Embed Size (px)
Citation preview
University of Iowa University of Iowa
Iowa Research Online Iowa Research Online
Theses and Dissertations
Fall 2017
Sites of CGRP action in light aversive behavior: implications for Sites of CGRP action in light aversive behavior: implications for
migraine migraine
Bianca Nicole Mason University of Iowa
Follow this and additional works at: https://ir.uiowa.edu/etd
Part of the Cell Biology Commons
Copyright © 2017 Bianca Nicole Mason
This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/5966
Recommended Citation Recommended Citation Mason, Bianca Nicole. "Sites of CGRP action in light aversive behavior: implications for migraine." PhD (Doctor of Philosophy) thesis, University of Iowa, 2017. https://doi.org/10.17077/etd.dk9g664u
Follow this and additional works at: https://ir.uiowa.edu/etd
Part of the Cell Biology Commons
!
!
SITES!OF!CGRP!ACTION!IN!LIGHT!AVERSIVE!BEHAVIOR:!!
IMPLICATIONS!FOR!MIGRAINE!
!
by!
Bianca!Nicole!Mason!!
!
!
A!thesis!submitted!in!partial!fulfillment!!of!the!requirements!for!the!Doctor!of!Philosophy!!
degree!in!Molecular!and!Cellular!Biology!in!the!Graduate!College!of!The!University!of!Iowa!
December!2017!
Thesis!Supervisor:!!Professor!Andrew!F.!Russo!!
!
!
!
!
! ! ! ! ! ! ! !!
!!
3
!
!
!
!
!
!
!
Copyright!by!!
BIANCA!NICOLE!MASON!!
2017!
All!Rights!Reserved!
!
!
!
!
!
!
!
!
! ! ! ! ! ! ! !!
!!
4
Graduate!College!!The!University!of!Iowa!!
Iowa!City,!Iowa!!
!
CERTIFICATE!OF!APPROVAL!!
______________________________!
PH.D!THESIS!!
______________________!
This!is!the!certify!that!the!Ph.D.!thesis!of!!
Bianca!Nicole!Mason!!
has!been!approved!by!the!Examining!Committee!for!!the!thesis!requirement!for!the!Doctor!of!Philosophy!degree!!in!Molecular!and!Cellular!Biology!at!the!December!2017!graduation.!!!!Thesis!Committee:!________________________________________!! ! ! Andrew!F.!Russo,!Thesis!Supervisor!!! ! ! !
!_______________________________________!Tina!L.!Tootle!!!_______________________________________!Pamela!K.!Geyer!!!_______________________________________!
! ! ! Donna!L.!Hammond!!!_______________________________________!
! ! ! C.!Andrew!Frank!!!
! ii!
!
!
!
!
!
!
To!my!mother!Andrea!and!my!heart!Michel!for!their!unwavering!love!and!support.!!
!
!
!
!
!
!
!
!
!
!
!
! iii!
!
!
Have!I!not!commanded!you?!Be!strong!and!courageous.!Do!not!be!afraidW!do!not!be!discouraged,!for!the!LORD!your!God!will!be!with!you!wherever!you!go.”!
YJoshua!1:9!
!
!
!
!
!
!
!!
!!
iv!
4
ACKNOWLEDGMENTS!
First!and!foremost,!I!would!like!to!thank!God.!Without!him!and!prayer,!I!
don’t!think!I!could!have!gotten!through!some!of!the!toughest!times!during!my!
graduate!studies.!In!addition,!it!would!have!been!impossible!for!me!to!make!it!
this!far!without!the!support!of!my!family,!especially!my!mom!who!sacrificed!a!lot!
for!me!to!get!the!education!she!believed!I!deserved!and!my!fiancé!Michel!who!
has!supported!me!and!told!me!how!proud!he!is!of!me!almost!every!day!since!we!
have!been!together.!
Second,!I!would!like!to!thank!my!mentor,!Andy.!I!do!not!think!I!can!put!in!
words!how!great!of!a!mentor,!person,!and!confident!he!has!been!to!me.!I!
appreciate!his!unwavering!support,!advice!and!encouragement!during!my!time!
as!a!member!of!the!lab.!He!has!not!only!given!me!the!tools!to!be!a!great!scientist,!
he!has!also!given!me!some!tools!to!be!a!great!person.!I!appreciate!the!fact!that!I!
could!walk!into!his!office!with!a!question!and!without!hesitation,!he!was!always!
willing!to!have!a!discussion.!Not!all!people!can!say!this!but!I!am!blessed!to!have!
had!a!mentor!like!him!in!my!life!for!these!past!years.!!
I!also!would!like!to!thank!both!Tina!Tootle!and!Jodi!Graff,!who!have!
supported!me!and!encouraged!me!when!I!had!doubts!in!myself!since!the!
moment!I!arrived!in!Iowa!City.!They!both!have!been!people!I!have!garnered!
advice!from!whether!it!was!personal!or!science!related.!They!have!always!had!
my!back!and!I!couldn’t!have!asked!for!better!people!to!be!there!for!me!
throughout!the!years.!!
!!
!!
v!
5
Lastly,!I!have!to!thank!my!labmates,!Adisa,!Sophie,!Brandon,!and!Levi.!
They!have!been!a!constant!support!system!and!an!awesome!form!of!
entertainment!throughout!my!graduate!studies.!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
vi!
6
ABSTRACT!
Migraine!is!a!complex!neurological!disorder!that!affects!approximately!38!
million!Americans.!For!over!25!years,!the!neuropeptide!calcitonin!geneYrelated!
peptide!(CGRP)!has!been!implicated!in!the!pathogenesis!of!migraine.!In!fact,!
several!pharmaceutical!companies!are!tailoring!treatments!to!antagonize!CGRP!
actions.!However,!due!to!the!complexity!of!migraine,!exactly!how!and!where!
CGRP!acts!to!contribute!to!migraine!have!remained!controversial:!whereas!
several!studies!suggest!that!CGRP!acts!in!the!central!nervous!system!(CNS)!in!
this!context,!others!have!indicated!a!role!in!the!periphery.!Central!nervous!
system!sites!of!action!include!the!trigeminal!nucleus!and!several!higher!brain!
regions,!and!peripheral!sites!include!the!vasculature!and!dural!mast!cells!in!the!
meninges.!Among!the!sites!of!CGRP!action,!the!trigeminal!nerve,!which!is!the!
major!somatosensory!structure!of!the!face,!is!of!particular!interest!because!it!
bridges!the!CNS!and!the!periphery.!!
Migraine!is!generally!thought!to!involve!abnormal!signaling!in!the!
trigeminovascular!system,!and!about!50%!of!trigeminal!neurons!have!CGRP!
immunoreactivity.!Although!the!notion!that!CGRP!has!a!central!site!of!action!in!
relation!to!migraine!had!gained!ground!over!the!past!decade,!the!recent!
discovery!that!monoclonal!antibodies!against!CGRP!can!prevent!migraine!
attacks!has!resurrected!the!possibility!that!a!peripheral!site!of!action!is!involved!
as!well.!Clarification!of!the!sites!of!CGRP!action!in!migraine!will!be!crucial!to!
developing!an!understanding!of!mechanisms!that!underlie!migraine!so!that!future!
treatments!can!be!rationally!designed.!
!!
!!
vii!
7
One!diagnostic!criterion!for!migraine!is!photophobia,!a!painful!and!often!
debilitating!response!to!nonYnoxious!levels!of!light.!Our!laboratory!previously!
developed!a!preclinical!model!of!migraine!in!which!the!lightYaversive!behavior!of!
mice!is!used!as!a!surrogate!of!photophobia.!Specifically,!mice!were!sensitized!to!
CGRP!by!introducing!a!nestin/hRAMP1!transgene.!In!these!mice!versus!control!
littermates,!light!aversion!in!response!to!central!(intracerebroventricular,!ICV)!
injection!of!CGRP!was!enhanced!in!dim!light.!In!wildYtype!mice,!CGRP!(ICV)!also!
elicited!aversion!to!very!bright!lightW!this!did!not!occur!in!vehicleYtreated!mice.!
Additionally,!I!have!shown!that!CGRP!injected!peripherally!(intraperitoneal,!IP)!
can!induce!significant!light!aversion!in!wildYtype!mice.!I!have!begun!to!identify!the!
sites!of!action!outside!of!the!central!nervous!system,!using!four!lines!of!
transgenic!mice!with!different!patterns!of!overexpression!of!CGRP!receptors:!
global!hRAMP1!mice!(expression!in!all!tissues),!nestin/hRAMP1!mice!
(expression!only!in!nervous!tissue),!tagln/hRAMP1!(expression!only!in!smooth!
muscle!cells),!and!tek2/hRAMP1!(expression!in!endothelial!cells).!As!predicted,!
in!the!global!hRAMP!mice!light!aversion,!in!response,!to!IPYinjected!CGRP!was!
enhanced.!However,!in!nestin/hRAMP1!mice,!only!ICVYinjected,!and!not!IPY
injected,!CGRP!induced!enhanced!light!aversion.!This!finding!suggests!that!
peripheral!CGRP!activates!neural!pathways!involved!in!light!aversion,!but!by!an!
indirect!mechanism.!
To!determine!where!in!the!periphery!CGRP!is!acting,!a!pharmacological!and!
genetic!approach!was!taken.!Since!CGRP!is!one!of!the!most!potent!vasodilators!
in!the!body,!it!is!well!positioned!to!have!vascular!effects!that!induce!light!aversive!
!!
!!
viii!
8
behavior.!This!hypothesis!was!based!on!findings!that!1)!intravenous!
administration!of!CGRP!in!human!subjects!can!cause!migraine!pain,!and!2)!
perivascular!CGRP!can!sensitize!the!trigeminal!nerve,!which!could!alter!synaptic!
transmission!to!the!central!nervous!system!and!3)!CGRP!monoclonal!antibodies!
are!effective!in!clinical!trial!and!likely!do!not!cross!the!blood!brain!barrier.!Thus,!
there!is!a!mechanism!by!which!CGRP!in!the!periphery!can!sensitize!the!
trigeminal!nerve!and!alter!sensory!perception,!leading!to!photophobia.!!
The!role!of!the!vasculature!in!migraine,!specifically!vasodilation,!has!been!
controversial!and!now!the!consensus!is!that!it!is!neither!necessary!nor!sufficient.!
First,!I!wanted!to!test!the!role!of!vasodilation!in!this!model.!I!pharmacologically!
inhibited!CGRPYinduced!vasodilation!using!two!vasoconstrictors,!phenylephrine!
and!endothelinY1.!Blocking!CGRPYinduced!vasodilation!partially!attenuates!the!
light!aversive!response.!Moreover,!mice!that!overexpress!the!CGRP!receptor!in!
smooth!muscle,!but!not!endothelial,!cells!exhibit!enhanced!light!aversion!
indicating!a!role!for!vascular!actions!of!CGRP!in!this!preclinical!model!of!
migraine.!These!results!present!clear!evidence!that!CGRP!has!actions!on!the!
vasculature!to!induce!light!aversion.!Additionally,!the!inability!of!blocking!
vasodilation!to!completely!rescue!the!light!aversion!suggests!that!the!vasculature!
may!not!be!the!only!peripheral!target!of!CGRP!in!migraine!pathophysiology.!
This!work!improves!the!understanding!of!peripheral!CGRP!actions!in!
migraine!and!raises!awareness!that!contribution!of!the!vasculature!in!migraine!
should!not!be!ignored.!The!identification!of!sites!of!CGRP!action!in!regions!inside!
!!
!!
ix!
9
and!outside!of!the!CNS!could!lead!to!improved!and!more!successful!therapeutics!
for!migraine.!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
x!
10
PUBLIC!ABSTRACT!
The!neuropeptide!calcitonin!geneYrelated!peptide!(CGRP)!is!a!key!player!in!migraine.!
While!migraine!can!be!induced!by!peripherally!administered!CGRP!(intravenous)!and!
can!be!treated!using!CGRP!antagonists!that!act!peripherally,!the!relevant!sites!of!CGRP!
action!remain!unknown.!To!address!the!role!of!CGRP!both!within!and!outside!the!central!
nervous!system,!we!used!a!mouse!model!of!photophobia.!Photophobia!is!abnormal!
discomfort!to!nonYnoxious!levels!of!light!and!is!experienced!by!approximately!90%!of!
migraine!patients.!We!have!previously!shown!that!peripheral!(intraperitoneal,!IP)!
injection!of!CGRP!resulted!in!light!aversive!behavior!in!wildYtype!CD1!mice!similar!to!
aversion!previously!seen!following!central!(intracerebroventricular,!ICV)!injection.!
Importantly,!two!clinically!effective!migraine!drugs,!the!5YHT1B/D!agonist!sumatriptan!and!
a!CGRPYblocking!monoclonal!antibody,!attenuated!the!peripheral!CGRPYinduced!light!
aversion!and!motility!behaviors.!Our!goal!for!this!study,!is!to!identify!the!mechanism!of!
action!of!peripheral!CGRP!using!light!aversion.!Previously!we!showed!that!ICV!CGRP,!
but!not!IP!CGRP,!in!nestin/hRAMP1!mice,!mice!that!overexpress!CGRP!receptors!in!the!
nervous!system,!causes!enhanced!light!aversion.!We!have!now!used!transgenic!CGRPY
sensitized!mice!that!have!globally!elevated!levels!of!hRAMP1!(global!hRAMP1)!in!all!
tissues.!Interestingly,!our!preliminary!data!show!sensitivity!to!low!light!after!IP!CGRP!in!
these!mice.!This!suggests!that!CGRP!actions!in!the!periphery!play!an!important!role!in!
the!induction!of!light!aversion!in!this!model.!CGRP!is!one!of!the!most!potent!
endogenous!vasodilators!known!and!receptors!are!on!vessels.!To!examine!the!
vasculature!as!a!potential!site!of!peripheral!CGRP!action,!we!took!two!approaches:!(1)!
genetic!overexpression!of!the!CGRP!receptor!in!the!vasculature!(2)!injection!of!
phenylephrine!to!minimize!vasodilation!induced!by!CGRP.!Blocking!CGRPYinduced!
vasodilation!partially!blocks!light!aversion!and!smooth!muscle,!not!endothelial,!cell!
!!
!!
xi!
11
overexpression!of!the!CGRP!receptor!induces!enhanced!light!aversion.!This!also!
suggests!that!peripheral!CGRP!actions!may!be!transmitted!to!the!CNS!via!indirect!
sensitization!of!peripheral!nerves!and!likely!not!on!CGRP!receptors!in!the!nervous!
system!to!cause!migraineYlike!photophobia.!This!work!improves!the!understanding!of!
CGRP!actions!in!migraine!and!raises!awareness!that!contribution!of!the!vasculature!in!
migraine!should!not!be!ignored.!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
xii!
12
TABLE!OF!CONTENTS!
LIST!OF!FIGURES!..............................................................................................!xv!LIST!OF!ABBREVIATIONS!...............................................................................!xviii!!
CHAPTER!!
! I.! INTRODUCTION!..................................................................................!1!
Migraine!in!the!clinic!and!its!burden!on!society!.............................!1!Influence!of!sex!hormones!in!migraine!..........................................!3!Vasculature!and!migraine!..............................................................!5!Triptans!..........................................................................................!8!Trigeminovascular!system!...........................................................!10! !CGRP!and!its!implications!in!migraine!........................................!11!Sensitivity!to!light!in!migraine,!a!rodent!model!of!photophobia!.................................................................................!14!Thesis!overview!and!specific!contributions!..................................!15!!!!
! II.! INDUCTION!OF!MIGRAINEYLIKE!PHOTOPHOBIC!BEHAVIOR!IN!MICE!BY!BOTH!PERIPHERAL!AND!CENTRAL!CGRP!MECHANISMS!...................................................................................!17!
!Abstract!........................................................................................!17!Significance!.................................................................................!18!Introduction!..................................................................................!18!!Materials!and!Methods!................................................................!21!
Animals!...............................................................................!21!Intraperitoneal!drug!and!antibody!administration!................!21!Intracerebroventricular!drug!administration!........................!22!Light!aversion!and!motility!assays!......................................!23!Open!field!assay!.................................................................!23!RNA!isolation!and!measurement!of!RNA!levels!.................!24!Statistical!analysis!...............................................................!25!
!Results!.........................................................................................!26!
Peripheral!CGRP!administration!elicits!light!aversion!in!mice!.....................................................................................!26!Sex!comparison!following!peripheral!administration!of!CGRP!..................................................................................!27!Peripheral!CGRP!administration!reduces!motility!only!in!the!dark!zone!..................................................................!28!Central!CGRP!administration!elicits!light!aversion!and!lightYdependent!resting!behavior!!........................................!29!Peripheral!CGRP!does!not!enhance!anxiety!behavior!in!the!open!field!!.....................................................................!30!
!!
!!
xiii!
13
Sumatriptan!attenuates!light!aversion!induced!by!peripheral!CGRP!.................................................................!30!A!CGRPYblocking!antibody!prevents!CGRPYinduced!!light!aversion!.......................................................................!31!Neural!expression!of!hRAMP1!in!nestin/hRAMP1!mice!.....!32!Central!CGRP,!but!not!peripheral!CGRP,!elicits!sensitized!light!aversion!in!nestin/hRAMP1!mice!...............!34!!!
Discussion!...................................................................................!35!!!
! III.! VASCULAR!CONTRIBUTIONS!OF!PERIPHERAL!CGRP!IN!MIGRAINEYLIKE!PHOTOPHOBIA!IN!MICE!......................................!81!
!!! ! !!!!!!!Abstract!.......................................................................................!81!! ! !!!!!!!Introduction!.................................................................................!82!
! ! !!!!!!!Materials!and!Methods!................................................................!84!! ! ! Animals!...............................................................................!85! ! ! !! ! ! Intraperitoneal!drug!administration!.....................................!86!! ! ! Light!aversion!and!motility!assays!......................................!86!! ! ! OpenYfield!Assay!.................................................................!88!! ! ! RNA!isolation!and!measurement!of!RAMP1!RNA!levels!....!88!! ! ! Animal!Surgery!and!Blood!pressure!assessment!
following!drug!administration!..............................................!89!! ! ! Statistical!Analysis!..............................................................!89!!! ! ! Results..…!...................................................................................!90!! ! ! Blood!pressure!changes!and!light!aversion!following!
administration!CGRP!and!vasoconstrictors!PE!..................!90!! ! ! Blood!pressure!changes!and!light!aversion!following!
administration!CGRP!and!vasoconstrictors!!! ! ! endothelinY1!........................................................................!92!! ! ! Blood!pressure!changes!and!light!aversion!following!
administration!PACAPY38!and!VIP!.....................................!95!! ! ! Pupil!diameter!changes!following!administration!of!
CGRP!!.................................................................................!96!! ! ! Sensitized!CGRPYinduced!light!aversive!behavior!of!
global!hRAMP1!mice!..........................................................!97!! ! ! Sensitized!CGRPYinduced!light!aversive!behavior!of!
smooth!muscle!hRAMP1!mice,!but!not!endothelial!hRAMP1!and!motility...........................................................!98!
!! ! !!!!!!!Discussion!...................................................................................!99!! ! ! !!
! IV.! CONCLUSIONS!AND!FUTURE!DIRECTIONS……….………….………………………………………..140!
!!!!!!!!! !!!!!!!Conclusions!...............................................................................!140!! ! !!!!!!!Future!Perspectives!..................................................................!143! ! ! Future!Perspectives!81! ! !!!!!!! ! ! Extracranial!arteries!in!CGRPYinduced!light!aversion!.......!143! ! ! !! ! ! The!role!of!mast!cells!in!CGRPYinduced!light!aversion!.....!144!
!!
!!
xiv!
14
! ! ! The!role!of!sex!hormones!in!CGRPYinduced!light!aversion!.............................................................................!145!
!! IV.! REFERENCES!……….………….……………………………………...148!
! ! !!!!!!!!! ! ! !
!!
!!
xv!
15
LIST!OF!FIGURES!
Figure!
II.1.! CGRP!induces!light!aversion!in!CD1!mice!..................................................!41!
II.2.! CGRP!induces!light!aversion!in!C57BL/6J!mice!.........................................!42!
II.3.! Comparison!of!CGRPYinduced!light!aversion!in!wildYtype!mice!..................!44!
II.4.! Gender!effects!of!0.1!mg/kg!CGRPYinduced!light!aversion!in!female!CD1!mice!....................................................................................................!45!
II.5.!!Gender!effects!of!0.5!mg/kg!CGRPYinduced!light!aversion!in!male!CD1!mice.!............................................................................................................!46!
II.6.!!Degree!of!CGRPYinduced!light!aversive!response!by!gender!in!CD1!mice!.............................................................................................................!48!
II.7.!!Gender!effects!of!0.1!mg/kg!CGRPYinduce!light!aversion!in!C57BL/6J!mice!.............................................................................................................!49!
II.8.!!Gender!effects!of!0.5!mg/kg!CGRPYinduced!light!aversion!in!C57BL/6J!mice!.............................................................................................................!50!
II.9.!!!Degree!of!CGRPYinduced!light!aversive!response!by!gender!in!C57BL/6J!mice!............................................................................................!52!
II.10.!Peripheral!CGRP!increases!resting!behavior!in!the!dark!...........................!54!
II.11.!Peripheral!CGRP!reduces!rearing!behavior...!............................................!56!
II.12.!Peripheral!CGRP!decreases!transitions!between!the!light!and!the!dark!zone!....................................................................................................!58!
II.13.!Central!injection!of!CGRP!through!a!cannula!elicits!light!aversion!............!59!
II.14.!Central!injection!of!CGRP!through!a!cannula!induces!an!increase!in!!!resting!behavior!...........................................................................................!60!
II.15.!Peripheral!CGRP!does!not!induce!anxiety!in!the!open!field!assay!in!CD1!mice!....................................................................................................!61!
II.16.!Peripheral!CGRP!does!not!induce!anxiety!in!the!open!field!assay!in!C57BL/6J!mice.!...........................................................................................!62!
II.17.!Sumatriptan!attenuates!light!aversion!induced!by!peripheral!CGRP!in!CD1!mice!....................................................................................................!64!
II.18.!Sumatriptan!partially!attenuates!light!aversion!induced!by!peripheral!CGRP!in!C57BL/6J!mice!.............................................................................!66!
II.19.!Sumatriptan!attenuates!CGRPYinduced!resting!behavior!..........................!68!
!!
!!
xvi!
16
II.20.!PreYtreatment!with!a!monoclonal!CGRP!antibody!prevents!light!aversion!induced!by!peripheral!CGRP!in!CD1!mice!...................................!70!
II.21.!PreYtreatment!with!monoclonal!CGRP!antibody!attenuates!CGRPYinduced!reduction!in!motor!activity!..............................................................!72!
II.22.!Generation!schematic!and!gross!GFP!expression!profile!in!nestin/hRAMP1!double!transgenic!mice!.....................................................!73!
II.23.!RAMP1!expression!in!nestin/hRAMP1!transgenic!mice!.............................!74!
II.24.!Peripheral!CGRP!does!not!induce!enhanced!light!aversion!in!CGRPYsensitized!nestin/hRAMP1!transgenic!mice!................................................!76!
II.25.!Central!CGRP!induces!enhanced!light!aversion!in!CGRPYsensitized!nestin/hRAMP1!transgenic!mice!.................................................................!78!
II.26.!Peripheral!CGRP!induced!light!aversion!using!wildYtype!testing!paradigm!in!nestin/hRAMP1!mice!...............................................................!80!
III.1.!Blood!pressure!changes!following!administration!of!CGRP!and!PE!.........!105!
III.2.!Phenylephrine!attenuates!CGRPYinduced!light!aversion!..........................!107!
III.3.!Gender!effects!in!the!light!aversion!assay!................................................!108!
III.4.!Resting!behavior!following!clamping!of!CGRPYinduced!vasodilation!in!the!light!aversion!assay!.............................................................................!109!
III.5.!Rearing!behavior!following!clamping!of!CGRPYinduced!vasodilation!in!the!light!aversion!assay!.............................................................................!110!
III.6.!Blood!pressure!changes!following!administration!of!CGRP!and!endothelinY1!..............................................................................................!111!
III.7.!EndothelinY1!attenuates!CGRPYinduced!light!aversion!.............................!112!
III.8.!Gender!effects!in!the!light!aversion!assay!................................................!113!
III.9.!Resting!behavior!following!clamping!of!CGRPYinduced!vasodilation!in!the!light!aversion!assay.!............................................................................!114!
III.10.!Rearing!behavior!following!clamping!of!CGRPYinduced!vasodilation!in!the!light!aversion!assay!.............................................................................!115!
III.11.!Peripheral!PACAP!induces!light!aversive!behavior!.................................!117!
III.12.!Peripheral!VIP!fails!to!induce!light!aversive!behavior!.............................!119!
III.13.!Retest!of!VIP!cohort!still!fails!to!induce!light!aversive!behavior!..............!120!
III.14.!Blood!pressure!changes!following!administration!of!VIP,!PACAP,!and!CGRP!........................................................................................................!122!
!!
!!
xvii!
17
III.15.!Representative!image!of!mouse!pupil!diameter!and!pupil!diameter!changes!following!CGRP!administration!...................................................!124!
III.16.!Generation!schematic!of!global!hRAMP1!transgenic!mice!.....................!125!
III.17.!Expression!pattern!of!RAMP1!in!global!hRAMP1!mice!...........................!127!
III.18.!Peripheral!CGRP!induces!light!aversive!behavior!in!global!hRAMP1!mice!...........................................................................................................!129!
III.19.!Generation!schematic!of!tagln/hRAMP1!and!tek2/hRAMP1!double!transgenic!mice!.........................................................................................!130!
III.20.!Peripheral!CGRP!induces!light!aversive!behavior!in!tagln/hRAMP1!mice!...........................................................................................................!132!
III.21.!Resting!behavior!following!administration!of!CGRP!in!tagln/hRAMP1!mice!...........................................................................................................!133!
III.22.!Rearing!behavior!in!tagln/hRAMP1!following!administration!of!CGRP!...!134!
III.23.!Peripheral!CGRP!does!not!induce!light!aversive!behavior!in!tek2/hRAMP1!mice!...................................................................................!136!
III.24.!Resting!behavior!following!administration!of!CGRP!in!tek2/hRAMP1!mice!...........................................................................................................!137!
III.25.!Rearing!behavior!in!tek2/hRAMP1!following!administration!of!CGRP!....!138!
III.26.!Model!of!peripheral!and!central!CGRP!intersection!in!migraine!.............!139!
!
!
!
!
!
!
!
!!
!!
xviii!
18
LIST!OF!ABBREVIATIONS!
5YHT1B/D! 5Yhydroxytryptamine!receptor!type!1B!or!1D,!a!serotonin!receptor!
ACE! angiotensin!converting!enzyme!!
AMPA! !YaminoY3YhydroxyY5YmethylY4Yisoxazoleproprionic!acid!receptor!
ANOVA! analysis!of!variance!!
ATP! adenosine!triphosphate!!
cAMP! cyclic!adenosine!monophosphate!!
CGRP! calcitonin!geneYrelated!peptide!!
CLR! calcitoninYlike!receptorYlike!receptor,!a!subunit!of!CGRP!receptor!
CNS! central!nervous!system!
CTR! calcitonin!receptor!
EndoY1! EndothelinY1!
GPCR! GYprotein!coupled!receptor!
GTN! nitroglycerin!!
ICV! intracerebroventricular!!
IP! intraperitoneal!
KCl! potassium!chloride!!
MAP! mean!arterial!pressure!
MCA! middle!cerebral!artery!
MRA! magnetic!resonance!angiography!
NMDA! NYmethylYdYaspartic!acid!
NO! nitric!oxide!!
PACAPY38! pituitary!adenylate!cyclaseYactivating!polypeptide!38!
PE! phenylephrine!
!!
!!
xix!
19
qPCR! quantitative!realYtime!polymerase!chain!reaction!
RAMP1! receptor!activityYmodifying!protein!1,!a!subunit!of!the!CGRP!receptor!
RCP! receptor!component!protein,!a!subunit!of!the!CGRP!receptor!!
SEM! standard!error!of!the!mean!!
TG! trigeminal!ganglion!
TRPV1! transient!receptor!potential!cation!channel!subfamily!V!member!1!
Veh!! vehicle!!
VIP! vasoactive!intestinal!peptide!
WT! wildYtype!
! !
! !
! 1!
CHAPTER!I!
INTRODUCTION!
Migraine(in(the(clinic(and(its(burden(on(society(
Migraine!is!a!prevalent!and!complex,!neurovascular!disorder!that!afflicts!
approximately!1!billion!people!worldwide.!It!affects!about!40!million!Americans,!
with!2K3!million!suffering!from!chronic!migraine1K5.!Strikingly,!migraine!affects!
approximately!one!in!four!households,!and!the!World!Health!Organization!lists!
this!condition!as!one!of!the!top!20!disabilities!when!expressed!as!years!of!
healthy!life!lost!to!a!disability1.!According!to!the!Migraine!Research!Foundation,!
almost!90%!of!sufferers!experience!disruptions!in!their!daily!activities!6.!
Additionally,!60%!of!migraine!sufferers!have!a!relative!that!has!also!been!
affected!by!a!migraine7.!!Furthermore,!migraine!experiences!are!mostly!episodic,!
however,!a!small!population!of!sufferers!experience!chronic!attacks!and!as!a!
result!they!are!usually!incapacitated!for!long!periods!of!time,!approximately!15!
days!per!month8.!An!estimated!1/3!of!workdays!are!lost!annually!due!to!migraine!
and!accounts!for!approximately!$100!billion!of!revenue!lost!to!employers9,10.!!
The!presentation!of!migraine!varies!between!each!individual!patient.!In!
order!for!headache!to!qualify!as!a!migraine,!it!must!present!as!a!unilateral!
headache!that!lasts!for!approximately!4K72!hours!accompanied!by!a!pulsating!
quality,!severe!pain!intensity,!and!alterations!in!sensory!perception.!Migraine!
attacks!can!begin!long!before!the!headache!starts.!It!involves!a!cascade!of!
events!that!progress!through!3K4!clinical!phases11.!!
! 2!
The!preKheadache!phase!comprises!of!two!subKphases.!The!prodrome!is!
the!initial!phase!that!is!experienced!by!almost!60%!of!sufferers11.!This!phase!is!
usually!identified!by!hyperalgesia!or!allodynia,!depression,!weight!loss,!fatigue,!
gastrointestinal!symptoms!and!many!other!vegetative!or!affective!symptoms.!
Additionally,!these!symptoms!can!be!present!24K72!hours!before!the!headache!
begins.!The!second!subKphase!in!the!preKheadache!stage!is!the!initiation!of!aura.!
Aura!manifests!with!neurological!features!such!as!visual!disturbances!and!affects!
approximately!20K30%!of!migraine!sufferers11.!!!
The!next!phase!to!occur!is!the!headache!stage,!the!most!debilitating!
stage.!During!this!phase!is!when!patients!experience!a!throbbing!headache!that!
is!severely!exacerbated!upon!movement.!Accompanied!with!the!headache!are!
sensory!alterations!such!as!phonophobia!and!photophobia!which!are!
experienced!by!an!overwhelming!majority!of!sufferers.!Other!clinical!features!
include!nauseas,!vomiting,!and!lacrimation.!The!last!phase!of!migraine,!the!
postdrome,!can!last!up!to!two!days!following!the!headache.!It!is!often!
accompanied!by!extreme!fatigue!and!exhaustion.!!
! Migraine!onset!can!be!triggered!by!a!multitude!of!factors.!Most!triggers!in!
migraineurs!are!usually!harmless!in!people!who!do!not!get!migraines.!An!
overwhelming!majority!of!migraine!sufferers!report!that!emotional!stress!can!
easily!cause!an!attack12K14.!Among!the!top!migraine!triggers,!women!report!that!
the!start!of!their!menstrual!cycle!is!a!definite!trigger13,15,16.!Indeed,!one!study!
found!that!88%!of!women!in!their!child!bearing!years!reported!menses!as!a!
trigger15K17.!
! 3!
!
Influence(of(sex(hormones(in(migraine((
A!gender!disparity!is!highly!prevalent!in!people!that!suffer!from!migraines!
and!has!been!recognized!since!ancient!times.!Mostly!a!women’s!health!issue,!
attacks!are!more!frequently!reported!in!women!than!in!men.!In!fact,!more!women!
seek!medical!attention!and!enroll!in!clinical!trials!than!men.!From!these!reports,!it!
is!presumed!that!migraine!is!three!times!more!common!in!women!compared!to!
men.!Additionally,!women!report!a!longer!attack!duration,!increased!risk!of!
reoccurrence,!more!severe!debilitation,!and!longer!postKdrome!periods,!making!it!
the!fourth!leading!cause!of!disability!in!women!worldwide.!!
Before!the!onset!of!puberty,!migraine!usually!affects!both!sexes!equally.!
Indeed,!a!Swedish!study!found!that!girls!and!boys!ages!7K9!had!a!prevalence!of!
2.4%!and!2.5%,!respectively18,19.!While!boys!had!a!trend!of!developing!childhood!
migraines!earlier!than!girls,!the!prevalence!of!occurrence!in!girls!dramatically!
increases!as!the!age!groups!get!older.!As!a!matter!of!fact,!this!same!study!
determined!by!the!time!sufferers!reached!their!early!teenage!years!and!at!the!
onset!of!menarche!in!females,!approximately!4%!of!males!and!6.4%!females!
reported!migraine!attacks.!At!full!maturity!and!childbearing!years,!ages!18K29,!
migraine!strikingly!affects!18%!of!women!and!6%!men20.!This!obvious!correlation!
between!migraine!prevalence!and!reproductive!milestones!in!women!and!men!is!
evident!that!changes!in!sex!hormones!may!play!a!role!in!its!occurrence!in!
women.!!
