+ All Categories
Home > Documents > Periaqueductal gray calcitonin

Periaqueductal gray calcitonin

Date post: 10-Sep-2015
Category:
Upload: robin-james-storer
View: 218 times
Download: 1 times
Share this document with a friend
Description:
AbstractBackground: Calcitonin gene-related peptide (CGRP) receptor antagonism is an approach to migraine therapy. The locusof action of antimigraine treatment is not resolved. The objective was to investigate CGRP receptors in the ventrolateralperiaqueductal gray (vlPAG) involved in the modulation of trigeminovascular nociception by descending influences onneurotransmission.Methods: The presence of calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1),which form functional CGRP receptors, was investigated. CGRP and its receptor antagonists, olcegepant and CGRP(8–37), were microinjected into the vlPAG while changes of neural responses in the trigeminocervical complex (TCC)were monitored.Results: Immunoreactivity indicated the presence of functional CGRP receptor components in the vlPAG and adjacentmesencephalic trigeminal nucleus. Inhibition of TCC responses to stimulation of dural afferents and ophthalmic cutaneousreceptive fields after microinjection of bicuculline into vlPAG indicated a connection between the vlPAG and TCCneurons. CGRP facilitated these TCC responses, whereas olcegepant and CGRP (8–37) decreased them.Conclusions: CGRP and its receptor antagonists act on neurons in the region of vlPAG to influence nociceptive transmissionin the TCC. This suggests CGRP receptor antagonists may act at loci outside of the TCC and reinforces theconcept of migraine as a disorder of the brain.
Popular Tags:
10
Original Article Periaqueductal gray calcitonin gene-related peptide modulates trigeminovascular neurons P Pozo-Rosich 1,a , RJ Storer 1,b , AR Charbit 1 and PJ Goadsby 1,2 Abstract Background: Calcitonin gene-related peptide (CGRP) receptor antagonism is an approach to migraine therapy. The locus of action of antimigraine treatment is not resolved. The objective was to investigate CGRP receptors in the ventrolateral periaqueductal gray (vlPAG) involved in the modulation of trigeminovascular nociception by descending influences on neurotransmission. Methods: The presence of calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1), which form functional CGRP receptors, was investigated. CGRP and its receptor antagonists, olcegepant and CGRP (8–37), were microinjected into the vlPAG while changes of neural responses in the trigeminocervical complex (TCC) were monitored. Results: Immunoreactivity indicated the presence of functional CGRP receptor components in the vlPAG and adjacent mesencephalic trigeminal nucleus. Inhibition of TCC responses to stimulation of dural afferents and ophthalmic cuta- neous receptive fields after microinjection of bicuculline into vlPAG indicated a connection between the vlPAG and TCC neurons. CGRP facilitated these TCC responses, whereas olcegepant and CGRP (8–37) decreased them. Conclusions: CGRP and its receptor antagonists act on neurons in the region of vlPAG to influence nociceptive trans- mission in the TCC. This suggests CGRP receptor antagonists may act at loci outside of the TCC and reinforces the concept of migraine as a disorder of the brain. Keywords Periaqueductal gray matter, trigeminovascular, migraine, CGRP Date received: 2 September 2014; revised: 24 December 2014; 6 February 2015; accepted: 13 February 2015 Introduction Calcitonin gene-related peptide (CGRP) plays a role in a variety of biologic functions including nocicep- tion. It has been widely implicated in the pathophysi- ology of migraine and other primary headaches (1–3). Clinical trials have shown that the CGRP receptor antagonists olcegepant (4), telcagepant (5), MK-3207 (6) and BI 44370 TA (7) are effective in the acute treatment of migraine. To understand better the mechanism of CGRP receptor antagonist actions in alleviating primary headache disorders, it is import- ant to identify their sites of action within cranial nociceptive systems (2). Because of the widespread distribution of CGRP and CGRP receptors in the brain, it appears possible that CGRP-receptor antag- onists have an action at supramedullary structures. We have seen effects of CGRP receptor modulation 1 Headache Group-Department of Neurology, University of California, San Francisco, CA, USA 2 Headache Group- Basic & Clinical Neuroscience, King’s College London, UK Corresponding author: PJ Goadsby, NIHR-Wellcome Trust Clinical Research Facility, King’s College Hospital, London SE5 9PJ, UK. Email: [email protected] a Current address: Headache and Neurological Pain Research Group, Vall d’Hebron Institute of Research (VHIR), Universitat Auto ` noma de Barcelona & Neurology Department, Vall d’Hebron University Hospital, Barcelona, Spain b Current address: Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Cephalalgia 0(0) 1–10 ! International Headache Society 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0333102415576723 cep.sagepub.com
Transcript
  • XML Template (2015) [18.3.201511:12am] [110]//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150030/APPFile/SG-CEPJ150030.3d (CEP) [PREPRINTER stage]

    Original Article

    Periaqueductal gray calcitoningene-related peptide modulatestrigeminovascular neurons

    P Pozo-Rosich1,a, RJ Storer1,b, AR Charbit1 and PJ Goadsby1,2

    Abstract

    Background: Calcitonin gene-related peptide (CGRP) receptor antagonism is an approach to migraine therapy. The locus

    of action of antimigraine treatment is not resolved. The objective was to investigate CGRP receptors in the ventrolateral

    periaqueductal gray (vlPAG) involved in the modulation of trigeminovascular nociception by descending influences on

    neurotransmission.

    Methods: The presence of calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1),

    which form functional CGRP receptors, was investigated. CGRP and its receptor antagonists, olcegepant and CGRP

    (837), were microinjected into the vlPAG while changes of neural responses in the trigeminocervical complex (TCC)

    were monitored.

