Behavioral and neuroimaging responses induced by mental imagery of threatening scenarios

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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

Behavioral

and

neuroimaging

responses

induced

by

mental

imagery

of

threatening

scenarios

Rosana

Shuhama

a,∗

_,

_Carlo

_Rondinoni

b

_,

_Draulio

_Barros

_de

_Araujo

c

_,

Gustavo

de

Freitas

Caetano

b

_,

_Antonio

_Carlos

_dos

_Santos

b

_,

_Frederico

_Guilherme

_Graeff

d,e

_,

Cristina

Marta

Del-Ben

a

a_Department_of_{Neurosciences}_and_Behavior,_Ribeirão_Preto_Medical_School,_University_of_São_Paulo,_Ribeirão_Preto,_SP,_Brazil b_Image_Sciences_and_Medical_Physics_Center_Laboratory,_Ribeirão_Preto_Medical_School,_University_of_São_Paulo,_Ribeirão_Preto,_SP,_Brazil c_Brain_Institute,_University_Hospital_Onofre_Lopes,_Federal_University_of_Rio_Grande_do_Norte,_Natal,_RN,_Brazil

d_{INeC—Institute}_of_Neuroscience_and_Behavior,_Ribeirão_Preto,_SP,_Brazil

e_{NAP-NuPNE—Neurobiology}_of_Emotions_Research_Center,_University_of_São_Paulo,_Ribeirão_Preto,_SP,_Brazil

h

i

g

h

l

i

g

h

t

s

•ImaginaryofimminentthreatscenariosincreasedfMRIsignalofthedorsalmidbrain.

•ActivityinvmPFCwasreducedduringtheimaginationofimminentthreatscenarios.

•Activationpatternwasconsistentwithanimaldataandactualthreatsituations.

•Imminentandpotentialthreatsmodulatebrainstructuresrelatedtofearandanxiety.

•Mentalimageryisareliablemethodtostudytheneurobiologyofbehavioralprocesses.

a

r

t

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c

l

e

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n

f

o

Articlehistory: Received26April2016

Receivedinrevisedform26June2016 Accepted29June2016

Availableonline22July2016 Keywords:

Mentalimagery Threatsituation Defensivebehavior

Functionalmagneticresonanceimaging Neuroimaging

a

b

s

t

r

a

c

t

Functionalneuroimaging studieshaveshownthatactualsituationsof uncertainor distantthreats

increasetheactivityofforebrainregions,whereasproximalthreatsincreasetheactivityofthe

dor-salmidbrain.Thisexperimentaimedattestingthehypothesisthatbrainactivityelicitedbyimagined

scenariosofthreatswithtwodifferentmagnitudes,potentialandimminent,resemblesthatfoundin

responsetoactualthreats.First,wemeasuredsubjectiveresponsestoimaginedscenariosofpotential

andimminentthreatscomparedwithneutralandpleasantscenarios.Thesamescenarioswereusedasa

paradigminafunctionalmagneticresonanceimagingexperiment.Behavioralresultsshowthatthe

sce-nariosdrawagradientofhedonicvalenceandarousaldimensions.Bothpotentialandimminentthreat

scenariosincreasedsubjectiveanxiety;theimminentthreatscenarioalsoincreasedfeelingsof

discom-fortandbodilysymptoms.Thefunctionalmagneticresonanceimagingresultsrevealedmodulationsof

BOLDsignalintheventromedialprefrontalcortexbypotentialthreatandintheperiaqueductalgray

matterbyimminentthreat.Theseresultsagreewithpreviouslyreportedevidenceusingactualthreat

situations,indicatingthatmentalimageryisareliablemethodforstudyingthefunctionalneuroanatomy

ofrelevantbehavioralprocesses.

1. Introduction

Studiesof nonhumananimal specieshaveprovided

substan-tialevidenceofspeciﬁcdefensivebehaviorsdependinguponthe

featuresof thethreateningsituation,which include magnitude,

∗ Correspondingauthorat:RiberãoPretoMedicalSchool.UniversityofSãoPaulo. E-mailaddress:roshuhama@gmail.com(R.Shuhama).

distanceandambiguityofthethreat,aswellastheavailabilityof

escapeorhidingplaces[1].Riskassessmentisanadaptive

defen-siveresponsewhenthethreathasahighlevelofambiguityand/or

isatasafedistance.Flightisanadaptivestrategywhenthethreat

isproximal,unambiguous,ofhighmagnitudeandanescaperoute

isavailable.Otherwise,whenthereisnoavailablerouteorhiding

place,defensivethreatandattackaretheadaptiveresponsesmost

oftenperformed[2–4].

http://dx.doi.org/10.1016/j.bbr.2016.06.059

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R.Shuhamaetal./BehaviouralBrainResearch313(2016)358–369 359

Aimingtoverifythecorrelationbetweenanimaland human

defensivestrategies,Blanchardetal.[4]proposedasetofimagery

scenariosthatvaryalongthementionedthreateningdimensions.