! 4!
At!least!50%!of!all!women!will!experience!migraine!once!during!their!
childbearing!years.!In!women,!it!is!thought!that!the!fluctuations!of!estrogen!
trigger!the!onset!of!migraine!15.!!For!example,!menarche,!menstruation,!
pregnancy,!menopause,!hormonal!contraceptives,!and!estrogen!replacement!
therapy!all!may!influence!the!manifestation!of!migraine!21.!Indeed,!the!menstrual!
cycle!has!long!been!linked!to!migraine!and!the!International!Criteria!of!Headache!
Diagnosis!uses!special!criteria!to!identify!menstrual!migraine!in!women22.!
Menstrual!migraine!is!defined!as!the!onset!of!migraine!that!occurs!within!48!
hours!of!the!beginning!of!menstruation!and!lasts!up!to!3!days.!This!headache!is!
commonly!associated!with!the!sudden!drop!in!estrogen!at!the!beginning!of!
menses!and!is!referred!to!as!estrogen!withdrawal23.!Similar!levels!of!estrogen!
withdrawal!also!occur!during!ovulation!and!have!been!associated!with!frequency!
of!migraine!in!women24.!In!a!study!by!Calhoun!and!Hutchinson,!the!incidence!of!
migraine!attacks!decreased!when!patients!were!placed!on!monophasic!
estrogen/progesterone!oral!contraceptive!pills!to!eliminate25.!!
During!the!first!trimester!of!pregnancy,!studies!did!not!find!a!change!in!the!
frequency!of!attacks!compared!to!preKpregnancy26.!Interestingly,!prevalence!of!
migraine!dramatically!decreases!and!many!women!report!complete!abrogation!
by!the!third!trimester26K30.!However,!in!a!study!by!Sances!et!al.,!migraine!
reoccurred!during!the!first!week!after!childbirth.!This!postKpartum!return!of!
migraine!is!closely!associated!with!the!drastic!drop!in!estrogen!following!
childbirth28.!!!
!
! 5!
!
Vasculature(and(migraine(
Theories!of!migraine!pathophysiology!have!been!debated!for!quite!some!
time.!In!particular,!whether!migraine!genesis!involves!a!primary!role!for!the!
vasculature!remains!controversial31K33.!The!vascular!theory!was!first!articulated!
by!Galen!in!the!second!century!and!later!reKproposed!by!Thomas!Willis!in!the!
late!17th!century34.!However,!it!wasn’t!until!the!early!1940’s!that!Harold!Wolff!first!
showed!that!intensity!of!migraine!was!closely!linked!to!the!pulsating!branches!of!
the!external!carotid!arteries!and!decreasing!the!amplitude!of!the!pulsations!also!
decreased!the!intensity!of!the!headache35.!In!the!same!study,!ergots!induced!
vasoconstriction!of!temporal!and!middle!meningeal!arteries!and!reduced!the!
throbbing,!coincidently!diminishing!the!intensity!of!the!headache.!These!
observations!were!the!genesis!of!what!would!later!be!known!as!the!vascular!
theory!of!migraine.!!!
! Using!the!vascular!hypothesis!as!inspiration,!several!studies!have!shown!
that!migraine!attacks!could!be!recapitulated!in!an!experimental!setting!using!
pharmacological!agents!that!have!vascular!actions.!Indeed,!CGRP,!pituitary!
adenylate!cyclaseKactivated!peptide!(PACAPK38)!and!nitroglycerin!(GTN)!all!can!
cause!sustained!cranial!vasodilation!and!can!induce!a!migraineKlike!attack!only!in!
patients!that!suffer!from!migraine36K43.!Earlier!studies!used!ultrasonography!to!
measure!blood!flow!velocity,!a!marker!for!increased!artery!lumen,!during!
migraine!attacks44.!More!recently,!studies!have!taken!advantage!of!magnetic!
resonance!angiography!(MRA),!a!technique!specifically!used!to!measure!the!
! 6!
circumference!of!arteries.!Using!this!technique,!several!studies!report!an!
increase!in!the!circumference!of!extracranial!arteries!during!attacks45.!!
It!is!possible!that!extracranial!arteries!play!a!nociceptive!role!in!migraine.!It!
has!been!reported!that!superficial!temporal!arteries!are!wider!on!the!pain!side!of!
the!head,!rather!than!the!side!where!the!pain!is!not!felt!and!compression!of!the!
artery!relieved!pain!in!approximately!30%!of!patients46,47.!Additionally,!Elkind!et!
al.!concluded!that!during!unilateral!headache,!phenotypically!similar!to!migraine,!
blood!flow!was!increased!only!on!the!painful!side.!More!to!the!point,!
frontotemporal!blood!flow!was!measured!in!small!pool!of!patients!with!a!severe,!
migraineKlike!headache48.!In!71%!of!patients,!ergotamine!tartrate,!a!potent!
vasoconstrictor,!reduced!blood!flow!and!consequently!headache!disappeared!
within!30!min!of!infusion48.!!
! Meningeal!arteries!are!thought!to!play!a!major!role!in!the!pain!experienced!
during!a!migraine.!The!dura!mater!is!a!highly!vascularized!membrane!that!
protects!the!brain!and!the!spinal!cord!and!has!been!shown!to!be!a!painKsensitive!
structure.!Dural!vessels!are!thought!to!contribute!to!neurogenic!inflammation,!an!
event!that!occurs!at!the!periphery!that!activates!sensory!neurons!and!is!
characterized!by!vasodilation,!plasma!extravasation,!and!release!of!proK
inflammatory!molecules!from!mast!cells49.!Intravenous!infusion!of!migraineK
inducing!substances!such!as!CGRP!cause!dilation!in!the!middle!meningeal!
artery!compared!to!pial!cerebral!arteries!and!blocking!this!vasodilation!with!
CGRP!antagonists!can!effectively!abort!migraine!attacks!50,51.!!
! 7!
Despite!all!of!the!evidence!above,!the!role!of!the!vasculature,!specifically!
vasodilation,!remains!controversial.!!Of!late,!the!vascular!theory!has!fallen!out!of!
favor!after!a!40Kyear!reign.!In!1981,!a!study!by!Olesen!and!colleagues!showed!
that!the!pain!experienced!during!a!migraine!was!manifested!only!after!the!
vasodilation!had!subsided52.!In!addition,!the!most!convincing!argument!against!a!
role!for!vasodilation!in!migraine!came!from!a!study!that!showed!vasoactive!
intestinal!peptide!(VIP)!induces!substantial!vasodilation!in!people!but!only!
produces!a!mild!headache!and!not!a!migraine!attack!like!other!vasodilators53.!
However,!the!conclusion!that!vasodilation!is!an!epiphenomenon!albiet!logical!due!
to!the!inability!of!VIP!to!induce!migraine!may!be!a!premature!conclusion.!CGRP,!
PACAPK38,!and!GTN!are!all!proKinflammatory!and!all!cause!sustained!
vasodilation!following!infusion.!!However,!VIP,!well!known!for!its!protective!and!
antiKinflammatory!role,!induces!marked!vasodilation!similar!the!other!vasodilators!
but!only!for!a!transient!amount!of!time!compared!to!CGRP,!PACAPK38,!and!
GTN.!It!is!possible!that!(1)!increasing!the!VIP!infusion!time!could!unveil!an!ability!
of!VIP!to!induce!migraineKlike!headaches!or!(2)!the!antiKinflammatory!properties!
of!VIP!play!a!role!in!its!inability!to!induce!migraine54,55.!In!contrast!to!CGRP,!VIP!
has!the!capacity!to!inhibit!mast!cell!degranulation,!although!controversial54K56.!
Future!studies!that!explore!the!morphology!of!cephalic!mast!cells!after!infusion!of!
known!mast!cell!activating!inducers!of!migraine!and!VIP!are!warranted.!!!
! Several!studies!have!linked!migraine!with!the!increased!risk!of!
cardiovascular!disease.!Indeed,!there!is!debate!on!whether!hypertension!and/or!
hypotension!have!a!relationship!with!migraine57K60.!It!has!been!suggested!that!
! 8!
migraine!and!hypertension!have!a!high!prevalence!of!coKmorbidity.!A!clinical!
study!by!Janeway!in!1913!reported!that!there!was!a!high!incidence!of!migraine!in!
patients!that!were!diagnosed!with!hypertension61.!Additionally,!in!a!retrospective!
study,!Grebe!et!al.!found!61%!of!patients!that!had!developed!medicine!overuse!
headaches!(MOH)!from!the!treatment!of!migraine!also!had!hypertension57.!The!
same!group!also!found!that!migraineurs!with!aura!had!an!increase!in!systolic!
pressure!compared!to!control!subjects.!The!reninKangiotensin!system,!which!is!
involved!in!hypertension,!has!long!been!thought!to!be!involved!in!migraine!
pathogenesis.!Indeed,!the!ability!of!angiotensin!converting!enzyme!(ACE)!
inhibitors!to!alter!several!processes!involved!in!migraine!and!be!effective!in!
migraine!treatment!is!evidence!that!there!may!be!a!possible!link!between!
migraine!and!hypertension62,63.!Conversely,!several!studies!have!found!no!
correlation!between!migraine!and!hypertension58,64,65.!In!fact,!one!study!
suggested!that!individuals!with!migraineKlike!episodes!had!a!higher!correlation!
with!lower!blood!pressure!than!individuals!without!headache66.!More!
comprehensive!studies!are!needed!to!determine!if!hypertension!or!hypotension!
contributes!to!a!subKpopulation!of!migraine!attacks.!!
! !
Triptans(!
Based!on!the!theory!that!cranial!vasodilation!was!the!sole!cause!of!
migraine!and!intravenous!infusion!of!serotonin!can!successfully!to!treat!migraine,!
a!new!evidenceKbased!target!for!migraine!therapy!was!born,!the!triptans.!Looking!
for!an!alternative!for!the!sideKeffect!inducing!ergotamines,!the!Humphrey!group!
! 9!
identified!a!subtype!of!serotonin!receptors!that!were!found!mostly!on!cranial!
vasculature.!!This!finding!lead!to!the!engineering!of!triptans67.!There!have!been!
over!seven!types!of!triptans!developed!since!their!original!production.!They!have!
now!become!the!goldKstandard!in!migraine!treatment!68K71.!!
Despite!the!wide!use!and!tolerability!of!the!triptans,!the!mechanism!of!
action!has!never!been!entirely!defined.!However,!it!is!known!that!triptans!are!5K
HT1B/D/F,!serotonin,!receptor!agonists,!that!can!inhibit!the!release!of!neuropeptides!
involved!in!migraine!and!act!as!vasoconstrictors!on!dilated!cranial!vessels68,72K75.!
A!study!by!the!Moskowitz!group!showed!that!sumatriptan,!the!goldKstandard!
triptan,!treatment!could!reduce!plasma!levels!of!calcitoninKgene!related!peptide!
(CGRP)!in!animal!models!of!migraine72,76,77.!Moreover,!vascular!studies!of!
triptans!have!given!us!insight!into!its!mechanism!of!action!and!the!roles!vessels!
might!play!in!migraine.!In!fact,!a!study!using!singleKphoton!emission!computed!
tomography!combined!with!Doppler!sonography!showed!that!if!sumatriptan!is!
infused!on!the!headache!side!in!patients,!only!the!abnormally!dilated!vessels,!
and!not!the!normal!ones,!of!the!middle!cerebral!artery!(MCA)!were!reversed!back!
to!normal78,79.!!
Although!triptans!have!been!the!best!option!for!migraine!treatment!for!
many!decades,!there!are!many!pitfalls!that!come!with!their!use!that!have!inspired!
a!race!to!create!best!migraine!therapy.!Unforturnately,!only!about!60%!of!
migraine!patients!experience!relief!from!triptans71.!Additionally,!triptans!can!only!
be!used!as!an!abortive!treatment!and!are!ineffective!when!used!as!a!
prophylactic.!Moreover,!a!study!using!tactile!responses!in!rats,!showed!that!
! 10!
sustained!systemic!administration!of!triptans!elicited!latent!sensitization!and!
enhanced!cutaneous!allodynia80.!To!this!end,!chronic!use!of!triptans!in!patients!
can!induce!the!onset!of!MOH!and!can!lead!to!untreatable!migraines81.!
!
Trigeminovascular(system((
Historically,!migraine!was!thought!to!be!a!vascular!disease32,82,83.!
However,!over!the!past!20!years,!the!emphasis!has!shifted!to!the!neural!
imbalances!associated!with!migraine,!and!vascular!contributions!are!now!
generally!viewed!as!an!epiphenomenon!that!are!neither!sufficient!nor!necessary!
to!induce!migraine.!Indeed,!persuasive!arguments!have!been!made!both!for!and!
against!vascular!contributions!to!migraine32,33,84,85.!About!20!years!ago,!a!
neurovascular!model!of!migraine!was!articulated.!It!posits!that!vascular!
inflammatory!signals!lead!to!an!altered!neural!state,!and!suggests!that!a!
peripheral!inflammatory!event!in!the!meninges,!which!are!innervated!by!the!
trigeminal!nerve,!lead!to!the!sensitization!and!activation!of!nociceptors.!The!
resulting!neuronal!hyperexcitability!and!central!sensitization!are!thought!to!lead!
to!migraine!pain.!While!there!is!controversy!regarding!the!origination!of!migraine!
pain,!all!can!agree!that!there!is!involvement!of!the!trigeminovascular!system31.!!
The!trigeminal!ganglia!provide!sensory!input!that!innervate!the!peripheral!
vasculature!of!the!dura!mater!and!central!terminals!project!to!the!trigeminal!
nucleus!caudalis!and!the!cortex,!where!the!processing!of!signals!is!perceived!as!
pain.!Although!many!studies!have!admittedly!pushed!the!vasculature!into!the!
curtains,!it!remains!possible!that!the!vasculature!can!influence!activity!of!the!
! 11!
trigeminal!nerve86.!Notably,!the!smooth!muscle!of!the!intracranial!and!
extracranial!vasculature!is!heavily!invested!with!CGRP!receptors,!and!CGRP!is!
present!in!approximately!50%!of!trigeminal!neurons87K92.!!
!
CGRP(and(its(implications(in(migraine(
CGRP!is!a!37Kamino!acid,!multifunctional!neuropeptide!that!has!two!
isoforms,!CGRP!!and!CGRP".!Both!isoforms!are!structurally!similar!and!widely!
expressed!in!the!body,!with!CGRP!!mostly!found!in!the!central!and!peripheral!
nervous!systems91K94.!CGRP!!is!a!splice!variant!of!the!CALCA!gene!and!is!
present!on!both!unmyelinated!C!and!myelinated!A#!sensory!nerve!fibers!which!
are!commonly!found!innervating!or!in!close!proximity!to!nearby!vasculature93.!
Relevant!to!migraine,!CGRP!!is!the!most!dominant!isoform!present!in!the!
cerebrovasculature.!CGRP"!is!a!product!of!the!CALCB!gene!and!is!biologically!
similar!to!CGRP!!but!differs!by!three!amino!acids!in!humans.!However,!CGRP"!
has!a!more!restricted!expression!and!is!found!mostly!in!the!enteric!system!and!in!
parts!of!the!central!nervous!system94,95.!This!chapter!will!focus!on!CGRP!!and!
will!from!herein!be!referred!to!as!CGRP.!!
The!canonical!CGRP!receptor!consists!of!a!seven!transmembrane!G!
proteinKcoupled!receptor!calcitonin!receptorKlike!receptor!(CLR),!receptor!
component!protein!(RCP),!and!single!transmembrane!receptor!activity!modifying!
protein!1(RAMP1)95K98.!RAMP1!is!required!for!both!ligand!recognition!and!
receptor!trafficking96.!Additionally,!it!has!been!shown!to!be!rateKlimiting!in!both!
trigeminal!neuron!and!vascular!smooth!muscle!cultures99,100.!RCP!is!required!for!
! 12!
gKprotein!coupling!which!activates!the!cyclic!adenosine!monophosphate!(cAMP)!
signaling!pathway101,102.!In!addition!to!the!canonical!receptor,!CGRP!can!also!
bind!to!a!nonKcanonical!complex!of!calcitonin!receptor!(CTR)!and!RAMP1!also!
known!as!the!AMY1!receptor!with!equal!affinity!and!can!be!activated!by!both!
CGRP!and!amylin103,104.!Additionally,!this!receptor!has!also!been!identified!in!
migraine!relevant!areas!such!as!the!trigeminal!ganglion!and!the!brainstem!and!it!
would!be!interesting!to!determine!if!amylin!plays!a!role!in!migraine101,105,106.!!!
CGRP!has!the!ability!to!regulate!the!cardiovascular!system!and!is!one!of!
the!bodies!most!potent!vasodilators.!In!congruity!with!its!role!in!vasodilation,!
CGRP!receptors!are!found!on!various!cell!types!including!smooth!muscle!cells,!
endothelial!cells,!mediate!neurogenic!inflammation,!and!facilitate!nociceptive!
transmission49,107K109.!Considering!these!neurovascular!and!nociceptive!
properties!of!CGRP!and!the!location!of!CGRP!and!its!receptor,!it!makes!CGRP!
well!positioned!to!play!a!role!in!migraine.!Additionally,!clinical!trials!performed!
over!the!last!decade,!have!established!the!importance!of!CGRP!in!migraine!
pathogenesis.!!
CGRP!is!present!in!nerve!fibers!that!innervate!almost!every!organ.!
Moreover,!CGRP!is!stored!and!transported!in!nerve!terminals!and!is!released!
upon!stimulation49,93,110,111.!CGRP!release!can!be!caused!by!a!number!of!
mechanisms!among!them!include!KCl!evoked!depolarization,!TRPV!channelK
evoked!release,!and!nitric!oxideKinduced!positive!feedback49,101.!All!of!which!
have!been!thought!to!contribute!in!migraine.!Conversely,!high!estrogen!states!
can!inhibit!the!release!of!CGRP!and!other!neuropeptides,!this!may!indicate!a!key!
! 13!
role!for!the!role!of!migraine!in!the!“estrogen!withdrawal”!stages!of!the!menstrual!
cycle112,113.!!
! CGRP!can!contribute!to!migraine!through!a!multitude!of!processes!such!
as!vasodilation,!neurogenic!inflammation,!central!sensitization.!CGRP!is!on!the!
most!potent!vasodilators!in!the!body,!especially!on!cranial!vessels111.!As!
previously!mentioned,!vasodilation!has!long!been!associated!with!the!pain!side!
of!a!migraine.!Therefore,!a!role!for!CGRP!actions!on!the!vasculature!seems!
likely.!CGRP!can!also!contribute!to!neurogenic!inflammation!by!way!of!mast!
cells.!CGRP!receptors!are!found!on!dural!mast!cells!and!has!been!shown!to!
activate!them!thus!releasing!their!contents49,114.!Granules!of!mast!cells!contain!
proKinflammatory!mediators!such!as!cytokines,!prostaglandins,!ATP!and!NO115K
117.!These!mediators!can!activate!and!sensitize!nearby!sensory!neurons!and!
cause!vasorelaxation!and!extravasation.!In!sensory!terminals!of!spinal!and!
trigeminal!neurons,!CGRP!can!also!increase!glutamate!signaling,!thus,!
modulating!and!enhancing!synaptic!transmission.!In!this!respect,!CGRP!
receptors!are!also!found!on!postKsynaptic!spinothalamic!neurons!and!colocalize!
with!glutamate!receptors.!This!may!lead!to!central!sensitization,!increased!
neuronal!firing!of!sensitized!neurons!can!lead!to!innocuous!signals!being!
perceived!as!painful118,119.!!
In!regards!to!clinical!studies,!there!are!three!lines!of!evidence!that!support!
the!role!of!CGRP!in!the!pathogenesis!of!migraine.!CGRP!levels!are!elevated!in!
the!external!jugular!and!cubital!veins!during!a!spontaneous!and!nitroglycerinK
induced!migraine!attack120K123.!Studies!have!elaborated!on!this!finding!further!
! 14!
reporting!an!increase!in!serum!and!saliva!during!attacks.!Second,!Intravenous!
infusion!of!CGRP!provokes!a!headache!phenotypically!indistinguishable!from!
spontaneous!migraine.!Additionally,!triptans!are!able!to!inhibit!release!of!CGRP!
and!are!effective!in!reversing!CGRPKinduced!migraines79,124K126.!Furthermore,!in!
preKclinical!studies,!central!and!peripheral!injection!of!CGRP!in!CGRPKsensitized!
mice!or!wildKtype!mice!elicits!migraineKlike!behavior!in!rodents119,127K130.!Lastly,!
CGRP!receptor!antagonists!monoclonal!CGRP!and!CGRP!monoclonal!receptor!
antibodies!are!effective!in!attenuating!migraine!pain!and!reducing!the!number!of!
attacks.!Given!that!monoclonal!antibodies!are!unlikely!to!cross!the!blood!brain!
barrier!and!peripheral!administration!of!CGRPKinduces!migraine!attacks,!
identifying!peripheral!targets!of!CGRP!may!prove!useful!in!finding!new!targets!
and!engineering!more!selective!drugs.!
!
Sensitivity(to(light(in(migraine,(a(rodent(model(of(photophobia(
! Photophobia!is!defined!as!an!aberrant!sensitivity!to!normal!levels!of!light!
that!commonly!leads!to!the!exacerbation!of!migraine,!eye!pain,!or!lightKinduced!
discomfort131.!It!is!present!in!approximately!80K90%!of!migraine!cases,!is!
debilitating!and!affects!quality!of!life132.!According!to!Vanagaite!et!al.!
“photophobia!seems!to!be!an!intrinsic!property!of!migraineurs”!based!on!a!study!
showing!that!migraine!sufferers!are!more!photosensitive!than!controls!(nonK
migraineurs)!during!and!between!migraine!attacks133.!Since!light!can!exacerbate!
pain!experienced!during!a!migraine,!it!is!fitting!to!say!that!photophobia!is!linked!
to!pain!sensation.!In!fact,!photosensitivity!has!been!linked!to!the!activation!of!
! 15!
afferents!within!the!trigeminal!nerve134.!Of!particular!interest,!afferents!from!the!
ophthalmic!branch!of!the!trigeminal!nerve!send!pain!signals!from!the!eye!to!the!
CNS134K137.!Importantly,!a!positron!emission!tomography!study!examining!the!
light!response!during!migraine!showed!that!lightKinduced!pain!was!experienced!
during!headache!and!was!attenuated!by!treatment!with!sumatriptan138K140.!
Therefore,!photophobia!is!excellent!marker!of!a!migraine!state!and!is!an!effective!
tool!to!study!pathophysiology!in!rodents.!
! The!studies!in!this!thesis!use!a!newly!developed!model!of!migraine!using!
photophobia!in!mice!to!revisit!the!vascular!basis!of!migraine.!Specifically,!I!tested!
the!following!hypothesish!(1)!Can!peripheral!administration!of!CGRP!have!actions!
to!induce!migraineKlike!photophobiah!(2)!Does!peripheral!CGRP!have!sites!of!
action!to!induce!photophobia!inside!or!outside!of!the!central!nervous!systemh!and!
(3)!Does!CGRPKinduced!vasodilation!play!a!role!in!this!model?!These!findings!
further!our!understanding!of!how!CGRP!may!contribute!and/or!interact!with!other!
contributors!of!migraine!and!will!aid!in!the!development!of!better!animal!models!
that!are!phenotypically!similar!to!migraine!presentation!in!the!clinic,!thus!better!
targets!for!drug!discovery.!
!
Thesis(overview(and(specific(contributions(
Chapter!II!focuses!on!peripheral!CGRP!actions!using!light!aversion!as!a!
surrogate!for!photophobia.!All!of!the!work!was!done!by!me.!Adisa!Kuburas!
assisted!with!central!CGRP!injections.!Leon!GarciaKMartinez,!MariaKCristina!
Loomis,!and!John!Latham!from!Alder!biopharmaceuticals!provided!me!with!
! 16!
CGRP!monoclonal!antibody.!Nestin/hRAMP1!QPCR!experiments!were!done!by!
myself!with!the!assistance!of!Adisa!Kuburas.!!Nestin/hRAMP1!gross!GFP!
expression!data!was!done!and!analyzed!by!myself!with!little!assistance!from!
AnneKSophie!Wattiez.!All!data!was!analyzed!by!myself.!All!of!the!work!in!this!
chapter!is!published!in!the!Journal!of!Neuroscience128!and!the!QPCR!data!has!
been!resubmitted!to!the!Journal!of!Cerebral!Blood!Flow!and!metabolism.!!
Chapter!III!examines!the!role!of!the!vasculature!in!light!aversion!induced!
by!peripheral!CGRP.!All!telemetry!experiments!were!done!by!both!myself!and!
William!Kutschke!in!the!cardiovascular!core.!All!light!aversion!assays!were!
mostly!done!by!myself!with!Adisa!Kuburas!and!AnneKSophie!Wattiez!testing!a!
small!cohort!of!mice!for!the!CGRP+endothelinK1!experiment.!Global!hRAMP1!
QPCR!experiments!were!done!by!both!myself!and!AnneKSophie!Wattiez.!The!
pupil!diameter!photos!were!taken!by!Brandon!Rea!but!measured!and!analyzed!
by!me.!The!data!in!this!chapter!will!be!submitted!for!publication.!!
!
!
!
!
!
!
!
!
!
! 17!
CHAPTER!II!
INDUCTION!OF!MIGRAINEKLIKE!PHOTOPHOBIC!BEHAVIOR!IN!MICE!BY!
BOTH!PERIPHERAL!AND!CENTRAL!CGRP!MECHANISMS!
!
ABSTRACTt(
The!neuropeptide!calcitonin!geneKrelated!peptide!(CGRP)!is!a!key!player!in!
migraine.!While!migraine!can!be!treated!using!CGRP!antagonists!that!act!
peripherally,!the!relevant!sites!of!CGRP!action!remain!unknown.!To!address!the!
role!of!CGRP!both!within!and!outside!the!central!nervous!system,!we!used!
CGRPKinduced!light!aversive!behavior!in!mice!as!a!measure!of!migraineK
associated!photophobia.!Peripheral!(intraperitoneal,!IP)!injection!of!CGRP!
resulted!in!light!aversive!behavior!in!wildKtype!CD1!mice!similar!to!aversion!
previously!seen!following!central!(intracerebroventricular,!ICV)!injection.!The!
phenotype!was!also!observed!in!C57BL/6J!mice,!although!to!a!lesser!degree!and!
with!more!variability.!Following!IP!CGRP,!motility!was!decreased!in!the!dark!only,!
similar!to!motility!changes!following!ICV!CGRP.!In!addition,!as!with!ICV!CGRP,!
there!was!no!general!increase!in!anxiety!as!measured!in!an!open!field!assay!
following!IP!CGRP.!Importantly,!two!clinically!effective!migraine!drugs,!the!5K
HT1B/D!agonist!sumatriptan!and!a!CGRPKblocking!monoclonal!antibody,!
attenuated!the!peripheral!CGRPKinduced!light!aversion!and!motility!behaviors.!To!
begin!to!address!the!mechanism!of!peripheral!CGRP!action,!we!used!transgenic!
CGRPKsensitized!mice!that!have!elevated!levels!of!the!CGRP!receptor!hRAMP1!
subunit!in!nervous!tissue!(nestin/hRAMP1).!Surprisingly,!sensitivity!to!low!light!
! 18!
was!not!seen!after!IP!CGRP!injection,!but!was!seen!after!ICV!CGRP!injection.!
These!results!suggest!that!CGRP!can!act!in!both!the!periphery!and!the!brain!by!
distinct!mechanisms!and!that!CGRP!actions!may!be!transmitted!to!the!CNS!via!
indirect!sensitization!of!peripheral!nerves.!
!
SIGNIFICANCE(
The!neuropeptide!CGRP!is!a!central!player!in!migraine!pathogenesis,!yet!its!
site(s)!of!action!remain!unknown.!Some!preclinical!studies!have!pointed!to!
central!sites!in!the!brain!and!brainstem.!However,!a!peripheral!site!of!action!is!
indicated!by!the!ability!of!intravenous!CGRP!to!trigger!migraine!in!humans!and!
the!efficacy!of!CGRP!receptor!antagonists!that!evidently!do!not!penetrate!the!
CNS!in!effective!amounts.!Resolving!this!issue!is!particularly!important!given!
recent!clinical!trials!showing!that!antiKCGRP!monoclonal!antibodies!can!reduce!
and!even!prevent!migraine!attacks.!In!this!study,!we!report!that!CGRP!can!act!in!
both!the!brain!and!the!periphery!of!the!mouse!to!cause!migraineKlike!
photophobia!by!apparently!distinct!mechanisms.!
!
INTRODUCTION!
The!neuropeptide!CGRP!is!now!recognized!as!a!key!player!in!the!
pathogenesis!of!migraine!101.!CGRP!is!found!in!neurons!of!both!the!central!and!
peripheral!nervous!systems!and!its!receptors!are!widespread!throughout!the!
body,!where!it!has!been!implicated!in!diverse!functions!141.!Most!notably,!in!the!
CNS,!CGRP!has!been!linked!to!nociceptive!signaling,!and!in!the!periphery,!it!is!
! 19!
the!most!potent!vasodilatory!peptide!and!contributes!to!neurogenic!inflammation,!
in!part!by!actions!on!mast!cells.!Both!central!and!peripheral!effects!of!CGRP!
action!are!consistent!with!migraine!symptoms.!
Early!clinical!studies!supported!a!peripheral!site!of!action!for!CGRP.!
Plasma!levels!were!increased!during!induced!and!spontaneous!migraineh!
moreover,!migraines!could!be!triggered!by!intravenous!injection!of!CGRP!in!the!
cubital!vein!124.!Later,!clinically!effective,!small!molecule!CGRP!receptor!
antagonists!were!shown!to!have!relatively!low!brain!penetranceh!despite!this,!the!
possibility!of!central!action!has!been!debated!50,142K144.!Furthermore,!therapeutic!
efficacy!of!CGRPKblocking!and!CGRP!receptorKblocking!monoclonal!antibodies!
145K149!strongly!supports!a!peripheral!site!of!CGRP!action!150,151.!Notwithstanding!
the!evidence!that!peripheralKacting!CGRP!antagonists!can!treat!migraine,!CGRP!
in!the!CNS!may!also!play!a!role!in!some!migraine!symptoms.!!For!example,!
preclinical!studies!have!demonstrated!central!modulation!of!trigeminal!nerve!
activity!by!CGRP!108,152K154.!Thus,!we!have!reasoned!that!CGRP!is!well!
positioned!to!act!in!both!the!CNS!and!on!peripheral!nerves!49h!hence,!either!
central!or!peripheral!administration!of!CGRP!might!be!sufficient!to!induce!at!least!
a!subset!of!migraineKlike!symptoms!in!mice.!
To!address!the!possible!sites!of!CGRP!in!migraine,!we!have!used!a!
mouse!model!of!photophobia.!Photophobia!is!an!abnormal!discomfort!to!nonK
noxious!levels!of!light!that!is!a!diagnostic!feature!of!migraine.!Our!lab!has!
previously!shown!that!intracerebroventricular!(ICV)!injection!of!CGRP!in!both!
wildKtype!and!CGRPKsensitized!transgenic!mice!induced!light!aversion,!a!
! 20!
surrogate!for!photophobia!127,129,130.!The!CGRPKsensitized!mice!have!the!human!
receptor!activityKmodifying!protein!1!(RAMP1)!subunit!of!the!CGRP!receptor!
overexpressed!in!the!nervous!system!(nestin/hRAMP1)!100.!RAMP1!is!an!
obligatory!subunit!that!helps!form!a!binding!pocket!for!CGRP!155,!and!was!shown!
to!be!the!rateKlimiting!component!of!the!receptor!in!both!trigeminal!neuron!and!
vascular!smooth!muscle!cultures!99,100.!RAMP1!interacts!with!both!the!canonical!
CGRP!receptor,!calcitonin!receptorKlike!receptor!(CLR)!94!and!a!second!CGRP!
receptor,!calcitonin!receptor!(CTR)!104.!The!key!behavioral!difference!between!
the!transgenic!and!wildKtype!mice!is!that!the!former!exhibits!light!sensitivity!by!
even!dim!light!following!ICV!CGRP!administration!129,156,!while!wildKtype!mice!
require!a!bright!light!stimulus!and!lowered!exploratory!drive!130.!However,!there!is!
a!caveat!to!these!prior!studies.!The!ICV!injections!were!done!by!direct!injection!
through!the!scalp,!which!allowed!leakage!onto!the!meninges.!Thus,!one!goal!of!
this!study!was!to!minimize!this!leakage!to!confirm!a!central!site!of!action.!!
In!this!chapter,!we!have!demonstrated!that!peripheral!injection!of!CGRP!
causes!light!aversion!in!wildKtype!mice!similar!in!many!respects!to!centrally!
administered!CGRP.!This!aversion!to!bright!light!could!be!pharmacologically!
attenuated!by!clinicallyKeffective!agents.!However,!unlike!centrally!administered!
CGRP,!peripheral!CGRP!did!not!induce!aversion!to!dim!light!in!the!CGRPK
sensitized!nestin/hRAMP1!mice.!These!data!suggest!that!peripheral!and!central!
CGRP!act!by!distinct,!possibly!overlapping,!mechanisms!to!cause!light!aversive!
behavior!in!mice.!
!
! 21!
MATERIALS!AND!METHODS!
Animals(
Two!strains!of!wildKtype!mice!were!used:!C57BL/6J!and!CD1.!Equivalent!
numbers!of!adult!male!and!female!mice,!aged!10K20!weeks,!were!used!in!all!
experiments.!The!transgenic!nestin/hRAMP1!line!has!been!previously!described!
