Cerebral responses and role of the prefrontal cortex in conditioned pain modulation: an fMRI study in healthy subjects

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ContentslistsavailableatScienceDirect

Behavioural

Brain

Research

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b b r

Research

report

Cerebral

responses

and

role

of

the

prefrontal

cortex

in

conditioned

pain

modulation:

an

fMRI

study

in

healthy

subjects

Volodymyr

B. Bogdanov

a,b,∗

_,

_Alessandro

_Viganò

b,c

_,

_Quentin

_Noirhomme

d,e

_,

Olena

V. Bogdanova

f

_,

_Nathalie

_Guy

g

_,

_Steven

_Laureys

d,e

_,

_Perry

_F.

_Renshaw

f

_,

Radhouane

Dallel

g,1

_,

_Christophe

_Phillips

d,h,1

_,

_Jean

_Schoenen

b,1

a_INRA,_Nutrition_et_{Neurobiologie}_Intégrée_and_Bordeaux_Segalen_University,_UMR_1286,₁₄₆_rue_{Léo-Saignat,}_Bordeaux_Cedex,_33076,_France b_Headache_Research_Unit,_Department_of_Neurology,_CHR_Citadelle,_University_of_Liège,_Liège,_Belgium

c_SAMILAL-_Dept._Anatomy,_Histology,_Forensic_Medicine,_Orthopaedics_-_La_Sapienza,_University_of_Rome;_Dept._of_Neurology_and_Psychiatry_-_La_Sapienza,

UniversityofRome

d_Cyclotron_Research_Centre,_University_of_Liège,_Liège,_Belgium

e_Coma_Science_Group,_Department_of_Neurology,_University_and_University_Hospital_of_Liège,_Liège,_Belgium f_University_of_Utah,_Salt_Lake_City,_USA

g_Clermont_Université,_Université_{d’Auvergne,}_BP_10448,_F-63000_{Clermont-Ferrand;}_Inserm,_UMR1107,_Trigeminal_pain_and_Migraine_F-63000

Clermont-Ferrand;CHU,Clermont-Ferrand

h_Department_of_electrical_engineering_and_computer_science,_University_of_Liège,_Liège,_Belgium

h

i

g

h

l

i

g

h

t

s

•Themechanismsunderlyingconditionedpainmodulationaremultifaceted.

•Duringcold,application-speciﬁccerebralactivationswerefoundinprecuneusandleftinsula. •Conditionedsuppressionofinsulatest-painresponsewasrelatedtohypoalgesia.

•Theinsularchangesarepredictedbyearlyprefrontalresponsetocoldconditioning. •Smallerearlyprefrontalresponsepredictsheterotopicnoxioushyperalgesia.

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received27June2014

Receivedinrevisedform28October2014 Accepted18November2014

Availableonline25November2014 Keywords:

Brainimaging Prefrontalcortex

Conditionedpainmodulation

a

b

s

t

r

a

c

t

Themechanismsunderlyingconditionedpainmodulation(CPM)aremultifaceted.Wesearchedforalink betweenindividualdifferencesinprefrontalcortexactivityduringmulti-trialheterotopicnoxiouscold conditioningandmodulationofthecerebralresponsetophasicheatpain.

In24healthyfemalesubjects,weconditionedlaserheatstimulitothelefthandbyapplying alter-nativelyice-coldorlukewarmcompressestotherightfoot.Wecomparedpainratingswithcerebral fMRIBOLDresponses.WealsoanalyzedtherelationbetweenCPMandBOLDchangesproducedbythe heterotopiccoldconditioningitself,aswellastheimpactofanxietyandhabituationofcold-painratings. Speciﬁccerebralactivationwasidentiﬁedinprecuneusandleftposteriorinsula/SII,respectively,during earlyandsustainedphasesofcoldapplication.Duringcoldconditioning,laserpaindecreased(n=7), increased(n=10)orstayedunchanged(n=7).Attheindividuallevel,thepsychophysicaleffectwas directlyproportionaltothecold-inducedmodulationofthelaser-inducedBOLDresponseinleftposterior insula/SII.ThelattercorrelatedwiththeBOLDresponserecorded80searlierduringtheinitial10-sphase ofcoldapplicationinanteriorcingulate,orbitofrontalandlateralprefrontalcortices.

Abbreviations: ACC,anteriorcingulatecortex;BOLD,bloodoxygenationlevel-dependentresponse;fMRI,functionalmagneticresonanceimaging;FWER,family-wise errorrating;NRS,numericratingscale;OFC,orbitofrontalcortex;PAG,periaqueductalgraymatter;PFC,prefrontalcortex;SMA,supplementarymotorarea;SIandSII, primaryandsecondarysomatosensorycortices;VOI,volumeofinterest.

∗ Correspondingauthorat:BordeauxSegalenUniversity,146rueLéo-Saignat,BordeauxCedex33076,France.Tel.:+33557571226;fax:+33557571227. E-mailaddress:vlabogd@yahoo.com(V.B.Bogdanov).

1 _Last_authors_contributed_equally_to_the_supervision_of_the_study.

http://dx.doi.org/10.1016/j.bbr.2014.11.028

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1. Introduction

Endogenouspaincontrolisacomplexmulti-stepprocess com-posedofseveralinteractingneuronal systemsand mechanisms: stimulation-inducedanalgesia[1],diffusenoxiousinhibitory con-trols[2],ascendingnociceptivecontrol[3]andplaceboanalgesia [4],distractionordisengagement[5,6],stress-inducedanalgesia [7],and“offset”analgesia[8,9].

Diffuse noxious inhibitory controls (DNIC) are a form of supraspinal endogenous analgesia where phasic test pain responses are attenuated by heterotopic noxious conditioning stimuli.

Theterm“conditionedpainmodulation”(CPM)wasproposed todeﬁne thepsychophysicalparadigm totest DNIC [10,11]. In humans,theconditioningstimulusmodulatingtheresponsetothe teststimuluscanbepainfulornon-painfulandCPMcaninhibitor facilitatethisresponse[11].CPMcanbeassociatedwithchanges inneuronalactivityatmultiplesitesfromspinalcorddorsalhorn, medullarynucleusreticularisdorsalis,periaqueductalgraymatter [12–14]toprefrontalcortex[15,16]

UnderstandingthemechanismsinvolvedinCPMisimportant because it is impaired in several hyperalgesic conditions (see forareview[17]:tension-typeheadache[18],migraine[19,20], ﬁbromyalgia[21,22],irritablebowelsyndrome[23],osteoarthritis [24]andchronicpost-surgicalpain[25].