    Results: Immunoreactivity indicated the presence of functional CGRP receptor components in the vlPAG and adjacent

    mesencephalic trigeminal nucleus. Inhibition of TCC responses to stimulation of dural afferents and ophthalmic cuta-

    neous receptive fields after microinjection of bicuculline into vlPAG indicated a connection between the vlPAG and TCC

    neurons. CGRP facilitated these TCC responses, whereas olcegepant and CGRP (837) decreased them.

    Conclusions: CGRP and its receptor antagonists act on neurons in the region of vlPAG to influence nociceptive trans-

    mission in the TCC. This suggests CGRP receptor antagonists may act at loci outside of the TCC and reinforces the

    concept of migraine as a disorder of the brain.

    Keywords

    Periaqueductal gray matter, trigeminovascular, migraine, CGRP

    Date received: 2 September 2014; revised: 24 December 2014; 6 February 2015; accepted: 13 February 2015

    Introduction

    Calcitonin gene-related peptide (CGRP) plays a rolein a variety of biologic functions including nocicep-tion. It has been widely implicated in the pathophysi-ology of migraine and other primary headaches (13).Clinical trials have shown that the CGRP receptorantagonists olcegepant (4), telcagepant (5), MK-3207(6) and BI 44370 TA (7) are eective in the acutetreatment of migraine. To understand better themechanism of CGRP receptor antagonist actions inalleviating primary headache disorders, it is import-ant to identify their sites of action within cranialnociceptive systems (2). Because of the widespreaddistribution of CGRP and CGRP receptors in thebrain, it appears possible that CGRP-receptor antag-onists have an action at supramedullary structures.We have seen eects of CGRP receptor modulation

    1Headache Group-Department of Neurology, University of California,

    San Francisco, CA, USA2Headache Group- Basic & Clinical Neuroscience, Kings College London,

    UK

    Corresponding author:

    PJ Goadsby, NIHR-Wellcome Trust Clinical Research Facility, Kings

    College Hospital, London SE5 9PJ, UK.

    Email: [email protected]

    aCurrent address: Headache and Neurological Pain Research Group, ValldHebron Institute of Research (VHIR), Universitat Auto`noma deBarcelona & Neurology Department, Vall dHebron University Hospital,Barcelona, SpainbCurrent address: Department of Physiology, Faculty of Medicine,Chulalongkorn University, Bangkok, Thailand

    Cephalalgia

    0(0) 110

    ! International Headache Society 2015Reprints and permissions:

    sagepub.co.uk/journalsPermissions.nav

    DOI: 10.1177/0333102415576723

    cep.sagepub.com

  • XML Template (2015) [18.3.201511:12am] [110]//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150030/APPFile/SG-CEPJ150030.3d (CEP) [PREPRINTER stage]

    on second- (8) and third-order (9) nociceptive trige-minovascular neurons, so we have considered thehypothesis that CGRP receptor activation may beoutside the direct trigeminovascular system, as fortriptans (10).

    Calcitonin receptor-like receptor (CLR) and recep-tor activity-modifying protein 1 (RAMP1), whichdetermines the binding of CGRP receptor antagonists(11), form a complex that can couple to receptor com-ponent protein (RCP) resulting in receptor activationand signal transduction after CGRP ligand binding(12). RCP and RAMP1 messenger RNAs (mRNAs)have been found in the periaqueductal gray (PAG),predicting this as a site of functional CGRP receptorexpression (13).

    Immunohistochemistry better determines the local-ization of translation products and abundant RCP-immunoreactive cells have been observed in the PAG,especially in its ventrolateral region, suggesting thepresence of functional CGRP receptors there.Moreover, brain imaging studies have also implicatedthe PAG in the pathophysiology of migraine (14),making the pharmacology of that region more interest-ing in a clinical context.

    Because of the critical importance of CGRP in painmodulation, and the prevalence of CGRP and itspotential receptor function in the caudal ventrolateralperiaqueductal gray (vlPAG), we sought to elucidatewhether CGRP and its receptor antagonists have asite of action in this region in relationship to intracra-nial nociceptive signaling. We extended the localiza-tion of the functional CGRP-receptor componentsRAMP1 and CLR to the PAG by immunohistochem-istry and characterized the eect of CGRP-receptoractivation and blockade by microinjecting CGRPand its receptor antagonists in this region. Weobserved modulation of neural activity recordedfrom second-order neurons in the trigeminocervicalcomplex (TCC) that receive nociceptive input fromdural and craniovascular aerents, and convergentinput from cutaneous receptor elds of the ophthalmicdivision of the trigeminal nerve, adopting a systems-level in vivo model (15).

    Materials and methods

    General

    Male Sprague Dawley rats (Charles RiverLaboratories, Hollister, CA) maintained under stand-ard conditions were used in experiments conductedunder anesthesia with terminal euthanasia in accord-ance with protocols approved by the InstitutionalAnimal Care and Use Committee of the University ofCalifornia, San Francisco.

    Immunohistochemistry

    Rats (n 3) weighing 350400 g were anesthetized withsodium pentobarbital (80mg/kg intraperitoneally (i.p.),Nembutal; Hospira, Lake Forest, IL) and euthanized bytranscardial perfusion with heparinized saline followedby xative containing 0.5% paraformaldehyde in phos-phate-buered saline (PBS) (pH 7.4). The brain and cer-vical spinal cord were resected and placed in the samexative overnight, then cryoprotected with 30% (w/v)sucrose in PBS. Coronal sections (40mm) were cutthrough the PAG (Paxinos and Watson, 2005) andcaudal spinal cord on a cryostat. Alternate serial sec-tions were collected into PBS, mounted onto slides andNissl-stained with cresyl violet (Biological StainCommission certied AcW-25 acetate, Sigma C5042)using conventional methods (16).