129.!Double!transgenic!progeny!from!crosses!of!the!parental!CX1KGFPKhRAMP1!
mice!and!nestinKcre!(Jackson!Labs,!stock!003771)!were!used.!Mice!were!housed!
in!groups!of!3!to!5!per!cage,!unless!otherwise!indicated,!on!a!12Kh!light!cycle!with!
food!and!water!ad!libitum.!All!behavioral!experiments!were!performed!between!8!
AM!and!2:30!PM.!For!all!experiments,!investigators!were!blinded!to!genotype!
and!drug!treatment.!Animal!procedures!were!approved!by!the!University!of!Iowa!
Animal!Care!and!Use!Committee!and!performed!in!accordance!with!the!
standards!set!by!the!National!Institutes!of!Health.!!!
!
Intraperitoneal(drug(and(antibody(administration(
All!drugs!that!required!dilution!were!prepared!with!Dulbecco!PBS!
(Hyclone)!as!the!vehicle.!The!amounts!injected!were:!0.1!mg/kg!(unless!
otherwise!indicated)!rat!αKCGRP!(Sigma),!0.6!mg/kg!sumatriptan!succinate!(APP!
Pharmaceuticals),!30!mg/kg!ALD405!(a!monoclonal!antiKCGRP!antibody),!and!30!
mg/kg!AD26K10v2!(a!monoclonal!IgG!control!antibody).!The!antibody!dose!
corresponded!to!8!nmol!per!mouse,!which!is!approximately!8Kfold!excess!
antibody!(16Kfold!excess!binding!sites)!over!exogenous!CGRP!(1!nmol).!CGRP,!
sumatriptan,!and!vehicle!were!administered!10!μl/g!bodyweight!with!a!30!g!x!0.5!
! 22!
in!needle.!Antibody!was!administered!with!BD!ultrafine!31g!insulin!syringes.!
Antibodies!were!administered!via!IP!injection!24!h!prior!to!treatment!with!CGRP!
or!vehicle.!All!injections!were!performed!by!either!B.M.!or!A.K.!Animals!were!
gently!held,!but!not!anesthesized!during!injection.!After!CGRP!or!vehicle!
injection,!mice!were!allowed!to!recover!for!30!min!in!their!home!cage!prior!to!
testing!based!on!original!studies!using!the!light!aversion!assay!157.!!
!
Intracerebroventricular(drug(administration(
Drugs!were!administered!via!ICV!injection!in!the!right!lateral!ventricle!with!
a!needle!as!previously!described!156!or!via!a!cannula.!Mice!were!given!CGRP!
(1μg/μl)!at!a!volume!of!2μl.!To!ensure!a!slow!delivery!through!the!cannula,!the!
rate!of!injection!was!0.5μl/min!for!4!min.!Cannulas!were!handKconstructed!from!
304!stainless!steel!24Kgauge!hypodermic!tubing!cut!to!8mm.!Obturators!used!to!
seal!the!opening!of!the!cannula!were!made!by!soldering!a!short!3mm!cannula!to!
a!12mm!piece!of!30Kgauge!wire!tubing.!The!cannula!was!implanted!using!a!
stereotaxis!frame!at!the!target!coordinates!(1mm!ML,!0.3mm!AP,!2.2mm!DV).!Once!
surgery!was!complete,!mice!were!housed!individually!to!reduce!the!risk!of!
cannula!displacement!and!allowed!to!recover!for!approximately!2!weeks!prior!to!
the!first!exposure!to!light!aversion!assay.!All!surgeries!were!performed!by!the!
same!person!(B.M.),!with!a!success!rate!of!>95%!as!demonstrated!by!injections!
of!dye!into!the!cannulated!ventricles.!B.M.!and!A.K.!performed!all!injections.!
Before!injections,!mice!were!anesthetized!with!3.5%!isofluorane!in!an!induction!
chamber.!During!injections,!anesthesia!was!sustained!with!1.5%!isofluorane!
! 23!
through!a!nose!cone.!After!administration!of!drugs,!mice!were!allowed!to!recover!
for!60!min!in!their!home!cage!prior!to!testing.!The!60!min!postKICV!injection!time!
frame!is!preferable!to!minimize!anesthesia!effects!130.!
!
Light(aversion(and(motility(assays(
LightKdark!boxes!with!infrared!beam!tracking!were!used!(Med!Associates,!
St.!Albans,!Vermont).!For!wildKtype!mice,!mice!were!preKexposed!to!the!chamber!
twice!every!three!days!prior!to!treatment!exposure.!For!the!postKexposure,!mice!
were!tested!in!the!lightKdark!boxes!three!days!following!treatment.!In!addition,!
these!mice!are!tested!using!bright!light!(27,000!lux),!as!described!130.!For!
transgenic!mice,!mice!naïve!to!the!chamber!were!tested!using!dim!light!(55!lux),!
as!described!130.!Data!were!collected!for!30!min!and!analyzed!in!sequential!5!min!
intervals,!as!well!as!average!time!spent!on!each!side!of!the!chamber!per!5!min!
interval.!
Motility!outcomes!were!measured!as!described!130.!To!account!for!the!
variation!in!the!amount!of!time!mice!spent!in!each!zone,!data!were!normalized!to!
time!spent!in!the!dark!and!light!zones.!
!
Open(field(assay(
This!assay!was!performed!as!described!130.!Mice!were!placed!in!the!
center!of!the!chamber!and!tested!for!30!min.!The!periphery!was!defined!as!4.22!
cm!from!the!border!with!the!remaining!18.56!x!18.56!cm!area!as!the!center.!
!
! 24!
RNA(isolation(and(measurement(of(human(and(mouse(RAMP1(RNA(levels!
RNA!was!isolated!by!homogenizing!tissues!in!Trizol!(Ambion!Life!
Technologies)!followed!by!70%!ethanol!precipitation!and!purification!using!
RNeasy!Micro!columns!(Qiagen).!To!examine!the!quality!of!RNA!integrity,!RNA!
agarose!gels!were!run!and!18S!and!28S!bands!were!scrutinized!for!band!
intensity.!A!spectrophotometer!was!used!for!quantifying!the!amount!of!RNA!in!
the!sample!and!260/280!ratio!was!used!to!determine!the!purity!of!each!sample.!
For!reverse!transcription,!approximately!50ngK150ng!of!RNA!was!reversed!
transcribed!using!a!Takara!Clontech!Reverse!Transcript!kit.!!
Human!RAMP1!(hRAMP1)!gene!expression!was!measured!by!real!time!
quantitative!PCR!(QKPCR)!using!primer!sequences!previously!described!and!
validated!by!our!team!158.!The!mRNA!levels!of!both!hRAMP1!and!endogenous!
mouse!RAMP1!(mRAMP1)!was!determined!using!Power!SYBR!Green!PCR!
Master!Mix!(Applied!Biosystems,!UK).!Reactions!were!performed!in!triplicate!
using!ABI!SDS!7900!HT!thermocycler.!!Analysis!was!performed!using!ABI!SDS!
v2.4!software.!The!mRAMP1!and!hRAMP1!Ct!values!were!converted!to!absolute!
copy!numbers!and!normalized!to!50,000!copies!of!"Kactin!using!standard!curves!
generated!with!1:10!serial!dilutions!of!pGEMKQmRAMP1,!pbsCX1KLELKhRAMP1,!
and!pGEMKQmbetaKactin!plasmids!in!10!ng/ml!yeast!tRNA.!Total!RAMP1!copies!
were!summed!and!results!reported!as!the!meanKfold!increase!relative!to!control!±!
SEM!from!5!pairs!of!control!and!hRAMP1!mice.!
(
(
! 25!
Statistical(analysis(
The!data!were!analyzed!between!treatment!groups!(e.g.!vehicle!vs!
CGRP)!within!each!exposure!to!the!chamber.!A!twoKway,!repeated!measures!
ANOVA!(factors:!treatment!and!observation!time)!was!used!with!a!Bonferroni!
multiple!comparisons!test!to!compare!treatment!groups!at!each!interval.!For!
experiments!with!mice!tested!in!the!chamber!multiple!times,!a!twoKway!repeated!
measures!ANOVA!was!also!used!to!compare!between!exposure!daysh!treatment!
day!was!compared!with!the!preKtreatment!exposure!and!postKtreatment!at!each!
interval.!A!oneKway!ANOVA!repeated!measures!was!used!to!determine!if!overall!
significant!effects!were!observed!in!bar!graphs!with!individual!points.!Bonferroni!
or!Tukey!multiple!comparisons!test!was!used!as!the!post(hoc!analysis.!Data!are!
reported!as!mean!±!standard!error!of!the!mean!(SEM).!Data!were!analyzed!using!
Graphpad!Prism!software.!!
Exclusions!were!applied!to!the!dataset!due!to!the!following!reasons:!never!
leaving!the!light!zone!during!30!min!of!testing,!mice!had!an!overall!resting!time!
greater!than!90%,!or!mice!were!considered!statistical!outliers!according!the!
Graphpad!Prism!criteria!(more!than!3!standard!deviations!from!the!mean).!For!all!
CD1!mice,!4!mice!were!excluded!for!resting!more!than!90%,!and!2!mice!were!
considered!statistical!outliers!for!a!total!of!six!mice!for!all!experiments.!For!all!
C57BL/6J!mice,!3!mice!were!excluded!due!to!loss!of!cannula,!and!2!mice!were!
excluded!for!resting!more!than!90%!for!a!total!of!five!excluded!for!all!
experiments.!
!
! 26!
RESULTS!
Peripheral(CGRP(administration(elicits(light(aversion(in(mice((
As!a!starting!point,!we!looked!at!the!effect!of!peripheral!CGRP!using!wildK
type!mice.!Two!wildKtype!strains!were!tested,!C57BL/6J!and!CD1.!The!C57BL/6J!
strain!was!chosen!to!match!our!previous!studies!with!wildKtype!mice!130!and!the!
transgenic!hRAMP1!mice,!which!are!on!a!predominantly!C57BL/6J!background!
156.!The!CD1!strain!was!chosen!based!on!blood!flow!observations!suggesting!the!
possibility!these!mice!might!be!more!responsive!to!sensory!neuropeptides!than!
C57BL/6J!mice!159,160.!!
CD1!and!C57BL/6J!mice!were!given!vehicle!or!CGRP!in!a!single!IP!
injection!after!two!preKexposures!to!the!chamber!to!reduce!exploratory!drive!130.!
Testing!began!30!min!after!the!mice!were!injected.!In!CD1!mice,!CGRP!elicited!
significant!light!aversion!in!all!six!5Kmin!intervals!compared!to!vehicle,!with!a!
significant!overall!effect!(p<0.0001h!F(1,36)=!26.93)!(Fig.!II.1).!The!CGRPKtreated!
mice!(Tx)!spent!significantly!less!time!in!the!light!compared!to!preKtreatment!
exposure!2!(Pre2)!and!postKtreatment!exposure!(Post)!(p<0.0001).!There!were!
no!significant!differences!for!vehicleKtreated!mice.!On!average,!the!vehicleK
treated!CD1!mice!spent!106!sec!in!the!light!per!each!5!min!interval!compared!to!
28!sec!for!CGRPK!treated!mice!(p<0.0001).!!
Similar!results!were!seen!with!C57BL/6J!mice!(p<0.0001h!F(1,84)=!24.18)!
(Fig.!II.2).!However,!the!C57BL/6J!responses!were!not!as!profound!as!those!
seen!with!CD1!mice.!Furthermore,!in!2!out!of!8!experiments!with!C57BL/6J!mice,!
IP!CGRP!(0.1!or!0.5!mg/kg)!did!not!yield!a!significant!light!aversive!response!(all!
! 27!
data!are!included!in!the!figure).!In!contrast,!with!CD1!mice!significant!responses!
were!seen!in!5!out!of!5!experiments.!These!data!suggest!that!the!CD1!strain!is!
more!sensitive!to!CGRP.!To!further!explore!differences!between!CD1!and!
C57BL/6J!mice,!a!doseKresponse!curve!was!generated!to!compare!the!
responses!of!C57BL/6J!and!CD1!mice.!These!data!demonstrate!that!CD1!mice!
are!significantly!more!susceptible!than!C57BL/6J!mice!to!administration!of!
peripheral!CGRP!at!both!0.1mg/kg!and!0.5mg/kg!CGRP!(F(1,238)=!26.33),!
whereas!the!vehicleKtreated!mice!were!not!significantly!different!between!the!two!
strains!(Fig.!II.3).!Thus,!peripheral!administration!of!CGRP!elicited!light!aversion!
in!both!wildKtype!strains!of!mice,!although!the!response!was!greater!and!more!
consistent!with!CD1!mice.!
!
Sex(comparison(following(peripheral(administration(of(CGRP(
Migraine!affects!women!approximately!three!times!more!than!men.!To!
determine!if!one!sex!contributed!more!to!the!light!aversive!phenotype,!we!sorted!
and!analyzed!the!data!in!Fig.!II.1!and!Fig.!II.2!by!male!and!female.!In!CD1!mice,!
peripheral!CGRP!at!0.1mg/kg!did!not!elicit!sex!differences!in!the!light!aversion!
assay.!However,!male!mice!trended!towards!significance!by!spending!an!
average!of!approximately!35!s!in!the!light!compared!to!an!average!of!58!s!for!
female!mice!(Fig.!II.4).!However,!when!CD1!mice!were!administered!0.5mg/kg!
CGRP!female!mice!trended!towards!significance!compared!to!the!male!mice!
(Fig.!II.5).!In!addition,!we!wanted!to!analyze!the!percent!change!in!the!time!spent!
in!light!after!CGRP!treatment!and!compare!it!to!the!baseline!of!each!individual!
! 28!
mouse.!Following!0.1mg/kg!the!degree!of!response!was!very!similar!between!
female!and!male!mice!(Fig.!II.6A).!However,!when!0.5!mg/kg!CGRP!was!
delivered!in!these!mice,!female!mice!had!approximately!a!90%!change!from!
baseline!compared!to!a!45%!change!seen!in!the!male!cohort!(Fig.!II6B).!!
On!treatment!day,!C57BL/6J!male!mice!spent!significantly!less!time!in!the!
light!compared!to!female!mice!at!0.1mg/kg!CGRP!(Fig.!II7)!and!trended!towards!
significant!at!0.5mg/kg!(Fig.!II.8).!However,!when!we!analyzed!percent!change!
from!baseline,!at!either!dose!both!male!and!female!mice!had!similar!changes!
from!baseline!ranging!from!15K30!percent!(FII.9A,B).!!
!
Peripheral(CGRP(administration(reduces(motility(only(in(the(dark(zone((
We!evaluated!the!effect!of!IP!CGRP!on!motility!as!measured!by:!resting!
time,!transitions,!ambulatory!distance,!and!vertical!rearing.!Consistent!with!ICV!
injection!of!CGRP!129,130,!IP!CGRP!reduced!motility!only!in!the!dark!zone!(Fig.!
II.10!and!Fig.II.11).!In!both!CD1!and!C57BL/6J!mice,!the!resting!time!was!
significantly!increased!in!the!dark!zone!(CD1,!F(1,36)=!16.52h!C57,!F(1,84)=!12.24)!
(Fig.!II.10A,B),!along!with!decreased!rearing!behavior!(CD1,!F(1,36)=!26.51h!C57,!
F(1,84)=24.20)!(Fig.!II.11A,B),!and!ambulatory!distance!(not!shown).!In!the!light!
zone,!while!there!was!a!trend!towards!decreased!rearing!in!both!genotypes,!this!
was!not!significant!for!CD1!and!was!significant!for!only!two!time!points!with!
C57BL/6J!mice.!Mice!also!transitioned!significantly!less!between!light!and!dark!
zones!after!CGRP!treatment!as!compared!to!vehicle!treatment!(CD1,!F(1,36)=!
27.17!h!C57,!F(1,84)=!10.09)!(Fig.!II.12A,B).!For!the!CD1!mice,!in!all!measures!of!
! 29!
motility!the!CGRP!cohort!was!significantly!different!from!the!Pre2!and!PostK
treatment!periods,!but!this!was!not!always!seen!for!the!C57BL/6J!mice!(Fig!
II.11B).!!Analysis!of!motility!data!comparing!C57!and!CD1!mice!revealed!that!
peripheral!CGRP!significantly!increased!resting!in!CD1!mice!more!than!in!
C57BL/6J!mice!when!calculated!as!the!increase!in!resting!compared!to!vehicle!
(F(3,122)=19.19hp<0.01)!or!change!from!Pre2!baseline!(p<0.001).!In!addition,!
CGRPKinduced!reduction!in!rearing!was!significantly!greater!in!CD1!than!in!
C57BL/6J!mice!when!calculated!as!the!decrease!in!rearing!compared!to!vehicle!
(F(3,122)!27.09hp<0.0001)!or!change!from!Pre!2!baseline!(p<0.05).!!
!
Central(CGRP(administration(elicits(light(aversion(and(lightMdependent(resting(
behavior(
Given!the!ability!of!peripheral!CGRP!administration!to!elicit!light!aversion!
and!lightKdependent!resting!behavior,!we!reKexamined!the!effect!of!centrallyK
administered!CGRP!under!conditions!to!minimize!leakage!of!CGRP!during!the!
ICV!injection.!This!was!a!potential!confounder!of!our!previous!ICV!studies!
129,130,156.!C57BL/6J!mice!were!used!in!this!experiment!because!our!previous!
reports!used!C57BL/6J!WT!mice,!and!our!transgenic!mice!are!on!the!C57BL/6J!
background.!To!reduce!peripheral!leakage,!we!inserted!a!cannula!to!allow!slow!
delivery!of!CGRP!into!the!ventricles.!Consistent!with!our!previous!findings,!ICV!
injection!of!CGRP!via!a!cannula!resulted!in!significant!light!aversive!behavior!(F(1,!
22)=!12.23)!(Fig.!II.13).!In!addition,!the!mice!showed!increased!resting!behavior!in!
the!dark,!but!not!light,!zone!(F(1,22)=!17.67)!(Fig.!II.14).!These!findings!are!
! 30!
consistent!with!observations!seen!with!C57BL/6J!injected!with!ICV!CGRP!via!
direct!CGRP!administration!without!a!cannula!130.!
!
Peripheral(CGRP(does(not(enhance(anxiety(behavior(in(the(open(field(
To!determine!whether!the!peripheral!CGRP!lightKaversive!phenotype!was!
being!driven!by!an!increased!anxiety!state,!we!used!the!open!field!test.!Mice!that!
had!previously!been!tested!in!the!lightKdark!assay!were!placed!in!an!open!field!2K
3!days!after!the!postKtreatment!exposure!(see!Fig.!II.1).!The!light!intensity!
remained!at!2.7x104!lux.!The!mice!were!tested!30!min!after!IP!injection!of!CGRP!
or!vehicle,!as!done!with!the!lightKdark!assay.!There!was!no!significant!difference!
in!the!time!the!mice!spent!in!the!center!of!the!open!field!between!treatments!in!
either!CD1!or!C57BL/6J!mice!(Fig.!II.15!and!F.II.16).!This!suggests!that!
peripheral!administration!of!CGRP!does!not!increase!anxiety!behaviors!in!mice!
to!influence!a!lightKaversive!phenotype,!which!is!consistent!with!prior!studies!
involving!ICV!CGRP!in!nestin/hRAMP1!mice!156!and!C57BL/6J!mice!130.!!!
!
Sumatriptan(attenuates(light(aversion(induced(by(peripheral(CGRP(
Sumatriptan!is!one!of!the!5KHT1B/D!receptor!agonists!that!are!considered!
the!gold!standard!in!migraine!treatment!and!administered!peripherally!to!abort!
migraine!161.!We!have!previously!reported!that!a!related!triptan,!rizatriptan,!was!
able!to!attenuate!light!aversion!in!mice!given!ICV!CGRP!130.!Mice!were!given!an!
IP!injection!of!CGRP,!vehicle,!sumatriptan,!or!coKadministration!of!CGRP!+!
sumatriptan!30!min!prior!to!light!aversion!testing!on!treatment!day.!As!with!
! 31!
centrally!administered!CGRP,!the!effect!of!peripheral!CGRP!in!mice!was!
attenuated!by!sumatriptan!(Fig.!II.17!and!Fig.!II.18).!CoKadministration!of!
sumatriptan!with!CGRP!via!IP!injection!in!CD1!mice!fully!rescued!the!phenotype!
seen!with!CGRP!treatment!alone!(F(3,79)=!8.91)!(Fig.!II.17).!Likewise,!in!C57BL/6J!
mice,!coKtreatment!of!CGRP!and!sumatriptan!also!reduced!light!aversion,!but!did!
not!fully!inhibit!the!CGRPKinduced!behavior!as!observed!with!CD1!mice!(F(3,!77)=!
10.59)!(Fig.!II.18).!However,!a!potential!confounder!to!the!C57BL/6J!data!was!
that!the!CGRPKtreated!mice!did!not!return!to!baseline!during!the!postKexposure!
period!in!this!experiment.!For!both!CD1!and!C57BL/6J!mice,!sumatriptan!
prevented!CGRPKmediated!increased!resting!in!the!dark!zone!(CD1,!F(3,79)=!4.31!
h!C57,!F(3,77)=!11.74)!(Fig.!II.19A,B)!and!decreased!vertical!rearing!(CD1,!F(3,79)=!
4.08,!p<0.05h!C57,!F(3,77)=!10.64!p<0.05).!CoKadministration!of!CGRP!and!
sumatriptan!also!partially!rescued!the!number!of!transitions!between!zones!for!
both!genotypes!(not!shown).!The!persistent!light!aversion!in!the!C57BL6/J!mice!
(Fig.!II.19B)!cannot!be!explained!in!this!one!experiment.!However,!in!4!other!
experiments!(Fig.!II.2),!the!C57BL/6J!mice!returned!to!baseline!levels!after!
CGRP!treatment.!Together,!these!data!indicate!sumatriptan!can!attenuate!
peripheral!CGRPKinduced!light!aversion.!
!
A(CGRPMblocking(antibody(prevents(CGRPMinduced(light(aversion(
We!then!tested!whether!a!monoclonal!antibody!that!binds!CGRP!would!be!
able!to!block!CGRPKinduced!light!aversion!in!CD1!mice!(Fig.!II.20).!For!this!
experiment,!we!used!a!sequential!treatment!paradigm.!The!first!treatment!with!IP!
! 32!
CGRP!or!vehicle!was!followed!by!IP!injection!with!antibodies!prior!to!a!second!
treatment!with!IP!CGRP!or!vehicle.!The!antibody!dosage!was!theoretically!
sufficient!to!attenuate!both!exogenous!and!endogenous!CGRP!actions.!This!
paradigm!established!the!responses!of!individual!mice!prior!to!treatment!with!
antibodies.!During!the!first!treatment!(Tx1),!CGRPKtreated!mice!spent!
significantly!less!time!in!the!light!compared!to!vehicleKtreated!mice!(F(2,42)=!11.83)!!
and!compared!to!Pre2!and!Post!periods.!PostKtreatment!was!used!to!ensure!that!
the!time!spent!in!light!by!the!treated!mice!had!returned!to!levels!prior!to!
treatment.!The!mice!that!were!given!CGRP!in!the!first!treatment!were!then!given!
either!the!control!antibody!or!the!antiKCGRP!antibody!24!h!prior!to!testing!with!a!
second!injection!of!CGRP.!Likewise,!the!vehicle!treated!mice!were!given!a!
second!injection!of!vehicle.!During!the!second!treatment!(Ab!Tx),!mice!treated!
with!CGRP!plus!the!control!antibody!spent!less!time!in!the!light!compared!to!
mice!that!received!CGRP!plus!antiKCGRP!antibody!or!vehicle!plus!antiKCGRP!
antibody!(F(2,42)=!6.35).!Antibody!treatment!also!abolished!the!change!in!resting!
activity!(Fig.!II.21A)!and!rearing!behavior!(Fig.!II.21B)!induced!by!CGRP.!!
!
Neural(expression(of(hRAMP1(in(nestin/hRAMP1(mice((
! In!these!experiments,!we!have!restricted!overexpression!of!hRAMP1!to!the!
nervous!system!with!nestinKcre!dependent!expression!of!hRAMP1.!The!
nestin/hRAMP1!transgenic!mice!have!previously!been!shown!to!display!a!CGRPK
sensitized!phenotype!for!two!migraineKassociated!symptoms,!photosensitivity!to!
central!CGRP!and!mechanical!allodynia.!GFPKhRAMP1!mice!were!crossed!with!
! 33!
mice!expressing!cre!recombinase!under!control!of!the!nestin!promoter,!which!is!
primarily!active!in!CNS!and!peripheral!nervous!system!neural!progenitor!cells.!
Expression!of!hRAMP1!is!dependent!on!cre,!which!is!needed!to!remove!an!
upstream!GFP!stop!cassette!(Fig.!II.22A).!Hence,!in!the!absence!of!cre!activity,!
there!should!still!be!GFP!fluorescence.!!
! Consistent!with!our!previous!studies,!the!double!transgenic!nestin/hRAMP1!
mice!did!not!express!GFP!in!the!brain!cortices!or!trigeminal!ganglion.!However,!
in!heart!and!aorta!(and!other!nonKneural!tissues,!data!not!shown)!GFP!was!still!
present!in!the!nestin/hRAMP1!mice.!As!controls,!GFP!was!detectable!in!tissues!
from!prarental!single!transgenic!GFPKhRAMP1!mice,!but!not!from!single!
transgenic!nestinKcre!mice!(Fig.!II.22B).!The!levels!of!hRAMP1!and!endogenous!
mRAMP1!gene!expression!were!measured!by!QKPCR!(Fig.!II.23).!Unfortunately,!
there!are!no!available!RAMP1!antibodies!for!quantitative!measurement!of!
RAMP1!protein!levels.!Consistent!with!the!GFP!expression,!there!was!very!little!
detectable!hRAMP1!RNA!present!in!nonKneural!tissues.!!While!there!was!some!
hRAMP1!detected!in!cerebral!arteries!(basilar,!middle!cerebral,!and!posterior!
communicating!arteries),!we!cannot!rule!out!contamination!from!the!neural!tissue!
during!the!dissecting!procedure.!There!were!no!statistical!differences!in!
mRAMP1!RNA!levels!between!sexes!or!between!genotypes!and!no!statistical!
difference!in!hRAMP1!levels!between!sexes!of!the!double!transgenic!
nestin/hRAMP1!mice.!!
!
! 34!
Central(CGRP,(but(not(peripheral(CGRP,(elicits(sensitized(light(aversion(in(
nestin/hRAMP1(mice(
To!begin!to!identify!the!site!of!CGRP!action!in!the!periphery,!we!took!a!
genetic!approach!using!the!CGRPKsensitized!nestin/hRAMP1!mice.!These!mice!
have!conditional!overKexpression!of!the!hRAMP1!subunit!of!the!CGRP!receptor!
in!both!the!CNS!and!peripheral!nervous!system!100.!We!have!previously!reported!
that!ICV!CGRP!induces!light!aversive!behavior!in!these!mice!at!much!lower!light!
intensity!(55!lux)!compared!to!the!27,000!lux!required!with!wildKtype!mice!
129,130,156.!Furthermore,!unlike!wildKtype!mice,!nestin/hRAMP1!mice!do!not!require!
preKexposure!to!the!chamber!in!order!to!demonstrate!CGRPKinduced!light!
aversion.!Surprisingly,!after!IP!administration!of!CGRP!to!naïve!mice,!the!
nestin/hRAMP1!mice!did!not!exhibit!enhanced!light!aversion!under!these!low!
light!conditions!of!55!lux!(Fig.!II.24).!As!a!control,!we!showed!that!ICV!injections!
of!CGRP!could!induce!light!aversion!under!these!experimental!conditions.!
Nestin/hRAMP1!mice!were!tested!by!both!ICV!and!IP!CGRP!injection!over!the!
same!period!of!time!and!in!one!experiment!cagemates!were!tested!on!the!same!
day.!As!predicted,!nestin/hRAMP1!mice!given!ICV!CGRP!were!light!aversive!
(F(3,86)=!10.44)!(Fig.!II.25).!In!addition,(the(nestin/hRAMP1!mice!given!IP!CGRP!
did!not!show!any!change!in!motility,!while!mice!given!ICV!CGRP!rested!more!in!
the!dark!(not!shown).!As!a!control,!we!wanted!to!show!that!nestin/hRAMP1!mice!
did!have!the!ability!to!respond!to!IP!CGRP!in!bright!light,!analogous!to!wildKtype!
mice.!!A!cohort!of!nestin/hRAMP1!mice!that!had!previously!not!responded!to!
CGRP!in!dim!light!(Fig.!II.24)!were!tested!at!27,000!lux.!Both!nestin/hRAMP1!
! 35!
and!control!littermates!responded!to!IP!CGRP!to!about!the!same!degree!(Fig.!
II.26).!!
!
DISCUSSION!
In!this!chapter,!we!report!that!peripherally!administered!CGRP!can!induce!
light!aversion!in!two!strains!of!wildKtype!mice.!This!peripheral!administration!is!
clinically!relevant,!because!CGRP!injection!studies!and!clinical!trials!with!CGRP!
receptor!antagonists!and!CGRPKblocking!antibodies!suggest!to!a!peripheral!site!
of!CGRP!action!in!migraine!124,150,151,162,163.!Yet,!the!neural!symptoms!of!migraine!
and!several!preclinical!studies!have!pointed!to!actions!in!the!CNS!108,152K154.!In!
this!regard,!we!have!now!confirmed!our!previous!finding!that!ICV!injection!of!
CGRP!induces!light!aversion!in!mice!albeit!this!time!with!the!use!of!a!cannula.!
This!confirmation!was!desired!as!the!previous!ICV!protocol!could!not!rule!out!the!
possibility!of!peripheral!actions!of!CGRP!in!the!meninges.!Importantly,!the!
abilities!of!an!antiKmigraine!drug,!sumatriptan,!and!a!CGRPKblocking!antibody!to!
attenuate!light!aversion!provide!validation!that!peripheral!CGRPKinduced!light!
aversion!in!mice!is!analogous!to!photophobia!experienced!by!migraineurs.!Taken!
together,!these!findings!support!our!hypothesis!that!CGRP!has!central!and!
peripheral!actions!that!cause!migraineKlike!light!aversion!in!mice,!notwithstanding!
that!blocking!CGRP!effects!peripherally!is!sufficient!to!treat!or!prevent!migraine.!
While!CGRP!elicited!light!aversion!in!both!wildKtype!strains!of!mice,!the!
response!was!greater!and!more!consistent!with!the!albino!CD1!mice.!In!addition!
to!light!aversion,!CD1!mice!also!displayed!a!greater!reduction!in!movement!than!
! 36!
C57BL/6J!mice.!Although!it!is!possible!that!the!intrinsic!lack!of!retinal!pigment!
could!contribute!to!the!phenotype,!it!does!not!seem!likely!to!be!a!major!cause!
since!both!genotypes!spent!similar!time!in!the!light!during!the!preKexposures!and!
in!response!to!vehicle.!This!suggests!that!CD1!may!have!greater!sensitivity!to!
CGRP!than!C57BL/6J.!This!conclusion!is!consistent!with!the!greater!blood!flow!
in!CD1!than!C57BL/6J!mice!observed!after!stimulation!of!peptidergic!sensory!
nerve!terminals!by!topical!mustard!oil!160.!While!that!study!did!not!explicitly!show!
CGRP!involvement,!it!seems!likely!given!CGRP’s!role!as!the!most!potent!
vasodilatory!peptide.!Indeed,!Mogil!and!colleagues!reported!strain!variability!in!
thermal!sensitivity!due!to!differences!in!CGRP!levels!164.!They!found!that!CD1!
mice!showed!a!trend!towards!lower!CGRP!expression!in!dorsal!root!ganglia!than!
C57BL/6J!mice,!and!suggest!that!mice!with!low!basal!CGRP!expression!have!
increased!heat!sensitivity!following!injection!of!CGRP.!Thus,!it!seems!possible!
that!differences!in!either!basal!CGRP!or!CGRP!receptor!levels!could!contribute!
to!the!strain!differences!observed!in!the!light!aversion!assay.!
The!ability!of!peripheral!CGRP!to!induce!a!fairly!rapid!light!aversive!
response!in!mice!is!consistent!with!recent!human!studies.!Ashina!and!colleagues!
reported!the!average!onset!of!photophobia!was!30!min!(range!19!minK9!h)!after!
intravenous!CGRP,!while!the!average!onset!of!migraine!headache!was!!~3!h!
(range!10!minK12!h)!165.!Thus,!it!seems!possible!that!aversion!to!light!may!not!be!
secondary!to!headache,!although!we!cannot!rule!out!the!possibility!of!a!biphasic!
(early!and!delayed)!light!aversive!response!in!mice.!Also,!while!migraine!is!more!
prevalent!in!women,!we!did!not!detect!a!significant!difference!in!the!time!spent!in!
! 37!
light!between!male!and!female!mice.!However,!there!was!a!trend!towards!CD1!
female!mice!spending!less!time!in!the!light!after!0.5!mg/kg!CGRP.!Future!tests!
during!the!estrous!cycle!may!reveal!sex!differences!during!CGRPKinduced!light!
aversion.!!
The!efficacy!of!triptans!to!attenuate!both!central!130!and!peripheral!CGRPK
induced!light!aversion!and!motility!in!mice!is!consistent!with!the!ability!of!triptans!
to!reduce!CGRPKinduced!migraine!in!humans!79.!Triptans!are!a!family!of!antiK
migraine!drugs!that!activate!5KHT1B/D!receptors!that!induce!vasoconstriction!
and!inhibit!the!release!of!CGRP!and!other!neuropeptides!from!nociceptors!161,!
including!possibly!inhibition!of!CGRP!release!by!trigeminal!afferents!in!this!study.!