Functionalneuroimagingstudieshaveshownthatactivationsof primarysomatosensory(SI),prefrontal(PFC)oranteriorcingulate cortices (ACC) are associated with conditioned pain modula-tion [15,16,26]. The PFC, for instance, is activated in healthy controls by heterotopic cold stimulation during rectal pain [23,27]. Sustained cold-induced responses in orbitofrontal cor-tex (OFC)are linked tocold-induced analgesia [15]. Functional couplingbetweenthesubgenualanteriorcingulatecortex(ACC) and PAG is related to stronger individual heterotopic noxious analgesia[16].

Themechanisms of endogenous analgesia are clearly multi-facetedanddespitethenumerousstudiesinhealthysubjectsand inpathologicalconditions,thepreciseunderlyingneuralprocesses arestilluncertain.Moreover,painisahighlysubjectiveexperience andcanbeinﬂuencedbydifferencesinindividualsusceptibilityas wellaspersonality.Theaimofourstudywasthereforetosearchfor alinkbetweenindividualdifferencesinactivityofcerebralregions ofinterestduringmulti-trialheterotopicnoxiousconditioningand modulationoftheresponsestotestpainstimuli.

Toassessbrainactivity,weanalyzedfMRIBOLDresponses pha-siclaserheat-inducedtestpainwithorwithoutsustainednoxious coldconditioningandtheircorrelationwithself-ratingsofpain lev-els.Wefocusedonthemodulationoftest-painresponsesinthe so-called“painmatrix”[28]andthelinksbetweentheindividual differencesinCPMandprecedingorconcurrentneuronalresponses tonoxiouscoldconditioninginprefrontalareas(ACC,lateralPFC andOFC).

Contrarytotheabove-mentionedstudies,weusedarepeated block paradigm of noxious cold conditioning alternating with lukewarm application, which allows identifyingmore precisely

responsesinPFCtonoxiouscoldstimulationandreducesnovelty effects.

We expected that continuous cold pain stimulation would reduceself-ratingsoflaserheat-inducedpainandassociated cere-bralresponsesinthepainmatrix.InthoseareasofPFCknownfrom previousstudiestobeinvolvedindescendinganalgesia(ACC, lat-eralPFCandOFC),wesearchedforcold-inducedactivation,butalso forpossibledeactivation,asreportedinACC[29]andPFCareas[30]. WehypothesizedthatindividualdifferencesinPFCresponsesto coldpainwouldcorrelatewithlevelsofCPM.

2. Materialandmethods 2.1. Subjects

Twenty-fourhealthyfemalevolunteers(meanage:31.3years, range:24–45years)tookpartinthemainexperimentandfour subjectsparticipatedinapilotstudy.

Noneofthesubjectswassufferingfrompain,neurologicalor psychiatricdisorders.Allsubjectssignedaninformedconsentform thatmentionedthegeneralobjective,i.e.tostudytheimpactofa coldstimulusonpainandphysiologicalbrainresponsesinducedby laserheatstimuli,andwerethusnaïveaboutourworking hypothe-sis.Theyreceivedaﬁnancialcompensationfortravelexpensesand timespentinthelaboratory.TheEthicsCommitteeoftheUniversity ofLiègeapprovedthestudy.

2.2. Experimentalprotocol

Laserheatpulsesdeliveredtothedorsumofthelefthandserved aspainfulteststimuli,whereassustainednoxiouscoldappliedon therightfootwasusedastheconditioningstimulus.Tenblocks ofcoldstimulation(120s)alternatedwith11blocksoflukewarm stimulation(100s).Singlelaserstimuliweredeliveredonce40s beforetheendofeachblock(Fig.1).

Fortheconditioningstimulation,weusedanapproachsimilar tothatofSprengeretal.,whoused12smallbags(600goficeand 250mlofwater)tocoverthewholesurfaceofthelegandthefoot [16].Weappliedtwolargerbags(2L ofcrushediceand200ml ofwater)tothefootonly.Therewereseveralbagspreparedin advance.Theywerekeptinfoamboxeswhennotappliedtothe sub-ject.Besidescompressbagssomeadditionalicewasalwayspresent inthecontainer.Wecontrolledthetemperatureoftheicebags.In general,thetemperatureinCPMexperimentsisadjustedbya cir-culationcircuit(5–8◦C)[31,32]orastillicewaterbath(4–6◦C) [15,33]suchastobetolerable.Inourexperiment,thetemperature wasabout+2◦Canddidnotchangesigniﬁcantlythroughoutthe experiment.Thiswasachievedbyalargeproportionoficeinthe bagsandthestoragecontainer,whichmaintainedalow temper-atureduringthemeltingprocess.Ourmodeofcoldconditioning resultedinarelativelylesspowerfulnoxiousstimulation,butall oursubjectswereabletotoleraterepetitive2-minnoxiouscold applicationsduringtheentireexperimentalsession.

Thesameinvestigator(A.V.)manuallyappliedthecompresses overtherightfootofthesubjectsinthescannerroom.

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Fig.1.Experimentalprotocol.Continuouslukewarm+35◦_C_(W)_and_cold₂_±₂◦_C_(C)_stimulation_is_applied_to_the_right_foot_of_the_subjects._At_the_end_of_each_lukewarm_and

coldblock,asinglelaserpulse(L)isappliedtothedorsumofthelefthand.

Laserpulses(testpain)weregeneratedbyathuliumYAGlaser (BaaselLasertech,Starnberg,Germany)thatemitscalibrated near-infraredradiation(wavelength1.96m;spotdiameter5mm;pulse duration1ms,energy600mJ).Tofamiliarizethesubjectswiththe procedure,threelaserpulseswereadministeredbeforethe begin-ningoftheexperiment.ThisexperimentwasrealizedusingCogent 2000(Cogent2000teamandCogentGraphicsdevelopedbyJohn RomayaattheWelcomeDepartmentofImagingNeuroscience). 2.3. Behavioraldataacquisition

BeforethefMRIstudy,thesubjectsﬁlledintheFrenchversionof Spielberger’straitanxietyinventory[34,35].Theywereinformed thattheyshouldbeattentivetothelevelofpainproducedbythe heatstimulusonthehandtheywouldhavetorateafterthe exper-imentalsession. Painratingswereperformedinpenandpencil formatonanumericratingscale(NRS)from0for‘nopain’to10for ‘worstimaginablepain’.Painratingsforlaserstimuliwereobtained separately during lukewarm and cold conditioning.“Behavioral CPM”wasdeﬁnedasadifferenceofatleastonepoint between laser-painratingsduringcoldandlukewarmconditioning.