    The other sections were double stained with primaryantibodies specic for CLR and RAMP1 (17). Sectionswere incubated in a blocking solution (10% normaldonkey serum and 0.2% Tween 20 in PBS), then over-night at 4C with goat anti-RAMP1 diluted 1:100 inPBS containing 2% normal donkey serum and 0.5%Triton X-100 (dilution buer) and for two hours withAlexa Fluor 488 donkey anti-goat immunoglobulin G(IgG) conjugate (Invitrogen, Molecular Probes,Eugene, OR) diluted 1:800. The sections were subse-quently incubated in blocking solution using 10%normal goat serum and then overnight at 4C withrabbit anti-CLR diluted 1:500 and for two hours withAlexa Fluor 568 goat anti-rabbit IgG conjugate(Invitrogen) diluted 1:800 before mounting on glassslides under no. 1 coverslips (Corning Life Sciences,Acton, MA) using antifade hard-set mountingmedium containing 40,6 diamidino-2-phenylindole(Vectorshield, Vector Laboratories, Burlingame, CA).At each step, sections were washed three times withPBS. Fluorescent staining was visualized at room tem-perature using a Zeiss Axioplan Universal microscope(Carl Zeiss Microscopy, Oberkochem, Germany) ttedwith an HBO 50 mercury short-arc lamp uorescenceilluminator and power supply (Carl ZeissMicroImaging, Gottingen, Germany) and MRc5Axiocam digital camera (Carl Zeiss Vision,Hallbergmoos, Germany). Filter set 09 (excitation450490 nm, dichrotic beam splitter 510 nm, emissionbarrier 520 nm) was used for Alexa Fluor 488 conjugate(green) and lter set 00 (excitation 530585 nm, dichro-tic beam splitter 600 nm, emission barrier 615 nm) forAlexa Fluor 568 conjugate (red). Plan-Neouar object-ives (magnication/numerical aperture: 40/0.65, 5/0.15, 2.5/0.075) and Pl 10/25 eyepieces were used.Images were processed using an AxioVision LE soft-ware (version 4.6, Carl Zeiss MicroImaging, Jena,Germany) microscope tted with a mercury arc lamp

    2 Cephalalgia 0(0)

  • XML Template (2015) [18.3.201511:12am] [110]//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150030/APPFile/SG-CEPJ150030.3d (CEP) [PREPRINTER stage]

    and an appropriate lter set (Zeiss Axioplan;Oberkochem, Germany). Images were processed usingAxioVision LE software (version 4.6, Carl ZeissMicroImaging, Jena, Germany) and imported intoAdobe Illustrator CS3 (version 13.0.1, Adobe,San Jose, CA) for gure layout and to generate TIFFs.

    Electrophysiology

    Surgery. Rats (315 26 g, range 278372 g, n 17) wereanesthetized with 3% (v/v) isourane (AbbottLaboratories, North Chicago, IL) and then sodiumpentobarbital during surgery (Nembutal, Hospira;6070mg/kg i.p. after induction, then 1020mg/kg/has required) followed by a-chloralose dissolved in45% 2-hydroxypropyl-b-cyclodextrin (18), initially6070mg/kg (intravenously (i.v.)) and then 1020mg/kg/h through a femoral venous catheter. Arterial bloodpressure was monitored through a cannulated femoralartery (DTX Plus DT-XX; Becton Dickinson, Sandy,UT) and maintained within physiologic limits. Duringelectrophysiologic recordings and test stimuli, the ratswere immobilized using pancuronium bromide (GensiaSicor, Irvine, CA), 1.11.6mg/kg, then 0.81.8mg/kg/h, and articially ventilated. End-tidal CO2was maintained between 3.5% and 4.5% and skin andmucous membrane color monitored to conrm ade-quate oxygenation. Core temperature was maintainedat 37.5 0.5C using a homeothermic blanket system(Harvard Apparatus). The rat was xed in a stereotaxicinstrument (Kopf, Tujunga, CA) and the incisor baradjusted until the heights of lambda and Bregma werein a horizontal plane. This at-skull position wasachieved when the incisor bar was lowered3.9 0.5mm as consistent with published data (19).

    The middle meningeal artery, its branches, and adja-cent dura mater (MMA) were exposed by performing asmall craniotomy over the parietal cortex. The vlPAGwas accessed through a burr hole in the skull adjacentto the Bregma suture and superior sagittal sinus allow-ing dorsal entry and thus avoiding tracts of relevance tothe pathways studied (Figure 1(a) from Akerman et al.(20)). A C1 partial hemilaminectomy was performed toallow access to the TNC and its C1 transition zone inthe TCC.

    Stimulation and recording

    We stimulated the MMA electrically and recorded mul-tiple units in the TCC where we found convergence ofinput to nociceptive neurons from periorbital cutane-ous receptive elds, thus providing a quantitative meas-urement of craniovascular nociception. The generalprocedure was similar to that described previously(10,15). In brief, a bipolar stimulating electrode was

    placed straddling the MMA, just touching the duramater, and trains of stimuli were applied.

    TCC neuron recording

    Extracellular multiunit-recordings were made fromneurons in C1 (about 3.23.8mm caudal to the obexand 1.21.9mm lateral to the midline) with tungstenmicroelectrodes (TM33A05; WPI, Sarasota, FL) pos-itioned in 5 mm steps. Signals from recording electrodeswere amplied, ltered (from 300Hz to 10 kHz), fed toa noise subtraction system (Hum Bug; Quest Scientic,Vancouver, BC, Canada); and further amplied to totalgain of around 50100 k. The signal was passed to adata acquisition system (Power 1401; CED,Cambridge, UK) and to a gated amplitude discrimin-ator, the output of which was fed to the data acquisi-tion system, and to an oscilloscope and audio amplierto assist with spike discrimination. A storage oscillo-scope triggered by the MMA stimulus further assistedwith spike discrimination by displaying post-stimulusactivity.

    Characterization of neurons

    Neurons in the TCC with convergent input from theMMA and cutaneous receptive elds on the face wereidentied in the region of C1. The receptive elds weremapped by applying innocuous (brush) and noxious(pinch) stimuli. We selected wide-dynamic range neu-rons (WDR) with dermatomes predominantly in therst division of the trigeminal nerve (V1) and with con-vergent input from the MMA. Cutaneous receptiveelds in all three trigeminal innervation territorieswere assessed.

