lee T.J.
        Origin and Co-localization of nitric oxide synthase, CGRP, PACAP, and VIP in the cerebral circulation of the rat.
        Microsc Res Tech. 2001; 53: 221-228
        • Edvinsson L.
        • Fredholm B.B.
        • Hamel E.
        • Jansen I.
        • Verrecchia C.
        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. 1985; 58: 213-217
        • Edvinsson L.
        • Gulbenkian S.
        • Barroso C.P.
        • Cunha e Sa M.
        • Polak J.M.
        • Mortensen A.
        • Jorgensen L.
        • Jansen-Olesen I.
        Innervation of the human middle meningeal artery: Immunohistochemistry, ultrastructure, and role of endothelium for vasomotility.
        Peptides. 1998; 19: 1213-1225
        • Edvinsson L.
        • Linde M.
        New drugs in migraine treatment and prophylaxis: Telcagepant and topiramate.
        Lancet. 2010; 376: 645-655
        • Edvinsson L.
        • Uddman R.
        Adrenergic, cholinergic and peptidergic nerve fibres in dura mater—Involvement in headache?.
        Cephalalgia. 1981; 1: 175-179
        • Edvinsson L.
        • Uddman R.
        Neurobiology in primary headaches.
        Brain Res Brain Res Rev. 2005; 48: 438-456
        • Eftekhari S.
        • Edvinsson L.
        Possible sites of action of the new calcitonin gene-related peptide receptor antagonists.
        Ther Adv Neurol Disord. 2010; 3: 369-378
        • Eftekhari S.
        • Edvinsson L.
        Calcitonin gene-related peptide (CGRP) and its receptor components in human and rat spinal trigeminal nucleus and spinal cord at C1-level.
        BMC Neurosci. 2011; 12: 112
        • Eftekhari S.
        • Salvatore C.A.
        • Calamari A.
        • Kane S.A.
        • Tajti J.
        • Edvinsson L.
        Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion.
        Neuroscience. 2010; 169: 683-696
        • Galli S.J.
        New concepts about the mast cell.
        N Engl J Med. 1993; 328: 257-265
        • Garcia M.L.
        • Lobsiger C.S.
        • Shah S.B.
        • Deerinck T.J.
        • Crum J.
        • Young D.
        • Ward C.M.
        • Crawford T.O.
        • Gotow T.
        • Uchiyama Y.
        • Ellisman M.H.
        • Calcutt N.A.
        • Cleveland D.W.
        NF-M is an essential target for the myelin-directed “outside-in” signaling cascade that mediates radial axonal growth.
        J Cell Biol. 2003; 163: 1011-1020
        • Goadsby P.J.
        • Charbit A.R.
        • Andreou A.P.
        • Akerman S.
        • Holland P.R.
        Neurobiology of migraine.
        Neuroscience. 2009; 161: 327-341
        • 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: 48-56
        • Goadsby P.J.
        • Edvinsson L.
        • Ekman R.
        Vasoactive peptide release in the extracerebral circulation of humans during migraine headache.
        Ann Neurol. 1990; 28: 183-187
        • Hess D.T.
        • Slater T.M.
        • Wilson M.C.
        • Skene J.H.
        The 25 kDa synaptosomal-associated protein SNAP-25 is the major methionine-rich polypeptide in rapid axonal transport and a major substrate for palmitoylation in adult CNS.
        J Neurosci. 1992; 12: 4634-4641
        • Ho T.W.
        • Edvinsson L.
        • Goadsby P.J.
        CGRP and its receptors provide new insights into migraine pathophysiology.
        Nat Rev Neurol. 2010; 6: 573-582
        • Hsieh Y.L.
        • Lin C.L.
        • Chiang H.
        • Fu Y.S.
        • Lue J.H.
        • Hsieh S.T.
        Role of peptidergic nerve terminals in the skin: Reversal of thermal sensation by calcitonin gene-related peptide in TRPV1-depleted neuropathy.