Subjectsratedcold-inducedpainforthe1standthelast con-ditioning, each time at onset and offset of the cold compress application.Theaverageofthesefourratingswastakenas mea-sureofcold-inducedpain.Habituationofcold-inducedpainwasthe differencebetweenratingsduringthe1standthelastapplication. 2.4. fMRIdataacquisitionandanalysis

MRIdatawereacquiredona3Thead-onlyscanner (Magne-tomAllegra,SiemensMedicalSolutions,Erlangen,Germany)using thestandard transmit-receivequadratureheadcoil.Astructural MRI was obtained in a separate session 20–30min beforethe functionalacquisition,usingahigh-resolutionT1-weightedimage [36]. Multislice T2*-weighted functional images were acquired with a gradient-echo echo-planar imaging sequence using an axial slice orientation and covering the whole brain (repeti-tion time=2040ms, echo time=30ms, flip angle=90◦, field of view=192×192mm,voxelsize3×3×3mm,34slices,25% inter-slicegap,matrixsize64×64×34).Thethreeinitialvolumeswere discardedtoavoidT1saturationeffects.Fieldmapdatawerealso acquiredtounwarpthefunctionalimages:acquisitiontime:TR/TE: 517/4.92&7.38ms, flip angle90◦, field of view: 220×220mm2_, resolution:64×64,voxelsize:3.4×3.4×3mm,slices=32(3mm thick,30%gap;bandwidth:260Hz/Px).

ThefMRI imageswereprocessed usingthe“Statistical Para-metric Mapping” software (SPM8; Wellcome Trust Centre for Neuroimaging,UniversityCollegeLondon,UK;http//www.ﬁl.ion. uce.ac.uk/spm)implementedinMATLAB(MathworksInc., Sher-born,MA,USA).Functionalimagetimeserieswerecorrectedfor

motionanddistortionusingthe“RealignandUnwarp”tool[37] togetherwiththeFieldmapToolbox[38].Thehigh-resolutionT1 image was co-registered with the functional images and seg-mentedintograymatter,whitematterandcerebrospinalﬂuid[39]. FunctionalimageswerespatiallynormalizedtotheMontreal Neu-rologicalInstitute(MNI)space(voxelsize:3×3×3mm)usingthe normalizationparametersobtainedfromthesegmentation pro-cedureand,subsequently,smoothedwithaGaussiankernelwith full-widthathalf-maximumof6mm.

2.5. Statistics

Dataanalysiswasperformedusingagenerallinearmodeland summarystatisticsapproach.

2.5.1. BOLDresponsestolaser-heatstimulationandtheir modulationbynoxiouscold

Atthefirstlevel,dataweresubjectedtohigh-passfilteringusing acut-offperiodof280s.Eachboxcarorimpulsestimulusfunction wasconvolvedwithacanonicalhemodynamicresponsefunction asimplementedinSPM8. Explicitmaskswerecreatedfromthe brainmaskimageavailableinSPM8.Thebinarymaskwascreated bythresholdingthebrainmaskimageatalevelof0.5intensity units.Thismaskfittedtheaverageanatomicalimageofoursubjects’ groupwell.

Preliminary experiments in four subjects with sustained cold/lukewarm stimulation onthe foot showed that the onset ofbothstimulationmodalitiesinducedhighamplitudebut tran-sientBOLDresponseswithmaximaindorsal SIcontralateral to thestimulatedside.Inordertodiscriminatethisearlyresponse, eachcoldconditionwasmodeledasthreesuccessiveblocks, last-ing10sfor“early”,10sfor“transitional”and100sfor“sustained” cold responses.For thesustainedlukewarm condition,weonly modeledthe1st10sfor“early”lukewarmandthe2nd10sfor “sec-ondary”lukewarmresponses.Thelaserresponsesweremodeledas instantaneouseventswithseparateregressorsforthoseoccurring duringcoldandthoseoccurringduringlukewarmstimulation.An autoregressivemodeloforder1,pluswhitenoise,accountedfor correlationoftheresiduals.

Aftermodelestimation,speciﬁceffectsweretestedwith appro-priatelinearcontrastsoftheparameterestimates,resultingina contrastvaluefor eachvoxel.We computedthefollowing con-trasts:(1)earlynon-speciﬁcBOLDresponsestolukewarmorcold applications; (2) early cold pain responses vs early lukewarm responses; (3) sustainedcold responses; (4) laser pain-induced responsesduringlukewarmcondition,asa measureofbaseline testpain-inducedcerebralchanges,(5)cold-inducedmodulationof laserresponses,i.e.changeofthelaser-inducedresponsesduring coldapplicationascomparedtothoseinthelukewarmcondition.

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Fig.2.Sequentialoutlineofthestatisticalanalysesusedinthestudyanddeﬁnitionofterms.

The contrast imageswere included in a second-level group analysisusingarandom-effectsapproach, treatinginter-subject variabilityasarandomfactor.Group-leveleffectsweretestedusing one-sidedt-tests.

2.5.2. CorrelationbetweenbehavioralCPMandmodulationof laserpain-inducedBOLDresponses

Therelationbetweennoxiouscoldconditioningoflaser-evoked cerebralresponsesandbehavioralCPMassessedwiththeNRSwas furthertestedinthesecond-levelgroupanalysis.Themodulation oflaserresponses(contrast5)andthebehavioralanalgesiaratings werecomparedinabetween-subjectcorrelationanalysis(Fig.2). Thisallowedﬁndingbrainareas where cold-induced changeof thelaserpain-inducedresponsewasdirectlycorrelatedwithpain ratings,i.e.behavioralCPM.Thisregionalbehaviorallycorrelated modulationoflaser-induced neuronalresponseswasdeﬁnedas “physiologicalCPM”.

2.5.3. Correlationbetweencold-inducedresponsesinprefrontal cortexandphysiologicalCPM

To verify our hypothesis on the relationship between cold-inducedphysiologicalCPMandcold-inducedresponsesinPFCareas, weanalyzedthecorrelationbetweenearly,sustained(negativeand positive)coldpainresponses(contrasts2and3)andphysiological CPM(seeFig.2).

Thevolumeofinterestwasderivedfromthestatisticalmapof physiologicalCPM.Clusterswereformedbythresholdingthevoxel statisticsatp<0.001uncorrected.Subsequently,thelargestcluster withinthe“painmatrix”wasbinarizedtocreateastudy-speciﬁc mask.ThismaskcoveredtheareaswherephysiologicalCPMwas observed.Theeigenvariatesofindividualcontrastimagesof cold-inducedmodulationoflaserresponses(contrast5)wereextracted fromabrainareaoutlinedwiththemaskofphysiologicalCPMand usedascovariateregressorsinfurthercorrelationanalyses,in par-ticular,inaone-samplet-testofindividualimagesofcontrasts2 and3(earlyandsustainedcoldresponses).

2.5.4. Signiﬁcancelevelanddirectedsearchforpainresponses andpainmodulation

First,weusedanundirectedsearch,lookingforresponseswith p<0.05familywiseerrorrate(FWER)correctedforthewhole-brain volumebothatvoxelandclusterlevels.Fortheclusterlevel infer-ence,wefirstuseda cluster-formingthresholdofp<0.001(not corrected).Significantresultsfromthisundirectedsearchare indi-catedinthetextandinthetablesby*WB(wholebrain).Hereandin otheranalyses,wetookintoaccountbothpeak-andcluster-level significanceandlabeledthemas*WB,respectively,nexttopeak coordinatesandclustersize.