These scenarios were tested in Hawaiian [4] and Brazilian [5]

healthy volunteers, and the results pointed to a resemblance

betweennonhumanandhumandefensiverepertoires[6].Perkins

andCorr[7]usedthesamescenariosetandfoundanassociation

betweenpsychoticism,asonecharacteristicofpersonality,andthe

evaluationofdefensivemagnitudewhich,initsturns,inﬂuenced

thedefensivechoicesandorientationtodirectionawayfromthreat

source.Animalstudieshavealsoprovidedevidenceforneural

sub-stratesofdefensivereactions[8].Riskassessmentbehaviorsare

relatedtoincreasedactivityinforebrainareas[9,10].Themidbrain

periaqueductalgraymatter(PAG),whereasalsoinvolvedin

medi-atingrisk assessment behavior [11,12], iscommonly associated

withurgentresponses,suchasﬂightandﬁght[9,10].

Aseriesofwell-designedfunctionalmagneticresonance

imag-ing(fMRI)studiesfoundaparallelbetweennonhumanandhuman

defensiveneuralprocessing.Inshort,theyhaveshownthatdistant

orpotentialthreatsareassociatedwithactivityinthe

ventrome-dialprefrontalcortex(vmPFC),butasthethreatbecomesnearer

and intense, brain activity shifts to the PAG [13–15]. In these

experiments,actualthreatsituationswereused.Onestudyuseda

predator-preyvideogame,controlledbytheexperimentalsubject

[13],andtheothersimulatedatarantulamovingbackandforth

fromoneofthesubject’sbarefoot[15].

Knowledgeabouttheneurobiologyofdefensivebehaviorscan

be relevant for the understanding of mental disorders, mainly

anxietydisorders.However,modelingthreatsituationsinhuman

subjectsisnotonlyexpensiveandpossiblypainstaking,butalso

hasethicalandpracticallimitations.Theseshortcomingscouldbe

surmountedifmentalimageryproveseffectiveinchangingbrain

activityinmeaningfulways.Becausereportedevidencehasshown

thatmentalimagerycanreproducesubjectiveresponsesandbrain

activitythatareexpectedinrealsituations[16,17],weaimedat

testingthehypothesisthatbrainactivityelicitedbyimagined

sce-nariosofdistalandimminentthreatswouldresemblethatfound

inresponsetoactualthreats.

Inthepresentstudy,weﬁrstmeasuredsubjectiveresponsesto

imaginedscenariosofpotentialandimminentthreats,compared

withneutralandpleasantones,inordertoestimatetheabilityof

thescenarios togeneratedifferentthreatdimensions.Thesame

scenarioswerethenusedasaparadigminanfMRIexperiment.

Wepredictedthattheimaginationofanaversivescenariofeatured

bypotentialthreatwouldelicit activityin forebrainareas,such

asvmPFCandamygdala, whereastheimaginationofascenario

ofproximalthreatwouldengagemidbrainareas,particularlythe

PAG.

2. Behavioralexperiment

2.1. Materialandmethods

2.1.1. Participants

Twentyhealthy volunteers(17 women,age=28.5±4.1)

par-ticipatedinthisstudy.Theparticipantswererecruitedbyposter

advertisements,electronicmessagesandpersonalcontact.

Individ-ualshadatleast12yearsofschooling,withnohistoryofpsychiatric

andneurologicalcondition.Participantswerenotincludedifthey

usedanypsychoactivesubstancesand/ormedicationwithintwo

weeksbeforetheexperiment (exceptcontraceptives). Thelocal

ResearchEthicsCommitteeofRibeirãoPretoMedicalSchool,

Uni-versityofSãoPaulo,RibeirãoPreto,SãoPaulo,Brazil,approvedthe

studyandwritteninformedconsentwasobtainedfromall

partici-pants.