Though!studied!for!over!20!years,!the!sites!of!action!of!triptans!remain!
controversial!166,!but!the!similar!efficacy!of!various!triptans!irrespective!of!their!
brain!penetrance!supports!the!importance!of!a!peripheral!site!of!action!unless!
there!is!bloodKbrain!barrier!breakdown,!which!has!yet!to!be!demonstrated!167.!In!
addition,!the!ability!of!sumatriptan!to!attenuate!the!effects!of!CGRP!on!both!light!
aversion!and!motility!suggests!these!responses!may!be!mediated!by!overlapping!
mechanisms.!This!speculation!is!consistent!with!both!light!aversion!and!motility!
being!more!pronounced!in!CD1!mice!and!the!ability!of!the!CGRP!antibody!to!
attenuate!all!these!responses.!!
Along!the!same!lines,!the!ability!of!a!CGRPKblocking!antibody!to!block!
light!aversion!in!mice!also!supports!a!peripheral!site!of!CGRP!action!in!migraine.!
Several!pharmaceutical!companies!have!engineered!humanized!monoclonal!
antibodies!against!CGRP!and!its!receptor!to!prophylactically!prevent!migraine.!
! 38!
Humanized!antibodies!are!preferred!to!avoid!immunogenicityh!furthermore,!
antibodies!also!have!a!prolonged!halfKlife!and!avoid!offKtarget!liver!toxicity!that!
has!hindered!the!development!of!small!molecule!receptor!antagonists!to!date!
101,150.!Currently,!all!phase!II!clinical!trials!with!CGRPKblocking!and!receptorK
blocking!antibodies!have!successfully!reduced!the!number!of!migraine!attacks!
145K149.!The!success!of!antibodies!suggests!a!peripheral!site!of!actionh!moreover,!
in!one!recent!report,!only!about!0.1%!of!one!of!the!antiKCGRP!antibodies!could!
be!detected!in!the!CNS!168.!Nonetheless,!the!relevant!sites!of!antibody!action!
remain!unknown.!The!mechanism!and!site!of!action!will!be!particularly!intriguing!
given!reports!that!a!single!injection!of!one!of!the!CGRPKblocking!antibodies!can!
prevent!migraine!attacks!for!at!least!several!months!147.!This!suggests!a!longK
lasting!desensitization!of!CGRP!actions!in!migraine.!Future!studies!with!
preclinical!mouse!models!should!be!able!to!address!the!mechanism!by!which!
inhibiting!CGRP!actions!can!prevent!migraine.!
The!unexpected!inability!of!peripheral!CGRP!to!induce!aversion!to!dim!
light!in!nestin/hRAMP1!mice!suggests!that!peripheral!CGRP!causes!light!
aversion!by!(1)!a!mechanism!that!is!most!likely!distinct!from!central!CGRP!and!
(2)!a!nonKneuronal!mechanism.!However,!we!cannot!exclude!the!alternative!
possibilities!that!the!lack!of!response!of!nestin/hRAMP1!mice!to!dim!light!is!due!
to!dim!light!aversion!being!an!exclusive!feature!of!CGRP!CNS!signaling!or!that!
aversion!to!dim!and!strong!lights!is!mediated!by!different!mechanisms.!In!either!
case,!in!the!CNS,!the!most!likely!mechanism!would!seem!to!involve!CGRP’s!role!
as!a!neuromodulator,!by!which!it!can!increase!synaptic!transmission!169,170.!For!
! 39!
example,!CGRP!has!been!reported!to!increase!both!NMDA!and!AMPA!receptor!
activity!171.!In!the!periphery,!a!modulatory!role!on!peripheral!neurons!also!seems!
likely,!but!by!an!indirect!mechanism.!An!indirect!mechanism!in!the!meninges!
would!be!consistent!with!studies!that!have!failed!to!identify!CLR/RAMP1!CGRP!
receptors!on!afferent!trigeminal!fibers!114!and!the!inability!of!direct!application!of!
CGRP!or!antagonists!onto!afferent!trigeminal!fibers!in!the!meninges!to!activate!
or!inhibit!nerve!signaling!to!the!spinal!cord!108,153,154.!However,!another!study!did!
find!CLR!and!RAMP1!immunoreactivity!on!trigeminal!AKfibers!in!the!dura!mater!
89,!and!the!possibility!of!CGRP!actions!within!the!trigeminal!ganglia!at!either!
CLR/RAMP1!or!CTR/RAMP1!receptors!on!neuronal!cell!bodies!88,89!must!be!
considered.!In!this!regard,!the!possibility!of!CTR/RAMP1!receptors!on!afferent!
fibers!has!not!been!examined!yet.!In!these!scenarios,!CGRP!could!sensitize!
trigeminal!nociceptors!by!autocrine!or!paracrine!mechanisms!via!CTR/RAMP1!or!
CLR/RAMP1!receptors,!although!the!lack!of!sensitization!in!the!nestin/hRAMP1!
mice!suggests!that!neuronal!CGRP!receptors!are!not!rateKlimiting!for!peripheral!
CGRP!induction!of!light!aversion!in!mice.!Hence,!our!results!suggest!that!CGRP!
actions!are!transmitted!to!the!CNS!via!indirect!sensitization!of!the!peripheral!
nerves.!!
If!CGRP!is!not!acting!directly!on!peripheral!neurons,!the!question!remains!
where!else!might!CGRP!act!outside!the!CNS.!The!neurovascular!model!of!
migraine,!articulated!over!20!years!ago!172,!suggests!that!vascular!inflammatory!
signals!in!the!meninges!lead!to!sensitization!and!activation!of!trigeminal!
nociceptors.!Within!the!trigeminovascular!system,!there!are!CGRP!receptors!on!
! 40!
satellite!glia!in!the!ganglia!88,114,!vascular!smooth!muscle!(Eftekhari!et!al.,!2013),!
possibly!endothelial!cells!87,!and!dural!mast!cells!114,!although!reportedly!not!on!
human!mast!cells!89.!While!the!weight!of!evidence!argues!against!a!causal!link!
between!vasodilation!and!migraine!33,!a!nonKvasodilatory!role!for!the!vasculature!
should!not!be!ruled!out!173.!It!is!possible!that!CGRP!actions!on!vessels!could!
sensitize!peripheral!nociceptors!by!release!of!nitric!oxide!from!endothelial!cells!
along!with!cytokines!and!inflammatory!agents!from!nearby!mast!cells!36,101,174.!
Such!a!mechanism!on!afferent!fibers!could!act!in!concert!with!CGRP!actions!in!
the!trigeminal!ganglia!that!could!also!sensitize!nociceptors,!for!example!by!
cytokine!release!from!satellite!glia.!In!all!these!mechanisms,!a!common!
prediction!is!that!peripheral!CGRP!acts!to!alter!the!microenvironment!of!the!
trigeminovascular!system.!Future!genetic!dissection!using!the!conditional!
transgenic!hRAMP1!expression!system!should!help!reveal!how!these,!still!poorly!
understood!mechanisms,!contribute!to!migraine.!
!
!
!
!
!
!
!
!
!"
! 41!
"""
"""Figure.!II.1.!CGRP!induces!light!aversion!in!CD1!mice.!Time!spent!in!the!light!zone!by!CD1!mice!during!sequential!exposures!in!the!lightKdark!chamber!at!2.7x104!lux!(left!panel).!Mice!were!preKexposed!to!the!chamber!twice!(Pre1,!Pre2)!at!3!day!intervals!to!reduce!exploratory!drive,!then!treated!with!vehicle!(Veh,!n=19)!or!0.1!mg/kg!CGRP!(n=19)!on!treatment!day!(Tx),!followed!by!a!postKtreatment!(Post)!measurement.!The!mean!±!SEM!is!shown,!with!significance!indicated!for!comparisons!of!vehicle!to!CGRP!at!each!time!point!and!comparisons!of!Tx!to!Pre2!and!Post!indicated!by!brackets,!*p<0.05,!**p<0.01,!***p<0.001,!****p<0.0001.!Right!panel!shows!the!mean!time!(±!SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day,!****p<0.0001.!Previously!published.!
! 42!
"!!
!!!Figure!II.2.!CGRP!induces!light!aversion!in!C57BL/6J!mice.!Time!spent!in!the!light!zone!by!C57BL/6J!mice!at!2.7x104!lux!(left!panel).!Mice!were!treated!as!described!in!Fig.!II.1!with!vehicle!(Veh,!n=42)!or!0.1!mg/kg!CGRP!(n=44).!Right!panel!shows!the!mean!time!(±!SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day,!****p<0.0001.!Previously!published.!!!!!!!
! 43!
!!!
!!!!!!!!!
! 44!
Figure!II.3.!Comparison!of!CGRPKinduced!light!aversion!in!wildKtype!mice.!DoseKdependent!effect!of!CGRP!on!time!in!light!in!CD1!and!C57BL/6J!(C57)!mice!(left!panel).!Mice!were!treated!with!vehicle!(CD1,!n=29,!5!experimentsh!C57BL/6J,!n=83,!8!experiments),!0.1!mg/kg!CGRP!(CD1,!n=19,!2!experimentsh!C57BL/6J,!n=44,!4!experiments),!0.5!mg/kg!CGRP!(CD1,!n=28,!3!experimentsh!C57BL/6J,!n=51,!4!experiments).!CGRP!treated!C57BL/6J!and!CD1!mice!spent!significantly!less!time!in!the!light!at!0.1!mg/kg!and!0.5!mg/kg!compared!to!vehicle!(****p<0.0001).!Right!panel!shows!the!mean!time!(±!SEM)!in!light!per!5!min!interval!for!individual!CD1!and!C57BL/6J!mice!on!treatment!day.!CD1!mice!spent!significantly!less!time!in!the!light!compared!to!C57BL/6J!mice!at!both!0.1!mg/kg!CGRP!(***p<0.001)!and!0.5!mg/kg!CGRP!(****p<0.0001),!and!were!not!significant!(ns)!with!vehicle.!!!!!!!!!!!!!!!!).!!!!!!!!!!!!!!!!!!!
! 45!
!!!
!!!Figure.!II.4.!Gender!effects!of!0.1mg/kg!CGRPKinduced!light!aversion!in!CD1!mice.!Data!from!Fig.!II.1!was!analyzed!by!sex!in!sequential!5!min!intervals!(top!panel)!as!well!as!average!time!spent!in!the!light!per!5min!interval!(bottom!panel!!!!!!!
! 46!
!!!
!!!Figure.!II.5.!Gender!effects!of!0.5!mg/kg!CGRPKinduced!light!aversion!in!CD1!mice.!Data!from!Fig.!II.3!was!analyzed!by!sex!in!sequential!5!min!intervals!(top!panel)!as!well!as!average!time!spent!in!the!light!per!5min!interval!(bottom!panel).!!!!!
! 47!
!!!!!
! 48!
Figure.!II.6.!Degree!of!CGRPKinduced!light!aversive!response!by!gender!in!CD1!mice.!Data!from!Fig.!II.1!and!II.3!was!analyzed!by!comparing!the!time!spent!in!light!following!at!baseline!to!the!time!spent!in!light!following!CGRP!treatment!at!0.1mg/kg!(top!panel)!and!0.5mg/kg!(bottom!panel).!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
! 49!
!!!
!!!Figure.!II.7.!Gender!effects!of!0.1!mg/kg!CGRPKinduce!light!aversion!in!C57BL/6J!mice.!Data!from!Fig.!II.1!was!analyzed!by!sex!in!sequential!5!min!intervals!(top!panel)!as!well!as!average!time!spent!in!the!light!per!5min!interval!(bottom!panel).!!!!!!!
! 50!
!!!
!!!Figure.!II.8.!Gender!effects!of!0.5!mg/kg!CGRPKinduced!light!aversion!in!C57BL/6J!mice.!Data!from!Fig.!II.3!was!analyzed!by!sex!in!sequential!5!min!intervals!(top!panel)!as!well!as!average!time!spent!in!the!light!per!5min!interval!(bottom!panel).!!!!!!
! 51!
!!!
!
! 52!
Figure.!II.9.!Degree!of!CGRPKinduced!light!aversive!response!by!gender!in!C57BL/6J!mice.!Data!from!Fig.!II.1!and!II.3!was!analyzed!by!comparing!the!time!spent!in!light!following!at!baseline!to!the!time!spent!in!light!following!CGRP!treatment!at!0.1mg/kg!(top!panel)!and!0.5mg/kg!(bottom!panel).!For!all!panels,!the!mean!±!SEM!is!shown,!*p<0.05,!**p<0.01,!***p<0.001,!****p<0.0001.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
! 53!
!!!
!!!!!!
! 54!
Figure.!II.10.!Peripheral!CGRP!increases!resting!behavior!in!the!dark.!Resting!time!in!light!and!dark!zones.!After!IP!injection,!CGRPKtreated!mice!(CD1,!n=19h!C57BL/6J,!n=44)!spent!significantly!more!time!resting!in!the!dark!zone!compared!to!vehicleKtreated!mice!(CD1,!n=19h!C57BL/6J,!n=42),!and!compared!to!preKtreatment!2!(Pre2)!and!postKtreatment!(Post)!periods.!There!were!no!significant!differences!in!the!light!zone.!For!all!panels,!the!mean!±!SEM!is!shown,!*p<0.05,!**p<0.01,!***p<0.001,!****p<0.0001.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
! 55!
!!!
!!!!
! 56!
Figure.!II.11.!Peripheral!CGRP!reduces!rearing!behavior.!Number!of!vertical!beam!breaks!per!min!in!light!and!dark!zones.!For!both!strains,!CGRP!treatment!decreased!the!number!of!vertical!beam!breaks!in!the!dark!and!to!a!lesser!degree!in!the!light.!For!all!panels,!the!mean!±!SEM!is!shown,!*p<0.05,!**p<0.01,!***p<0.001,!****p<0.0001.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
! 57!
!!!
!!!!!!
! 58!
Figure.!II.12.!Peripheral!CGRP!decreases!transitions!between!the!light!and!the!dark!zone.!Number!of!transitions!between!the!light!and!dark!zones.!For!both!strains!CGRP!decreased!transitions!between!zones,!although!to!a!greater!degree!with!CD1!mice.!For!all!panels,!the!mean!±!SEM!is!shown,!*p<0.05,!**p<0.01,!***p<0.001,!****p<0.0001.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
! 59!
!!!
!!!Figure!II.13.!Central!injection!of!CGRP!through!a!cannula!elicits!light!aversion.!A.!Time!spent!in!the!light!zone!by!C57BL/6J!mice!at!2.7x104!lux!(left!panel).!!The!mice!were!preKexposed!to!the!chamber!twice!(Pre1,!Pre2),!then!treated!with!vehicle!(Veh,!n=11)!or!CGRP!(2!�g/mouse,!ICV,!n=13)!on!treatment!day!(Tx),!followed!by!a!postKtreatment!(Post)!measurement.!After!ICV!injection,!CGRPKtreated!mice!spent!less!time!in!light!compared!with!vehicle!(*p<0.05,!***p<0.001)!and!compared!to!Pre!2!and!Post!as!indicated!by!brackets!(****p<0.0001).!Right!panel!shows!the!mean!time!(±!SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day,!**p<0.01.!!!
! 60!
!!!Figure!II.14.!Central!injection!of!CGRP!through!a!cannula!induces!an!increase!in!resting!behavior.!Resting!time!in!light!and!dark!zones!measured!at!the!same!time!as!light!aversion!with!mice!shown!in!panel!A.!CGRPKtreated!mice!spent!significantly!more!time!resting!in!the!dark!compared!to!vehicleKtreated!mice!and!Pre2!and!Post!periods!(mean!±!SEM,!**p<0.01,!***p<0.001,!****p<0.0001).!!!!!!!!!!!!!!!!!!!!!
! 61!
!!!
!!!Figure!II.15.!Peripheral!CGRP!does!not!induce!anxiety!in!the!open!field!assay!in!CD1!mice.!Time!spent!in!the!center!of!the!open!field!after!IP!injection!of!vehicle!(n=10)!or!CGRP!(0.1!mg/kg,!n=10)!or!in!CD1!mice!over!the!30!min!testing!period!(left!panel).!Right!panel!shows!the!mean!percent!time!(±!SEM)!in!center!per!5!min!interval!for!individual!mice.!
! 62!
!!!
!!!Figure!II.16.!Peripheral!CGRP!does!not!induce!anxiety!in!the!open!field!assay!in!C57BL/6J!mice.!Time!spent!in!the!center!of!the!open!field!after!IP!injection!of!vehicle!(n=9)!or!CGRP!(0.1!mg/kg,!n=9)!or!in!C57BL/6J!mice!over!the!30!min!testing!period!(left!panel).!Right!panel!shows!the!mean!percent!time!(±!SEM)!in!center!per!5!min!interval!for!individual!mice!on!treatment!da!
! 63!
A
!!!!!!
B!
!!!!!!!!!!!!!
! 64!
Figure!II.!17.!Sumatriptan!attenuates!light!aversion!induced!by!peripheral!CGRP!in!CD1!mice.!A.!Time!spent!in!light!zone!by!CD1!mice!at!2.7x104!lux.!Mice!were!injected!IP!with!vehicle!(Veh,!n=19),!CGRP!(0.1!mg/kg,!n=22),!sumatriptan!(Suma,!0.6!mg/kg,!n=19),!or!CGRP!+!sumatriptan!(0.1!mg/kg!CGRP,!0.6!mg/kg!Suma,!n=23).!Data!are!from!2!independent!experiments.!B.!The!mean!time!(±!SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day.!For!both!panels!A!and!B,!mean!±!SEM!is!shown,!with!significance!indicated!for!CGRP!compared!to!vehicle!as!#p<0.05,!##p<0.01,!###p<0.001,!####p<0.0001h!CGRP!compared!to!Suma!as!^p<0.05,!^^p<0.01,!^^^^p<0.0001h!CGRP!compared!to!CGRP+Suma!as!*p<0.05,!**p<0.01.!Time!spent!in!the!light!between!treatment!days!with!significance!is!indicated!for!CGRP!(*)!and!CGRP+Suma!(#).!!!!!!!!!!!!!!!!!!!!!!
! 65!
A
!!!B!
!!!!!!!!!!!!
! 66!
Figure!II.18.!Sumatriptan!partially!attenuates!light!aversion!induced!by!peripheral!CGRP!in!C57BL/6J!mice.!A.!Time!spent!in!light!zone!by!C57BL/6J!mice!at!2.7X104!lux!(left!panel).!Mice!were!injected!IP!with!either!vehicle!(n=20),!CGRP!(0.1!mg/kg,!n=21),!Suma!(0.6!mg/kg,!n=19),!or!CGRP+Suma!(0.1!mg/kg!CGRP,!0.6!mg/kg!sumatriptan,!n=21).!Data!are!from!2!independent!experiments,!although!the!postKexposure!is!from!only!1!experiment.!B.!The!mean!time!(±!SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day.!For!both!panels!A!and!B,!mean!±!SEM!is!shown,!with!significance!indicated!for!CGRP!compared!to!vehicle!as!#p<0.05,!##p<0.01,!###p<0.001,!####p<0.0001h!CGRP!compared!to!Suma!as!^p<0.05,!^^p<0.01,!^^^^p<0.0001h!CGRP!compared!to!CGRP+Suma!as!*p<0.05,!**p<0.01.!Time!spent!in!the!light!between!treatment!days!with!significance!is!indicated!for!CGRP!(*)!and!CGRP+Suma!(#).!!!!!!!!!!!!!!!!!
! 67!
!!!
!!
! 68!
Figure!II.19.!Sumatriptan!attenuates!CGRPKinduced!resting!behavior.!Resting!time!was!measured!concurrently!with!light!aversion!for!the!same!mice!in!Fig.!II.17!and!II.18.!CGRPKtreated!CD1!(panel!A)!and!C57!(panel!B)!mice!spent!significantly!more!time!(mean!±!SEM)!resting!in!the!dark!compared!to!mice!treated!with!vehicle,!Suma,!or!CGRP+Suma.!For!both!panels!A!and!B,!mean!±!SEM!is!shown,!with!significance!indicated!for!CGRP!compared!to!vehicle!as!#p<0.05,!##p<0.01,!###p<0.001,!####p<0.0001h!CGRP!compared!to!Suma!as!^p<0.05,!^^p<0.01,!^^^^p<0.0001h!CGRP!compared!to!CGRP+Suma!as!*p<0.05,!**p<0.01.!Time!spent!in!the!light!between!treatment!days!with!significance!is!indicated!for!CGRP!(*)!and!CGRP+Suma!(#).!!!!!!!!!!!!!!!!!!!!!!!!!!
! 69!
!!!!
!!!!!!!!!!!!!!
! 70!
Figure!II.20.!PreKtreatment!with!a!monoclonal!CGRP!antibody!prevents!light!aversion!induced!by!peripheral!CGRP!in!CD1!mice.!"Time!spent!in!the!light!zone!during!sequential!exposures!in!the!lightKdark!chamber!at!2.7x104!lux!(left!panel).!Mice!were!preKexposed!to!the!chamber!twice!(Pre1,!Pre2)!at!3!day!intervals!to!reduce!exploratory!drive,!prior!to!the!first!treatment!(Tx1)!with!an!IP!injection!of!vehicle!(n=11)!or!CGRP!(0.1!mg/kg,!two!random!cohorts:!CGRP(1)!n=14,!CGRP(2)!n=14).!CGRPKtreated!mice!spent!less!time!in!light!compared!to!vehicle!(for!CGRP(1)!*p<0.05,!**p<0.01,!***p<0.001,!****p<0.0001h!CGRP(2)!#p<0.05,!###p<0.001,!####p<0.0001)!and!compared!to!Pre2!and!postKtreatment!(Post)!indicated!by!brackets,!***p<0.001,!###p<0.001,!####p<0.0001.!Four!days!after!postKtreatment,!antibody!treatment!(Ab!Tx)!was!done!by!IP!injection!of!control!antibody!(Con!Ab)!or!CGRPKblocking!antibody!(CGRP!Ab).!The!following!day,!mice!were!injected!with!IP!vehicle!or!CGRP!(0.1!mg/kg).!Mice!that!received!CGRP!following!Con!Ab!(CGRP!(2)!+Con!Ab,!n=14)!spent!less!time!in!the!light!compared!to!CGRP!following!CGRP!Ab!(CGRP!(1)+CGRP!Ab,!n=14),!*p<0.05,!**p<0.01,!and!compared!to!vehicle!following!CGRP!antibody!(Veh+CGRP!Ab,!n=11),!#p<0.05.!Data!are!from!2!independent!experiments!(top!panel).!The!mean!time!(±!SEM)!in!light!per!5!min!interval!for!individual!mice!during!the!first!treatment!(Tx1)!and!second!treatment!(Ab!Tx)!following!injection!of!Con!Ab!or!CGRP!Ab!is!shown!(bottom!panel).!!
! 71!
!!!!!!!!!!!!
! 72!
Figure!II.21.!PreKtreatment!with!monoclonal!CGRP!antibody!attenuates!CGRPKinduced!reduction!in!motor!activity.!A.!Resting!time!was!measured!concurrently!with!light!aversion!for!the!same!mice!in!Fig.!II.20.!CGRPKtreated!mice!and!CGRP(2)+Con!Ab!mice!spent!more!time!(mean!±!SEM)!resting!in!the!dark!during!Tx1!and!Ab!Tx,!respectively!(CGRP(1)!*p<0.05,!**p<0.01,!***p<0.001,!****p<0.0001h!CGRP(2)!#p<0.05,!####p<0.0001).!B."Number!of!vertical!beam!breaks!per!min!in!light!and!dark!zones.!CGRPKtreated!mice!and!CGRP+Con!Ab!had!significantly!less!vertical!beam!breaks!in!the!dark!during!Tx1!(CGRP(1)!**p<0.01,!***p<0.001h!CGRP(2)!#p<0.05,!##p<0.01,!!####p<0.0001)!and!Ab!Tx!compared!to!Veh+CGRP!(*p<0.05)!and!compared!to!CGRP(1)+CGRP!Ab!(#p<0.05).!!!!!!!!!!!!!!!
! 73!
!!!Figure!II.22.!Generation!schematic!and!gross!GFP!expression!profile!in!nestin/hRAMP1!double!transgenic!mice.!A.!Schematic!of!the!GFPKhRAMP1!expression!vector.!The!loxP!sites!that!flank!the!GFP!reporter!allow!for!CreKmediated!excision!of!GFP!and!associated!stop!sequences!when!the!parental!mice!are!crossed!with!mice!expressing!Cre!recombinase.!B.!Following!mating!with!nestinKcre!mice,!GFP!expression!was!observed!in!the!aorta!and!heart,!but!not!brain!or!trigeminal!ganglia!of!nestin/hRAMP1!double!transgenic!(R+C+)!mice,!indicating!nestinKdriven!Cre!removal!of!the!GFP!stop!sequence!in!the!brain!and!ganglia,!but!not!the!aorta!and!heart.!As!controls,!GFP!was!detected!in!both!tissues!of!single!transgenic!mice!containing!only!the!parental!GFPKhRAMP1!transgene!(R+CK)!and!not!in!single!transgenic!mice!containing!only!the!nestinKcre!transgene!(RK/C+).!!!!!!!!
! 74!
!!!Figure!II.23.!RAMP1!expression!in!nestin/hRAMP1!transgenic!mice.!!QKPCR!measurement!of!RAMP1!levels!in!nestin/hRAMP1!(R+C+)!mice!and!control!littermates!(both!R+/CK!and!RKC+).!The!copies!of!mouse!and!human!RAMP1!RNAs!were!calculated!from!standard!curves!and!normalized!to!100,000!copies!of!"Kactin!mRNAh!n=6!for!each!tissue!per!genotype.!!!!!!!!!!!!!!!!!!!
! 75!
!!!
!!!!!!!!
! 76!
Figure!II.24.!Peripheral!CGRP!does!not!induce!enhanced!light!aversion!in!CGRPKsensitized!nestin/hRAMP1!transgenic!mice.!Time!spent!in!light!by!naïve!mice!(no!preKexposure!to!the!lightKdark!chamber)!at!55!lux!after!IP!injection!of!vehicle!or!CGRP!(0.1!mg/kg)!(left!panel).!Nestin/hRAMP1!mice!treated!with!CGRP!(hRAMP1/CGRP,!n=29)!had!no!significant!reduction!in!time!spent!in!light!compared!to!vehicle!(hRAMP1/Veh,!n=28)!or!control!littermates!treated!with!vehicle!(Control/Veh,!n=29),!or!with!CGRP!(Control/CGRP,!n=30).!Data!are!from!3!independent!experiments.!Right!panel!shows!the!mean!time!(±!SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day,!not!significant!(ns).!!!!!!!!!!!!!!!!!!!!!!!!!
! 77!
!!!!
!!!!!!!
! 78!
Figure!II.25.!Central!CGRP!induces!enhanced!light!aversion!in!CGRPKsensitized!nestin/hRAMP1!transgenic!mice.!Time!spent!in!light!by!naïve!mice!at!55!lux!after!ICV!injection!of!vehicle!or!CGRP!(2!�g/mouse)!(left!panel).!Nestin/hRAMP1!mice!treated!with!CGRP!(hRAMP1/CGRP,!n=30)!spent!less!time!in!the!light!compared!to!vehicle!(hRAMP1/Veh,!n=16)!or!control!littermates!treated!with!vehicle!(Control/vehicle,!n=15)!or!with!CGRP!(Control/CGRP,!n=29),!***!p<0.001,!****!p<0.0001.!Data!are!from!3!independent!experiments.!Right!panel!shows!the!mean!time!(±!SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day,!****!p<0.0001.""""""""""""""""""""""""
! 79!
""""
!!!!!!!!!
! 80!
Figure!II.26.!Peripheral!CGRP!induced!light!aversion!using!wildKtype!testing!paradigm!in!nestin/hRAMP1!mice.!Time!spent!in!light!by!mice!at!27,000!lux!(bright!light)!after!IP!injection!of!vehicle!or!CGRP!(0.1!mg/kg)!(left!panel).!Nestin/hRAMP1(mice!that!had!previously!been!tested!at!55!lux!in!the!chamber!(panel!A)!were!reKtested!in!bright!light.!The!mice!were!treated!with!CGRP!(hRAMP1/CGRP,!n=11)!spent!less!time!in!the!light!compared!to!vehicle!(hRAMP1/Veh,!n=11),!***!p<0.001,!****!p<0.0001.!Control!littermates!treated!with!CGRP!(Con/CGRP,!n=12)!spent!less!time!in!the!light!compared!to!vehicle!(Con/Veh,!n=12),!#p<0.05.!Right!panel!shows!the!mean!time!(±!SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day,!hRAMP1/CGRP!compared!to!hRAMP1/Veh,!****!p<0.0001h!Con/CGRP!compared!to!Con/Veh,!#p<0.05.!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
! 81!
CHAPTER!III!
VASCULAR!CONTRIBUTIONS!OF!PERIPHERAL!CGRP!IN!MIGRAINEKLIKE!
PHOTOPHOBIA!IN!MICE!
ABSTRACt!
CGRP!is!now!recognized!as!a!key!player!in!the!pathogenesis!of!migraine,!yet!the!
mechanisms!and!sites!of!CGRP!actions!remain!unknown.!CGRP!is!known!to!act!
directly!on!neurons!as!a!neuromodulator,!yet!it!is!also!the!most!potent!
vasodilatory!peptide!with!receptors!on!vascular!smooth!muscle.!While!the!role!of!
the!vasculature!in!migraine!has!been!questioned!over!the!past!20!years,!the!
recent!efficacy!of!CGRPKblocking!antibodies!as!preventative!migraine!drugs!has!
reignited!the!debate!regarding!CGRP!actions!on!the!vasculature.!The!goal!of!this!
study!was!to!test!the!role!of!vasodilation!in!peripheral!CGRPKinduced!light!
aversive!behavior!of!CD1!mice!by!coKadministering!a!vasoconstrictive!dose!of!
phenylephrine!or!endothelinK1!to!systemically!blunt!vasodilation!caused!by!
CGRP.!We!found!that!prevention!of!CGRPKinduced!vasodilation!partially!
attenuated!the!light!aversive!response.!However,!light!aversion!was!apparently!
not!simply!due!to!vasodilation!since!blunting!dilatory!response!to!CGRP!did!not!
completely!block!light!aversion.!Differential!responses!in!the!light!aversion!assay!
following!administration!of!PACAP!and!VIP!suggest!that!the!length!of!
vasodilation,!rather!than!the!degree!of!vasodilation,!may!be!important!in!this!
migraine!model.!Additionally,!this!may!also!highlight!the!importance!of!CGRPK
receptor!activation.!To!begin!to!identify!the!site(s)!of!CGRP!action,!we!
! 82!
overexpressed!the!rateKlimiting!subunit!of!CGRP!receptors,!human!receptor!
activityKmodifying!protein!1!(hRAMP1).!Transgenic!mice!were!engineered!to!
overexpress!hRAMP1!globally!in!all!tissues!and!selectively,!in!smooth!muscle!
and!endothelial!cells,!including!the!vasculature.!Both!the!global!and!smooth!
muscle!hRAMP1,!but!not!endothelial!hRAMP1,!mice!displayed!a!sensitized!
CGRP!light!aversive!phenotype!in!dim!light.!!This!is!in!contrast!to!our!previously!
reported!nervous!system!hRAMP1!mice!that!do!not!exhibit!enhanced!light!
aversion!following!peripheral!CGRP.!Together,!these!results!are!consistent!with!
a!vascular!site!of!peripheral!CGRP!action.!We!propose!that!peripheral!and!
central!CGRP!actions!can!intersect!at!pial!and!parenchymal!vessels,!with!local!
vasodilation!activating!central!neurons,!which!in!turn!release!CGRP!to!modulate!
neural!activity!and!further!dilate!vessels!in!a!positive!feedback!mechanism.(
!
INTRODUCTION!
Over!the!past!several!decades,!the!role!of!vasodilation!in!the!genesis!of!
migraine!pain!has!had!its!share!of!considerable!controversy.!Towards!the!end!of!
the!17th!century!the!first!theory!that!migraine!pain!was!due!to!vessel!relaxation!
was!articulated!by!Thomas!Willis.!However,!the!growing!view!is!that!migraine!is!
largely!a!neural!disorder!and!not!a!vascular!disorder33,131,175.!Indeed,!it!is!
suggested!that!vasodilation!is!an!epiphenomenon!and!is!not!important!in!the!
genesis!of!migraine!pain!121.!Conversely,!other!studies!speculate!that!
vasodilation!may,!in!fact,!contribute!to!the!origin!of!migraine!pain!32,84.!Now,!
studies!have!considered!the!possibility!of!migraine!being!both!a!vascular!and!
! 83!
neural!disorder!giving!rise!to!the!neurovascular!model!of!migraine!173,176,177.!!This!
model,!articulated!about!25!years!ago,!suggests!that!an!inflammatory!event!
happens!at!the!meningeal!vasculature!which!leads!to!an!altered!neural!state!that!
is!the!result!of!sensitization!and!activation!of!nearby!nociceptors.!Even!though!
there!is!intense!debate!on!the!origin!of!migraine!pain,!all!can!agree!that!migraine!
does!involve!activation!of!the!trigeminal!vascular!system31,101,131,178.!!