    Substance preparation

    ()-Bicuculline methiodide (BIC), a-CGRP (rat), andCGRP-(837) (rat) were freshly obtained from Tocris(Ellisville, MO). We were particularly conscious of thelability of the Cys2Cys7 disulde bridge and, to avoidits oxidation, peptides were stored as solids below20C in sealed vials before their reconstitution insaline (United States Pharmacopeial Convention(USP) grade) that had been purged of oxygen by nitro-gen sparging. CGRP (316mg/ml) and CGRP-(837)(2mg/ml) pH< 7; BIC (0.4mM, pH 5.05.5) and olce-gepant (2.0mM BIBN4096BS, pH 6.07.0; BoehringerIngelheim, Biberach, Germany) were dissolved insaline. All drug solutions were freshly prepared andltered to 0.02 mm. Chicago (Pontamine) Sky Blue(Color Index 24410; Alfa Aesar; Heysham,Lancashire, UK) (2% w/v) was dissolved in 100mMsodium acetate, pH 6.0.

    Pozo-Rosich et al. 3

  • XML Template (2015) [18.3.201511:12am] [110]//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150030/APPFile/SG-CEPJ150030.3d (CEP) [PREPRINTER stage]

    vlPAG microinjection

    A seven-barreled glass capillary assembly pulled tolong taper (PMP107; MicroData Instruments, SouthPlaineld, NJ) was used for the microinjection ofdrugs. The position of the multibarrel pipette assem-bly tip was aimed at coordinates for the vlPAG ipsi-lateral to the spinal recording site (16): 1.36mmrostral and 4.08 0.29mm dorsal to the interauralline, and 0.6 0.15mm from the midline as used byothers (10,15).

    Experimental protocol

    Inhibition of the neural responses to MMA and V1receptive eld stimulation after microinjections of theGABAA receptor antagonist BIC into the vlPAG wasconsidered the most reliable evidence of a functionalprojection between neurons in the region of the PAGmicroinjection and the recorded trigeminal neurons(15) and was used as a search stimulus. The antinoci-ceptive responses to the small amounts of BIC (20 pmolin 50 nl) microinjected into the vlPAG have a rapid

    (a) (b)

    (c) (d) (e)

    Figure 1. Microscopy results.

    (a) Cresyl violet-stained 40mm section of the PAG showing the ventrolateral portion. The aqueduct can be seen at the top left of thesection; surrounding this is the cell-dense region clearly revealed by the Nissl staining (black bar 0.5mm). (b) Some large cell bodies

    are seen to the right of the micrograph toward the edge of the vlPAG (white bar, 200mm) and the detail is shown in (c)(e). Anti-CLR((c); red, white bar 50mm) and anti-RAMP1 ((d); green, white bar 50mm) staining with double immunolabeling of consecutive serial40mm sections in panel (e) (white bar 50 mm). Note the significant colocalization of immunoreactivities (yellow). Original magnifica-tions 50 ((a), (b)), 400 ((c)(e)). PAG: periaqueductal gray; vlPAG: ventrolateral periaqueductal gray; CLR: calcitonin receptor-likereceptor; RAMP1: receptor activity modifying protein 1.

    4 Cephalalgia 0(0)

  • XML Template (2015) [18.3.201511:12am] [110]//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150030/APPFile/SG-CEPJ150030.3d (CEP) [PREPRINTER stage]

    onset and were transient and reversible, allowing sub-sequent testing with other substances (21).

    The responses of WDR neurons in the TCC to elec-trical stimulation of the MMA were monitored beforeand after microinjecting CGRP, CGRP-(837), olcege-pant, or saline vehicle control into the vlPAG. Thesetest substances and controls were microinjected with avolume range of 150250 nl in a random order over aperiod of 30 to 120 seconds as previously described (22).Responses to stimulation were analyzed using post-sti-mulus histograms (PSTH), which were determined andanalyzed with the assistance of Spike 5 software(Cambridge Electronic Design, UK). Trains of 25 sti-muli at 0.5Hz (0.5ms duration) were applied at inter-vals (1, 5, 10, 15, 20, 25, 30minutes) after interventions,before which a stable baseline ( 5%) of at least threePSTH at ve-minute intervals was established. Up to 90minutes was allowed for activity to return to baselinebetween drug interventions. Eects on trigeminal activ-ity were calculated as a percentage of the mean of threebaseline responses before an intervention.

    Recording and microinjection sites

    At the end of the protocols, Chicago Sky Blue dye solu-tion (250 nl) was microinjected into the vlPAG to esti-mate the spread of the test substances and mark themicroinjection site. The recording sites in the TCCwere marked with electrothermolytic lesions. Themarked tissue was excised, xed in neutral buered10% formalin, saturated with 30% sucrose (w/v) inPBS, and cryostat-cut serial sections (40 mm) mountedon glass slides. Images of the dye and lesion marks werevisualized and processed using AxioVision LE software(Carl Zeiss Microscopy).

    Statistical analyses

    PSTH responses were normalized and expressed as apercentage of the mean change in regard to baselineresponse. Data were analyzed using statistical software(SSPS version 20.0; Chicago, IL) with an analysis ofvariance (ANOVA) for repeated measurements toevaluate changes over time with respect to the stimula-tion intervals. If Mauchlys test of sphericity was vio-lated we made appropriate corrections to degrees offreedom according to GreenhouseGeisser (23).Where applicable, a Bonferroni post hoc procedurewas applied for multiple comparisons. At the time ofmaximal eect, Students two-sample t test was used toevaluate statistical signicance compared with baseline.Data are expressed as mean standard error of themean (SEM) or mean standard deviation (SD) forweight and anatomic location observations; p< 0.05was considered statistically signicant.