Second,weperformedadirectedsearchfocusingonvolumesof interest(VOI).Weapplieda“smallvolumecorrection”inorderto obtainFWER-correctedp-valuesatbothvoxeland clusterlevels (withacluster-formingthresholdofp<0.001).“Painmatrix”VOI includedbrainstem,thalamus,striatumandcorticalareasknown torespondtonoxiousstimulation(seeSupplementaryMaterial1). Signiﬁcantresultsforlaserandcoldresponsesin“Painmatrix”VOI

werelabeledwith*PM.Whenalargeclusterencompassing multi-plebrainareaswasfound,itwasmaskedwiththepainmatrixto identifyspecificbrainareasincluded.WedefinedfivebilateralVOI includingPFCareasmostoftenreportedtobesourcesof descen-dingCPMeffects:medialOFC,lateralOFC,ACC,rostrallateralPFC andcaudallateralPFC(seeSupplementaryMaterial2).Thosefive masksofPFCVOIwereusedforcoldresponsesandthecorrelation analysisofcoldresponsesandphysiologicalCPM.Significantresults ofthisdirectedsearchwereindicatedwith*DS(directedsearch).

Finally,smallvolumecorrectionwasappliedtospheresaround physiologicalCPMpeakswitharadiusof10mm(seeTable2)to test whethertheindividualratingsofanxietyor across-session coldhabituationcouldinﬂuencetheabove-mentionedresponses. Signiﬁcantresultsoftheseanalyseswerelabeled*SV(small vol-ume).Pearson’scorrelationwasestimatedfortherelationbetween behavioralandphysiologicalvariables.

3. Results

3.1. BehavioralCPM

Althoughbothlaserstimuliandcoldapplicationproducedpain, thelatterwasperceivedasmorepainfulontheNRSthantheformer (5.6±0.4vs3.8±0.3,respectively,mean±SEM)(Fig.3).

Wefoundnosigniﬁcantdifferenceinaverageratingsof laser-inducedpainbetweenthelukewarmandcoldconditions,e.g.no signiﬁcantmean cold-induced behavioralCPM. Thiswasdue to thefactthat,attheindividuallevel,7subjectshadhypoalgesia duringcoldconditioning(NRSreductionof≥1),10subjectshad hyperalgesia(increase of ≥1) and 7 subjects had equal ratings oflaser-induced painduring thelukewarm and coldconditions (Fig.3).

3.2. CerebralBOLDresponses

3.2.1. Non-specificcerebralresponsestoconditioningstimuli Non-specific responses to the application on the right foot of eithercold orlukewarm compresseswere significant(FWER p<0.001voxellevel)in bilateral,but predominantly left,SI,SII, SMA,thalamus,insula, precuneus,ACC,MCCanddorsal premo-torcortex,aswellasin centralcerebellum,midbrain(including PAG),bilateralamygdala,predominantlyrighthippocampusand dorsolateralPFC.

Lesssigniﬁcantactivationwasalsofoundincaudatenucleus bilaterally, left putamen, frontal operculum and cuneus (FWER p<0.05voxellevel)(seeSupplementaryMaterial3).

3.2.2. Speciﬁccerebralresponsestoearlycoldstimulation

Atthewholebrainlevel,whenonlytheresponsetothefirst10s ofcoldstimulationwasconsideredandcontrastedtothatduring thefirst10soflukewarmstimulation,wefoundsignificant activa-tioninalargeclusterincludingbilateralprecuneus,dorsalposterior cingulateandpredominantlyleftoccipitalcortex(1259voxelsor 34cm3_,_p_<_0.005_FWER_cluster_level)₍_Table₁_A_and_Fig.₄_)._In addi-tion,threeothersignificantlylargeclusters(p<0.05FWERcluster

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Fig.3.Distributionofscoresonanumericratingscale(NRS,0-10)fornoxiouspain anditsmodulationbythermalconditioning.Thetotalnumberofsubjects(abscissa) is24.(A)Coldpainscores.(B)Laserpainscoresduringlukewarmstimulation.(C) Variationoflaserpainscoresduringnoxiouscoldstimulationexpressedasthe differencebetweenindividuallaserpainscoresduringlukewarmandcold con-ditioning.Positivevaluesrepresentcold-inducedanalgesiawhilenegativevalues indicatecold-inducedhyperalgesia.

level)encompassedlefttemporo-occipitalcortex,rightdorsal pre-motor/motorcortexandrightputamen/amygdala.

UsingthedirectedsearchwithinVOIofthe“painmatrix”,the precuneussignalwasconfirmedtobesignificant(p<0.05FWER voxellevel)and significantclusterswereidentified inpremotor cortexandventralposteriorcingulate(bothp<0.05FWERcluster level).InprefrontalVOI,therewerethreesmall,butsignificant clus-tersinmedialOFCandrightcaudo-lateralPFC(Table1AandFig.4).

Table1

Brainareasshowingaresponsetonoxiousstimuli.

Clustersize(kE) andclusterlevel signiﬁcance Peaklocation ([X,Y,Z]inMNI coordinates)and voxellevel signiﬁcance 1A.Earlynoxiouscold-inducedBOLDresponses

Precuneus [–3–7349]*PM

Temporo-occipitalcortex 152*WB [–57–70–75]

Dorsalpremotor/motorcortex 124*WB(#65*PM) [45558]

Caudalputamen,amygdala, hippocampus

119*WB [27–4–8]

Dorsalposteriorcingulate #72*WB [6–4046]

Ventralposteriorcingulate #68*PM [3–4316]

MedialOFC 7*DS [1247–11]*DS

8*DS [–929–17]*DS

Caudo-lateralPFC 10*DS [482328]*DS

1B.Sustainednoxiouscold-inducedBOLDresponses Whitematter,caudalcorona

radiata

15*WB [–21–2525]*WB

Caudatetailandwhitematter adjacenttoit

5*WB [18–1628]*WB

1*WB [–18–125]*WB

1*WB [–18–1328]*WB

Whitematter,spleniumof corpuscallosum

3*WB [12–3713]*WB

Posteriorinsula 4*WB [–33–2219]*WB

Largecaudalwhitematter cluster

3350*WB [–21–2525]*WB

Rostro-dorsalthalamus #102*WB [–12–1919]

1C.Sustainednegativenoxiouscold-inducedBOLDresponse

MedialOFC 11*DS [–1229–17]*DS

7*DS [335–17]