! CalcitoninKgene!related!peptide!(CGRP)!is!a!37Kamino!acid,!vasodilatory!
neuropeptide!that!is!present!in!approximately!50!%!of!trigeminal!neurons!49,179K
181.!Additionally,!the!vascular!smooth!muscle!of!cerebral!and!extracranial!
vasculature!are!heavily!invested!with!CGRP!receptors!182.!Peripheral!blockade!of!
CGRP!receptor!activation!using!CGRPKreceptor!antagonists!and!monoclonal!
antibodies!have!been!successful!in!the!amelioration!of!migraine!pain!and!number!
of!attack!days!and!strongly!supports!a!peripheral!site!of!action!150,151,183K185.!
Nevertheless,!reports!suggest!CGRP!also!has!central!actions!that!contribute!to!
migraine!symptoms.!For!example,!our!group!along!with!other!studies!have!
shown!that!CGRP!has!central!targets!that!may!contribute!to!
migraine100,119,128,156,186K189.!Hence,!we!have!reasoned!that!peripheral!CGRP!
actions!on!the!vasculature!are!sufficient!to!induce!migraineKlike!photophobia!in!
this!model.!Thus,!possibly!modulating!neural!activity!in!the!CNS!via!indirect!
sensitization!of!peripheral!nerves.!!
! To!address!whether!peripheral!CGRP!has!vasculature!actions!to!induce!
migraineKlike!symptoms,!we!have!used!a!mouse!model!of!photophobia128.!
Photophobia!is!a!common,!often!debilitating,!aberrant!sensitivity!to!nonKpainful!
! 84!
levels!of!light.!We!have!previously!shown!that!central!and!peripheral!
administration!of!CGRP!is!sufficient!to!induce!light!aversion,!a!surrogate!for!
photophobia,!in!wildKtype!mice!when!exposed!to!bright!light128,130.!Moreover,!
central,!but!not!peripheral,!administration!of!CGRP!can!induce!enhanced!light!
aversion,!with!exposure!to!dim!light,!in!CGRPKsensitized!mice!that!express!the!
humanized!receptor!activity!modifying!protein!1(RAMP1)!component!of!the!
CGRP!receptor!in!the!nervous!system128,129,156.!!These!data!suggested!that!
central!and!peripheral!CGRP!have!actions!distinct!from!each!other,!however,!
with!potentially!overlapping!mechanisms.!Therefore,!the!goal!of!this!study!was!to!
identify!possible!sites!of!action!of!peripheral!CGRP!in!light!aversive!behavior.!!
! In!this!study,!we!have!demonstrated!that!pharmacologically!inhibiting!
vasodilation!induced!by!peripheral!administration!of!CGRP!can!partially!attenuate!
light!aversion.!In!addition,!pituitary!adenylate!cyclase!activating!polypeptide!
(PACAP),!but!not!vasoactive!intestinal!peptide!(VIP),!two!potent!vasodilators,!
induce!different!effects!in!the!light!aversion!assay.!Furthermore,!peripheral!
administration!of!CGRP!in!mice!that!overexpress!the!RAMP1!subunit!in!smooth!
muscle!cells,!but!preliminarily!not!in!endothelial!cells,!is!sufficient!to!induce!
migraineKlike!photophobia.!These!data!suggest!that!CGRP!in!the!periphery,!by!
way!of!vascular!smooth!muscle,!may!indirectly!activate!neural!pathways!in!the!
periphery.!!
!
MATERIALS!AND!METHODS!
(
! 85!
Animals(
WildKtype!CD1!mice!were!used.!Equivalent!numbers!of!adult!male!and!
female!mice,!aged!10K20!weeks,!were!used!in!all!experiments.!The!transgenic!
global/hRAMP1!line!has!been!described!previously190.!The!parental!GFPK
hRAMP1!transgenic!mice!with!an!upstream!stop!sequence!and!the!GFP!reporter!
gene!flanked!by!loxP!sites!followed!by!the!hRAMP1!cDNA!have!been!described!
100.!!Hemizygous!GFPKhRAMP1!mice!(strain!28412/3)!were!crossed!with!mice!
expressing!cre!recombinase!under!the!control!of!the!ubiquitous!adenovirus!EIIa!
promoter!(B6.FVBKTg(EIIaKcre)C5379Lmgd/J,!Stock!No.!003724,!Jackson!
Laboratory)!to!produce!double!transgenic!EIIa/hRAMP1!mice.!The!resultant!
double!transgenic!mice!were!then!bred!with!nonKtransgenic!littermates!to!
generate!progeny!with!the!hRAMP1!transgene,!but!without!cre!recombinase.!
PCR!genotyping!using!primers!detecting!hRAMP1!and!cre!was!performed!as!
described!100.!The!tagln/hRAMP1!and!tek2/hRAMP1!mice!used!the!same!
conditional!overexpression!strategy!as!the!global!hRAMP1!mice.!DoubleK
transgenic!progeny!from!crosses!of!the!parental!CX1KGFPKhRAMP1!mice!and!
tek2Kcre!or!taglnKcre!were!used.!Mice!were!housed!in!groups!of!three!to!five!per!
cage,!unless!otherwise!indicated,!on!a!12!h!light!cycle!with!food!and!water!ad!
libitum.!All!behavioral!experiments!were!performed!between!8:00!A.M.!and!2:30!
P.M.!For!all!telemetry!experiments,!blood!pressure!was!measured!between!8AM!
and!11:30!for!each!recording.!For!all!experiments,!investigators!were!blinded!to!
genotype!and!drug!treatment.!Animal!procedures!were!approved!by!the!
! 86!
University!of!Iowa!Animal!Care!and!Use!Committee!and!performed!in!
accordance!with!the!standards!set!by!the!National!Institutes!of!Health.!!
!
Intraperitoneal(drug(administration(
All!drugs!that!required!dilution!were!prepared!with!Dulbecco!PBS!
(Hyclone)!as!the!vehicle.!The!amounts!injected!were!as!follows:!0.1mg/kg!rat!!K
CGRP!(SigmaK!Aldrich),!1mg/kg!phenylpherine!(PE),!2mg/kg!PE,!0.1mg/kg!
endothelinK1,!0.01mg/kg!endothelinK1,!0.3mg/kg!pituitary!adenylate!cyclaseK
activating!polypeptide!(PACAP),!0.1mg/kg!vasoactive!intestinal!peptide!(VIP),!
and!0.3mg/kg!VIP.!All!drugs!were!administered!at!10µl/g!bodyweight!with!a!30g!x!
0.5!needle.!All!injections!were!performed!by!B.N.M.!Animals!were!gently!held!but!
not!anesthetized!during!injection.!For!telemetry!experiments,!following!injection!
mice!were!placed!back!in!their!home!cage!and!blood!pressure!recordings!started!
immediately.!For!the!behavioral!experiments,!mice!were!allowed!to!recover!for!
30!min!in!their!home!cages!before!testing!based!as!previously!described128.!!
!
Light(aversion(and(motility(assays(
The!light/dark!boxes!were!made!out!of!Plexiglas!(27cm!x!27cm!x!20.3cm)!
and!composed!of!three!sets!of!16!beam!infrared!arrays.!The!testing!field!was!
dividing!into!equal!sized!zones!by!a!removable,!opaque!dark!insert.!The!insert!
had!five!sides!with!a!hinged!top,!no!floor,!and!a!small!mouseKsized!door.!The!
Plexiglas!allowed!for!the!infrared!beams!to!pass!through!the!chamber!without!
interruption.!This!allowed!for!the!infrared!beams!to!track!the!movement!of!the!
! 87!
animals!in!the!both!the!light!zone!and!the!dark!zone.!Each!testing!chamber!was!
located!inside!a!soundKproof!chamber!(56cm!x!38cm!x!38cm)!with!a!ventilation!
fan.!Data!were!collected!on!a!computer!that!had!the!Activity!Monitor!version!7.06!
was!used!for!recording!data!from!the!twelve!chambers128,130.!
CD1!mice!were!preKexposed!to!the!chamber!twice!every!3!days!before!
treatment!exposure!to!habituate!the!mice!to!the!environment.!After!preKexposure!
to!the!chambers,!mice!were!then!tested!following!drug!administration.!In!addition,!
the!wildKtype!mice!were!tested!in!the!light/dark!boxes!with!bright!light!(27,000!lux)!
For!transgenic!hRAMP1!mice!and!their!control!littermates,!mice!were!tested!
naïve!to!chamber,!no!prior!habituation!paradigm,!using!dim!light!(70!lux),!as!
previously!described128,130.!Data!were!collected!for!30!min!and!analyzed!as!
average!time!spent!on!each!side!of!the!chamber!per!5!min!interval.!!
Motor!activity!was!measured!to!assess!the!effects!of!the!administered!
drugs!by!several!different!parameters.!Motility!was!measured!in!concert!with!the!
light!aversion!assay!using!the!testing!chambers,!conditions,!and!software!
described!above.!Rearing,!or!vertical!beam!breaks,!were!measured!by!the!
animal!crossing!the!beam!at!the!height!of!7.3cm.!Resting!time!was!calculated!as!
the!percentage!of!time!spent!not!moving!or!breaking!any!new!beams.!Transitions!
were!calculated!as!the!number!of!times!the!animal!crossed!the!center!beam,!
which!is!where!the!chamber!transitions!from!the!light!to!the!dark!zone.!To!
account!for!variation!in!the!amount!of!time!mice!spent!in!each!zone,!data!were!
normalized!to!time!spent!in!the!dark!zone!and!the!light!zones.!!
!
! 88!
OpenMfield(Assay(
The!openKfield!assay!was!performed!to!assess!the!contribution!of!anxiety!
in!the!light!aversion!assay!following!drug!administration.!Thirty!minutes!following!
injection,!the!mice!were!placed!in!the!lightKdark!chamber!but!the!dark!inserts!
were!removed,!leaving!the!chamber!as!an!open!field.!Animals!were!placed!in!the!
center!of!the!open!field!and!tested!for!30!minutes.!The!periphery!was!defined!as!
a!4.22!cm!border!along!the!inside!of!the!testing!chamber,!leaving!an!18.56cm!x!
18.56cm!square!which!defined!the!center!of!the!chamber.!The!amount!of!time!
the!animal!spent!in!the!center!of!the!open!field!was!assed.!!
!
RNA(isolation(and(measurement(of(human(and(mouse(RAMP1(RNA(levels!
RNA!was!isolated!by!homogenizing!tissues!in!Trizol!(Ambion!Life!
Technologies)!followed!by!70%!ethanol!precipitation!and!purification!using!
RNeasy!Micro!columns!(Qiagen).!To!examine!the!quality!of!RNA!integrity,!RNA!
agarose!gels!were!run!and!18S!and!28S!bands!were!scrutinized!for!band!
intensity.!A!spectrophotometer!was!used!for!quantifying!the!amount!of!RNA!in!
the!sample!and!260/280!ratio!was!used!to!determine!the!purity!of!each!sample.!
For!reverse!transcription,!approximately!50ngK150ng!of!RNA!was!reversed!
transcribed!using!a!Takara!Clontech!Reverse!Transcript!kit.!!
Human!RAMP1!(hRAMP1)!gene!expression!was!measured!by!real!time!
quantitative!PCR!(QKPCR)!using!primer!sequences!previously!described!and!
validated!by!our!team!158.!The!mRNA!levels!of!both!hRAMP1!and!endogenous!
mouse!RAMP1!(mRAMP1)!was!determined!using!Power!SYBR!Green!PCR!
! 89!
Master!Mix!(Applied!Biosystems,!UK).!Reactions!were!performed!in!triplicate!
using!ABI!SDS!7900!HT!thermocycler.!!Analysis!was!performed!using!ABI!SDS!
v2.4!software.!The!mRAMP1!and!hRAMP1!Ct!values!were!converted!to!absolute!
copy!numbers!and!normalized!to!50,000!copies!of!"Kactin!using!standard!curves!
generated!with!1:10!serial!dilutions!of!pGEMKQmRAMP1,!pbsCX1KLELKhRAMP1,!
and!pGEMKQmbetaKactin!plasmids!in!10!ng/ml!yeast!tRNA.!Total!RAMP1!copies!
were!summed!and!results!reported!as!the!meanKfold!increase!relative!to!control!±!
SEM!from!5!pairs!of!control!and!hRAMP1!mice.!
!
Animal(surgery(and(blood(pressure(assessment(following(drug(administration(
! Mice!were!anesthesized!with!intraperitoneal!ketamine!(91!µg/g)!and!
xylazine!(9.1!µg/g)!for!implantation!of!a!radiotelemetry!probe!(PC10,!DSI)!that!
enabled!measurement!of!arterial!blood!pressure,!heart!rate!(HR)!and!locomotor!
activity!were!inserted!into!the!thoracic!aorta!through!the!left!common!carotid!
artery.!Each!animal!was!housed!in!individual!cages!in!the!Animal!Care!Facility,!
and!monitored!daily!to!ensure!animals!were!healthy.!Thereafter,!changes!in!MAP!
and!heart!rate!were!measured!for!approximately!2.5!hrs!at!a!sampling!rate!of!500!
Hz!every!20!seconds!using!the!DSI!Acquisition!software.!!
!
Statistical(analysis((
The!data!were!analyzed!between!treatment!groups!(e.g.!vehicle!vs!
CGRP)!within!each!exposure!to!the!chamber.!A!twoKway,!repeated!measures!
ANOVA!(factors:!treatment!and!observation!time)!was!used!with!a!Bonferroni!
! 90!
multiple!comparisons!test!to!compare!treatment!groups!at!each!interval.!A!oneK
way!ANOVA!repeated!measures!was!used!to!determine!if!overall!significant!
effects!were!observed!in!bar!graphs!with!individual!points.!Bonferroni!or!Tukey!
multiple!comparisons!test!was!used!as!the!post(hoc!analysis.!Data!are!reported!
as!mean!±!standard!error!of!the!mean!(SEM).!Data!were!analyzed!using!
Graphpad!Prism!software.!
!
RESULTS!
Blood(pressure(changes(and(light(aversion(following(administration(CGRP(and(
vasoconstrictors(PE((
To!begin!to!address!the!question!whether!vasodilation!plays!a!role!in!
CGRPKinduced!light!aversion,!we!wanted!to!start!by!blocking!the!vasodilatory!
effects!of!CGRP!pharmacologically!by!using!vasoconstrictors.!We!used!arterial!
blood!pressure!as!a!readout!for!changes!in!vascular!tone191.!First,!we!used!the!
potent!selective!!1Kadrenergic!receptor!agonist!phenylephrine!to!minimize!the!
vasodilation!induced!by!CGRP.!CD1!mice!were!implanted!with!radiotelemeters!
and!allowed!to!recover!for!at!least!one!week.!Baseline!was!recorded!for!30!min!
afterwards!mice!were!administered!intraperitoneal!CGRP,!vehicle,!phenylephrine!
(PE),!or!coKadministration!of!CGRP!and!PE.!After!administration!of!the!drugs,!
animals!were!placed!back!in!their!home!cage!and!recorded!for!2!hours!(Fig.!
III.1).!Mice!treated!with!vehicle!had!an!average!mean!arterial!blood!pressure!of!
100!mm!Hg!compared!to!a!significant!decrease!to!63!mm!Hg!for!CGRPKtreated!
mice.!We!started!with!2!mg/kg!PE!coKadministered!with!CGRP!to!minimize!
! 91!
CGRPKinduced!vasodilation.!This!dose!blocked!CGRPKinduced!vasodilation!well,!
however,!we!wanted!to!determine!the!lowest!dose!possible!that!could!block!
CGRP!effects.!We!then!coKadministered!1mg/kg!PE!with!CGRP!and!determined!
this!dose!blocked!CGRPKinduced!vasodilation!equally!as!well.!Similar!to!the!
average!blood!pressure!seen!with!vehicle!administration,!CGRP!and!1mg/kg!PE!
administration!had!average!mean!arterial!blood!pressure!of!101!mm!Hg.!
Additionally,!since!1mg/kg!PE!was!sufficient!to!block!CGRP!effects,!we!chose!to!
use!this!dose!for!the!remainder!of!the!study.!!The!average!blood!pressure!
observed!following!1mg/kg!PE!alone!was!approximately!126!mm!Hg.!This!was!
significantly!higher!than!vehicle,!CGRP,!and!CGRP+PE!at!several!time!points.!!
We!then!tested!whether!the!blockage!of!CGRPKinduced!vasodilation!
would!abolish!the!light!aversion!elicited!by!peripheral!CGRP.!For!this!experiment,!
mice!were!treated!with!the!same!doses!given!for!the!telemetry!experiments!in!
(Fig.!III.2).!Consistent!with!previous!reports128,!CGRPKtreated!mice!spent!an!
average!of!31s!in!the!light!compared!with!94!s!for!vehicleKtreated!mice.!However,!
when!CGRP!is!coKadministered!with!PE,!the!light!aversion!is!partially!attenuated!
though!still!significantly!different!from!both!CGRPKtreated!and!vehicleKtreated!
mice.!In!addition,!administration!of!PE!alone!produced!a!significant!light!aversive!
response.!This!response!is!consistent!with!reports!that!phenylephrine!can!be!
used!to!dilate!the!iris!and!induce!mydriasis192,193.!The!increased!amount!of!bright!
light!that!enters!the!eye!following!PE!administration!in!mice!would!promote!an!
affinity!towards!the!dark!zone.!!
! 92!
Since!migraine!affects!women!more!than!me,!we!wanted!to!determine!the!
contribution!of!each!sex!in!the!induction!of!light!aversion!following!treatment.!In!
these!studies,!we!analyzed!the!data!obtained!in!Fig.!III.2!by!gender!of!the!mice.!
In!this!cohort,!male!mice!that!received!CGRP!spent!significantly!less!time!in!the!
light!compared!to!the!CGRPKtreated!female!mice.!In!contrast,!there!was!no!
significant!difference!between!male!and!female!mice!in!response!to!CGRP!coK
administered!with!PE.!However,!PE!administration!in!these!mice!elicited!a!
stronger!light!aversive!response!in!females!than!males!(Fig.!III.3).!!
During!the!time!we!assessed!light!aversive!behavior,!we!also!evaluated!
the!effect!of!coKadministration!of!PE!and!CGRP!on!motor!activity!as!measured!by!
resting!time!and!vertical!beam!breaks!(rearing).!Consistent!with!previous!reports,!
peripheral!CGRP!reduced!motor!activity!only!in!the!dark!zone!(Fig.!III.4!and!III.5).!
In!the!dark!zone,!CGRP!and!PE!administration!significantly!increased!resting!
behavior!(Fig.III.4)!and!decreased!rearing!behavior!(Fig.III.5)!compared!to!
vehicle.!Interestingly,!blocking!CGRPKinduced!vasodilation!does!not!attenuate!
the!resting!behavior!or!rearing!elicited!by!CGRP.!!Additionally,!in!the!light!zone,!
there!was!no!difference!in!either!resting!or!rearing!behavior!among!all!
treatments.!
!
Blood(pressure(changes(and(light(aversion(following(administration(CGRP(and(
vasoconstrictors(endothelinM1(((
We!wanted!to!confirm!these!findings!by!using!another!vasoconstrictor!to!
minimize!the!CGRPKinduced!changes!in!vascular!tone.!For!the!next!set!of!
! 93!
experiments,!we!chose!endothelinK1,!a!potent!vasoconstrictor!produced!by!
endothelial!cells.!In!the!body,!endothelinK1!is!the!most!potent!vasoconstrictor!
known!and!regulates!vascular!tone!by!acting!on!vascular!smooth!muscle!cells!
and!by!coordinating!the!release!of!several!vasoactive!molecules194,195.!It!has!
been!shown!that!0.01mg/kg!intravenous!injection!of!endothelinK1!can!induce!
hypertension!in!rats196.!As!a!starting!point,!we!administered!0.01mg/kg!
intraperitoneal!endothelinK1!to!minimize!CGRPKinduced!vasodilation.!CoK
administration!of!0.01!mg/kg!endothelinK1!and!CGRP!did!not!block!the!initial!
blood!pressure!decrease!induced!by!CGRP,!however,!after!30!min!arterial!blood!
pressure!is!similar!to!the!levels!of!vehicleKtreated!mice!but!not!baseline!
measurements!(Fig.!III.6).!To!determine!if!a!higher!dose!of!endothelinK1!could!
block!the!initial!reduction!in!blood!pressure,!we!coKadministered!0.1!mg/kg!
endothelinK1!with!CGRP.!Indeed,!with!this!injection,!coKadministration!of!CGRP!
and!endothelinK1!abrogated!any!significant!change!in!mean!arterial!blood!
pressure.!Additionally,!the!initial!blood!pressure!decrease!in!the!first!30!min!was!
slight,!however,!more!transient!than!the!0.01mg/kg!dose.!In!this!study,!the!
average!blood!pressure!readout!for!vehicle!slightly!decreased!from!baseline.!Due!
to!this,!we!chose!to!continue!to!use!the!0.1!mg/kg!endothelinK1!dose!which!
maintained!the!blood!pressure!levels!close!to!baseline.!!
! Similar!to!the!results!seen!in!Fig.!III.2,!CGRP!induced!significant!light!
aversion!compared!to!vehicle.!Additionally,!coKadministration!of!CGRP!and!
endothelinK1!partially!blocked!the!CGRPKinduced!light!aversion!to!the!same!
degree!as!coKadministration!of!CGRP!and!PE!(Fig.!III.7).!Interestingly,!there!
! 94!
seems!to!be!two!populations!that!respond!and!do!not!respond!to!blocking!CGRPK
induced!vasodilation!in!this!cohort.!In!addition,!endothelinK1!alone!induced!
significant!light!aversion!similar!to!phenylephrine!alone.!!
! VehicleKtreated!mice!spent!significantly!less!time!resting!compared!to!
CGRP,!CGRP+EndothelinK1,!and!endothelinK1!alone.!Consistent!with!the!inability!
of!PEKinduced!vasoconstriction!to!attenuate!resting!behavior,!coKadministration!of!
CGRP!and!endothelinK1!also!failed!to!ameliorate!resting!behavior!in!these!mice.!
There!was!also!no!significant!difference!among!each!treatment!group!in!the!
amount!of!resting!time!in!the!light!zone!(Fig.!III.8).!Mice!treated!with!vehicle!
exhibited!a!higher!incidence!of!rearing!at!all!time!points!compared!to!CGRP,!
CGRP+endothelinK1,!and!endothelinK1!alone!(Fig.!III.9).!Additionally,!there!was!
no!significant!difference!in!rearing!in!the!light!zone.!!
! Although!preliminary,!male!mice!that!were!administered!CGRP!in!these!
studies!spent!less!time!in!the!light!compared!to!female!mice!(Fig.!III.10).!
However,!the!CGRPKtreated!female!mice!displayed!a!more!variable!response!
than!the!male!mice!treated!with!CGRP.!Additionally,!coKadministration!of!CGRP!
and!endothelinK1!displayed!no!significant!sex!differences!between!male!and!
female!mice!in!the!light!aversion!assay.!Moreover,!the!response!to!endothelinK1!
was!equal!between!both!genders!of!mice.!However,!more!experiments!are!need!
to!increase!the!number!of!animals!needed!for!comparison!
(
(
(
! 95!
Blood(pressure(changes(and(light(aversion(following(administration(PACAPM38(
and(VIP(
Since!the!observations!seen!in!Figs.!III.2!and!III.7!suggest!that!changes!in!
vascular!tone!may!play!a!role!in!our!model!of!migraineKlike!photophobia,!we!
wanted!to!determine!if!any!vasodilator!could!induce!light!aversion!in!this!assay.!
First,!we!chose!to!determine!if!we!could!induce!light!aversion!by!intraperitoneal!
administration!of!pituitary!adenylate!cyclaseKactivating!polypeptide!(PACAPK38).!
Previous!reports!have!shown!that!infusion!of!PACAPK38!can!cause!sustained!
dilation!in!humans55!and!rats56!and!induce!a!migraineKlike!headache55,!indicating!
that!PACAPK38!may!play!a!role!in!migraine!pathophysiology.!In!these!studies,!
mice!were!injected!with!0.3mg/kg!PACAPK38.!PACAPK38!induced!marked!light!
aversion!similar!to!what!has!been!observed!with!CGRP.!PACAPKtreated!mice!
spent!an!average!of!34!s!in!the!light!compared!to!vehicleKtreated!mice!which!
spent!an!average!of!106!s!in!the!light!(Fig.III.11).!!
! Next,!we!wanted!to!determine!if!inducing!light!aversion!was!a!property!of!
any!vasodilator.!We!chose!to!test!vasoactive!intestinal!peptide!(VIP),!a!
vasodilator!that!is!in!the!same!family!as!PACAPK38.!In!clinical!studies,!infusion!of!
VIP!induces!marked!vasodilation!but!only!has!the!ability!to!induce!a!mild!
headache!and!not!a!migraine!in!patients.!Intraperitoneal!administration!of!VIP!at!
both!0.1!mg/kg!and!0.3!mg/kg!failed!to!induce!light!aversion.!VIPKtreated!mice!
spent!86!s!and!93!s,!respectively,!compared!to!90!s!vehicleKtreated!mice!spent!in!
the!light!(Fig.III.12).!As!a!control,!we!wanted!to!ensure!that!the!VIP!response!was!
indeed!due!to!incapacity!to!respond!and!not!just!a!random!observation.!A!cohort!
! 96!
of!mice!that!had!previously!not!responded!to!VIP!were!tested!again.!Both!doses!
of!VIP!continued!to!not!respond!whereas!CGRP!continued!to!induce!a!significant!
light!aversive!response!(Fig.III.13).!!
! Since!PACAPK38!and!VIP!had!very!different!effects!in!the!light!aversion!
assay,!we!wanted!to!assess!the!blood!pressure!changes!following!administration!
of!either!peptide.!Mice!that!were!administered!PACAPK38!had!a!significant,!
sustained!decrease!in!mean!arterial!blood!pressure!similar!to!the!decrease!
observed!with!CGRP!administration!(Fig.!III.14).!Interestingly,!when!VIP!was!
administered,!the!blood!pressure!decrease!was!very!similar!to!both!CGRP!and!
PACAP.!However,!the!vasodilation!induced!by!VIP!was!shortKlived!compared!to!
both!CGRP!and!PACAPK38.!!
!
Pupil(diameter(changes(following(administration(of(CGRP((
As!a!control,!we!wanted!to!ensure!that!CGRP!was!not!inducing!light!aversion!
due!to!mydriasis,!sustained!abnormal!pupil!dilation.!To!determine!if!CGRP!
induces!mydriasis,!we!analyzed!the!pupils!of!C57BL/6J!mice!30!min!after!CGRP!
injection.!We!have!previously!shown!that!IP!CGRP!in!the!C57B/6J!strain!induces!
significant!light!aversion,!so!we!chose!to!use!this!strain!since!CD1!mice!are!
albino!and!their!pupils!are!difficult!to!detect!with!our!software.!Following!
intrapertiontal!injection,!there!was!an!overall!trend!of!CGRP!decreasing!pupil!
diameter!(p=0.0265)!(Fig.!III.15,!III.16),!contrary!to!what!has!been!previously!
seen!with!intracerebroventricular!(ICV)!injection!of!CGRP.!This!suggests!that!ICV!
! 97!
and!IP!injection!of!CGRP!have!very!different!modes!of!action!and!possibly!
different!targets.!!
!
Sensitized(CGRPMinduced(light(aversive(behavior(of(global(hRAMP1(mice((
Using!mice!that!overexpressed!the!humanized!rateKlimiting!RAMP1!
component!of!the!CGRP!receptor!in!the!nervous!system!(nestin/hRAMP1),!we!
have!previously!reported!that!administration!of!central!CGRP,!but!not!peripheral!
CGRP,!elicited!light!aversion!at!a!much!lower!light!intensity!compared!to!the!
bright!light!required!for!wildKtype!mice.!These!data!suggested!that!central!and!
peripheral!CGRP!had!different!targets.!To!begin!to!identify!where!peripheral!
CGRP!could!be!acting,!other!than!on!nervous!tissue,!we!used!mice!that!
overexpressed!the!activated!hRAMP1!allele!in!the!germline!allowing!ubiquitous!
expression!of!hRAMP1!and!this!strain!is!herein!referred!to!as!global!hRAMP1!
mice!(Fig.!III.17).!These!mice!have!a!combined!total!mRAMP1!and!hRAMP1!
approximately!10K300Kfold!greater!than!endogenous!mRAMP1!levels190!in!all!
tissues!(Fig.III.18).!!
Contrary!to!intraperitoneal!injection!of!CGRP!in!nestin/hRAMP1!mice,!
global!hRAMP1!mice!exhibited!enhanced!light!aversion!compared!to!control!
littermates!that!also!received!CGRP!(Fig.!III.19).!Although!significant,!the!degree!
of!light!aversion!induced!by!CGRP!seems!to!be!not!as!strong!as!what!we!have!
previously!seen!with!ICV!injections!in!the!nestin/hRAMP1!strain!of!mice128,129,156.!
It!is!possible!that!there!is!a!compensatory!mechanism!to!counteract!the!actions!
of!the!RAMP1!since!there!is!a!significant!amount!of!overexpression.!!
! 98!
(
Sensitized(CGRPMinduced(light(aversive(behavior(of(smooth(muscle(hRAMP1(
mice,(but(not(endothelial,(hRAMP1(and(motility(
As!previously!mentioned,!CGRP!is!one!of!the!most!potent!vasodilators!in!
the!body.!Blocking!vasodilation!induced!by!CGRP!partially!blocks!light!aversion!
and!suggests!that!the!vasculature!may!play!in!role!in!our!migraine!model.!CGRP!
receptors!have!been!reported!on!both!vascular!smooth!muscle89!and!endothelial!
cells87.!To!determine!whether!peripheral!CGRP!has!actions!on!smooth!muscle!
cells!or!endothelial!cells!to!induce!light!aversion,!we!generated!double!transgenic!
mice!that!overexpress!hRAMP1!in!either!cell!type.!These!lines!were!generated!
by!crossing!CX1KGFPKhRAMP1!mice!with!either!Tagln(SM22!)Kcre!mice!(smooth!
muscle)!or!Tek2(Tie2)Kcre!mice!(endothelial!cell).!Expression!of!hRAMP1!in!
either!cell!type!requires!Cre!to!excise!a!floxed!GFP!upstream!cassette!
(Fig.III.20).!!
! Double!transgenic!tagln/hRAMP1!mice!and!their!control!littermates!were!
administered!either!vehicle!or!CGRP!and!subjected!to!the!light!aversion!assay.!
Tagln/hRAMP1!mice!that!received!CGRP!spent!an!average!of!approximately!43!
s!in!the!light,!which!was!statistically!significant!from!the!94!s!the!CGRPKtreated!
control!littermates!spent!in!the!light.!In!this!cohort!of!mice,!vehicleKtagln/hRAMP1!
mice!spent!less!time!in!the!light!(83!s)!compared!to!vehicleKtreated!control!
littermates!(119!s)!(Fig.!III.21),!however,!this!was!not!statistically!significant.!!
! Similar!to!wildKtype!mice,!peripheral!CGRP!in!tagln/hRAMP1!mice!
increased!resting!time!in!the!dark!zone!compared!to!mice!that!received!vehicle!
! 99!
and!CGRPKtreated!control!littermates.!!Consistent!with!previous!reports,!there!
was!no!difference!in!the!amount!of!time!the!mice!rested!among!either!treatment!
condition!(Fig.!III.22).!However,!only!the!25!min!and!30!min!time!points!are!
significant!in!the!CGRPKtreated!hRAMP1!mice.!In!addition,!tagln/hRAMP1!mice!
that!were!administered!CGRP!had!less!vertical!beam!breaks!at!the!15!min!and!
25!min!time!point!in!the!dark!compared!to!CGRPKtreated!control!littermates!(Fig.!
III.23).!!
To!determine!if!endothelial!cellKspecific!overexpression!of!hRAMP1!is!
sufficient!to!induce!enhanced!light!aversion!with!peripheral!CGRP!administration,!
we!injected!CGRP!in!Tek2/hRAMP1!mice.!Surprisingly,!in!this!preliminary!study,!
administration!of!CGRP!in!Tek2/hRAMP1!mice!failed!to!exhibit!enhanced!light!
aversion!under!normal!experimental!conditions!(Fig.!III.24).!Consistent!with!the!
inability!to!induce!light!aversion!in!these!mice,!CGRPKtreated!hRAMP1!mice!
exhibited!no!significant!change!in!their!resting!behavior!compared!to!CGRPK
treated!control!littermates!or!vehicle!treated!mice!in!either!the!dark!zone!or!the!
light!zone!(Fig.!III.25).!Moreover,!rearing!activity!in!these!mice!was!unchanged!in!
both!zones!(Fig.!III.26).!
"
DISCUSSION!
A!role!for!the!vasculature!in!migraine!has!largely!been!dismissed!in!recent!
years.!In!fact,!most!studies!suggest!that!vasodilation!is!considered!an!
epiphenomenon,!simply!due!to!studies!that!report!the!migraine!pain!state!begins!
after!the!vasodilation!has!arrested!and!that!VIP,!a!potent!vasodilator,!cannot!
! 100!
induce!migraine!in!people53.!The!consensus!is!now!that!migraine!is!a!disorder!of!
neural!imbalances.!However,!migraine!has!been!studied!for!over!a!century!now!
and!we!are!still!a!long!way!from!completely!understanding!its!pathophysiology.!It!
is!now!necessary!to!take!a!step!back!and!determine!what!could!be!the!missing!
piece!that!links!the!vascular!theory!to!the!neural!theory.!In!this!chapter,!we!focus!
on!identifying!possible!sites!of!peripheral!CGRP!action!in!migraineKlike!
photophobia.!We!have!previously!reported!that!peripheral!CGRP!can!induce!light!
aversion!in!wildKtype!mice128.!In!the!same!study,!we!also!reported!that!central!