    Results

    Immunohistochemistry

    Within the PAG, we found CLR immunoreactivity toappear more intense in more medial locations, while theintensity of RAMP1 immunoreactivity increasedventrolaterally. Neuronal cell bodies in the vlPAG, par-ticularly near its ventrolateral edge, showed colocalizedCLR and RAMP1 immunoreactivity (Figure 1).Homogeneous staining was observed in the cytoplasmof positive cells with a spared nucleus. We noted largecells with particularly intense immunoreactive doublelabeling in the area of the mesencephalic trigeminalnucleus (rMTN). Colocalization of CLR and RAMP1receptor components suggesting the presence of func-tional CGRP receptors was prevalent on larger neu-rons, but there were exceptions, with some cellsexpressing only RAMP1. A positive control of spinalcord neurons in the trigeminal nucleus at the level of C1also showed double staining immunoreactivity for CLRand RAMP1, whereas a vlPAG negative control inwhich incubation with primary antibodies was omitteddid not show staining (data not shown).

    Electrophysiology

    Basic characteristics. All neurons tested were classied asWDR and had cutaneous receptive elds, primarily inthe rst division of the trigeminal nerve, although someoverlap with the second trigeminal division was some-times observed. Where overlap occurred, the ophthal-mic division was the most sensitive to stimulation.

    Cells responded to stimulation of the MMA aer-ents with mostly short latencies consistent with conduc-tion velocities of A-ber aerents: 530ms for thedistance between the MMA and the TCC recordingsite. However, when higher stimulus intensities wereused they sometimes elicited long latency responses(30100ms) consistent with C-ber aerent activation.To prevent temporal summation or wind-up of neu-rons recorded in the TCC, we avoided excessive stimu-lation of cutaneous receptive elds and of the dural andperivascular aerents by restricting stimulation volt-ages (currents) to those just supramaximal for elicitingA-ber responses: in the range 824V depending on theinherent resistance across the bipolar stimulating elec-trode. Recording sites at C1 were at depths correspond-ing to Rexed laminae III and IV (Figure 2).

    Pharmacology

    Bicuculline

    Microinjection of BIC into the vlPAG. A maximalinhibitory eect of 35 7% (F6,30 10.4; post

    Pozo-Rosich et al. 5

  • XML Template (2015) [18.3.201511:12am] [110]//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150030/APPFile/SG-CEPJ150030.3d (CEP) [PREPRINTER stage]

    hoc: p 0.009; n 6) with respect to baseline wasobserved at ve minutes of BIC microinjection(Figure 3). This activity increased to values that werenot signicantly dierent from baseline. A decrease inblood pressure was observed in some rats. This decreaseof 1020mmHg over 60100 s after microinjection of

    BIC is characteristic of activation of the vlPAG (15).The blood pressure decrease was considered indicativeof micropipette tip placement in the vlPAG, whereas ablood pressure increase suggested a more dorsal tiplocation and a need to plunge the micropipettedeeper (26).

    dmPAG

    dlPAG

    lPAG

    vlPAGDRd

    Aq

    DRl

    DRv

    V1

    V2

    V3

    0.5 mm

    pDR

    12

    34

    5

    7

    989

    9

    10

    (a)

    (c) (d)

    (e)

    (b)

    Figure 2. Receptive fields of recording sites in the trigeminocervical complex (TCC).

    (a) Schematic coronal section through the periaqueductal gray (PAG) onto which are mapped microinjection sites (blue dots)

    determined by recovery of Chicago Sky Blue delivered by microinjection at the end of experiments. The schematic has been adapted

    from the atlas of Paxinos and Watson (16) from the section 1.44mm rostral to the interaural point (or 7.56mm caudal of Bregma). Aq:

    aqueduct; dm: dorsomedial; dl: dorsolateral PAG; lPAG: lateral PAG; DRd: dorsal raphe dorsal; DRl: dorsal raphe lateral; pDR:

    postdorsal raphe; DRv: dorsal raphe ventral. Bar, 0.5mm. (b) Unstained coronal cryosection (40 mm) 1.36mm rostral to the interauralpoint showing a Chicago Sky Blue microinjection mark at the ventrolateral edge of the PAG. Staining is most intense at the micro-

    injection site and diffusion of the dye (and therefore presumably of the microinjected substances) can be seen around it. Bar, 0.5mm.

    (c) Extracellular recordings made from wide-dynamic range neurons in the spinal trigeminal nucleus had cutaneous or corneal

    receptive fields in at least the ophthalmic division of the trigeminal nerve. The variously shaded areas indicate the approximate

    sensory somatotopical organization of the rat head (adapted from a drawing by A.R.L. Fritchle (24)). V1, ophthalmic; V2, maxillary; V3,

    mandibular. (d) The electrical stimulation of afferent nerves from the MMA or its branches and periarterial dura mater (MMA). The

    middle meningeal artery showing its branches is represented in red here on an idealized skull schematic indicating the site of the

    craniectomy. A bipolar electrode (NE-200; Rhodes Medical Instruments, Summerland, CA) was placed typically straddling the artery at

    the point below the division of the artery (adapted from Paxinos and Watson (16)). (e) Sites of recorded neurons in the TCC were

    marked by electrolytic lesions (anodal DC 25 mA 2030 s; DC-LM5, Grass Instruments). Images of lesion marks were visualized andprocessed using AxioVision LE software to maintain a permanent record, with the positions determined using digital caliper software

    and from micropositioner readings (n 10) reconstructed on atlas maps (16) in 10 rats. The Rexed laminae are indicated by Arabicnumerals (25).