2.1.2. Mentalimageryparadigm

Thementalimageryparadigmwascomposedoffourscenarios,

twoofthemextractedfromtheDefensiveBehaviorQuestionnaire

[4].Onescenariorepresentedanimminentthreat(scenario

Eleva-tor)andanotherpotentialthreat(scenarioNoise).Thesescenarios

havebeenchosenbecause,inpreviousstudies[4,5],theformer

wasevaluatedasthehighestmagnitudeof threatandthe

low-estpossibilityofescapeandofﬁndinga hidingplace;thelatter

wasratedasthemostambiguousscenario.Themostfrequently

recordedresponsetotheElevatorscenariowasdefensiveattack

andtoNoise,checkingout.Theothertwoscenariosrepresentedan

intendedneutralscenario(Meeting)andapleasantscenario(Beach),

situationsconstructedtothestudyinordertocontrastwiththe

threatscenarios.

a)Meeting(neutralscenario):“Itisnightandyouarealoneathome

watchingTV.Thephoneringsandwhenyouanswerthephoneitis

aco-workerconﬁrmingthemeetingthenextmorning”.

b)Noise(potentialthreatscenario):“Lateatnight,itisdarkandyou

aresleepingaloneinyourbed.Yousuddenlywakeupfeelingthat

youheardasuspiciousnoise”.

c)Elevator(imminentthreatscenario):“Lateatnight,you’realone

inanelevator.Whenitstopsandthedoorsopen,aroughlooking

strangergetsinfasttoattackyou,blockingyourexit”.

d)Beach (pleasant scenario):“You arealone andslowly walking

alongabeach.Itistheendofthedayand,onthehorizon,youcan

seethesunset.Somechildreninviteyoutocompletetheirgroup

thatisplayingvolleyball”.

Astandardsetofinstructionswasprovidedtoallparticipants

asfollows:“Youwill hearthescript ofasituationthatwecango

through.Listentoitcarefullyandfocusonit.Imagineyourselftaking

partinthesituation,asifitwerereal.Imaginethemostlikelyfeelings

andreactionsyouwouldhavetoit,themorespontaneousresponses.

Onceyouareimaginingthedepictedscene,stiraﬁngerofyourright

hand.Remainfocusedandimaginingyourselfinthescenariountilyou

receiveinstructionstostop.”

Toelicit emotional arousal,a professionalactor recordedan

audiodescribingeachscriptaloud,givingemotionaltonetoeach

situation.

2.1.3. Hedonicvalenceandarousalandqualityofthemental

imagery

Assessmentsofthehedonicvalenceandthearousalcausedby

each scenario were madewith Likert-typescalesbased on the

conceptthatunderliestheworkofLangandcoworkers[18].The

anchorsofthevalencescalevariedfrom1(unpleasant)to9

(pleas-ant),andofthearousalscalefrom1(calm)to9(excited).

Anestimativeofqualityofthegeneratedmentalimageswas

assessedthroughaLikert-typescale,anchoredfrom1to9,as

previ-ouslyproposed[19].Sevenqualitieswereevaluated:(a)theeasein

formingthementalimagery(1=veryeasy,9=verydifﬁcult);(b)the

amountofdetailsincludedinthementalimagerytask(1=highly

detailed,9=withoutanydetail);(c)theclarityintheimage

dis-played(1=verysharp,9=veryvague);(d)theeaseofmaintenance

oftheimageryduringtheperiodproposed(1=veryeasy,9=very

difﬁcult);(e) thechanges in thedetail amountduring thetask

(1=nothingatall,9=dramaticchange);(f)howmuchofthe

infor-mationprovidedbythescriptwasusedintheimaginationofthe

situationdescribedineachscenario(1=allthescript,9=justasmall

portionofthescript),and(g)thesizeofthementalimageryset

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2.1.4. Subjectivemoodmeasures

Subjectivestateswereassessedbytwoeasytounderstandand

quicktoadministerscales,theVisualAnalogueMoodScale(VAMS)

[20,21]andtheBodilySymptomsScale(BSS)[22].

The VAMS is composed of 16 items, each item featured

by opposite adjectives, separated by a 100mm line. The

volunteer is asked to draw a dash on the line to

repre-sent his/her current feeling. The scale is composed of four

factors: (a) anxiety (3 items: calm–excited, relaxed–tense;

tranquil–troubled); (b) sedation (2 items: alert–drowsy;

attentive–dreamy); (c) cognitive impairment (7 items: clear

headed–muzzy; energetic–lethargic; gregarious–withdrawn;

proﬁcient–incompetent; quick witted–mentally slow;

strong–feeble, well-coordinated–clumsy), and (d) discomfort

(4 items: amicable–antagonistic; contented–discontented;

happy–sad;interested–bored)[22,23].