CGRP,!but!not!peripheral!CGRP,!induced!enhanced!light!aversion!in!mice!that!
overexpress!RAMP1!in!neural!tissue.!These!data!combined!suggested!that!
peripheral!CGRP!has!actions!outside!of!the!CNS!and!peripheral!neurons.!In!this!
study,!we!reKexamined!the!possibility!of!a!role!for!the!vasculature!in!migraine!
pathogenesis.!
Considering!the!wide!expression!of!CGRP!receptors!on!the!vasculature!
and!the!critical!role!of!CGRP!in!the!cardiovascular!system,!we!wanted!to!
determine!if!peripheral!CGRP!elicited!light!aversion!via!a!vascular!site!of!action!
in!this!model!of!migraine87,89.!The!ability!of!pharmacologically!blocking!CGRPK
induced!vasodilation!to!partially!attenuate!light!aversion!strongly!suggests!that!
peripheral!CGRP!has!actions!at!the!vasculature!to!induce!migraineKlike!
symptoms.!This!is!consistent!with!migraine!studies!in!humans!that!suggest!
vasodilation!of!the!middle!meningeal!artery!(MMA)!are!particularly!important!in!
migraine45,79.!Additionally,!these!data!are!also!in!agreement!with!the!ability!of!
extracranial!and!cerebral!vasoconstrictors!(ergots,!trptans,!and!gepants)!to!be!
! 101!
clinically!effective!in!reducing!migraine!attacks!and!pain197.!Based!on!these!data!
future!studies!should!reKexamine!the!role!of!extracranial!vessels!in!migraine!
pathophysiology.!
Additionally,!the!inability!of!clamping!vascular!tone!changes!induced!by!
CGRP!is!also!in!agreeance!with!the!notion!that!vascular!events!alone!are!not!
sufficient!to!induce!migraine198,199.!CGRP!receptors!are!found!on!other!peripheral!
cell!types,!outside!of!vessels,!and!may!play!a!role!in!migraine.!One!cell!type!of!
particular!interest!are!the!mast!cells.!The!role!of!mast!cells!in!migraine!has!been!
a!subject!of!speculation!for!years.!CGRP!receptors!have!been!identified!on!dura!
materKresident!mast!cells!and!activation!can!induce!degranulation115,117,200,201.!
Activation!of!mast!cells!can!result!in!release!of!a!plethora!of!mediators!such!as!
histamines!and!nitric!oxide!and!effect!vascular!permeability.!Therefore,!it!seems!
possible!that!peripheral!administration!of!CGRP!can!act!on!both!vessels!and!
mast!cells!simultaneously.!The!actions!on!mast!cells!may!contribute!to!the!
inflammatory!event!that!can!lead!to!increased!vessel!permeability!and!
subsequent!extrusion!of!proKinflammatory!neuropeptides!from!both!the!mast!cells!
and!vessels,!possibly!sensitizing!nearby!nociceptors115,202,203.!Future!studies!with!
mast!cell!degranulator!compound!48/80!and!mast!cell!stabilizers!may!give!insight!
into!the!possible!role!of!mast!cell!and!vessel!communication!in!nociceptor!
sensitization!in!this!model.!!
Hypertension!can!induce!a!subset!of!migraineKlike!symptoms!in!people!
and!poor!control!of!blood!pressure!may!exacerbate!the!frequency!of!migraine.!
However,!hypertension!has!only!been!considered!as!a!comordity!of!migraine.!
! 102!
Additionally,!in!a!study!by!Hamed!et!al.,!the!authors!found!that!subjects!
diagnosed!with!migraine!with!aura!had!higher!systolic!blood!pressure!than!
control!subjects!59,204.!The!ability!of!both!vasoconstrictors!phenylephrine!and!
endothelinK1!to!elicit!a!light!aversive!response!in!these!studies!futher!indicate!a!
role!for!the!vasculature!in!migraine.!On!the!contrary,!hypertension!has!been!
closely!linked!with!anxiety!in!people.!Indeed,!epinephrine,!a!potent!
vasoconstrictor,!has!been!shown!to!induce!a!significant!increase!in!anxiety!in!
normotensive!subjects205,206.!In!another!study!by!Alcantara!et!al.,!they!found!that!
anxiety!was!strongly!associated!with!patients!who!had!uncontrolled!blood!
pressure207.!Further!studies!examining!the!role!of!elevated!blood!pressure!and!
anxiety!can!help!explain!this!unexpected!light!aversive!response!of!
vasoconstrictors!in!this!study.!!
While!migraine!is!primarily!a!disorder!that!affects!women,!CGRP!did!not!
induce!a!more!significant!effect!in!female!mice!more!than!male!mice!in!the!light!
aversion!assay.!Contradictorily,!in!these!cohorts!of!mice!we!observed!a!greater!
effect!of!CGRP!in!male!mice.!However,!blunting!CGRPKinduced!vasodilation!had!
very!similar!effects!in!both!male!and!female!mice.!Future!studies!that!segregate!
female!mice!based!on!their!stage!of!the!estrous!cycle!could!potentially!uncloak!
sex!differences!during!CGRPKinduced!light!aversion.!!
PACAPK38!is!a!vasoactive!neuropeptide!that!has!been!used!in!
experimental!studies!to!cause!prolonged!dilation!of!the!MMA!and!induce!
migraine!in!people55.!!Conversely,!VIP!is!another!vasoactive!peptide!in!the!same!
family!as!PACAPK38!that!can!cause!extensive,!however!brief!in!comparison!to!
! 103!
PACAPK38!and!CGRP,!vasodilation!but!has!only!been!able!to!induce!a!mild!
headache!in!people53.!The!ability!of!PACAPK38,!but!not!VIP,!to!induce!light!
aversion!in!these!studies!is!consistent!with!previous!reports.!However,!
conclusions!that!VIP!is!incapable!of!causing!a!migraine!is!one!of!the!reasons!the!
vascular!theory!lost!its!steam.!Authors!of!these!reports!failed!to!consider!that!
CGRP,!PACAPK38,!NO!synthase!and!other!vasoactive,!migraineKprovoking!
agents!have!the!ability!to!induce!vasodilation!for!an!extended!period!of!time,!
whereas,!VIP!does!not!have!this!capability.!Moreover,!systemically!administered!
VIP!gets!degraded!at!a!much!faster!rate!than!PACAPK38.!In!fact,!similar!to!
CGRP,!PACAP!has!a!halfKlife!of!about!10!min!whereas!the!halfKlife!of!VIP!is!less!
than!3!min208,209.!It!is!possible!that!extended!infusion!times!of!VIP!in!experimental!
models!of!migraine!might!reveal!the!capability!of!VIP!to!induce!headache!and!
symptoms!phenotypically!similar!to!migraine.!Additionally,!PACAPK38,!but!not!
VIP,!can!induce!concentrationKdependent!release!of!CGRP!from!the!trigeminal!
nucleus!caudalis!and!both!are!coKexpressed!in!trigeminal!ganglion!neurons56.!It!
is!suggested!that!this!is!not!due!to!PACAPK38!receptor!activation,!thus,!it!is!also!
possible!that!CGRP!receptor!activation!may!play!a!role!in!PACAPKinduced!light!
aversion.!!
In!mesenteric,!cerebral,!and!coronary!arteries,!CGRP!has!the!ability!to!
increase!intracellular!cAMP!and!induce!vasorelaxation!via!an!endotheliumK
indedpendent!pathway111.!The!capacity!of!smooth!muscleKspecific!hRAMP1,!and!
not!endotheliumKspecific!hRAMP1,!to!induce!enhanced!light!aversion!following!
CGRP!administration!is!consistent!with!these!studies!and!clearly!suggests!a!role!
! 104!
for!CGRPKinduced!vasodilation!in!this!model!of!migraine.!!In!addition,!CGRP!is!
found!in!and!released!from!endothelial!cells210.!It!is!possible!that!the!sterile!
inflammatory!event!occurs!and!provokes!release!of!CGRP!and!other!mediators!
from!endothelial!cells!which!thereby!act!on!receptors!on!smooth!muscle!cells!
resulting!in!vasodilation!and!plasma!extravasation.!More!studies!with!the!
Tagln/hRAMP1!and!Tek2/hRAMP1!mice!are!necessary!to!reveal!how!CGRP!
actions!on!vessels!can!influence!neurotransmission.!!
Two!receptors!can!be!activated!by!CGRP!with!both!having!nearly!equal!
affinity!for!the!peptide.!CGRP1!consists!of!calcitonin!receptorKlike!receptor!and!
RAMP1!and!has!been!shown!to!be!the!primary!receptor!activated!for!the!
vasodilatory!effects!evoked!by!CGRP104.!However,!the!CGRP2,or!AMY1,!receptor!
consists!of!calcitonin!receptor!and!RAMP1!and!has!been!shown!to!impair!the!
responsiveness!of!endothelial!cells!causing!a!defect!in!endotheliumKdependent!
vasodilation!following!amylin!administration211.!In!this!regard,!future!studies!
should!dissect!whether!activation!of!CGRP2!receptor,!in!response!to!peripheral!
CGRP,!can!also!induce!vasodilation!and!if!blockade!of!this!receptor,!using!small!
molecule!antagonist!AC187,!can!attenuate!CGRPKinduced!migraine!symptoms.!
Together,!these!findings!support!the!neurovascular!hypothesis!of!migraine!and!
suggest!that!vascular!signals!can!affect!neurons!and!vice!versa!in!a!positive!
feedback!loop!(Fig.!III.28).!Future!studies!that!dissect!this!model!of!intersection!
of!peripheral!and!central!actions!of!CGRP!at!the!vasculature!should!help!reveal!
how!this!poorly!understood!mechanism!may!contribute!to!migraine.!!
! 105!
!!!Figure!III.1.!Blood!pressure!changes!following!administration!of!CGRP!and!PE.!The!mean!arterial!pressure!(MAP)!was!measured!by!telemetry!from!the!carotid!artery!of!CD1!at!baseline!and!following!administration!of!vehicle!(n=9),!CGRP!(0.1mg/kg)!(n=8),!PE,!and!CGRP+PE!(1mg/kg)!(n=8),!and!CGRP+!PE!(2!mg/kg)(n=8).!For!all!panels,!the!time!corresponding!to!behavior!monitoring!(30K60!min!postKinjection)!of!parallel!mouse!cohorts!is!indicated.!Data!are!the!mean!±SEM.!!!!!!!!!!!!!!!!!!!!!
! 106!
!!!
!!!!!!
! 107!
Figure.!III.2.!Phenylephrine!attenuates!CGRPKinduced!light!aversion.!Time!spent!in!the!light!zone!by!CD1!mice!on!treatment!day.!Mice!were!treated!with!vehicle!(n=16),!CGRP!(0.1mg/kg)!(n=20),!CGRP+PE!(n=17),!PE!(n=17).!The!mean!±SEM!is!shown!(top!panel).!Bottom!panel!shows!the!mean!(±SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day.!!!!!!!!!!!!!!!!!!!!!!!!!!!!
! 108!
!!!
!!!Figure.!III.3.!Gender!effects!in!the!light!aversion!assay.!Data!from!Fig.!III.2!was!analyzed!by!sex.!Top!panel!is!CGRP!and!vehicle!treatment.!Bottom!panel!is!CGRP+PE!treatment!and!PE!treatment.!Data!are!the!mean!±SEM.!!!!!
! 109!
!!!Figure.!III.4.!Resting!behavior!following!clamping!of!CGRPKinduced!vasodilation!in!the!light!aversion!assay.!Percentage!of!time!spent!resting!in!both!the!dark!and!light!zone!per!5!min!intervals!following!treatment!with!vehicle,!CGRP,!CGRP+PE,!and!PE.!Data!are!from!mice!in!Fig.!III.2.!Data!are!the!mean!±SEM.!!!!!!!!!!!!!!!!!!!
! 110!
!!!Figure.!III.5.!Rearing!behavior!following!clamping!of!CGRPKinduced!vasodilation!in!the!light!aversion!assay.!Average!time!spent!rearing!in!both!the!dark!and!light!zone!per!5!min!intervals!following!treatment!with!vehicle,!CGRP,!CGRP+PE,!and!PE.!Data!are!from!mice!in!Fig.!III.2.!Data!are!the!mean!±SEM.!"!!!!!!!!!!!!!!!!!
! 111!
!!!Figure.!III.6.!Blood!pressure!changes!following!administration!of!CGRP!and!endothelinK1.!The!mean!arterial!pressure!(MAP)!was!measured!by!telemetry!from!the!carotid!artery!of!CD1!at!baseline!and!following!administration!of!vehicle!(n=11),!CGRP!(0.1mg/kg)!(n=11),!endoK1,!and!CGRP+endoK1!(1mg/kg)!(n=6),!and!CGRP+!endoK1!(2!mg/kg)!(n=6).!For!all!panels,!the!time!corresponding!to!behavior!monitoring!(30K60!min!postKinjection)!of!parallel!mouse!cohorts!is!indicated.!Data!are!the!mean!±SEM.!!!!!!!!!!!!!!!!!!!!!!
! 112!
!!!
!!!Figure.!III.7.!EndothelinK1!attenuates!CGRPKinduced!light!aversion.!Time!spent!in!the!light!zone!by!CD1!mice!on!treatment!day.!Mice!were!treated!with!vehicle!(n=10),!CGRP!(0.1mg/kg)!(n=10),!CGRP+endoK1!(n=16),!endoK1!(n=10).!The!mean!±SEM!is!shown!(top!panel).!Bottom!panel!shows!the!mean!(±SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day.!!!
! 113!
!!!
!!!Figure.!III.8.!Gender!effects!in!the!light!aversion!assay.!Data!from!Fig.!III.7!was!analyzed!by!sex.!Top!panel!is!CGRP!and!vehicle!treatment.!Bottom!panel!is!CGRP+endoK1!treatment!and!endoK1treatment.!Data!are!the!mean!±SEM.!!!!!!
! 114!
!!!Figure.!III.9.!Resting!behavior!following!clamping!of!CGRPKinduced!vasodilation!in!the!light!aversion!assay.!Percentage!of!time!spent!resting!in!both!the!dark!and!light!zone!per!5!min!intervals!following!treatment!with!vehicle,!CGRP,!CGRP+endoK1,!and!endoK1.!Data!are!from!mice!in!Fig.!III.7.!!!!!!!!!!!!!!!!!!!!!
! 115!
!!!Figure.!III.10.!Rearing!behavior!following!clamping!of!CGRPKinduced!vasodilation!in!the!light!aversion!assay.!Average!time!spent!rearing!in!both!the!dark!and!light!zone!per!5!min!intervals!following!treatment!with!vehicle,!CGRP,!CGRP+endoK1,!and!endoK1.!Data!are!from!mice!in!Fig.!III.7.!!!!!!!!!!!!!!!!!!!!!
! 116!
!!!
!!!!!
! 117!
Figure.!III.11.!Peripheral!PACAP!induces!light!aversive!behavior.!Time!spent!in!the!light!zone!by!CD1!mice!on!treatment!day.!Mice!were!treated!with!vehicle!(n=10),!CGRP!(0.1mg/kg)!(n=22),!and!PACAP!(n=25)The!mean!±SEM!is!shown!(top!panel).!Bottom!panel!shows!the!mean!(±SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day.!!!!!!!!!!!!!!!!!!!!!!!!
! 118!
!!!
!!!!!!
! 119!
Figure!III.12.!Peripheral!VIP!fails!to!induce!light!aversive!behavior.!Time!spent!in!the!light!zone!by!CD1!mice!on!treatment!day.!Mice!were!treated!with!vehicle!(n=19),!CGRP!(0.1mg/kg)!(n=11),!VIP!(0.1mg/kg)!(n=16),!and!VIP!(0.3!mg/kg)!(n=13).!Bottom!panel!shows!the!mean!(±SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day.!!!!!!!!!!!!!!!!!!!!!!!!!!!
! 120!
!!!Figure!III.13.!Retest!of!VIP!cohort!still!fails!to!induce!light!aversive!behavior.!Time!spent!in!the!light!zone!by!CD1!mice!on!treatment!day.!Mice!were!treated!with!vehicle!(n=10),!CGRP!(0.1mg/kg)!(n=9),!VIP!(0.1mg/kg)!(n=6),!and!VIP!(0.3!mg/kg)!(n=13).!Data!are!the!mean!±SEM.!!!!!!!!!!!!!!!!!!!!!!!!
! 121!
!
!
!
! 122!
Figure!III.14.!Blood!pressure!changes!following!administration!of!VIP,!PACAP,!and!CGRP.!A.!The!mean!arterial!pressure!(MAP)!was!measured!by!telemetry!from!the!carotid!artery!of!CD1!at!baseline!and!following!administration!of!vehicle!(n=5),!CGRP!(0.1mg/kg)!(n=5)!and!PACAP(n=5).!B.!The!mean!arterial!pressure!(MAP)!was!measured!by!telemetry!from!the!carotid!artery!of!CD1!at!baseline!and!following!administration!of!vehicle!(n=5),!CGRP!(0.1mg/kg)!(n=5)!and!VIP(n=5).!C.!Both!graphs!from!panels!A!and!B!are!superimposed.!For!all!panels,!the!time!corresponding!to!behavior!monitoring!(30K60!min!postKinjection)!of!parallel!mouse!cohorts!is!indicated.!Data!are!the!mean!±SEM!!!!!!!!!!!!!!
! 123!
!!!C!
!!!!!!!!!
! 124!
Figure!III.15.!Representative!image!of!mouse!pupil!diameter!and!pupil!diameter!changes!following!CGRP!administration.!A.!Pupil!diameter!of!mouse!treated!with!vehicle.!B.!Pupil!diameter!of!mouse!treated!with!CGRP.!C.!Left!panel,!left!and!right!pupil!averages!per!mice.!Right!panel,!individual!eye!diameters!of!both!right!and!left!pupil.!Data!are!the!mean±SEM.!For!panel!C,!CGRPKtreated!mice!pupil!diameter!was!significantly!decreased!compared!to!vehicle!(left!panel,!p=0.0265).!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
! 125!
!!!Figure!III.!16.!Generation!schematic!of!global!hRAMP1!transgenic!mice.!The!loxP!sites!that!flank!the!GFP!reporter!allow!for!CreKmediated!excision!of!GFP!and!associated!stop!sequences!when!the!parental!mice!are!crossed!with!mice!expressing!Cre!recombinase.!Following!mating,!the!hRAMP1!transgene!is!incorporated!in!the!germline!of!progeny.!!!!!!!!!!!!!!
! 126!
!!!!!!!!
! 127!
Figure!III.17.!Expression!pattern!of!RAMP1!in!global!hRAMP1!mice.!A.!QKPCR!measurement!of!RAMP1!levels!in!global!hRAMP1!mice!and!control!littermates.!The!levels!of!hRAMP1,!mRAMP1,!and!"Kactin!were!determined!by!realKtime!PCR.!The!copies!of!RAMP1!RNAs!were!calculated!from!standard!curves!and!normalized!to!50,000!copies!of!"Kactin!mRNA.!Tissues!are!as!follows:!brain,!spinal!cord!(SC),!trigeminal!ganglion!(TG),!liver,!kidney,!spleen,!lungs,!heart,!and!aorta.!B.!The!fold!increase!of!total!RAMP1!(combined!mouse!and!human)!in!global!hRAMP1!mice!tissues!relative!to!mRAMP1!levels!is!indicated.!Data!are!the!mean!±SEM!from!five!mice!of!each!genotype.!!!!!!!!!!!!!!!!!!!!!
! 128!
!!!
!!!!!!!
! 129!
Figure!III.18.!Peripheral!CGRP!induces!light!aversive!behavior!in!global!hRAMP1!mice.!Time!spent!in!the!light!zone!by!mice!on!treatment!day.!Global!hRAMP1!mice!were!treated!with!either!vehicle!(hRAMP1!vehh!n=18)!or!CGRP!(0.1mg/kg)!(hRAMP1!CGRPh!n=18),!and!control!littermates!were!treated!with!vehicle!(Control!vehh!n=16)!or!CGRP!(0.1mg/kg)!(Control!CGRPh!n=16).!The!mean!±SEM!is!shown!(top!panel).!Bottom!panel!shows!the!mean!(±SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day.!!!!!!!!!!!!!!!!!!!!!!!!!!!
! 130!
!!!Figure!III.19.!Generation!schematic!of!tagln/hRAMP1!and!tek2/hRAMP1!double!transgenic!mice.!The!lox!P!sites!that!flank!the!GFP!reporter!allow!for!CreKmediated!excision!of!GFP!and!associated!stop!sequences!when!the!parental!mice!are!crossed!with!mice!expressing!Cre!recombinase.!Following!mating,!the!hRAMP1!transgene!is!incorporated!in!either!smooth!muscle!(tagln/hRAMP1)!or!endothelial!(tek2/hRAMP1)!cells.!!!!!!!!!!!!!!!!!!!!!!!!
! 131!
!!!
!!!!!!!!
! 132!
Figure!III.20.!Peripheral!CGRP!induces!light!aversive!behavior!in!tagln/hRAMP1!mice!in!which!hRAMP1!is!overexpressed!in!smooth!muscle!cells.!Time!spent!in!the!light!zone!by!mice!on!treatment!day.!Tagln/hRAMP1!mice!were!treated!with!either!vehicle!(hRAMP1!vehh!n=13)!or!CGRP!(0.1mg/kg)!(hRAMP1!CGRPh!n=19),!and!control!littermates!were!treated!with!vehicle!(Control!vehh!n=18)!or!CGRP!(0.1mg/kg)!(Control!CGRPh!n=18).!The!mean!±SEM!is!shown!(top!panel).!Bottom!panel!shows!the!mean!(±SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day!(*p<0.05,!**p<0.01).!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
! 133!
!
!!!Figure!III.21.!Resting!behavior!following!administration!of!CGRP!in!tagln/hRAMP1!mice.!Percentage!of!time!spent!resting!in!both!the!dark!and!light!zone!per!5!min!intervals!following!treatment!with!vehicle!or!CGRP!in!either!tagln/hRAMP1!mice!or!control!littermates.!!!!!!!!!!!!!!!!!!!
! 134!
!!!Figure.!III.22.!Rearing!behavior!in!tagln/hRAMP1!following!administration!of!CGRP.!Average!time!spent!rearing!in!both!the!dark!and!light!zone!per!5!min!intervals!following!treatment!with!vehicle!or!CGRP!in!either!tagln/hRAMP1!mice!or!control!littermates.!!!!!!!!!!!!!!!!!!!!!
! 135!
!!!
!!!!!!!!
! 136!
Figure.!III.23.!Peripheral!CGRP!does!not!induce!light!aversive!behavior!in!tek2/hRAMP1!mice!in!which!hRAMP1!is!overexptessed!in!endothelia!cells.!Time!spent!in!the!light!zone!by!mice!on!treatment!day.!Tagln/hRAMP1!mice!were!treated!with!either!vehicle!(hRAMP1!vehh!n=5)!or!CGRP!(0.1mg/kg)!(hRAMP1!CGRPh!n=5),!and!control!littermates!were!treated!with!vehicle!(Control!vehh!n=6)!or!CGRP!(0.1mg/kg)!(Control!CGRPh!n=5).!The!mean!±SEM!is!shown!(top!panel).!Bottom!panel!shows!the!mean!(±SEM)!in!light!per!5!min!interval!for!individual!mice!on!treatment!day.!!!!!!!!!!!!!!!!!!!!!!!!!
! 137!
!!!Figure!III.24.!Resting!behavior!following!administration!of!CGRP!in!tek2/hRAMP1!mice.!Percentage!of!time!spent!resting!in!both!the!dark!and!light!zone!per!5!min!intervals!following!treatment!with!vehicle!or!CGRP!in!either!tek2/hRAMP1!mice!or!control!littermates.!!!!!!!!!!!!!!!!!!!!!!
! 138!
!!!Figure!III.25.!Rearing!behavior!in!tek2/hRAMP1!following!administration!of!CGRP.!Average!time!spent!rearing!in!both!the!dark!and!light!zone!per!5!min!intervals!following!treatment!with!vehicle!or!CGRP!in!either!tek2/hRAMP1!mice!or!control!littermates.!!!!!!!!!!!!!!!!!!!
! 139!
!!!Figure!III.26.!Model!of!peripheral!and!central!CGRP!intersection!in!migraine.!Trigeminal!microenvironment!changes!due!to!neurogenic!inflammation,!thus!causing!local!decrease!in!blood!pressure!and!sensitizing!dural!and!pial!afferents.!Sensitized!afferents!can!release!CGRP!and!other!meidators,!thereby,!inducing!changes!in!cerebral!blood!flow!(CBF)!and!extavasation!of!proKinflammatory!mediators.!This!sensitizes!neurons!and!causes!neuromodulation!in!feed!forward!loop.!!!!!!!!!""
"
"
"
"
"
! 140!
CHAPTER!IV!
CONCLUSIONS!AND!FUTURE!DIRECTIONS!
CONCLUSIONS!
Migraine!is!a!multifactorial!neurovascular!disorder.!Despite!the!attention!
paid!to!migraine!pathogenesis!over!the!recent!years,!understanding!of!this!
disorder!remains!limited,!thus!rendering!it!difficult!to!treat!sufferers!effectively.!
Over!the!past!century,!several!treatments!including!ergots,!triptans,!gepants,!and!
now!CGRP!monoclonal!antibodies!have!been!used!effectively!in!patients.!
Interestingly,!though!blatantly!ignored,!a!common!theme!among!all!of!the!most!
effective!migraine!treatments!is!that!they!all!have!vasoactive!properties.!Indeed,!
they!all!have!ability!to!induce!vasoconstriction!in!dilated!extracranial!vessels!and!
ameliorate!migraine!symptoms35,212,213.!While!these!drugs!are!effective,!they!are!
not!without!contraindications!and!must!be!used!with!caution.!For!patients!that!are!
hypertensive,!it!seems!that!using!vasoconstrictive!drugs!will!only!increase!more!
ischemic!complications!such!as!coronary!artery!disease,!uncontrollable!
hypertension,!cerebrovascular!and!peripheral!vascular!disease,!and!etc.!Triptans!
are!only!effective!in!about!60%!of!migraine!patients!and!can!only!be!used!once!
symptoms!of!migraine!have!commenced.!Furthermore,!Ergots!have!been!
reported!to!have!severe!side!effects!such!as!hallucinations!and!can!unfortunately!
cause!miscarriage!in!pregnant!women.!Additionally,!for!chronic!migraineurs,!
medication!overuse!headache!tends!to!be!a!concerning!problem!and!provokes!
migraineKlike!headaches!that!are!no!longer!treatable.!In!contrast,!the!efficacy!and!
! 141!
tolerability!of!CGRP!monoclonal!antibodies!in!combination!with!their!low!side!
effects!in!clinical!trials!have!garnered!positive!reviews.!However,!use!of!the!
CGRP!antibody!also!raises!a!few!concerns!especially!since,!in!some!cases,!
CGRP!plays!a!protective!role!in!the!cardiovascular!system!and!blocking!CGRP!
may!be!detrimental!in!some!subpopulations!of!migraine!patients.!While!the!
attention!is!on!targeting!CGRP!for!drug!development,!it!is!still!necessary!to!
expand!our!understanding!of!CGRP!biology!and!its!sites!of!action!so!that!
therapeutics!can!be!engineered!to!minimize!off!target!effects.!!
In!this!thesis,!I!present!evidence!that!peripheral!and!central!CGRP!may!
have!a!clear!role!in!the!induction!of!migraineKlike!symptom!photophobia.!
Peripheral!CGRP!can!induce!light!aversiveness!via!endogenous!CGRP!
receptors!in!two!strains!of!wildKtype!mice,!CD1!and!C57BL/6J!with!habituation!to!
the!chamber!and!exposure!to!bright!light.!!Additionally,!peripheral!injection!of!
migraine!treatment!drug!Sumatriptan!and!preKtreatment!of!CGRP!monoclonal!
antibody!attenuates!CGRPKinduced!light!aversion!in!wildKtype!mice.!Consistent!
with!previous!reports!of!CGRP!affecting!motor!activity,!peripheral!injection!of!
CGRP!also!decreases!several!measures!of!motility!only!in!the!dark!zone.!This!is!
particularly!interesting!since,!in!humans,!movement!and!light!both!exacerbate!
headache!so!sufferers!often!seek!a!dark!place!such!as!a!bathroom!and!rest!for!
hours.!Furthermore,!we!report!that!there!were!not!any!differences!between!sexes!
in!responses!to!CGRP.!Since!fluctuations!in!the!estrous!cycle!are!important!
contributor!to!migraine!in!women,!further!studies!that!track!the!response!to!
CGRP!during!stages!of!the!estrous!cycle!are!needed!to!reveal!a!role!for!sex!
! 142!
hormones!in!this!assay.!Lastly,!using!mice!that!overexpress!the!obligatory!
component!of!the!CGRP!receptor,!hRAMP1,!in!the!nervous!system!we!report!
that!central,!but!not!peripheral,!CGRP!induces!light!aversion.!These!data!suggest!
that!central!and!peripheral!CGRP!have!distinct,!but!possibly!overlapping,!sites!of!
action!to!induce!migraineKlike!symptoms.!!
I!also!present!strong!evidence!for!a!role!for!the!vasculature,!specifically!
vasodilation,!in!migraine.!This!is!an!important!finding!since!it!reignites!the!
conversation!about!the!role!of!changes!in!vascular!tone!in!migraine.!For!several!
decades,!the!origin!of!migraine!pain!was!thought!to!just!be!vasodilation,!
however,!this!view!is!far!too!simplistic.!Unfortunately,!most!studies!have!
attempted!to!deem!the!vascular!contributions!neither!sufficient!nor!necessary.!I!
report!that!blocking!CGRPKinduced!vasodilation!with!two!potent!vasoconstrictors,!
phenylephrine!and!endothelinK1,!partially!blocks!CGRPKinduced!light!aversion,!
strongly!supporting!a!role!for!vasodilation!in!this!model.!However,!the!inability!of!
blocking!CGRPKinduced!vasodilation!to!completely!reverse!the!light!aversion,!
leaves!the!door!open!for!another!peripheral!site!of!action.!Another!likely!site!that!
may!contribute!to!peripheral!CGRPKinduced!light!aversion!is!mast!cells.!CGRP!
receptors!are!found!on!mast!cells!and!can!cause!degranulation!thus!causing!
plasma!extravasation!and!sensitizing!nearby!neurons.!Further!studies!that!
determine!the!contribution!of!these!cells!in!peripheral!CGRP!induced!light!
aversion!are!warranted.!!
Additionally,!using!mice!that!have!restricted!hRAMP1!expression!in!the!
vasculature,!I!also!report!that!CGRP!has!actions!on!vascular!smooth!muscle,!but!
! 143!
not!endothelial,!cells!to!induce!light!aversion.!This!further!confirms!a!role!of!
CGRPKinduced!vasodilation!in!this!assay!since!CGRP!is!known!to!induce!
vasodilation!in!an!endotheliumKindependent!manner.!Nevertheless,!contributions!
of!endothelial!cells!in!migraine!should!not!be!ruled!out.!Endothelial!and!smooth!
muscle!cell!activation!can!release!substances!that!can!also!sensitize!neurons.!
Further!investigations!into!the!role!of!endothelial!cells!in!migraine!should!
considered.!All!of!these!data!together!support!a!mechanism!whereby!peripheral!
and!central!actions!of!CGRP!intersect!to!cause!migraine!symptoms.!!
!
FUTURE!PERSPECTIVES!
Extracranial(arteries(in(CGRPMinduced(light(aversion(
! While!my!findings!in!Chapter!III!suggest!a!role!for!vasodilation!in!this!
model,!there!are!several!questions!that!remain.!I!report!that,!using!blood!
pressure!as!a!readout!for!vascular!tone,!CGRPKinduced!vasodilation!was!
pharmacologically!blocked!using!vasoconstrictors.!A!caveat!to!this!study!is!that!
the!blood!pressure!measurements!were!recorded!from!the!carotid!artery.!
Additional!experiments!should!assess!the!physiological!state!of!the!extracranial!
arteries!such!as!the!MMA!following!peripheral!administration!of!CGRP.!Additional!
experiments!should!also!address!whether!administration!of!the!vasoconstrictors!
phenylephrine!and!endothelinK1!blocks!CGRPKinduced!cranial!dilation.!Future!
studies!using!either!thinnedKskull!cranial!windows!or!nonKinvasive!ultra!highK
resolution!in!vivo!computed!tomography!imaging!of!mouse!intra!and!extracranial!
! 144!
vasculature!can!help!elucidate!whether!these!vessels!are!affected!by!CGRP!and!
contribute!the!effects!seen!in!the!light!aversion!assay.!!
!
The(role(of(mast(cells(in(CGRPMinduced(light(aversion(
! While!the!ability!of!blocking!CGRP!induced!vasodilation!to!partially!block!
light!aversion!suggest!a!role!for!the!vasculature,!the!inability!of!blocking!
vasodilation!to!completely!restore!the!time!in!light!to!baseline!levels!suggest!
another!peripheral!site!of!CGRP!action!may!contribute!to!this!behavior.!For!a!
while!now,!there!has!been!speculation!that!a!role!for!mast!cell!activation!in!
migraine!pathology!exists.!CGRP!receptors!have!been!reported!on!murine!dural!
mast!cells114!and!peripheral!release!of!CGRP!can!induce!mast!cell!
degranulation115,117.!Additionally,!duralKresident!mast!cells!lie!adjacent!to!vessels!
and!neurons!present!within!the!dura!mater!and!activation!can!release!vasoactive!
and!proKinflammatory!substances,!such!as!CGRP,!possibly!leading!to!a!positive!
feedback!loop!that!contributes!to!neurogenic!inflammation.!!