    6 Cephalalgia 0(0)

  • XML Template (2015) [18.3.201511:12am] [110]//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150030/APPFile/SG-CEPJ150030.3d (CEP) [PREPRINTER stage]

    CGRP and CGRP receptor antagonists

    Microinjection of CGRP into the vlPAG. CGRP-inducedfacilitation was shown by increased neuronal responsesto electrical stimulation of the MMA aerents occur-ring within minutes of the microinjection and wasobserved for response latencies corresponding to Adbers. A maximum increase of 32 5% above base-line (F6,30 2.9; post hoc: p 0.020; n 6) wasobserved at 10minutes of CGRP injection.

    Microinjection of olcegepant into the vlPAG. Olcegepant is aCGRP receptor antagonist. When microinjected intothe vlPAG, olcegepant inhibited the excitability of theTCC neurons to the MMA aerent stimulation with amaximum response of 25 6% (F6,54 10.4; post hoc:p< 0.001; n 10). A maximum response was observedat one minute after olcegepant microinjection and wassustained at a plateau for 30minutes.

    Microinjection of CGRP-(837) into the vlPAG. CGRP-(837)is a CGRP receptor antagonist. When microinjectedinto the vlPAG, CGRP-(837) inhibited the excitabilityof the TCC neurons to the MMA aerent stimulationwith a maximum response of 23 12% (F6,30 3.5;post hoc: p 0.092; n 6). A maximum response wasobserved 15minutes after CGRP-(837) injection,although the excitability of the TCC neurons wasdecreased from the rst minute.

    Control microinjections

    Microinjection of saline into the vlPAG. Saline vehicle controlmicroinjections did not have a signicant eect on TCCactivity over 30minutes recording (maximallyincreased 3 1%; F6,36 1.1; post hoc: p 1.000;n 7), indicating that the observed test substance eectswere not the result of mechanical or vehicle eects.

    Microinjection of BIC outside the vlPAG. For ve WDR neu-rons BIC was injected in areas unrelated to descendinginhibition outside the PAG and did not aect the eli-cited responses or spontaneous activity in second-orderneurons (p> 0.05).

    Discussion

    We report on the coexistence of potentially functionalCGRP receptor complex components, CLR andRAMP-1, using immunohistochemical methods, andfacilitation of trigeminal nociceptive activity aftermicroinjection of CGRP into the region of the caudalvlPAG. The eect is similar to what was seen aftermicroinjection of the P/Q calcium channel blocker aga-toxin (15) and implicates both CGRP and the PAG in

    50

    30

    10

    10

    30

    50

    0

    0

    25

    20

    15

    10

    5

    0

    25

    20

    15

    10

    5

    0

    25

    Firin

    g pe

    r 0.2

    ms

    bin

    20

    15

    10

    5

    0

    25

    20

    15

    10

    5

    00 10 20 30 40

    Saline

    CGRP-(837)

    9.0

    8.3

    BIBN40967.1

    CGRP13.6

    Baseline10.7

    50Firing latency (ms)

    25

    20

    15

    10

    5

    CGRP

    BIC

    Saline

    4096 (837)BIBN CGRP

    % B

    asel

    ine

    resp

    onse

    (a)

    (b)

    Figure 3. Effects of CGRP and CGRP receptor antagonists in

    the PAG.

    Panel (a) represents the maximal effect of saline, BIC, CGRP,

    BIBN4096 and CGRP 837) injected into the PAG for the first

    22ms of the data collection sweep without the stimulus artifact.

    For the time course of the maxima see Results. Error bars show 1

    standard error of mean. Panel (b) illustrates typical effects onMMA

    stimulus-evoked responses on baseline post-stimulus histograms

    after microinjections of the substances CGRP, BIBN4096, CGRP-

    (837) and saline into the PAG. Mean firing for the individual

    neuron is specified. Not all substances were tested on all neurons.

    CGRP: calcitonin gene-related peptide; PAG: periaqueductal gray;

    MMA: middle meningeal artery or its branches and periarterial

    dura mater; BIC: bicuculline; BIBN4096: olcegepant.

    Pozo-Rosich et al. 7

  • XML Template (2015) [18.3.201511:12am] [110]//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150030/APPFile/SG-CEPJ150030.3d (CEP) [PREPRINTER stage]

    migraine. The CGRP receptor antagonists, olcegepantand CGRP-(837) inhibited trigeminal nociceptiveactivity, as seen with the 5-HT1B/1D receptor agonistnaratriptan in the same region (10). The data suggestboth descending inhibitory and facilitatory inuencesfrom the PAG targets neurons of the TCC and involveCGRP-mediated components.

    PAG inuences on trigeminovascular nociceptionare regulated by presynaptic GABAergic and glutama-tergic, serotonergic eects (10), orexinergic inputs (27),and cannabinoid receptors (20). Our new ndings sup-port a hypothesis for the involvement of the caudalvlPAG region in trigeminal pronociception afterCGRP release in this area and a potentially novel siteof action for CGRP receptor antagonists. An import-ant issue that arises from clinical trials is the extent towhich CGRP receptor antagonists eective in migraineenter the brain. Positron-emission tomography (PET)studies suggest such entry is modest, although not zero(28), although the studies have been small (28). Theanity of the clinical drugs and the doses employedsuggests there is a complex question not yet completelyunderstood (2).

    Deletion of the CGRP gene prevents the develop-ment of hyperalgesia in mice (29), while in wild-typeanimals, levels of CGRP in the brain and cerebralspinal uid (CSF), and blood have been reported tobe raised in models of head pain (30). CGRP hasbeen demonstrated to be pronociceptive in the dorsalhorn of the spinal cord, where it facilitates glutamater-gic neurotransmission (8), and when administeredintracerebroventricularly (29). These pronociceptiveeects can be blocked by CGRP-(837) (8,3133) andolcegepant (8,31,33). In contrast, CGRP was appar-ently antinociceptive after other intracerebroventricular

    administration and in other parts of the brain (forexample, see Bates et al. (34)), an eect that couldalso be antagonized by CGRP-(837). Some of this dif-ference may simply be placement of injections withinthe PAG. As an example, in studies noted above,microinjections made in the rostral PAG, before thebeginning of the ventrolateral column, were analgesic(35). Taken together the data suggest a complex role forsupraspinal CGRP systems in the modulation ofnociception.