1D.Laserpain-inducedresponses

Insula/frontaloperculum 514*WB [3617–2]*WB 79*WB [–3020–5]*WB 17*WB [45–1616]*WB 18*WB [–39–7-2]*WB 2*WB [–30147]*WB 1*WB [42–710]*WB Premotor #29*WB [481119]*WB Amygdala #10*WB [18–4–14]*WB #4*WB [–21–4–14]*WB ventralputamen 64*WB [–158–11]*WB anteriorthalamus 28*WB [924]*WB PCC 26*WB [3–2231]*WB SII 25*WB [–60–2822]*WB 1*WB [63–2222]*WB ACC 19*WB [02025]*WB angularcortex 18*WB [–60–4943]*WB 4*WB [33–4943]*WB 6*WB [60–4037]*WB Dorso-lateralPFC 9*WB [48387]*WB Hippocampus 2*WB [–15–34-8]*WB #48*PM [–18–7–14]*WB Caudo-ventralthalamus 1*WB [6–284]*WB Thalamus 1*WB [12–1010]*WB SI 1*WB [54–1016]*WB OFC 1*WB [2729–17]*WB Precuneus 287*WB [12–6431]*PM Rostro-lateralPFC 255*WB [–483819] 81*WB [365331] SMA #215*WB [61161]*PM midbrain #231*WB [0–31–5]*PM

Middletemporalgyrus 60*WB [–60–587]

Cerebellum 52*WB [–18–73–32]

57*WB [12–91–26]

Caudatenucleus #8 [–984]*PM

#24 [957]*PM

Note:WB,wholebrainvolumecorrectedundirectedsearch,*PM,“painmatrix”VOI directedsearch;*DS,directedsearchatfivesmallerPFCandACCVOI.Ifindicated nexttoclustersizeornexttopeakcoordinatepointsatclusterlevelandvoxellevel FWER<0.05significance;#,inclusiveanatomicalmaskwasappliedtostatistical imageinordertoseparatesignificantresponseinasmallbrainareawhichisapart ofalargercluster.

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Fig.4. Cerebralresponsestoearly(shadedareaswithbackdots)andsustained(shadedareaswithwhitedots)cold-inducedpain.p<0.001uncorrectedmapsoverlaidon averageofindividualnormalizedstructuralT1images(n=24).Crosshairsarelocatedatpeaksofearlycold-painresponseinprecuneus(A),OFC(B),dorsolateralprefrontal cortex(C)andapeakofsustainedcoldpainresponseinposteriorinsula(D).VOIsofdirectedsearchareoutlinedwithgreyframes.

3.2.3. Speciﬁccerebralresponsestosustainedcoldstimulation

SustainedcoldstimulationinducedsustainedBOLDresponses

inonlyafewclustersmostlyextendingintoposteriorwhite

mat-ter(Table1B).Responsesweresignificantinposteriorinsulaand tailofcaudate(FWER<0.05 voxellevel).Thosepeakswerepart ofalargesignificantwhitemattercluster(30cm3_,_p_<_0.001_FWER clusterlevel)(Fig.4).Whenmaskingwiththe“painmatrix”VOI wasapplied,this30-cm3_cluster_included_a_significant_large_cluster inrostro-dorsalthalamus(p<0.01FWERclusterlevel)(Table1B). None of thedirected searches in prefrontal cortex VOI yielded significantsustained positiveresponsestocoldstimulation, but negativesustainedcold responses weredisclosedin mOFC VOI (Table1CandFig.5).

3.2.4. Laser-inducedcerebralresponsesduringlukewarm stimulation

Laserheatstimuliinducedasigniﬁcantresponseinmostareas ofthe“painmatrix”mask(seeSupplementaryMaterial1):insula, premotorcortex,amygdala,ventralputamen,anteriorthalamus, PCC,SII,ACC,and dorso-lateralPFC(peak levelFWERp<0.005) (Table1D).Outside of the“painmatrix”,there weresigniﬁcant laser-inducedresponsesbilaterallyinangulargyrusand cerebel-lum.

3.2.5. Changesinlaser-inducedcerebralresponsesduringcold stimulation

Theoveralldifferenceinresponsestolaserstimuliwithor with-outcontinuouscoldapplication(contrast 5)wasnotsigniﬁcant, whichwasduetothecontrastingCPMeffectbetweensubjects. Individualdifferencesincold-inducedbehavioralCPMscoreswere

infactproportionaltothecold-inducedchangeinlaser-induced responsesintherightanteriorinsularclusterpartiallyincluding whitematter,ventralpremotorcortexanddorsalstriatum.A sim-ilarrelationshipwasfoundinanotherclustercomprisingmostly

Fig.5. NegativeresponsestosustainednoxiouscoldstimulationinmedialOFC (out-linedwithwhite).Suprathreshodp<0.001uncorrectedT-maps.VOIsofdirected searchareoutlinedingrey.

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Table2

PhysiologicalCPM:Coldmodulationoflaser-inducedresponsescorrelatedwith behavioralCPM.

Clustersize(kE) andclusterlevel signiﬁcance Peaklocation ([X,Y,Z]inMNI coordinates)and voxellevel signiﬁcance Anteriorinsula/white matter/premotor cortex 122*WB [302616]*WB Posteriorinsula/SII 90**WB [–36–1619]*WB LateralOFC 64*WB [2414–20] Fusiform gyrus/parahippocampal 84**WB [–21–52–11] Posteriorinsula/SII 58**PM [–36–1619]*PM

Note:seeTable1.

graymatterwithintheanatomicallydeﬁned“painmatrix”mask: leftposteriorinsulaandSII.Thereweretwoothersigniﬁcantbut smallerclustersinlateralOFCandfusiformgyrus/parahippocampal cortex(Table2).However,whenwe appliedsmallvolume cor-rectionstothe“painmatrix”VOI,onlytheleftposteriorinsula/SII clustersurviveddirectedsearch(Table2andFig.6).

We used therefore the insula/SII cluster in the “pain matrix” (cluster size=58 voxels) to extract the eigenvariates of cold-inducedmodulationof laser responses(contrast 5). The cold-induced change of laser-induced responses in this cluster was directlycorrelated with individual behavioral CPM ratings. As mentioned, we called “physiological CPM” this concordant behavioral-BOLDresponsemodulation(Fig.2).