TheBSSiscomposedof21itemsthatdescribesomatic

symp-tomsrelated to anxiety: fatigue,weakness,lethargy, headache,

muscletension,tremor,hunger,thirst,coordinationdifﬁculty,

per-spiration,palpitation,dyspnea,agitation,urinaryurgency,nausea,

drymouth,blurredvision,dizziness,defecationurgency,dysuria

and paresthesia.There aresix possibleresponsesto each item,

whichvaryfrom0(nosymptom)to5(extremelymarked

symp-toms)[22].

2.1.5. Experimentaldesign

For setting habituation, participants wereasked to sit

com-fortablyandrelaxfor15min.Theyweretoldthattheywouldbe

listeningtodifferentscenariovignettes,andthattheyshould

imag-ine(eyesclosed)asiftheywerethemaincharacterofthedescribed

situations.

Eachscenariowasplayedtwice(iPodNano®,AppleInc.),ina

pseudorandomorder.Thescriptslastedfrom12to19s(listening

phase),whichwasfollowedby30sofmentalimagery(imagery

phase).Thevolunteersignaledwiththerighthandthathe/shewas

picturingthescene.Restingintervalsofﬁveminuteswereprovided

afterimaginingeachscenario.

VAMSandBSSwerescoredbytheparticipantsbeforethe

lis-teningphaseandattheendoftheimageryphaseforeachscenario.

Thequalityofthegeneratedmentalimage,andthehedonicvalence

andarousalwereassessedimmediatelyaftertheimageryphase.

2.1.6. Dataanalysis

DatawereanalyzedwiththeStatisticalPackageforSocial

Sci-ences(SPSS,version17.0forWindows).Qualityofmentalimagery,

hedonicvalenceandarousal,subjectivemeasures(VAMSandBSS)

weresubmittedtorepeatedmeasuresANOVAwith

Greenhouse-Geissercorrection.Posthoccomparisonswereperformedwiththe

Bonferronicorrection.Scenario(four)andtime(two:before

listen-ingandafterimageryphase)constitutedthewithinfactors.Paired

Student’st-testswereusedforposthoccomparisonswhenthe

sce-nariovs.timeinteractionwassigniﬁcant.Valuesofp<0.05were

consideredstatisticallysigniﬁcant.

2.2. Results

2.2.1. Hedonicvalenceandarousalandqualityofthemental

imagery

AsseeninFig.1A,thereweresigniﬁcantdifferencesregarding

thevalenceattributedtoeachscenario[F(3,50)=62.35,p<0.001].

Pleasantscenariowasperceivedasthemostappetitive(vs.

neu-tral,p<0.001;vs.potentialthreatp<0.001;vs.imminentthreat

p<0.001).Imminentthreatwasdescribedasmoreaversivethan

neutralscenario(p<0.001),andtherewasatrendtosigniﬁcant

differencefrompotentialthreat(p=0.059).Neutralscenariowas

signiﬁcantlymoreappetitivethanpotentialthreat(p=0.007).

Arousalcausedbythementalimagery(Fig.1B)wasalso

signiﬁ-cantlydifferentamongthescenarios[F(2,42)=19.49,p<0.001].Post

hocanalysesshowedthatthesedifferencesstemfrompleasant

sce-nario,whichwasratedbytheparticipantsasthelessexciting,near

theexperienceoffeelingcalm(vs.imminentthreat,p<0.001;vs.

neutralscenario,p=0.028;vs.potentialthreat,p<0.001).

Qualitiesofthescenarioswerewellevaluatedsuggestingthat

thesettingsofthescenarioswereappropriate.AsseeninTable1,

signiﬁcantdifferenceswerefoundin oneof thesevenassessed

qualitiesofthementalimageryofthescenarios.Allscenarioswere

perceivedaseasilyimagined,detailedandeasilymaintainedalong

withtheexperimentalprocedures.Thereweresomechangesinthe

detailsacrosstime,butthemajorityoftheinformationprovidedby

thescriptwasusedinthementalimageryofthescenarios,which

wereimaginedinmediumsize.Onlytheclarityofthemental

repre-sentationsdifferedsigniﬁcantlyamongthescenarios[F(2,45)=4.84,

p=0.009]._The_imagination_of_the_pleasant_scenario_was_reported_to

besharperthanthescenariowiththepotentialthreat(p=0.025),

andtheimminentthreatwasimaginedmoreclearlythanneutral

situation(p=0.016)andpotentialthreat(p=0.010).