    Functional CGRP receptors, i.e. CLR and RAMP-1, seemed curiously located to the region of the rMTN.This region receives input from at least serotonergicand dopaminergic neurons in limbic structures andthe dorsal raphe nucleus that also target the PAG(36). However, the functional signicance of theseinputs remains largely unknown (37). The rMTN maycontrol trigeminal motor activity through serotonergicand dopaminergic mechanisms (36).

    In summary, our data show CGRP and its receptorantagonists microinjected into the region of the caudalvlPAG modulate the activity of neurons in the TCCthat receive meningeal, craniovascular and facialinput. We found immunoreactivity for CLR andRAMP1 colocalized in the region of the caudalvlPAG, suggesting the presence of functional CGRPreceptors here and notably on neurons in the adjacentregion of the rMTN. Together, this suggests centraldescending mechanisms from these regions for theaction for CGRP and its receptor antagonists in thecontrol of head pain. A fuller understanding ofthe pronociceptive eects of CGRP and the regions inwhich it is active could have signicant clinical impli-cations for the renement of antinociceptive therapiesdesigned to treat migraine.

    Article highlights

    . Calcitonin gene-related peptide (CGRP) mechanisms have been implicated in migraine, and their blockadehas established antimigraine eects.

    . Potentially functional CGRP receptors, calcitonin-like receptor (CLR) and receptor activity modifyingprotein 1 (RAMP1), are identied in the ventrolateral periaqueductal gray (vlPAG).

    . Direct injection of CGRP receptor antagonists into the vlPAG inhibits nociceptive trigeminovascularactivation.

    Conflict of interest

    PPR, RJS and ARC have nothing to declare.

    DrGoadsby reports grants and personal fees fromAllergan,eNeura and Amgen, and personal fees from AutonomicTechnologies Inc, Bristol-Myers Squibb, AlderBio, Pzer,

    Zogenix, Nevrocorp, Impax, Dr Reddy, Zosano, Colucid,Eli Lilly, Medtronic, Avanir, Gore, Ethicon, Heptares,Nupathe, Ajinomoto and Teva, outside the submitted work.

    Funding

    This work was supported by the Sandler Foundation.

    Acknowledgments

    The authors are grateful for the generous gifts of antisera 844and 3155 from Merck Research Laboratories, West Point,PA, and olcegepant from Boehringer Ingelheim, Germany.

    We thank Michele Lasalandra for technical assistance with

    8 Cephalalgia 0(0)

  • XML Template (2015) [18.3.201511:12am] [110]//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150030/APPFile/SG-CEPJ150030.3d (CEP) [PREPRINTER stage]

    the histology and Drs Knight, Holland, and Akerman fortheir helpful advice. Dr Pozo-Rosich received a travel grantfrom the Headache Study Group of the Spanish Neurological

    Society.PPR, RJS and PJG were involved in the design, conduct,

    and analysis of the study, as well as manuscript preparation.ARC was involved in the conduct and analysis of the

    study, as well as manuscript preparation.

    References

    1. Goadsby PJ, Edvinsson L and Ekman R. Vasoactive pep-tide release in the extracerebral circulation of humans

    during migraine headache. Ann Neurol 1990; 28: 183187.2. Ho TW, Edvinsson L and Goadsby PJ. CGRP and its

    receptors provide new insights into migraine pathophysi-

    ology. Nat Rev Neurol 2010; 6: 573582.3. Durham PL and Vause CV. Calcitonin gene-related pep-

    tide (CGRP) receptor antagonists in the treatment of

    migraine. CNS Drugs 2010; 24: 539548.4. Olesen J, Diener HC, Husstedt IW, et al. Calcitonin gene-

    related peptide receptor antagonist BIBN 4096 BS for theacute treatment of migraine. N Engl J Med 2004; 350:

    11041110.5. Ho TW, Ferrari MD, Dodick DW, et al. Efficacy and

    tolerability of MK-0974 (telcagepant), a new oral antag-

    onist of calcitonin gene-related peptide receptor, com-pared with zolmitriptan for acute migraine: Arandomised, placebo-controlled, parallel-treatment trial.

    Lancet 2008; 372: 21152123.6. Hewitt DJ, Aurora SK, Dodick DW, et al. Randomized

    controlled trial of the CGRP receptor antagonist, MK-

    3207, in the acute treatment of migraine. Cephalalgia2011; 31: 712722.

    7. Diener H-C, Barbanti P, Dahlof C, et al. BI 44370 TA, anoral CGRP antagonist for the acute treatment of

    migraine attacks: Results from a phase II study.Cephalalgia 2011; 31: 573584.

    8. Storer RJ, Akerman S and Goadsby PJ. Calcitonin gene-

    related peptide (CGRP) modulates nociceptive trigemino-vascular transmission in the cat. Br J Pharmacol 2004;142: 11711181.

    9. Summ O, Charbit AR, Andreou AP, et al. Modulation ofnocioceptive transmission with calcitonin gene-relatedpeptide receptor antagonists in the thalamus. Brain2010; 133: 25402548.

    10. Bartsch T, Knight YE and Goadsby PJ. Activationof 5-HT1B/1D receptors in the periaqueductal grey inhibitsmeningeal nociception. Ann Neurol 2004; 56: 371381.

    11. Mallee JJ, Salvatore CA, LeBourdelles B, et al. Receptoractivity-modifying protein 1 determines the species select-ivity of non-peptide CGRP receptor antagonists. J Biol

    Chem 2002; 277: 1429414298.12. Walker CS, Conner AC, Poyner DR, et al. Regulation of

    signal transduction by calcitonin gene-related peptide

    receptors. Trends Pharmacol Sci 2010; 31: 476483.13. Oliver KR, Kane SA, Salvatore CA, et al. Cloning, char-

    acterization and central nervous system distribution ofreceptor activity modifying proteins in the rat. Eur J

    Neurosci 2001; 14: 618628.