3.2.6. Correlationwithspeciﬁccold-inducedprefrontalcortex responses

ThephysiologicalCPMparameterwasusedasacovariate regres-sorinaone-samplet-testofindividualimagesofcontrasts2and3 (earlyandsustainedcoldresponses).Thistestedthehypothesis thataspeciﬁcpatternofcold-inducedresponsesinPFCpreceeds subsequentphysiologicalCPMandhencemayexplaintheindividual differencesinCPM(seeFig.2fortheanalysisﬂow).When behav-ioralCPMratingswereincludedasacovariate,weobtainedsimilar

Fig.6.CerebralareasthatwereactivatedbytheLaser-teststimulationduring luke-warm,i.e.control,conditioning(shadedareawithblackdots)orsustainedcold conditioning(shadedareawithlight-greydots)andareaswherecold-induced inhi-bitionoflaserresponsescovariatedwithindividualanalgesiaratings(“physiological analgesia”,outlinedinwhite).p<0.001uncorrectedmapsareshownona sag-ittalsectionofaverageofindividualnormalizedstructuralT1images(n=24)at X=−36mmMNI.Crosshairlocationisatthemaximumofgreencontrast([–36–16 19]*PM).

Table3

Brainareaswherecold-inducedBOLDresponsesco-variatewithphysiologicalCPM (sourceofthephysiologicalCPMcovariateinleftinsula/SII).

Clustersizeand clusterlevel signiﬁcance

Peaklocation ([X,Y,Z]inMNI coordinates) andvoxellevel signiﬁcance 3A.Earlycold-inducedBOLDresponsesrelatedtophysiologicalCPM

Dorso-lateralPFC 29*WB [–155025]*WB 4*WB [–212531]*WB RostralACC 18*WB [94410]*WB 3*WB [–94116]*WB CaudalACC 12*WB [–122931]*WB Anterior-laterallingual/medial fusiformgyrus 11*WB [–24–55-2]*WB MedialPFC 8*WB [65616]*WB

Whitematter,caudalcorpus callosum

8*WB [–15–1331]*WB

Caudatehead 7*WB [–15201]*WB

Whitematterprecentralgyrus 4*WB [–36–728]*WB

Caudo-lateralOFC/rostralinsula 3*WB [–2723–14]*WB

1*WB [–3029–11]*WB 1*WB [–2411–17]WB Ventralstriatum/accumbens 2*WB [68–14]*WB SubgenualACC 2*WB [026–8]*WB Putamen 2*WB [2111–8]*WB 1*WB [27510]*WB Frontalpole 1*WB [186513]*WB 1*WB [–275910]*WB ACC 223*DS [94410]*DS Rostro-lateralPFC 178*DS [–155322]*DS MedialOFC 68*DS [635–2]*DS 11*DS [–944–8]*DS Caudo-lateralPFC 26*DS [452022]*DS LateralOFC 20*DS [–3029–11]*DS 4 [3929–17]*DS

3B.Positivesustainedcold-inducedBOLDresponsesrelatedto physiologicalCPM

Whitematter,internalcapsula, coronaradiata,putamen,insula

172*WB [–18114]

Cerebellum 146*WB [33–58–38]

Whitematteranteriorcorona radiata,adjacenttorostro-lateral PFC

117*WB [243816]

Rostro-lateralPFC 17*DS [274710]

3C.Negativesustainedcold-inducedBOLDresponsesrelatedto physiologicalCPM

LateralOFC,whitemattercorona radiata,insula,ACC,rostro-lateral PFC 229*WB [–3023–17] Cerebellum 83*WB [21–64–41] 64*WB [36–73–75] 73*WB [–27–64–41] Rostro-lateralPFC 41*DS [–244716]*DS LateralOFC 24*DS [–3023–17]*DS ACC 8*DS [–122919]*DS 5*DS [–124422] Caudo-lateralPFC 23*DS [453516]*DS

Note:seeTable1.

thoughlesssigniﬁcantresultsthatarenotpresentedbecausewe considerthemredundant.

EarlycoldresponsesindorsolateralandmedialPFC,ACC, lin-gual/fusiformgyrus,whitematteratprecentralgyrusandcaudal corpus callosum, caudo-lateral OFC/rostral insula, striatum and frontalpoleweredirectlycorrelatedwithphysiologicalCPM(FWER p<0.05 voxel level) (Table 2A and Fig.7). We also observed a large (64cm3₎ _signiﬁcant _cluster _(FWER _p_<_0.05 _cluster _level) that included perigenual ACC, rostral bilateral ACC, left lateral OFC,frontaloperculum, DLPFC,posteriorACC and headof cau-dateaswellasrightdorso-medialPFCandfrontalpole(Table3A

andFig.7).

ThedirectedsearchwithinﬁveVOIindicatedthatphysiological CPMwassigniﬁcantlypredictedbyearlycold-inducedresponses

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Fig.7. Positiveresponsestoearly(outlinedwithwhiteframes)andnegativeresponsestosustainedcold-pain(greyshading)covariatingwith“physiologicalanalgesia”in posteriorinsula/SII.p<0.001uncorrectedmapsareshown.VOIsofdirectedsearchareoutlinedwithblackframes.Greatercold-inducedanalgesiaisrelatedtopositiveearly ornegativesustainedcold-painresponsesinACC,OFCandrostro-lateralPFC.

inallﬁveVOI(medialandlateralOFC,ACC,leftrostralandright caudallateralPFC)(Table3A).

AsillustratedinFig.8formedialsubgenualACC,rostralACCand lateralOFCvoxels,BOLDresponseswerepositiveduringtheearly phaseofcoldconditioninginsubjectswhobecamehypoalgesicand hadreducedlaser-inducedactivationinleftposteriorinsula/SII,but negative(orlesspositive)inthosewhodevelopedhyperalgesia. Inthoseareastherewasaclearpositivecorrelationbetweenthe hemodynamicresponsesandphysiologicalCPM,asshowninFig.9 forthesubgenualACCVOI.

The correlationbetween the cerebralresponses during sus-tainedcoldconditioningandphysiologicalCPMweremorecomplex andvariablebetweenareas.Activationduringsustainedcold con-ditioning was positively related to physiological CPM in a few areasincludingcerebellum,coronaradiata,internalcapsula,dorsal putamenandinsula,andawhitematterclustercomprising ante-riorcoronaradiataandadjacentrostro-lateralPFC.(FWERp<0.05 clusterlevel).Thedirectedsearchconﬁrmeda singlesigniﬁcant cluster(FWERp<0.05clusterlevel)inrightrostro-lateralPFCVOI (Table3B).

By contrast, there was a negative correlation of sustained cold-inducedbrainactivitywithphysiologicalCPMinmostofthe prefrontalVOI(Table3C).Withthedirectedsearch,deactivation wassignificantlyrelatedtohypoalgesiaduringcoldconditioning inlateralOFC,ACC,leftrostro-lateralandrightcaudo-lateralPFC (Table3C).Atthewholebrainlevel,therewasasignificant clus-ter(6cm3_,_FWER_p_<_0.05_cluster_level)_including_lateral_OFC,_white matteranddorsolateralPFC(seeFig.7),aswellasthreesignificant clustersincerebellum.

3.3. Correlationswithanxietyandhabituation

Themean(trait)anxietyscoreinoursubjectswas36±1,range 26–51.Greatertraitanxietyscorescorrelatedoverallwith behav-ioralhyperalgesiaduringCPM(r=−0.46,p<0.05).