Compound!48/80,!a!potent!mast!cell!degranulator,!has!been!reported!to!
activate!meningeal!nociceptors!in!electrophysiological!recordings!and!increase!cK
fos!expression!in!the!trigeminal!nucleus!caudalis,!however,!nociceptor!activation!
resulting!from!mast!cell!degranulation!have!not!been!used!in!studies!that!use!in!
vivo!models!of!migraineKlike!behavior.!Future!studies!using!dural!application!of!
compound!48/80!in!light!aversion,!mechanical!allodynia,!mouse!grimace!scale!
assays!may!reveal!a!role!for!dural!mast!cell!degranulation!in!migraine.!
Additionally,!it!would!be!interesting!to!determine!if!peripheral!administration!of!
! 145!
CGRP!and!dural!application!of!compound!48/80!induce!an!additive!effect!in!light!
the!aversion!assay.!!!
Furthermore,!if!mast!cells!are!a!contributor!in!CGRPKinduced!light!
aversion,!it!would!be!beneficial!to!inhibit!mast!cell!degranulation.!A!report!from!
Oka!et!al.!determined!that!cromolyn,!a!mast!cell!stabilizer,!was!effective!in!rats,!
but!not!in!mice214.!However,!investigation!using!mast!cellKdeficient!mice,!
C57BL/6JKKitWMsh/WMsh,!and!peripheral!CGRP!can!further!reveal!another!
mechanism!of!CGRPKinduced!light!aversion.!Along!this!line,!if!mast!cells!indeed!
play!a!role!in!CGRPKinduced!light!aversion,!it!would!be!interesting!to!determine!if!
blocking!both!vasodilation!with!a!vasoconstrictor!and!the!dural!CGRP!receptor!
activation!with!Olcegepant!or!CGRP8K37,!CGRP!receptor!antagonists,!!or!mast!
cellKdeficient!mice!could!abolish!CGRPKprovoked!light!aversion.!Moreover,!it!has!
been!reported!that!CGRP!receptor!activation!on!duraKresident!mast!cells,!and!not!
peritoneal!mast!cells,!can!induce!release!of!histamine.!As!a!control,!it!might!
prove!useful!to!determine!if!peripheral!CGRP!increases!plasma!histamine!in!
mice!that!have!been!subjected!and!responded!to!treatment!in!the!light!aversion!
assay.!!
!
The(role(of(sex(hormones(in(CGRPMinduced(light(aversion(
In!my!studies,!I!was!unable!to!uncover!any!sex!differences!in!the!light!
aversion!assay.!Contrary!to!our!results,!migraine!is!a!disorder!that!afflicts!women!
three!times!more!than!men.!However,!one!caveat!to!our!studies!is!that!the!stage!
of!the!estrous!cycle!during!testing!was!unknown.!In!chapters!II!and!III,!I!have!
! 146!
noticed!that!female!mice!have!a!more!widespread!response!to!CGRP,!whereas!
male!mice!have!a!more!uniform!and!consistent!response!to!CGRP!in!the!light!
aversion!assay.!The!incidence!of!migraine!is!more!common!in!women!during!
their!reproductive!years!and!it!is!suggested!that!the!fluctuations!in!estrogen!and!
other!female!sex!hormones!play!a!role.!One!report!suggests!that!migraine!is!
significantly!associated!with!the!falling!of!estrogen!in!the!late!luteal/early!follicular!
phase!of!menses.!Contrarily,!it!has!been!reported!that!migraine!also!is!
significantly!associated!with!the!rising!of!estrogen!during!the!ovulation!phase.!
The!rodent!estrous!cycle!is!divided!into!4!stages—metestrus,!diestrus,!proestrus,!
estrus.!Future!studies!that!segregate!CGRPKinduced!responses!in!the!light!
aversion!assay!may!reveal!that!the!degree!of!light!aversion!is!possibly!
dependent!on!the!stage!of!the!estrus!cycle.!!
A!study!by!Dan!Levy!and!colleagues!reported!that!dural!mast!cell!density!
and!phenotype!can!fluctuate!during!the!estrous!cycle!in!rats215.!Furthermore,!
they!reported!that!administration!of!estradiol!and!progesterone,!mimicking!the!
proestrus!phase,!in!ovariectomized!rats!induced!a!significant!amount!of!dural!
mast!cell!degranulation.!It!would!be!interesting!to!determine!if!mice!that!are!in!the!
proestrus!stage!exhibit!a!greater!response!in!the!light!aversion!assay.!PostK
mortem!analysis!would!be!necessary!to!determine!mast!cell!phenotype!and!
percentage!of!degranulated!cells!following!CGRP!and!vehicle!treatment!in!mice!
in!the!proestrus!stage!after!light!aversion!testing.!!!
As!previously!mentioned!migraine!is!more!prevalent!in!women!that!are!in!
their!reproductive!years.!Interestingly,!during!pregnancy!women!have!a!lower!
! 147!
incidence!of!migraine!and!some!report!complete!abrogation!by!the!third!
trimester.!Unfortunately,!nearly!all!women!report!their!migraine!return!postK
partum.!There!are!several!changes!that!occur!during!pregnancy!that!may!
contribute!to!its!protective!effects!from!migraine.!One!major!change,!and!often!
overlooked,!is!that!the!estrogen!metabolites!switch!forms!during!pregnancy.!
Indeed,!estradiol,!the!normal!and!most!abundant!form!of!estrogen,!dramatically!
decreases!to!almost!undetectable!levels!and!estriol!becomes!the!dominant!form!
of!estrogen!by!the!third!trimester216,217.!!Additionally,!it!has!been!reported!that!
varying!estrogen!levels!have!been!shown!to!have!an!effect!on!trigeminal!CGRP!
release,!indicating!a!role!for!CGRP!and!estrogen!cross!talk112,113.!Future!studies!
that!incorporate!pregnant!dams,!ovariectomized!mice!with!estrogen!replacement,!
and!postKpartum!mice!following!injection!of!CGRP!may!uncover!a!role!for!sex!
hormones,!specifically!estrogens,!in!the!role!for!nociception!and!protection!in!
migraine.!!
"
"
"
"
"
"
"
"
"
! 148!
REFERENCES!
!
1! Lipton,!R.!B.,!Stewart,!W.!F.,!Diamond,!S.,!Diamond,!M.!L.!&!Reed,!M.!Prevalence!and!burden!of!migraine!in!the!United!States:!data!from!the!American!Migraine!Study!II.!Headache!41,!646K657!(2001).!
2! Lipton,!R.!B.,!Stewart,!W.!F.,!Sawyer,!J.!&!Edmeads,!J.!G.!Clinical!utility!of!an!instrument!assessing!migraine!disability:!the!Migraine!Disability!Assessment!(MIDAS)!questionnaire.!Headache!41,!854K861!(2001).!
3! Smitherman,!T.!A.,!Burch,!R.,!Sheikh,!H.!&!Loder,!E.!The!prevalence,!impact,!and!treatment!of!migraine!and!severe!headaches!in!the!United!States:!a!review!of!statistics!from!national!surveillance!studies.!Headache!53,!427K436,!doi:10.1111/head.12074!(2013).!
4! Buse,!D.!C.(et(al.!Chronic!migraine!prevalence,!disability,!and!sociodemographic!factors:!results!from!the!American!Migraine!Prevalence!and!Prevention!Study.!Headache!52,!1456K1470,!doi:10.1111/j.1526K4610.2012.02223.x!(2012).!
5! Manack,!A.!N.,!Buse,!D.!C.!&!Lipton,!R.!B.!Chronic!migraine:!epidemiology!and!disease!burden.!Curr(Pain(Headache(Rep!15,!70K78,!doi:10.1007/s11916K010K0157Kz!(2011).!
6! Silberstein,!S.!D.(et(al.!The!impact!of!migraine!on!daily!activities:!effect!of!topiramate!compared!with!placebo.!Curr(Med(Res(Opin!22,!1021K1029,!doi:10.1185/030079906X104731!(2006).!
7! Ottman,!R.,!Hong,!S.!&!Lipton,!R.!B.!Validity!of!family!history!data!on!severe!headache!and!migraine.!Neurology!43,!1954K1960!(1993).!
8! Silberstein,!S.!D.,!Lipton,!R.!B.!&!Sliwinski,!M.!Classification!of!daily!and!nearKdaily!headaches:!field!trial!of!revised!IHS!criteria.!Neurology!47,!871K875!(1996).!
9! Lipton,!R.!B.(et(al.!Migraine!prevalence,!disease!burden,!and!the!need!for!preventive!therapy.!Neurology!68,!343K349,!doi:10.1212/01.wnl.0000252808.97649.21!(2007).!
10! Messali,!A.(et(al.!Direct!and!Indirect!Costs!of!Chronic!and!Episodic!Migraine!in!the!United!States:!A!WebKBased!Survey.!Headache!56,!306K322,!doi:10.1111/head.12755!(2016).!
11! Dodick,!D.!W.!&!Gargus,!J.!J.!Why!migraines!strike.!Sci(Am!299,!56K63!(2008).!
12! Martin,!V.!T.!&!Behbehani,!M.!M.!Toward!a!rational!understanding!of!migraine!trigger!factors.!Med(Clin(North(Am!85,!911K941!(2001).!
13! AndressKRothrock,!D.,!King,!W.!&!Rothrock,!J.!An!analysis!of!migraine!triggers!in!a!clinicKbased!population.!Headache!50,!1366K1370,!doi:10.1111/j.1526K4610.2010.01753.x!(2010).!
14! Kelman,!L.!The!triggers!or!precipitants!of!the!acute!migraine!attack.!Cephalalgia!27,!394K402,!doi:CHA1303![pii]!10.1111/j.1468K2982.2007.01303.x!(2007).!
! 149!
15! MacGregor,!E.!A.,!Frith,!A.,!Ellis,!J.,!Aspinall,!L.!&!Hackshaw,!A.!Incidence!of!migraine!relative!to!menstrual!cycle!phases!of!rising!and!falling!estrogen.!Neurology!67,!2154K2158,!doi:10.1212/01.wnl.0000233888.18228.19!(2006).!
16! Macgregor,!E.!A.,!Rosenberg,!J.!D.!&!Kurth,!T.!SexKrelated!differences!in!epidemiological!and!clinicKbased!headache!studies.!Headache!51,!843K859,!doi:10.1111/j.1526K4610.2011.01904.x!(2011).!
17! Peterlin,!B.!L.,!Gupta,!S.,!Ward,!T.!N.!&!Macgregor,!A.!Sex!matters:!evaluating!sex!and!gender!in!migraine!and!headache!research.!Headache!51,!839K842,!doi:10.1111/j.1526K4610.2011.01900.x!(2011).!
18! Vetvik,!K.!G.!&!MacGregor,!E.!A.!Sex!differences!in!the!epidemiology,!clinical!features,!and!pathophysiology!of!migraine.!Lancet(Neurol!16,!76K87,!doi:10.1016/S1474K4422(16)30293K9!(2017).!
19! Bille,!B.!S.!Migraine!in!school!children.!A!study!of!the!incidence!and!shortKterm!prognosis,!and!a!clinical,!psychological!and!electroencephalographic!comparison!between!children!with!migraine!and!matched!controls.!Acta(Paediatr(Suppl!136,!1K151!(1962).!
20! Stewart,!W.!F.(et(al.!Cumulative!lifetime!migraine!incidence!in!women!and!men.!Cephalalgia!28,!1170K1178,!doi:10.1111/j.1468K2982.2008.01666.x!(2008).!
21! Sacco,!S.,!Ricci,!S.,!Degan,!D.!&!Carolei,!A.!Migraine!in!women:!the!role!of!hormones!and!their!impact!on!vascular!diseases.!The(journal(of(headache(and(pain!13,!177K189,!doi:10.1007/s10194K012K0424Ky!(2012).!
22! Olesen,!J.!Problem!areas!in!the!International!Classification!of!Headache!Disorders,!3rd!edition!(beta).!Cephalalgia!34,!1193K1199,!doi:10.1177/0333102414534327!(2014).!
23! Pavlovic,!J.!M.(et(al.!Sex!hormones!in!women!with!and!without!migraine:!Evidence!of!migraineKspecific!hormone!profiles.!Neurology!87,!49K56,!doi:10.1212/WNL.0000000000002798!(2016).!
24! Sulak,!P.,!Willis,!S.,!Kuehl,!T.,!Coffee,!A.!&!Clark,!J.!Headaches!and!oral!contraceptives:!impact!of!eliminating!the!standard!7Kday!placebo!interval.!Headache!47,!27K37,!doi:10.1111/j.1526K4610.2007.00650.x!(2007).!
25! Calhoun,!A.!H.!&!Hutchinson,!S.!Hormonal!therapies!for!menstrual!migraine.!Curr(Pain(Headache(Rep!13,!381K385!(2009).!
26! Maggioni,!F.,!Alessi,!C.,!Maggino,!T.!&!Zanchin,!G.!Headache!during!pregnancy.!Cephalalgia!17,!765K769,!doi:10.1046/j.1468K2982.1997.1707765.x!(1997).!
27! Granella,!F.(et(al.!Migraine!without!aura!and!reproductive!life!events:!a!clinical!epidemiological!study!in!1300!women.!Headache!33,!385K389!(1993).!
28! Sances,!G.(et(al.!Course!of!migraine!during!pregnancy!and!postpartum:!a!prospective!study.!Cephalalgia!23,!197K205,!doi:480![pii]!(2003).!
29! Aegidius,!K.,!Zwart,!J.!A.,!Hagen,!K.!&!Stovner,!L.!The!effect!of!pregnancy!and!parity!on!headache!prevalence:!the!HeadKHUNT!study.!Headache!49,!851K859,!doi:10.1111/j.1526K4610.2009.01438.x!(2009).!
! 150!
30! Kvisvik,!E.!V.,!Stovner,!L.!J.,!Helde,!G.,!Bovim,!G.!&!Linde,!M.!Headache!and!migraine!during!pregnancy!and!puerperium:!the!MIGRAKstudy.!J(Headache(Pain!12,!443K451,!doi:10.1007/s10194K011K0329K1!(2011).!
31! Messlinger,!K.!Migraine:!where!and!how!does!the!pain!originate?!Exp(Brain(Res!196,!179K193,!doi:10.1007/s00221K009K1756Ky!(2009).!
32! Shevel,!E.!The!extracranial!vascular!theory!of!migraineKKa!great!story!confirmed!by!the!facts.!Headache!51,!409K417,!doi:10.1111/j.1526K4610.2011.01844.x!(2011).!
33! Goadsby,!P.!J.!The!vascular!theory!of!migraineKKa!great!story!wrecked!by!the!facts.!Brain!132,!6K7,!doi:10.1093/brain/awn321!(2009).!
34! Isler,!H.!The!Galenic!tradition!and!migraine.!J(Hist(Neurosci!1,!227K233,!doi:10.1080/09647049209525535!(1992).!
35! TfeltKHansen,!P.!C.!&!Koehler,!P.!J.!History!of!the!use!of!ergotamine!and!dihydroergotamine!in!migraine!from!1906!and!onward.!Cephalalgia!28,!877K886,!doi:10.1111/j.1468K2982.2008.01578.x!(2008).!
36! Olesen,!J.,!Burstein,!R.,!Ashina,!M.!&!TfeltKHansen,!P.!Origin!of!pain!in!migraine:!evidence!for!peripheral!sensitisation.!Lancet(Neurol!8,!679K690,!doi:10.1016/S1474K4422(09)70090K0!(2009).!
37! Baun,!M.,!HayKSchmidt,!A.,!Edvinsson,!L.,!Olesen,!J.!&!JansenKOlesen,!I.!Pharmacological!characterization!and!expression!of!VIP!and!PACAP!receptors!in!isolated!cranial!arteries!of!the!rat.!Eur(J(Pharmacol!670,!186K194,!doi:10.1016/j.ejphar.2011.08.016!(2011).!
38! 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!endotheliumKderived!relaxing!factor!in!the!cat.!Neurosci(Lett!58,!213K217!(1985).!
39! JansenKOlesen,!I.,!Goadsby,!P.!J.,!Uddman,!R.!&!Edvinsson,!L.!Vasoactive!intestinal!peptide!(VIP)!like!peptides!in!the!cerebral!circulation!of!the!cat.!J(Auton(Nerv(Syst!49"Suppl,!S97K103!(1994).!
40! JansenKOlesen,!I.,!Mortensen,!A.!&!Edvinsson,!L.!Calcitonin!geneKrelated!peptide!is!released!from!capsaicinKsensitive!nerve!fibres!and!induces!vasodilatation!of!human!cerebral!arteries!concomitant!with!activation!of!adenylyl!cyclase.!Cephalalgia!16,!310K316!(1996).!
41! Uddman,!R.,!Edvinsson,!L.,!Ekblad,!E.,!Hakanson,!R.!&!Sundler,!F.!Calcitonin!geneKrelated!peptide!(CGRP):!perivascular!distribution!and!vasodilatory!effects.!Regul(Pept!15,!1K23!(1986).!
42! Uddman,!R.,!Goadsby,!P.!J.,!Jansen,!I.!&!Edvinsson,!L.!PACAP,!a!VIPKlike!peptide:!immunohistochemical!localization!and!effect!upon!cat!pial!arteries!and!cerebral!blood!flow.!J(Cereb(Blood(Flow(Metab!13,!291K297,!doi:10.1038/jcbfm.1993.36!(1993).!
43! Iversen,!H.!K.!&!Olesen,!J.!Headache!induced!by!a!nitric!oxide!donor!(nitroglycerin)!responds!to!sumatriptan.!A!human!model!for!development!of!migraine!drugs.!Cephalalgia!16,!412K418,!doi:10.1046/j.1468K2982.1996.1606412.x!(1996).!
! 151!
44! Thomsen,!L.!L.,!Iversen,!H.!K.!&!Olesen,!J.!Cerebral!blood!flow!velocities!are!reduced!during!attacks!of!unilateral!migraine!without!aura.!Cephalalgia!15,!109K116,!doi:10.1046/j.1468K2982.1995.015002109.x!(1995).!
45! Amin,!F.!M.(et(al.!Magnetic!resonance!angiography!of!intracranial!and!extracranial!arteries!in!patients!with!spontaneous!migraine!without!aura:!a!crossKsectional!study.!Lancet(Neurol!12,!454K461,!doi:10.1016/S1474K4422(13)70067KX!(2013).!
46! Drummond,!P.!D.!&!Lance,!J.!W.!Extracranial!vascular!changes!and!the!source!of!pain!in!migraine!headache.!Ann(Neurol!13,!32K37,!doi:10.1002/ana.410130108!(1983).!
47! Blau,!J.!N.!&!Dexter,!S.!L.!The!site!of!pain!origin!during!migraine!attacks.!Cephalalgia!1,!143K147,!doi:10.1046/j.1468K2982.1981.0103143.x!(1981).!
48! Elkind,!A.!H.,!Friedman,!A.!P.!&!Grossman,!J.!Cutaneous!Blood!Flow!in!Vascular!Headaches!of!the!Migraine!Type.!Neurology!14,!24K30!(1964).!
49! Raddant,!A.!C.!&!Russo,!A.!F.!Calcitonin!geneKrelated!peptide!in!migraine:!intersection!of!peripheral!inflammation!and!central!modulation.!Expert(Rev(Mol(Med!13,!e36,!doi:10.1017/S1462399411002067!(2011).!
50! Petersen,!K.!A.,!Birk,!S.,!Doods,!H.,!Edvinsson,!L.!&!Olesen,!J.!Inhibitory!effect!of!BIBN4096BS!on!cephalic!vasodilatation!induced!by!CGRP!or!transcranial!electrical!stimulation!in!the!rat.!Br(J(Pharmacol!143,!697K704!(2004).!
51! Gozalov,!A.,!JansenKOlesen,!I.,!Klaerke,!D.!&!Olesen,!J.!Role!of!BK(Ca)!channels!in!cephalic!vasodilation!induced!by!CGRP,!NO!and!transcranial!electrical!stimulation!in!the!rat.!Cephalalgia!27,!1120K1127,!doi:10.1111/j.1468K2982.2007.01409.x!(2007).!
52! Olesen,!J.,!Larsen,!B.!&!Lauritzen,!M.!Focal!hyperemia!followed!by!spreading!oligemia!and!impaired!activation!of!rCBF!in!classic!migraine.!Ann(Neurol!9,!344K352,!doi:10.1002/ana.410090406!(1981).!
53! Rahmann,!A.(et(al.!Vasoactive!intestinal!peptide!causes!marked!cephalic!vasodilation,!but!does!not!induce!migraine.!Cephalalgia!28,!226K236,!doi:10.1111/j.1468K2982.2007.01497.x!(2008).!
54! Tuncel,!N.,!Tore,!F.,!Sahinturk,!V.,!Ak,!D.!&!Tuncel,!M.!Vasoactive!intestinal!peptide!inhibits!degranulation!and!changes!granular!content!of!mast!cells:!a!potential!therapeutic!strategy!in!controlling!septic!shock.!Peptides!21,!81K89!(2000).!
55! Schytz,!H.!W.(et(al.!PACAP38!induces!migraineKlike!attacks!in!patients!with!migraine!without!aura.!Brain!132,!16K25,!doi:10.1093/brain/awn307!(2009).!
56! JansenKOlesen,!I.(et(al.!PACAPK38!but!not!VIP!induces!release!of!CGRP!from!trigeminal!nucleus!caudalis!via!a!receptor!distinct!from!the!PAC!receptor.!Neuropeptides,!doi:10.1016/j.npep.2014.01.004!(2014).!
57! Grebe,!H.!P.,!Nunes!da!Silva,!M.!J.!&!DiogoKSousa,!L.![Role!of!arterial!hypertension!in!comorbidity!of!chronic!headaches].!Rev(Neurol!33,!119K122!(2001).!
! 152!
58! Hagen,!K.(et(al.!Blood!pressure!and!risk!of!headache:!a!prospective!study!of!22!685!adults!in!Norway.!J(Neurol(Neurosurg(Psychiatry!72,!463K466!(2002).!
59! Hamed,!S.!A.,!Hamed,!E.!A.,!Ezz!Eldin,!A.!M.!&!Mahmoud,!N.!M.!Vascular!risk!factors,!endothelial!function,!and!carotid!thickness!in!patients!with!migraine:!relationship!to!atherosclerosis.!J(Stroke(Cerebrovasc(Dis!19,!92K103,!doi:10.1016/j.jstrokecerebrovasdis.2009.04.007!(2010).!
60! Low,!N.!C.!&!Merikangas,!K.!R.!The!comorbidity!of!migraine.!CNS(Spectr!8,!433K434,!437K444!(2003).!
61! Janeway,!H.!H.!Intratracheal!Anaesthesia:!A.!By!Nitrous!Oxide!and!Oxygen.!B.!By!Nitrous!Oxide!and!Oxygen!Under!Conditions!of!Differential!Pressure.!Ann(Surg!58,!927K933!(1913).!
62! Paterna,!S.(et(al.!AngiotensinKconverting!enzyme!gene!deletion!polymorphism!determines!an!increase!in!frequency!of!migraine!attacks!in!patients!suffering!from!migraine!without!aura.!Eur(Neurol!43,!133K136,!doi:8151!(2000).!
63! Schrader,!H.,!Stovner,!L.!J.,!Helde,!G.,!Sand,!T.!&!Bovim,!G.!Prophylactic!treatment!of!migraine!with!angiotensin!converting!enzyme!inhibitor!(lisinopril):!randomised,!placebo!controlled,!crossover!study.!BMJ!322,!19K22!(2001).!
64! Wiehe,!M.,!Fuchs,!S.!C.,!Moreira,!L.!B.,!Moraes,!R.!S.!&!Fuchs,!F.!D.!Migraine!is!more!frequent!in!individuals!with!optimal!and!normal!blood!pressure:!a!populationKbased!study.!J(Hypertens!20,!1303K1306!(2002).!
65! Tzourio,!C.,!Gagniere,!B.,!El!Amrani,!M.,!Alperovitch,!A.!&!Bousser,!M.!G.!Relationship!between!migraine,!blood!pressure!and!carotid!thickness.!A!populationKbased!study!in!the!elderly.!Cephalalgia!23,!914K920,!doi:10.1046/j.1468K2982.2003.00613.x!(2003).!
66! Secil,!Y.(et(al.!Blood!pressure!changes!in!migraine!patients!before,!during!and!after!migraine!attacks.!Pain(Pract!10,!222K227,!doi:10.1111/j.1533K2500.2009.00349.x!(2010).!
67! Humphrey,!P.!P.,!Feniuk,!W.,!Perren,!M.!J.,!Connor,!H.!E.!&!Oxford,!A.!W.!The!pharmacology!of!the!novel!5KHT1Klike!receptor!agonist,!GR43175.!Cephalalgia!9"Suppl"9,!23K33,!doi:10.1111/J.1468K2982.1989.TB00069.X!(1989).!
68! Knight,!Y.!E.,!Edvinsson,!L.!&!Goadsby,!P.!J.!4991W93!inhibits!release!of!calcitonin!geneKrelated!peptide!in!the!cat!but!only!at!doses!with!5HT(1B/1D)!receptor!agonist!activity?!Neuropharmacology!40,!520K525!(2001).!
69! Carmichael,!N.!M.,!Charlton,!M.!P.!&!Dostrovsky,!J.!O.!Activation!of!the!5KHT1B/D!receptor!reduces!hindlimb!neurogenic!inflammation!caused!by!sensory!nerve!stimulation!and!capsaicin.!Pain!134,!97K105,!doi:10.1016/j.pain.2007.03.037!(2008).!
70! Limmroth,!V.,!Kazarawa,!Z.,!Fritsche,!G.!&!Diener,!H.!C.!Headache!after!frequent!use!of!serotonin!agonists!zolmitriptan!and!naratriptan.!Lancet!353,!378!(1999).!
! 153!
71! Ferrari,!M.!D.,!Roon,!K.!I.,!Lipton,!R.!B.!&!Goadsby,!P.!J.!Oral!triptans!(serotonin!5KHT(1B/1D)!agonists)!in!acute!migraine!treatment:!a!metaKanalysis!of!53!trials.!Lancet!358,!1668K1675,!doi:10.1016/S0140K6736(01)06711K3!(2001).!
72! Nozaki,!K.,!Moskowitz,!M.!A.!&!Boccalini,!P.!CPK93,129,!sumatriptan,!dihydroergotamine!block!cKfos!expression!within!rat!trigeminal!nucleus!caudalis!caused!by!chemical!stimulation!of!the!meninges.!Br(J(Pharmacol!106,!409K415!(1992).!
73! Williamson,!D.!J.,!Hargreaves,!R.!J.,!Hill,!R.!G.!&!Shepheard,!S.!L.!Sumatriptan!inhibits!neurogenic!vasodilation!of!dural!blood!vessels!in!the!anaesthetized!ratKKintravital!microscope!studies.!Cephalalgia!17,!525K531!(1997).!
74! Jansen,!I.,!Edvinsson,!L.,!Mortensen,!A.!&!Olesen,!J.!Sumatriptan!is!a!potent!vasoconstrictor!of!human!dural!arteries!via!a!5KHT1Klike!receptor.!Cephalalgia(:(an(international(journal(of(headache!12,!202K205!(1992).!
75! Wackenfors,!A.,!Jarvius,!M.,!Ingemansson,!R.,!Edvinsson,!L.!&!Malmsjo,!M.!Triptans!induce!vasoconstriction!of!human!arteries!and!veins!from!the!thoracic!wall.!J(Cardiovasc(Pharmacol!45,!476K484!(2005).!
76! Buzzi,!M.!G.!&!Moskowitz,!M.!A.!The!antimigraine!drug,!sumatriptan!(GR43175),!selectively!blocks!neurogenic!plasma!extravasation!from!blood!vessels!in!dura!mater.!Br(J(Pharmacol!99,!202K206!(1990).!
77! Buzzi,!M.!G.,!Carter,!W.!B.,!Shimizu,!T.,!Heath,!H.,!3rd!&!Moskowitz,!M.!A.!Dihydroergotamine!and!sumatriptan!attenuate!levels!of!CGRP!in!plasma!in!rat!superior!sagittal!sinus!during!electrical!stimulation!of!the!trigeminal!ganglion.!Neuropharmacology!30,!1193K1200!(1991).!
78! Asghar,!M.!S.(et(al.!Dilation!by!CGRP!of!middle!meningeal!artery!and!reversal!by!sumatriptan!in!normal!volunteers.!Neurology!75,!1520K1526,!doi:10.1212/WNL.0b013e3181f9626a!(2010).!
79! Asghar,!M.!S.(et(al.!Evidence!for!a!vascular!factor!in!migraine.!Ann(Neurol!69,!635K645,!doi:10.1002/ana.22292!(2011).!
80! De!Felice,!M.(et(al.!TriptanKinduced!latent!sensitization:!a!possible!basis!for!medication!overuse!headache.!Ann(Neurol!67,!325K337,!doi:10.1002/ana.21897!(2010).!
81! Veitenhansl,!M.(et(al.!40th!EASD!Annual!Meeting!of!the!European!Association!for!the!Study!of!Diabetes!:!Munich,!Germany,!5K9!September!2004.!Diabetologia!47,!A1KA464,!doi:10.1007/BF03375463!(2004).!
82! Wolff,!H.!G.!Headache!mechanisms.!Bull(U(S(Army(Med(Dep!8,!641K653!(1948).!
83! Wolff,!H.!G.,!Marcussen,!R.!M.!&!Kunkle,!E.!C.!Studies!on!headacheh!analysis!of!the!contractile!state!of!the!cranial!vascular!tree!in!migraine.!Trans(Am(Neurol(Assoc!73,!14K17!(1948).!
84! Shevel,!E.!The!extracranial!vascular!theory!of!migraine:!an!artificial!controversy.!J(Neural(Transm((Vienna)!118,!525K530,!doi:10.1007/s00702K010K0517K1!(2011).!
85! Shevel,!E.!Migraine!painKKintracranial!or!extracranial?!Headache!47,!1458,!doi:10.1111/j.1526K4610.2007.00942.x!(2007).!
! 154!
86! Charles,!A.!Vasodilation!out!of!the!picture!as!a!cause!of!migraine!headache.!Lancet(Neurol!12,!419K420,!doi:10.1016/S1474K4422(13)70051K6!(2013).!
87! Hirata,!Y.(et(al.!Calcitonin!geneKrelated!peptide!receptor!in!cultured!vascular!smooth!muscle!and!endothelial!cells.!Biochem(Biophys(Res(Commun!151,!1113K1121!(1988).!
88! Eftekhari,!S.(et(al.!Differential!distribution!of!calcitonin!geneKrelated!peptide!and!its!receptor!components!in!the!human!trigeminal!ganglion.!Neuroscience!169,!683K696,!doi:10.1016/j.neuroscience.2010.05.016!(2010).!
89! Eftekhari,!S.,!Warfvinge,!K.,!Blixt,!F.!W.!&!Edvinsson,!L.!Differentiation!of!nerve!fibers!storing!CGRP!and!CGRP!receptors!in!the!peripheral!trigeminovascular!system.!J(Pain!14,!1289K1303,!doi:10.1016/j.jpain.2013.03.010!(2013).!
90! Tajti,!J.,!Uddman,!R.,!Moller,!S.,!Sundler,!F.!&!Edvinsson,!L.!Messenger!molecules!and!receptor!mRNA!in!the!human!trigeminal!ganglion.!J(Auton(Nerv(Syst!76,!176K183!(1999).!
91! Mulderry,!P.!K.(et(al.!Differential!expression!of!alphaKCGRP!and!betaKCGRP!by!primary!sensory!neurons!and!enteric!autonomic!neurons!of!the!rat.!Neuroscience!25,!195K205!(1988).!
92! Amara,!S.!G.(et(al.!Expression!in!brain!of!a!messenger!RNA!encoding!a!novel!neuropeptide!homologous!to!calcitonin!geneKrelated!peptide.!Science!229,!1094K1097!(1985).!
93! Russo,!A.!F.!&!Dickerson,!I.!M.!in!Handboook(Neurochem(Molec(Neurobiol!!Neuroactive(Proteins(and(Peptides!(ed!A.!Lajtha)!!391K426!(Springer,!2006).!
94! Poyner,!D.!R.!Calcitonin!geneKrelated!peptide:!multiple!actions,!multiple!receptors.!Pharmacol(Ther!56,!23K51!(1992).!
95! Poyner,!D.!R.(et(al.!International!Union!of!Pharmacology.!XXXII.!The!mammalian!calcitonin!geneKrelated!peptides,!adrenomedullin,!amylin,!and!calcitonin!receptors.!Pharmacol(Rev!54,!233K246!(2002).!
96! McLatchie,!L.!M.(et(al.!RAMPs!regulate!the!transport!and!ligand!specificity!of!the!calcitoninKreceptorKlike!receptor.!Nature!393,!333K339!(1998).!
97! Chakravarty,!P.(et(al.!CGRP!and!adrenomedullin!binding!correlates!with!transcript!levels!for!calcitonin!receptorKlike!receptor!(CRLR)!and!receptor!activity!modifying!proteins!(RAMPs)!in!rat!tissues.!Br(J(Pharmacol!130,!189K195,!doi:10.1038/sj.bjp.0702975!(2000).!
98! Archbold,!J.!K.,!Flanagan,!J.!U.,!Watkins,!H.!A.,!Gingell,!J.!J.!&!Hay,!D.!L.!Structural!insights!into!RAMP!modification!of!secretin!family!G!proteinKcoupled!receptors:!implications!for!drug!development.!Trends(Pharmacol(Sci!32,!591K600,!doi:10.1016/j.tips.2011.05.007!(2011).!