        PLoS One. 2012; 7: e50805
        • Jansen I.
        • Uddman R.
        • Ekman R.
        • Olesen J.
        • Ottosson A.
        • Edvinsson L.
        Distribution and effects of neuropeptide Y, vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide in human middle meningeal arteries: Comparison with cerebral and temporal arteries.
        Peptides. 1992; 13: 527-536
        • Keller J.T.
        • Marfurt C.F.
        Peptidergic and serotoninergic innervation of the rat dura mater.
        J Comp Neurol. 1991; 309: 515-534
        • Lariviere R.C.
        • Julien J.P.
        Functions of intermediate filaments in neuronal development and disease.
        J Neurobiol. 2004; 58: 131-148
        • Lennerz J.K.
        • Ruhle V.
        • Ceppa E.P.
        • Neuhuber W.L.
        • Bunnett N.W.
        • Grady E.F.
        • Messlinger K.
        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. 2008; 507: 1277-1299
        • Levy D.
        • Burstein R.
        • Kainz V.
        • Jakubowski M.
        • Strassman A.M.
        Mast cell degranulation activates a pain pathway underlying migraine headache.
        Pain. 2007; 130: 166-176
        • Li J.
        • Vause C.V.
        • Durham P.L.
        Calcitonin gene-related peptide stimulation of nitric oxide synthesis and release from trigeminal ganglion glial cells.
        Brain Res. 2008; 1196: 22-32
        • Liu-Chen L.Y.
        • Liszczak T.M.
        • King J.C.
        • Moskowitz M.A.
        Immunoelectron microscopic study of substance P-containing fibers in feline cerebral arteries.
        Brain Res. 1986; 369: 12-20
        • Martin J.H.
        3rd ed. McGraw-Hill, New York2003
        • McLatchie L.M.
        • Fraser N.J.
        • Main M.J.
        • Wise A.
        • Brown J.
        • Thompson N.
        • Solari R.
        • Lee M.G.
        • Foord S.M.
        RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor.
        Nature. 1998; 393: 333-339
        • Messlinger K.
        • Hanesch U.
        • Baumgartel M.
        • Trost B.
        • Schmidt R.F.
        Innervation of the dura mater encephali of cat and rat: Ultrastructure and calcitonin gene-related peptide-like and substance P-like immunoreactivity.
        Anat Embryol (Berl). 1993; 188: 219-237
        • Moller K.
        • Zhang Y.Z.
        • Hakanson R.
        • Luts A.
        • Sjolund B.
        • Uddman R.
        • Sundler F.
        Pituitary adenylate cyclase activating peptide is a sensory neuropeptide: Immunocytochemical and immunochemical evidence.
        Neuroscience. 1993; 57: 725-732
        • Moskowitz M.A.
        Neurogenic inflammation in the pathophysiology and treatment of migraine.
        Neurology. 1993; 43: S16-S20
      1. Newswire P: Labrys Biologics Secures $31 Million Series A Financing from Venture Capital Firms; Acquires Phase 2 Ready Antibody for Chronic Migraine from Pfizer. Available at: Accessed February 25, 2013

        • Nozaki K.
        • Uemura Y.
        • Okamoto S.
        • Kikuchi H.
        • Mizuno N.
        Origins and distribution of cerebrovascular nerve fibers showing calcitonin gene-related peptide-like immunoreactivity in the major cerebral artery of the dog.
        J Comp Neurol. 1990; 297: 219-226
        • Olesen J.
        • Burstein R.
        • Ashina M.
        • Tfelt-Hansen P.
        Origin of pain in migraine: Evidence for peripheral sensitisation.
        Lancet Neurol. 2009; 8: 679-690
        • Oliver K.R.
        • Wainwright A.
        • Edvinsson L.
        • Pickard J.D.
        • Hill R.G.
        Immunohistochemical localization of calcitonin receptor-like receptor and receptor activity-modifying proteins in the human cerebral vasculature.