Accordingtotheretrospectivepainrating,therewasaslight decrease in cold pain during the application (from 6.0_±0.5 to 5.1±0.4)andbetweentheﬁrstapplicationandthelastapplication (from6.7±0.4to4.5±0.4).CPMcorrelatedpositivelywith habit-uationofcoldpain,i.e.thedeclineofperceivedpaininthefootat theendoftheexperiment,(r=+0.49,p<0.05)andnegativelywith coldpainratingsattheendofthecompressapplication(r=−0.45, p<0.05).

Habituationofcoldpainratingscorrelatedwithcold-induced responsesinareasrelatedtophysiologicalCPM.Inparticular,like thelatter,habituationwasrelatedtoearlycold-inducedactivation inrostro-medialPFC[–66522],mOFC/sgACC[–1229–8]and cere-bellum[3–73–17](FWERp<0.05,clusterlevel)andothercluster inmOFCVOIconﬁrmedbydirectedsearch[632–14].Therewas,in addition,amedulla/caudalponscluster[6–40–35]wherenegative responsestosustainedcoldcorrelatedwithgreaterhabituationto cold(FWERp<0.05,clusterlevel).

4. Discussion

Theobjectiveofourstudywastobetterunderstandthe neu-ronalmechanismsof conditionedpainmodulation(CPM) using theheterotopicnoxious stimulationparadigm.Forthis purpose,

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Fig.8.AveragetimeseriesofBOLDsignalsinprefrontalareaswhereearlycoldresponsescorrelatewith“physiologicalCPM”.VOIare6mmradiusspheresaroundthepeaks atmedialsubgenualanteriorcingulatecortex(sgACC[026−8]),anteriorcingulatecortex(ACC[94410])andlateralorbitofrontalcortex(lOFC[−3029−11])(Table3A). Eigenvariateswereextracted,normalizedtothemedianvalueofagiventimeseries,epochedandindividuallyaveragedforcoldleft)andlukewarm(right)conditionand thegroupaveragewasestimated.Thecriteriafordeﬁning“physiologicalanalgesia”(dottedline)and“physiologicalhyperalgesia”(solidline)groupswereupperandlower quartilesofthephysiologicalanalgesiaparameterestimateextractedfromposteriorleftinsula/SIIVOI,anareawithbehaviorally-correlatedmodulationoflaser-induced neuronalresponses(seemethods2.5.2andresults3.2.5).

wesearchedwhetherindividualdifferencesbetweenconditioned modulationofbehavioralandcerebralresponsestothetestheat painwererelatedtothecerebralresponsesinducedbythecold conditioningstimulation.

4.1. Behavioralpainratings

Onaverage,overthe10consecutivetrials,therewasno signif-icantdecreaseinlaserpainratingsduringtheconditioningcold stimulation.FacilitatoryCPM(i.e.hyperalgesia)wasobservedin otherstudies,thoughoverallinasmallerproportionofsubjects. Inpre-operativepain-freepatients,facilitatoryCPMpredicts sub-sequentpost-surgerychronicpain[40]andhigheranalgesic con-sumption[41].ThelikelihoodtohavefacilitatoryCPMofaheattest stimulusbytourniquetischemicnoxiousconditioningwashigher insubjectswithlow5-HTTexpression[42].Inourstudy,only29% ofsubjectshadpainreduction,while29%hadnochangeand42% increasedlaserpain,clearlydemonstratinginter-individual differ-ences.ThiscontrastswithotherCPMstudies[15,16]andcouldhave severalnon-mutuallyexclusiveexplanations.

ThedegreeofCPMvariesbetweenstudiesanddependsonthe studydesign. For instance,CPM wasmarkedly greater(50%) in Pichéetal.[15]thaninSprengeretal.(20%)[16].Inourstudy,the conditioningcoldstimulationwasappliedrepeatedlycomparedto

a singlecounterstimulation inthetwopreviousstudies[15,16]. Individualdifferencesinhabituationtorepeatednoxiousstimuli maythusplayaroleinourparadigm,themoresothatwefound a positivecorrelationbetweenhabituation ofcold-inducedpain acrosssessionsand cold-inducedlaserpainreduction.Testpain producedbythetrainsofelectricshockslikeinPichéetal.’s pro-tocolmaybemoreamenabletoabehavioralCPMeffectthanpain inducedbyathermalstimuluswithsloweronsetandoffsetslopes usedinSprengeretal.’s[16]andinourstudy.Also,oursubjects ratedtheteststimulusaslesspainful(3.8/10NRS)comparedto thoseoftheabove-mentionedstudies.Thismayhavecontributed tothelackofconditionedhypoalgesiainsomesubjects,astheCPM effectcan beproportionaltothetest pain intensity[43]. How-ever,itisknownthatmoderatelypainfulteststimuli[15,44](about 40–50/100VAS),andevenonlyslightlypainfulones[31]are sufﬁ-cienttoobtainreliableCPM.

Othermethodologicalissues arediscussedin Supplementary Material4.

Besides methodological aspects related to the experimental design, psychobehavioral factors might contribute to the large interindividualvariabilitywefoundforCPM.

Amongthem, apriori expectationis wellknowntostrongly correlate with drug-induced analgesia [45] and pain modula-tion[32,33,44].Inthecase ofpharmacologicalopioidanalgesia,

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[33]. Stronger analgesia expectation and reduced stress during noxiouscoldconditioningwerelinkedtogreaterinhibitoryCPM [32].Althoughwekeptoursubjectsblindaboutthedirectionof expectedpainintensitychangesinducedbythecoldapplications, wecannotexcludethatexpectationplayedaroleinsomeofthem. Uncertainty about upcoming pain intensity also has a speciﬁc andpotenthyperalgesiceffect[46].Anxietyisknowntopartially mediatetheeffectofexpectationonthedirectionofCPM[44].Our ﬁndingthatanxietywasnegativelyrelatedwithbehavioralCPM supportsaroleofpsychologicalmodulators.

4.2. Cerebralresponsestotheconditioningcoldstimulation

Wefoundhighlysignificant(voxellevelFWERp<0.05) non-specificactivationsatonsetandoffsetofbothcoldandlukewarm stimulationsinmanyareasbelongingtotheso-called“painmatrix”. Themaximumofthisnon-specificresponsewasinleftSI,whichis consistentwithsomatosensoryresponsestothealternating sen-sorystimulationoftherightfoot.

By contrast, when early responses to cold were contrasted againstearlyresponsestolukewarm applicationtodetect pain-speciﬁcactivation,aclustercomprisingmainlybilateralprecuneus wassigniﬁcantly activatedbut not “pain matrix”areas suchas ACCandinsula.Theprecuneusisknowntorespondtomultimodal salientstimuli[47].