99! Zhang,!Z.,!Dickerson,!I.!M.!&!Russo,!A.!F.!Calcitonin!geneKrelated!peptide!receptor!activation!by!receptor!activityKmodifying!proteinK1!gene!transfer!to!vascular!smooth!muscle!cells.!Endocrinology!147,!1932K1940!(2006).!
!
! 155!
100! Zhang,!Z.,!Winborn,!C.!S.,!Marquez!de!Prado,!B.!&!Russo,!A.!F.!Sensitization!of!calcitonin!geneKrelated!peptide!receptors!by!receptor!activityKmodifying!proteinK1!in!the!trigeminal!ganglion.!J(Neurosci!27,!2693K2703!(2007).!
101! Russo,!A.!F.!Calcitonin!GeneKRelated!Peptide!(CGRP):!A!New!Target!for!Migraine.!Annual(review(of(pharmacology(and(toxicology!55,!533K552,!doi:10.1146/annurevKpharmtoxK010814K124701!(2015).!
102! Heroux,!M.,!Hogue,!M.,!Lemieux,!S.!&!Bouvier,!M.!Functional!calcitonin!geneKrelated!peptide!receptors!are!formed!by!the!asymmetric!assembly!of!a!calcitonin!receptorKlike!receptor!homoKoligomer!and!a!monomer!of!receptor!activityKmodifying!proteinK1.!J(Biol(Chem!282,!31610K31620,!doi:10.1074/jbc.M701790200!(2007).!
103! Walker,!C.!S.,!Conner,!A.!C.,!Poyner,!D.!R.!&!Hay,!D.!L.!Regulation!of!signal!transduction!by!calcitonin!geneKrelated!peptide!receptors.!Trends(Pharmacol(Sci!31,!476K483,!doi:10.1016/j.tips.2010.06.006!(2010).!
104! Walker,!C.!S.(et(al.!A!second!trigeminal!CGRP!receptor:!function!and!expression!of!the!AMY1!receptor.!Annals(of(Clinical(and(Translational(Neurology!2,!595K608!(2015).!
105! Hay,!D.!L.!&!Walker,!C.!S.!CGRP!and!its!receptors.!Headache!57,!625K636,!doi:10.1111/head.13064!(2017).!
106! Walker,!C.!S.!&!Hay,!D.!L.!CGRP!in!the!trigeminovascular!system:!a!role!for!CGRP,!adrenomedullin!and!amylin!receptors?!Br(J(Pharmacol!170,!1293K1307,!doi:10.1111/bph.12129!(2013).!
107! Rosenfeld,!M.!G.(et(al.!Production!of!a!novel!neuropeptide!encoded!by!the!calcitonin!gene!via!tissueKspecific!RNA!processing.!Nature!304,!129K135!(1983).!
108! Storer,!R.!J.,!Akerman,!S.!&!Goadsby,!P.!J.!Calcitonin!geneKrelated!peptide!(CGRP)!modulates!nociceptive!trigeminovascular!transmission!in!the!cat.!Br(J(Pharmacol!142,!1171K1181!(2004).!
109! Nuki,!C.,!Kawasaki,!H.,!Takasaki,!K.!&!Wada,!A.!Pharmacological!characterization!of!presynaptic!calcitonin!geneKrelated!peptide!(CGRP)!receptors!on!CGRPKcontaining!vasodilator!nerves!in!rat!mesenteric!resistance!vessels.!J(Pharmacol(Exp(Ther!268,!59K64!(1994).!
110! Recober,!A.!&!Russo,!A.!F.!Calcitonin!geneKrelated!peptide:!an!update!on!the!biology.!Curr(Opin(Neurol!22,!241K246!(2009).!
111! Brain,!S.!D.!&!Grant,!A.!D.!Vascular!actions!of!calcitonin!geneKrelated!peptide!and!adrenomedullin.!Physiol(Rev!84,!903K934!(2004).!
112! Gupta,!S.,!McCarson,!K.!E.,!Welch,!K.!M.!&!Berman,!N.!E.!Mechanisms!of!pain!modulation!by!sex!hormones!in!migraine.!Headache!51,!905K922,!doi:10.1111/j.1526K4610.2011.01908.x!(2011).!
113! Cairns,!B.!E.!The!influence!of!gender!and!sex!steroids!on!craniofacial!nociception.!Headache!47,!319K324,!doi:10.1111/j.1526K4610.2006.00708.x!(2007).!
!!
! 156!
114! Lennerz,!J.!K.(et(al.!Calcitonin!receptorKlike!receptor!(CLR),!receptor!activityKmodifying!protein!1!(RAMP1),!and!calcitonin!geneKrelated!peptide!(CGRP)!immunoreactivity!in!the!rat!trigeminovascular!system:!differences!between!peripheral!and!central!CGRP!receptor!distribution.!J(Comp(Neurol!507,!1277K1299!(2008).!
115! Ottosson,!A.!&!Edvinsson,!L.!Release!of!histamine!from!dural!mast!cells!by!substance!P!and!calcitonin!geneKrelated!peptide.!Cephalalgia!17,!166K174!(1997).!
116! Theoharides,!T.!C.!&!Kalogeromitros,!D.!The!critical!role!of!mast!cells!in!allergy!and!inflammation.!Ann(N(Y(Acad(Sci!1088,!78K99,!doi:1088/1/78![pii]!10.1196/annals.1366.025!(2006).!
117! Theoharides,!T.!C.,!Donelan,!J.,!KandereKGrzybowska,!K.!&!Konstantinidou,!A.!The!role!of!mast!cells!in!migraine!pathophysiology.!Brain(Res(Brain(Res(Rev!49,!65K76!(2005).!
118! Gu,!X.!L.!&!Yu,!L.!C.!The!colocalization!of!CGRP!receptor!and!AMPA!receptor!in!the!spinal!dorsal!horn!neuron!of!rat:!a!morphological!and!electrophysiological!study.!Neurosci(Lett!414,!237K241,!doi:10.1016/j.neulet.2006.12.056!(2007).!
119! Marquez!de!Prado,!B.,!Hammond,!D.!L.!&!Russo,!A.!F.!Genetic!enhancement!of!calcitonin!geneKrelated!peptideKinduced!central!sensitization!to!mechanical!stimuli!in!mice.!J(Pain!10,!992K1000,!doi:S1526K5900(09)00477K5![pii]!10.1016/j.jpain.2009.03.018!(2009).!
120! Goadsby,!P.!J.,!Edvinsson,!L.!&!Ekman,!R.!Vasoactive!peptide!release!in!the!extracerebral!circulation!of!humans!during!migraine!headache.!Ann(Neurol!28,!183K187!(1990).!
121! Ho,!T.!W.,!Edvinsson,!L.!&!Goadsby,!P.!J.!CGRP!and!its!receptors!provide!new!insights!into!migraine!pathophysiology.!Nat(Rev(Neurol!6,!573K582,!doi:nrneurol.2010.127![pii]!10.1038/nrneurol.2010.127!(2010).!
122! TfeltKHansen,!P.!&!Le,!H.!Calcitonin!geneKrelated!peptide!in!blood:!is!it!increased!in!the!external!jugular!vein!during!migraine!and!cluster!headache?!A!review.!J(Headache(Pain!10,!137K143,!doi:10.1007/s10194K009K0112K8!(2009).!
123! Ashina,!M.,!Bendtsen,!L.,!Jensen,!R.,!Schifter,!S.!&!Olesen,!J.!Evidence!for!increased!plasma!levels!of!calcitonin!geneKrelated!peptide!in!migraine!outside!of!attacks.!Pain!86,!133K138!(2000).!
124! Lassen,!L.!H.(et(al.!CGRP!may!play!a!causative!role!in!migraine.!Cephalalgia!22,!54K61!(2002).!
125! Hansen,!J.!M.!&!Ashina,!M.!Calcitonin!geneKrelated!peptide!and!migraine!with!aura:!A!systematic!review.!Cephalalgia!34,!695K707,!doi:10.1177/0333102413520084!(2014).!
!!
! 157!
126! Hansen,!J.!M.,!Thomsen,!L.!L.,!Olesen,!J.!&!Ashina,!M.!Calcitonin!geneKrelated!peptide!does!not!cause!migraine!attacks!in!patients!with!familial!hemiplegic!migraine.!Headache!51,!544K553,!doi:10.1111/j.1526K4610.2011.01861.x!(2011).!
127! Recober,!A.(et(al.!CGRPKinduced!photophobia!blocked!by!olcegepant!and!rizatriptan!in!a!transgenic!!migraine!model.!Cephalalgia!29,!1!(2009).!
128! Mason,!B.!N.(et(al.!Induction!of!migraineKlike!photophobic!behavior!in!mice!by!both!peripheral!and!central!CGRP!mechanisms.!J(Neurosci!37,!204K216,!doi:10.1523/JNEUROSCI.2967K16.2017!(2017).!
129! Recober,!A.,!Kaiser,!E.!A.,!Kuburas,!A.!&!Russo,!A.!F.!Induction!of!multiple!photophobic!behaviors!in!a!transgenic!mouse!sensitized!to!CGRP.!Neuropharmacology!58,!156K165,!doi:S0028K3908(09)00214K7![pii]!10.1016/j.neuropharm.2009.07.009!(2010).!
130! Kaiser,!E.!A.,!Kuburas,!A.,!Recober,!A.!&!Russo,!A.!F.!Modulation!of!CGRPKinduced!light!aversion!in!wildKtype!mice!by!a!5KHT(1B/D)!agonist.!J(Neurosci!32,!15439K15449,!doi:10.1523/JNEUROSCI.3265K12.2012!(2012).!
131! Goadsby,!P.!J.,!Lipton,!R.!B.!&!Ferrari,!M.!D.!MigraineKKcurrent!understanding!and!treatment.!N(Engl(J(Med!346,!257K270!(2002).!
132! Mulleners,!W.!M.(et(al.!SelfKreported!photophobic!symptoms!in!migraineurs!and!controls!are!reliable!and!predict!diagnostic!category!accurately.!Headache!41,!31K39,!doi:hed01004![pii]!(2001).!
133! Vanagaite,!J.(et(al.!LightKinduced!discomfort!and!pain!in!migraine.!Cephalalgia(:(an(international(journal(of(headache!17,!733K741!(1997).!
134! Okamoto,!K.,!Tashiro,!A.,!Chang,!Z.!&!Bereiter,!D.!A.!Bright!light!activates!a!trigeminal!nociceptive!pathway.!Pain!149,!235K242,!doi:10.1016/j.pain.2010.02.004!(2010).!
135! Okamoto,!K.,!Thompson,!R.,!Tashiro,!A.,!Chang,!Z.!&!Bereiter,!D.!A.!Bright!light!produces!FosKpositive!neurons!in!caudal!trigeminal!brainstem.!Neuroscience!160,!858K864,!doi:S0306K4522(09)00323K6![pii]!10.1016/j.neuroscience.2009.03.003!(2009).!
136! Drummond,!P.!D.!&!Woodhouse,!A.!Painful!stimulation!of!the!forehead!increases!photophobia!in!migraine!sufferers.!Cephalalgia!13,!321K324!(1993).!
137! Kowacs,!P.!A.(et(al.!Influence!of!intense!light!stimulation!on!trigeminal!and!cervical!pain!perception!thresholds.!Cephalalgia!21,!184K188,!doi:cha178![pii]!(2001).!
138! Boulloche,!N.(et(al.!Photophobia!in!migraine:!an!interictal!PET!study!of!cortical!hyperexcitability!and!its!modulation!by!pain.!J(Neurol(Neurosurg(Psychiatry!81,!978K984,!doi:10.1136/jnnp.2009.190223!(2010).!
139! Denuelle,!M.(et(al.!A!PET!study!of!photophobia!during!spontaneous!migraine!attacks.!Neurology!76,!213K218,!doi:10.1212/WNL.0b013e3182074a57!(2011).!
140! Winner,!P.(et(al.!A!randomized,!doubleKblind,!placeboKcontrolled!study!of!sumatriptan!nasal!spray!in!the!treatment!of!acute!migraine!in!adolescents.!Pediatrics!106,!989K997!(2000).!
! 158!
141! Russell,!F.!A.,!King,!R.,!Smillie,!S.!J.,!Kodji,!X.!&!Brain,!S.!D.!Calcitonin!geneKrelated!peptide:!physiology!and!pathophysiology.!Physiol(Rev!94,!1099K1142,!doi:10.1152/physrev.00034.2013!(2014).!
142! Durham,!P.!L.!&!Vause,!C.!V.!Calcitonin!geneKrelated!peptide!(CGRP)!receptor!antagonists!in!the!treatment!of!migraine.!CNS(Drugs!24,!539K548,!doi:10.2165/11534920K000000000K00000!(2010).!
143! Edvinsson,!L.!CGRP!blockers!in!migraine!therapy:!where!do!they!act?!Br(J(Pharmacol!155,!967K969,!doi:bjp2008346![pii]!10.1038/bjp.2008.346!(2008).!
144! Edvinsson,!L.!CGRPKreceptor!antagonism!in!migraine!treatment.!Lancet!372,!2089K2090,!doi:S0140K6736(08)61710K9![pii]!10.1016/S0140K6736(08)61710K9!(2008).!
145! Bigal,!M.!E.(et(al.!Safety,!tolerability,!and!efficacy!of!TEVK48125!for!preventive!treatment!of!highKfrequency!episodic!migraine:!a!multicentre,!randomised,!doubleKblind,!placeboKcontrolled,!phase!2b!study.!Lancet(Neurol!14,!1081K1090,!doi:10.1016/S1474K4422(15)00249K5!(2015).!
146! Bigal,!M.!E.(et(al.!Safety,!tolerability,!and!efficacy!of!TEVK48125!for!preventive!treatment!of!chronic!migraine:!a!multicentre,!randomised,!doubleKblind,!placeboKcontrolled,!phase!2b!study.!Lancet(Neurol!14,!1091K1100,!doi:10.1016/S1474K4422(15)00245K8!(2015).!
147! Dodick,!D.!W.(et(al.!Safety!and!efficacy!of!ALD403,!an!antibody!to!calcitonin!geneKrelated!peptide,!for!the!prevention!of!frequent!episodic!migraine:!a!randomised,!doubleKblind,!placeboKcontrolled,!exploratory!phase!2!trial.!Lancet(Neurol!13,!1100K1107,!doi:10.1016/S1474K4422(14)70209K1!(2014).!
148! Dodick,!D.!W.(et(al.!Safety!and!efficacy!of!LY2951742,!a!monoclonal!antibody!to!calcitonin!geneKrelated!peptide,!for!the!prevention!of!migraine:!a!phase!2,!randomised,!doubleKblind,!placeboKcontrolled!study.!Lancet(Neurol!13,!885K892,!doi:10.1016/S1474K4422(14)70128K0!(2014).!
149! Sun,!H.(et(al.!Safety!and!efficacy!of!AMG!334!for!prevention!of!episodic!migraine:!a!randomised,!doubleKblind,!placeboKcontrolled,!phase!2!trial.!Lancet(Neurol!15,!382K390,!doi:10.1016/S1474K4422(16)00019K3!(2016).!
150! Bigal,!M.!E.,!Walter,!S.!&!Rapoport,!A.!M.!Therapeutic!antibodies!against!CGRP!or!its!receptor.!British(journal(of(clinical(pharmacology!79,!886K895,!doi:10.1111/bcp.12591!(2015).!
151! Edvinsson,!L.!CGRP!receptor!antagonists!and!antibodies!against!CGRP!and!its!receptor!in!migraine!treatment.!British(journal(of(clinical(pharmacology!80,!193K199,!doi:10.1111/bcp.12618!(2015).!
152! Levy,!D.!Migraine!pain!and!nociceptor!activationKKwhere!do!we!stand?!Headache!50,!909K916,!doi:10.1111/j.1526K4610.2010.01670.x!(2010).!
153! Levy,!D.,!Burstein,!R.!&!Strassman,!A.!M.!Calcitonin!geneKrelated!peptide!does!not!excite!or!sensitize!meningeal!nociceptors:!implications!for!the!pathophysiology!of!migraine.!Ann(Neurol!58,!698K705!(2005).!
!!
! 159!
154! Fischer,!M.!J.,!Koulchitsky,!S.!&!Messlinger,!K.!The!nonpeptide!calcitonin!geneKrelated!peptide!receptor!antagonist!BIBN4096BS!lowers!the!activity!of!neurons!with!meningeal!input!in!the!rat!spinal!trigeminal!nucleus.!J(Neurosci!25,!5877K5883!(2005).!
155! Booe,!J.!M.(et(al.!Structural!Basis!for!Receptor!ActivityKModifying!ProteinKDependent!Selective!Peptide!Recognition!by!a!G!ProteinKCoupled!Receptor.!Mol(Cell!58,!1040K1052,!doi:10.1016/j.molcel.2015.04.018!(2015).!
156! Recober,!A.(et(al.!Role!of!calcitonin!geneKrelated!peptide!in!lightKaversive!behavior:!implications!for!migraine.!J(Neurosci!29,!8798K8804,!doi:29/27/8798![pii]!10.1523/JNEUROSCI.1727K09.2009!(2009).!
157! Crawley,!J.!&!Goodwin,!F.!K.!Preliminary!report!of!a!simple!animal!behavior!model!for!the!anxiolytic!effects!of!benzodiazepines.!Pharmacol(Biochem(Behav!13,!167K170!(1980).!
158! Sabharwal,!R.(et(al.!Receptor!activityKmodifying!protein!1!increases!baroreflex!sensitivity!and!attenuates!angiotensinKinduced!hypertension.!Hypertension,!doi:HYPERTENSIONAHA.109.148171![pii]!10.1161/HYPERTENSIONAHA.109.148171!(2010).!
159! Markovics,!A.(et(al.!Pituitary!adenylate!cyclaseKactivating!polypeptide!plays!a!key!role!in!nitroglycerolKinduced!trigeminovascular!activation!in!mice.!Neurobiology(of(Disease!45,!633K644,!doi:10.1016/j.nbd.2011.10.010!(2012).!
160! Botz,!B.(et(al.!Role!of!Pituitary!AdenylateKCyclase!Activating!Polypeptide!and!Tac1!gene!derived!tachykinins!in!sensory,!motor!and!vascular!functions!under!normal!and!neuropathic!conditions.!Peptides!43,!105K112,!doi:10.1016/j.peptides.2013.03.003!(2013).!
161! Loder,!E.!Triptan!therapy!in!migraine.!N(Engl(J(Med!363,!63K70,!doi:10.1056/NEJMct0910887!(2010).!
162! Hansen,!J.!M.,!Hauge,!A.!W.,!Olesen,!J.!&!Ashina,!M.!Calcitonin!geneKrelated!peptide!triggers!migraineKlike!attacks!in!patients!with!migraine!with!aura.!Cephalalgia!30,!1179K1186,!doi:10.1177/0333102410368444!(2010).!
163! Doods,!H.,!Arndt,!K.,!Rudolf,!K.!&!Just,!S.!CGRP!antagonists:!unravelling!the!role!of!CGRP!in!migraine.!Trends(Pharmacol(Sci!28,!580K587!(2007).!
164! Mogil,!J.!S.(et(al.!Variable!sensitivity!to!noxious!heat!is!mediated!by!differential!expression!of!the!CGRP!gene.!Proc(Natl(Acad(Sci(U(S(A!102,!12938K12943!(2005).!
165! Guo,!S.,!Vollesen,!A.!L.!H.,!Olesen,!J.!&!Ashina,!M.!Premonitory!and!nonKheadache!symptoms!induced!by!CGRP!and!PACAP38!in!migraine!patients.!Pain!In"Press,!1,!doi:10.1097/j.pain.0000000000000702!(2016).!
166! Ahn,!A.!H.!&!Basbaum,!A.!I.!Where!do!triptans!act!in!the!treatment!of!migraine?!Pain!115,!1K4,!doi:10.1016/j.pain.2005.03.008!(2005).!
167! Edvinsson,!L.!&!TfeltKHansen,!P.!The!bloodKbrain!barrier!in!migraine!treatment.!Cephalalgia!28,!1245K1258,!doi:CHA1675![pii]!10.1111/j.1468K2982.2008.01675.x!(2008).!
! 160!
168! Johnson,!M.!P.(et(al.!Peripheral!and!central!nervous!system!distribution!of!a!CGRP!neutralizing!antibody![125I]KLY2951742!in!male!rats.!Neurology!78!(2016).!
169! Seybold,!V.!S.!The!role!of!peptides!in!central!sensitization.!Handb(Exp(Pharmacol!194,!451K491,!doi:10.1007/978K3K540K79090K7_13!(2009).!
170! Han,!J.!S.,!Adwanikar,!H.,!Li,!Z.,!Ji,!G.!&!Neugebauer,!V.!Facilitation!of!synaptic!transmission!and!pain!responses!by!CGRP!in!the!amygdala!of!normal!rats.!Mol(Pain!6,!10,!doi:1744K8069K6K10![pii]!
10.1186/1744K8069K6K10!(2010).!171! Benarroch,!E.!E.!CGRP:!sensory!neuropeptide!with!multiple!neurologic!
implications.!Neurology!77,!281K287,!doi:10.1212/WNL.0b013e31822550e2!(2011).!
172! Moskowitz,!M.!A.!&!Macfarlane,!R.!Neurovascular!and!molecular!mechanisms!in!migraine!headaches.!Cerebrovasc(Brain(Metab(Rev!5,!159K177!(1993).!
173! Jacobs,!B.!&!Dussor,!G.!Neurovascular!contributions!to!migraine:!Moving!beyond!vasodilation.!Neuroscience!338,!130K144,!doi:10.1016/j.neuroscience.2016.06.012!(2016).!
174! Olesen,!J.!&!Ashina,!M.!Emerging!migraine!treatments!and!drug!targets.!Trends(Pharmacol(Sci!32,!352K359,!doi:10.1016/j.tips.2011.02.016!(2011).!
175! Goadsby,!P.!J.!Can!we!develop!neurally!acting!drugs!for!the!treatment!of!migraine?!Nat(Rev(Drug(Discov!4,!741K750!(2005).!
176! Buzzi,!M.!G.,!Bonamini,!M.!&!Moskowitz,!M.!A.!Neurogenic!model!of!migraine.!Cephalalgia!15,!277K280!(1995).!
177! BurgosKVega,!C.!C.,!Quigley,!L.!D.,!Avona,!A.,!Price,!T.!&!Dussor,!G.!Dural!stimulation!in!rats!causes!brainKderived!neurotrophic!factorKdependent!priming!to!subthreshold!stimuli!including!a!migraine!trigger.!Pain!157,!2722K2730,!doi:10.1097/j.pain.0000000000000692!(2016).!
178! Pietrobon,!D.!&!Striessnig,!J.!Neurobiology!of!migraine.!Nat(Rev(Neurosci!4,!386K398!(2003).!
179! Vause,!C.!V.!&!Durham,!P.!L.!Calcitonin!geneKrelated!peptide!differentially!regulates!gene!and!protein!expression!in!trigeminal!glia!cells:!findings!from!array!analysis.!Neurosci(Lett!473,!163K167,!doi:10.1016/j.neulet.2010.01.074!(2010).!
180! Sixt,!M.!L.,!Messlinger,!K.!&!Fischer,!M.!J.!Calcitonin!geneKrelated!peptide!receptor!antagonist!olcegepant!acts!in!the!spinal!trigeminal!nucleus.!Brain!132,!3134K3141,!doi:awp168![pii]!10.1093/brain/awp168!(2009).!
181! Li,!J.,!Vause,!C.!V.!&!Durham,!P.!L.!Calcitonin!geneKrelated!peptide!stimulation!of!nitric!oxide!synthesis!and!release!from!trigeminal!ganglion!glial!cells.!Brain(Res!1196,!22K32,!doi:S0006K8993(07)02963K0![pii]!10.1016/j.brainres.2007.12.028!(2008).!
182! McCulloch,!J.,!Uddman,!R.,!Kingman,!T.!A.!&!Edvinsson,!L.!Calcitonin!geneKrelated!peptide:!functional!role!in!cerebrovascular!regulation.!Proc(Natl(Acad(Sci(U(S(A!83,!5731K5735!(1986).!
! 161!
183! Dolgin,!E.!Antibody!drugs!set!to!revive!flagging!migraine!target.!Nature(reviews.(Drug(discovery!12,!249K250,!doi:10.1038/nrd3991!(2013).!
184! Mitsikostas,!D.!D.!&!Reuter,!U.!Calcitonin!geneKrelated!peptide!monoclonal!antibodies!for!migraine!prevention:!comparisons!across!randomized!controlled!studies.!Curr(Opin(Neurol!30,!272K280,!doi:10.1097/WCO.0000000000000438!(2017).!
185! Russo,!A.!F.!CGRP!as!a!neuropeptide!in!migraine:!lessons!from!mice.!British(journal(of(clinical(pharmacology!80,!403K414,!doi:10.1111/bcp.12686!(2015).!
186! Thompson,!S.(et(al.!Light!aversion!in!mice!depends!on!nonimageKforming!irradiance!detection.!Behav(Neurosci!124,!821K827,!doi:10.1037/a0021568!(2010).!
187! Zhang,!X.(et(al.!Activation!of!central!trigeminovascular!neurons!by!cortical!spreading!depression.!Ann(Neurol!69,!855K865,!doi:10.1002/ana.22329!(2011).!
188! Yamamura,!H.,!Malick,!A.,!Chamberlin,!N.!L.!&!Burstein,!R.!Cardiovascular!and!neuronal!responses!to!head!stimulation!reflect!central!sensitization!and!cutaneous!allodynia!in!a!rat!model!of!migraine.!J(Neurophysiol!81,!479K493!(1999).!
189! Malick,!A.!&!Burstein,!R.!Peripheral!and!central!sensitization!during!migraine.!Funct(Neurol!15"Suppl"3,!28K35!(2000).!
190! Bohn,!K.!J.(et(al.!CGRP!receptor!activity!in!mice!with!global!expression!of!human!receptor!activityKmodifying!proteinK1.!Br(J(Pharmacol,!doi:10.1111/bph.13783!(2017).!
191! Sabharwal,!R.(et(al.!Receptor!activityKmodifying!protein!1!increases!baroreflex!sensitivity!and!attenuates!angiotensinKinduced!hypertension.!Hypertension!55,!627K635,!doi:HYPERTENSIONAHA.109.148171![pii]!10.1161/HYPERTENSIONAHA.109.148171!(2010).!
192! EyesonKAnnan,!M.!L.,!Hirst,!L.!W.,!Battistutta,!D.!&!Green,!A.!Comparative!pupil!dilation!using!phenylephrine!alone!or!in!combination!with!tropicamide.!Ophthalmology!105,!726K732,!doi:10.1016/S0161K6420(98)94030K1!(1998).!
193! Ostrin,!L.!A.!&!Glasser,!A.!The!effects!of!phenylephrine!on!pupil!diameter!and!accommodation!in!rhesus!monkeys.!Invest(Ophthalmol(Vis(Sci!45,!215K221!(2004).!
194! La,!M.!&!Reid,!J.!J.!EndothelinK1!and!the!regulation!of!vascular!tone.!Clin(Exp(Pharmacol(Physiol!22,!315K323!(1995).!
195! Sandoo,!A.,!van!Zanten,!J.!J.,!Metsios,!G.!S.,!Carroll,!D.!&!Kitas,!G.!D.!The!endothelium!and!its!role!in!regulating!vascular!tone.!Open(Cardiovasc(Med(J!4,!302K312,!doi:10.2174/1874192401004010302!(2010).!
196! Cahill,!P.!A.(et(al.!Increased!expression!of!endothelin!receptors!in!the!vasculature!of!portal!hypertensive!rats:!role!in!splanchnic!hemodynamics.!Hepatology!28,!396K403,!doi:10.1002/hep.510280216!(1998).!
197! Antonaci,!F.,!Ghiotto,!N.,!Wu,!S.,!Pucci,!E.!&!Costa,!A.!Recent!advances!in!migraine!therapy.!Springerplus!5,!637,!doi:10.1186/s40064K016K2211K8!(2016).!
! 162!
198! Bolay,!H.(et(al.!Intrinsic!brain!activity!triggers!trigeminal!meningeal!afferents!in!a!migraine!model.!Nat(Med!8,!136K142!(2002).!
199! Moskowitz,!M.!A.!Neurogenic!inflammation!in!the!pathophysiology!and!treatment!of!migraine.!Neurology!43,!S16K20!(1993).!
200! Theoharides,!T.!C.!Mast!cells!and!migraines.!Perspect(Biol(Med!26,!672K675!(1983).!
201! ReynierKRebuffel,!A.!M.(et(al.!Substance!P,!calcitonin!geneKrelated!peptide,!and!capsaicin!release!serotonin!from!cerebrovascular!mast!cells.!Am(J(Physiol!267,!R1421K1429!(1994).!
202! Schwenger,!N.,!Dux,!M.,!de!Col,!R.,!Carr,!R.!&!Messlinger,!K.!Interaction!of!calcitonin!geneKrelated!peptide,!nitric!oxide!and!histamine!release!in!neurogenic!blood!flow!and!afferent!activation!in!the!rat!cranial!dura!mater.!Cephalalgia!27,!481K491,!doi:10.1111/j.1468K2982.2007.01321.x!(2007).!
203! Massaad,!C.!A.(et(al.!Involvement!of!substance!P,!CGRP!and!histamine!in!the!hyperalgesia!and!cytokine!upregulation!induced!by!intraplantar!injection!of!capsaicin!in!rats.!J(Neuroimmunol!153,!171K182!(2004).!
204! Hamed,!S.!A.!The!vascular!risk!associations!with!migraine:!relation!to!migraine!susceptibility!and!progression.!Atherosclerosis!205,!15K22,!doi:10.1016/j.atherosclerosis.2008.10.016!(2009).!
205! Vlachakis,!N.!D.,!Schiavi,!R.,!Mendlowitz,!M.,!De!Guia,!D.!&!Wolf,!R.!L.!Hypertension!and!anxiety.!Am(Heart(J!87,!518K526!(1974).!
206! Vlachakis,!N.!D.,!De!Guia,!D.,!Mendlowitz,!M.,!Antram,!S.!&!Wolf,!R.!L.!Hypertension!and!anxiety.!A!trial!with!epinephrine!and!norepinephrine!infusion.!Mt(Sinai(J(Med!41,!615K625!(1974).!
207! Alcantara,!C.(et(al.!Anxiety!sensitivity!and!medication!nonadherence!in!patients!with!uncontrolled!hypertension.!J(Psychosom(Res!77,!283K286,!doi:10.1016/j.jpsychores.2014.07.009!(2014).!
208! Li,!M.,!Maderdrut,!J.!L.,!Lertora,!J.!J.!&!Batuman,!V.!Intravenous!infusion!of!pituitary!adenylate!cyclaseKactivating!polypeptide!(PACAP)!in!a!patient!with!multiple!myeloma!and!myeloma!kidney:!a!case!study.!Peptides!28,!1891K1895,!doi:10.1016/j.peptides.2007.05.002!(2007).!
209! Boissard,!C.,!Marie,!J.!C.,!Hejblum,!G.,!Gespach,!C.!&!Rosselin,!G.!Vasoactive!intestinal!peptide!receptor!regulation!and!reversible!desensitization!in!human!colonic!carcinoma!cells!in!culture.!Cancer(Res!46,!4406K4413!(1986).!
210! Fang,!L.(et(al.!Calcitonin!geneKrelated!peptide!released!from!endothelial!progenitor!cells!inhibits!the!proliferation!of!rat!vascular!smooth!muscle!cells!induced!by!angiotensin!II.!Mol(Cell(Biochem!355,!99K108,!doi:10.1007/s11010K011K0843K0!(2011).!
211! El!Assar,!M.(et(al.!Differential!effect!of!amylin!on!endothelialKdependent!vasodilation!in!mesenteric!arteries!from!control!and!insulin!resistant!rats.!PLoS(One!10,!e0120479,!doi:10.1371/journal.pone.0120479!(2015).!
212! Nilsson,!F.,!Nilsson,!T.,!Edvinsson,!L.,!Bjorkman,!S.!&!Nordstrom,!C.!H.!Effects!of!dihydroergotamine!and!sumatriptan!on!isolated!human!cerebral!and!peripheral!arteries!and!veins.!Acta(Anaesthesiol(Scand!41,!1257K1262!(1997).!
! 163!
213! Edvinsson,!L.!Neurogenic!vs!vascular!mechanisms!of!sumatriptan!and!ergot!alkaloids!in!migraine.!Cephalalgia(:(an(international(journal(of(headache!18,!5!(1998).!
214! Oka,!T.,!Kalesnikoff,!J.,!Starkl,!P.,!Tsai,!M.!&!Galli,!S.!J.!Evidence!questioning!cromolyn's!effectiveness!and!selectivity!as!a!'mast!cell!stabilizer'!in!mice.!Lab(Invest!92,!1472K1482,!doi:10.1038/labinvest.2012.116!(2012).!
215! Boes,!T.!&!Levy,!D.!Influence!of!sex,!estrous!cycle,!and!estrogen!on!intracranial!dural!mast!cells.!Cephalalgia!32,!924K931,!doi:10.1177/0333102412454947!(2012).!
216! Mathur,!R.!S.,!Leaming,!A.!B.!&!Williamson,!H.!O.!An!assessment!of!the!total!estrone,!estradiolK17beta!and!estriol!in!high!risk!pregnancy!plasma.!J(Steroid(Biochem!6,!1421K1427!(1975).!
217! NevesKeKCastro,!M.!The!possible!role!of!high!estriol!levels!in!pregnancy.!Med(Hypotheses!1,!132K134!(1975).!
!