    14. Maniyar FH, Sprenger T, Monteith T, et al. Brain acti-vations in the premonitory phase of nitroglycerin-trig-gered migraine attacks. Brain 2014; 137: 232242.

    15. Knight YE, Bartsch T, Kaube H, et al. P/Q-type calciumchannel blockade in the PAG facilitates trigeminal noci-ception: A functional genetic link for migraine?J Neurosci 2002; 22: RC213.

    16. Paxinos G and Watson C. The rat brain in stereotaxiccoordinates. San Diego: Elsevier Academic Press, 2005.

    17. Eftekhari S, Salvatore CA, Calamari A, et al. Differential

    distribution of calcitonin gene-related peptide and itsreceptor components in the human trigeminal ganglion.Neuroscience 2010; 169: 683696.

    18. Storer RJ, Butler P, Hoskin KL, et al. A simple method,using 2-hydroxypropyl-beta-cyclodextrin, of administer-ing alpha-chloralose at room temperature. J Neurosci

    Methods 1997; 77: 4953.19. Paxinos G, Watson C, Pennisi M, et al. Bregma, lambda

    and the interaural midpoint in stereotaxic surgery withrats of different sex, strain and weight. J Neurosci Meth

    1985; 13: 139143.20. Akerman S, Holland PR, Lasalandra M, et al. Endocan-

    nabinoids in the brainstem modulate dural trigeminovas-

    cular nociceptive traffic via CB1 and triptan receptors:implications in migraine. J Neurosci 2013; 33:1486914877.

    21. Roychowdhury S and Fields H. Endogenous opioidsacting at a medullary mu-opioid receptor contribute tothe behavioral antinociception produced by GABAantagonism in the midbrain periaqueductal gray.

    Neuroscience 1996; 74: 863872.22. Sandkuhler J, Willmann E and Fu QG. Characteristics of

    midbrain control of spinal nociceptive neurons and non-

    somatosensory parameters in the pentobarbital-anesthe-tized rat. J Neurophysiol 1991; 65: 3348.

    23. Field A. In: Wright DB (ed.) Discovering statistics using

    SPSS for Windows. London: SAGE Publications, 2000.24. Waite PME. Trigeminal sensory system. In: Paxinos G

    (ed.) The rat nervous system. San Diego: Elsevier Aca-

    demic Press, 2004, pp.817851.25. Molander C and Grant G. Spinal cord cytoarchitecture.

    In: Paxinos G (ed.) The rat nervous system. San Diego:Academic Press, 1995.

    26. Waters AJ and Lumb BM. Inhibitory effects evoked fromboth the lateral and ventrolateral periaqueductal grey areselective for the nociceptive responses of rat dorsal horn

    neurones. Brain Res 1997; 752: 239249.27. Holland PR, Akerman S, Lasalandra MP, et al. Orexin A

    antinociceptive effects in the ventrolateral periaqueductal

    gray are blocked by 5HT1B/1D receptor antagonism. AnnNeurol 2008; 64(Suppl): S26.

    28. Vermeersch SGG, de Hoon J, De Saint-Hubert B, et al.PET imaging in healthy subjects and migraineurs suggests

    CGRP receptor antagonists do not have to act centrallyto achieve clinical efficacy. J Headache Pain 2013;1(Suppl 1): P224.

    29. Zhang L, Hoff AO, Wimalawansa SJ, et al. Arthritic cal-citonin/alpha calcitonin gene-related peptide knockoutmice have reduced nociceptive hypersensitivity. Pain

    2001; 89: 265273.

    Pozo-Rosich et al. 9

  • XML Template (2015) [18.3.201511:12am] [110]//blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150030/APPFile/SG-CEPJ150030.3d (CEP) [PREPRINTER stage]

    30. Goadsby PJ and Edvinsson L. The trigeminovascularsystem and migraine: Studies characterizing cerebrovas-cular and neuropeptide changes seen in humans and cats.

    Ann Neurol 1993; 33: 4856.31. Schorscher-Petcu A, Austin JS, Mogil JS, et al. Role of

    central calcitonin gene-related peptide (CGRP) in loco-motor and anxiety- and depression-like behaviors in two

    mouse strains exhibiting a CGRP-dependent difference inthermal pain sensitivity. J Mol Neurosci 2009; 39:125136.

    32. Ebersberger A, Charbel Issa P, Vanegas H, et al. Differ-ential effects of calcitonin gene-related peptide and calci-tonin gene-related peptide 8-37 upon responses to

    N-methyl-D-aspartate or (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate in spinal nociceptive neu-rons with knee joint input in the rat. Neuroscience 2000;

    99: 171178.33. Mogil JS, Miermeister F, Seifert F, et al. Variable sensi-

    tivity to noxious heat is mediated by differential

    expression of the CGRP gene. Proc Natl Acad Sci USA2005; 102: 1293812943.

    34. Bates R, Buckley G and McArdle C. Comparison of the

    antinociceptive effects of centrally administered calci-tonins and calcitonin gene-related peptide. Br JPharmacol 1984; 82(Suppl): 295.

    35. Xu S, Lundeberg T and Yu L. Antinociceptive effects of

    calcitonin gene-related peptide injected into periaqueduc-tal grey of rats with mononeuropathy. Brain Res 2000;859: 358360.

    36. Lazarov N. Neurobiology of orofacial proprioception.Brain Res Rev 2007; 56: 362383.

    37. Mor D, Bembrick A, Austin P, et al. Evidence for cellular

    injury in the midbrain of rats following chronic constric-tion injury of the sciatic nerve. J Chem Neuroanat 2011;41: 158169.

    10 Cephalalgia 0(0)


Recommended