Wefoundnosignificantactivationinmostareasofthe“pain matrix”duringthesubsequentsustainedphaseofcoldapplication, withthenotableexceptionofposteriorinsula/SII.Thelatter activa-tionisconsistentwithacentralrolefortheoperculo-insularcortex inspecificpainprocessingandcodingofpainintensity[48–53]. Althoughourstudywasnotspecificallydesignedtoaddressthis question,itsresultsfavornonethelesstheconceptthatthe“pain matrix”ismostlya“saliencematrix”ofareasthatprocesssalient multimodalsensoryinputs[54]andthattheposteriorinsulamay bemorepain-specific[50,53].

Duringthesustainedphaseofcoldapplication,wefound deacti-vationinmedialOFC.Thisareaisknowntobeactivatedbyvaluation ofarewardoutcome[55]andwespeculatethatitsdeactivation couldbe related to the negative affective response due tothe sustainednoxiousstimulation.Theobservedclustersofsustained BOLDresponseinthewhitematterarediscussedinSupplementary Material5.

4.3. Cerebralresponsestothelaserteststimulusandtheir conditioningbycoldstimulation

Laserpain-inducedactivationsweresigniﬁcantinvariousareas of the “pain matrix” in line with a number of prior studies [47,56,57].Thisindicatesthattheintensityofthelaserstimuliwas sufﬁcienttoinducecerebralresponsestypicallyreportedafter nox-iousstimulation.

Similartothebehaviorallaserpainratings(seeSection4.1),the laser-inducedBOLDresponsesdidnotdifferonaveragebetween cold and lukewarm conditioning.The inter-individual variation in CPM, however, that ranged from reduction to increase in painwascorrelatedwiththemodulationoftheBOLDresponse. Behavioralcold-inducedhypoalgesiawasassociatedwiththe sup-pression of laser-induced responses in posterior insula and SII contralateraltothecoldstimulation.Likesustainedcold-induced

Fig.9.RelationshipbetweenactivationofsgACCandbehavioralconditionedpain modulation.Eigenvariatesofbetaimagesforonlyearlycoldresponsewereextracted fromasphereof6mmradiusaroundthepeakofearlycoldvsearlywarmcontrast signiﬁcantlycorrelatedto“physiologicalconditionedpainmodulation”(the eigen-variatesofthelaserwarmvslasercoldcontrastcorrelatedtobehavioralconditioned painmodulation)(Table3A).Thescatterpotisonlyforillustrationpurposes.

activation,thephysiologicalCPMthusinvolvedtheposteriorinsula, supportingagainitscriticalroleinpainaswellasCPMprocessing [50,53].

4.4. Correlationofcold-inducedcerebralresponseswith physiologicalCPM

In medial and lateral OFC, ACC, rostral and caudal lateral PFC,theearlycold-inducedBOLDresponsesweredirectlyrelated to subsequent CPM (see Figs. 8 and 9). These ﬁndings are in agreementwithotherstudies,where CPM wasassociated with activityevoked by thecold painstimulus inmPFC, lateralOFC [15,26]androstralACC[16].Bycontrast,BOLDactivationduring thelaterphaseofcoldstimulationwaslesspredictiveof physio-logicalCPM,asthecorrelationwassigniﬁcantonlyinrostro-lateral PFC.

Thecorrelationbetweenearlycold-inducedPFCresponsesand subsequentphysiological CPMcouldbe due tothefact thatthe conditioningstimulusisabletostrengthenconnectivitybetween corticalsourcesofdescendinganalgesiaandtheirtargetsin sub-cortical structures [58]. For instance, increased connectivity of subgenualACCwithmidbrainandamygdalawascorrelatedwith CPM[16].

Wealsofoundanunexpectedcorrelationbetween physiolog-icalCPManddeactivationinlateralOFC,ACC,lateralrostraland caudalPFCareasduringsustainedcoldapplication,whichwasnot reportedinothersstudies[15,16].Thismaybeduetothedifferent experimentaldesignofourstudy,butdifferencesinexpectation

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orso-called“off-setanalgesia”couldbealternativeexplanations. Inamodelofvisceralpain,expectation-mediatedplacebo analge-siawasassociatedwithfMRIdeactivationinthedorsolateralPFC [59].NegativeBOLDresponsesinventro-medialPFC/OFCwerealso recordedduringlargereductionsofpainratinginducedbyaslight decreaseofa noxiousstimulation,i.e. off-setanalgesia[8].This paradigmcouldberelevantforourstudywheretherewas habit-uationofcoldpainratingsbetweenthe1standlastapplications, andhenceadecreaseinperceivedpain.

4.5. Correlationswithcoldpainhabituation

Habituationofcoldpainperceptioncorrelatedpositivelywith behavioral CPM, with early cold-induced lateral OFC activation thatisalsorelatedtophysiologicalCPM,andwithsustained cold-induceddeactivationinpons/cerebellum.Habituationofpainover severaldayswasfoundassociatedwithsubgenualACCactivation [60].Habituationthuslikelyinvolvescentralneuronalnetworks thatitsharesatleastpartiallywithCPM.Diffusenoxiousinhibitory control,non-noxiousinhibitorycontrolandhabituationareallpart ofendogenousanalgesiaandthoughttoberelatedtoeachother [43].

Toconclude,ourstudyconfirms that sustainednoxiouscold stimulationchieflyactivatesposteriorinsula/SII.Activationinother areasoftheso-called“painmatrix”appearstobemerely deter-minedbysaliencyandnoveltyratherthanbythenoxiouscharacter ofthestimulus.Wefindgreatinter-individualdifferencesinCMP thatrangesfromdecreasestoincreasesofthetestlaser-heatpain. LargerprefrontalpositiveBOLDresponsestoearlycold stimula-tionseemtopredictsubsequentconditionedanalgesia.Bycontrast, inadjacentprefrontalcorticesconditionedanalgesiaisassociated withdeactivationduringthesustainedconditioningcold stimula-tion.WeshowthatCPMisnegativelycorrelatedwithtraitanxiety, butpositivelywithhabituationofcoldpain.Theindividual differ-encesinprefrontalcortexactivityandCPMneedtobeconsidered infuturestudiesofdiseasedsubjects,includingmigraineurs. Acknowledgements

Thisstudywassupportedbytheresearchconvention3.4.650.09 fromtheNationalFundforScientiﬁcResearch–BelgiumtoJ.S.,and byaresearchgrantfromtheCHUofClermont-Ferrand–Franceto R.D.andJ.S.WearegratefultoDrsAndréLuxen,EricSalmonand PierreMaquetfororganizational andtoChristianDegueldrefor technicalassistance.

Theauthorsdeclarenoconﬂictsofinterest. AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.bbr.2014.11.028. References

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