        J Cereb Blood Flow Metab. 2002; 22: 620-629
        • Orr E.L.
        • Pace K.R.
        The significance of mast cells as a source of histamine in the mouse brain.
        J Neurochem. 1984; 42: 727-732
        • Ottosson A.
        • Edvinsson L.
        Release of histamine from dural mast cells by substance P and calcitonin gene-related peptide.
        Cephalalgia. 1997; 17: 166-174
        • Perry M.J.
        • Lawson S.N.
        • Robertson J.
        Neurofilament immunoreactivity in populations of rat primary afferent neurons: A quantitative study of phosphorylated and non-phosphorylated subunits.
        J Neurocytol. 1991; 20: 746-758
        • Ramachandran R.
        • Ploug K.B.
        • Hay-Schmidt A.
        • Olesen J.
        • Jansen-Olesen I.
        • Gupta S.
        Nitric oxide synthase (NOS) in the trigeminal vascular system and other brain structures related to pain in rats.
        Neurosci Lett. 2010; 484: 192-196
        • Ray B.S.
        • Wolff H.G.
        Experimental studies on headache: Pain-sensitive structures of the head and their significance in headache.
        Arch Surg. 1940; 41: 813-856
        • Rozniecki J.J.
        • Dimitriadou V.
        • Lambracht-Hall M.
        • Pang X.
        • Theoharides T.C.
        Morphological and functional demonstration of rat dura mater mast cell-neuron interactions in vitro and in vivo.
        Brain Res. 1999; 849: 1-15
        • Schwenger N.
        • Dux M.
        • de Col R.
        • Carr R.
        • Messlinger K.
        Interaction of calcitonin gene-related peptide, nitric oxide and histamine release in neurogenic blood flow and afferent activation in the rat cranial dura mater.
        Cephalalgia. 2007; 27: 481-491
        • Shi L.
        • Rao S.
        • Sun H.
        • Wild K.
        • Xu C.
        In vitro characterization of AA71, a potent and selective human monoclonal antibody against CGRP receptor.
        European Headache and Migraine Trust International Congress, London, UKSeptember 20–23, 2012
        • Silberstein S.D.
        Migraine pathophysiology and its clinical implications.
        Cephalalgia. 2004; 24: 2-7
        • Simone D.A.
        • Nolano M.
        • Johnson T.
        • Wendelschafer-Crabb G.
        • Kennedy W.R.
        Intradermal injection of capsaicin in humans produces degeneration and subsequent reinnervation of epidermal nerve fibers: Correlation with sensory function.
        J Neurosci. 1998; 18: 8947-8959
        • Sollner T.
        • Whiteheart S.W.
        • Brunner M.
        • Erdjument-Bromage H.
        • Geromanos S.
        • Tempst P.
        • Rothman J.E.
        SNAP receptors implicated in vesicle targeting and fusion.
        Nature. 1993; 362: 318-324
        • Strassman A.M.
        • Raymond S.A.
        • Burstein R.
        Sensitization of meningeal sensory neurons and the origin of headaches.
        Nature. 1996; 384: 560-564
        • Tajti J.
        • Uddman R.
        • Moller S.
        • Sundler F.
        • Edvinsson L.
        Messenger molecules and receptor mRNA in the human trigeminal ganglion.
        J Auton Nerv Syst. 1999; 76: 176-183
        • Tsai S.H.
        • Tew J.M.
        • McLean J.H.
        • Shipley M.T.
        Cerebral arterial innervation by nerve fibers containing calcitonin gene-related peptide (CGRP): I. Distribution and origin of CGRP perivascular innervation in the rat.
        J Comp Neurol. 1988; 271: 435-444
        • Weiller C.
        • May A.
        • Limmroth V.
        • Juptner M.
        • Kaube H.
        • Schayck R.V.
        • Coenen H.H.
        • Diener H.C.
        Brain stem activation in spontaneous human migraine attacks.
        Nat Med. 1995; 1: 658-660