2.2.2. Subjectivemoodmeasures

Fig.1C shows that thescenarios changed subjective anxiety

[F(2,37)=3.77,p=0.03],andthatthesechangesweredependenton

timeandscenario[timevs.scenarioF(1,28)=10.04,p=0.01].Post

hoct-testsshowedadecreaseinanxiety,measuredbyVAMS,after

mentalimageryofthescenariopleasant(p<0.01),andanincrease

afterimminent(p<0.02)andpotentialthreats(p=0.01).

The remaining VAMSfactors are shown in Table 2. Overall,

participantsreportedhigher cognitiveimpairmentsbeforethan

afterimaginingeveryscenario[timeF(1,16)=7.86,p=0.013].

Sce-nariosaffectedfeelingsofdiscomfortdifferently[timevs.scenario

F(2,34)=3.83, p=0.029]. Post hoc t-tests showed that imagining

imminentthreatcausedanincreaseinthefeelingsofdiscomfort

(p=0.031).Therewerenosigniﬁcantdifferencesamongthe

scenar-iosregardingthesedationfactor[timeF(1,17)=2.73,p=0.12;time

vs.scenarioF(2,31)=0.10,p=0.89].

Participants alsoscoredin BSSdifferently [time vs.scenario

F(2,35)=3.51, p=0.04]. Imminent threat wasassociated withan

increaseinreportedsomaticsymptomsaftertheimageryphase

(p=0.02;Fig.1D).

3. Neuroimagingexperiment

3.1. Materialsandmethods

3.1.1. Participants

Thirty-six volunteers were initially screened for the study.

Tenwere excludeddue to useofillicit substances [2], medical

conditions [1],useof psychotropic medication[1], useof ﬁxed

orthodonticappliances[1] and impedimentsfor schedulingthe

scanning[5].

Thescanningsessionwasscheduledfor26participants.Five

participantsdidnotattendonthescheduleddateandtwowere

notabletoﬁnishthescanningduetoclaustrophobicreactions.

Nineteen[19]healthyparticipants(12women,26.1±4.3years

old)tookpartinthefMRIexperiment.Theyhad aminimumof

13yearsofformalschoolingand reportednouseofmedication

otherthancontraceptives.Afterexclusionduetoexcessivehead

movements,analysiswasconductedwith16participants.

3.1.2. Experimentaldesign

During image acquisition, participants were asked to stay

awake, still, and as comfortable as possible, with eyes closed

throughouttheexperiment.E-Prime1.1software[24]wasused

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Fig.1. Subjectiveassessmentofscenarios.Columnsrepresentmeanandbars,standarderrorofthemeanof:(A)Valence(1=unpleasant;9=pleasant).*Pleasantscenario>all.# Imminent<neutralscenario.&Neutral>potentialthreat;(B)Arousal(1=calm;9=excited).*Pleasantscenario<all;(C)SubjectiveanxietyfactoroftheVAMSbefore(empty columns)andafter(ﬁlledcolumns)thementalimagerytask.*Before /=aftertoimminent,potentialthreatsandpleasantscenario;(D)Bodilysymptomsbefore(empty columns)andafter(ﬁlledcolumns)mentalimagerytask.*Before<aftertoimminentthreat.Falsepositiveprobabilitysetto0.05.

Table1

Subjectivequalitiesattributedbyhealthyvolunteerstomentalimageryscenariosrelatedtoaneutralcontent,apotentialthreat,animminentthreatandapleasantsituation.

Imagequalities Scenarios p

Neutral Potential Imminent Pleasant

Easiness 2.45(0.34) 2.50(0.32) 3.25(0.40) 2.30(0.36) 0.163 Details 3.45(0.40) 3.10(0.31) 4.05(0.37) 3.25(0.26) 0.112 Clarity 2.75(0.34) 2.45(0.25) 3.85(0.41) 3.25(0.35) 0.009 Maintenance 3.00(0.41) 3.05(0.36) 3.35(0.41) 2.65(0.37) 0.448 Changes 3.50(0.46) 3.35(0.37) 4.25(0.42) 3.80(0.46) 0.306 Informationuse 2.55(0.37) 2.40(0.43) 2.65(0.42) 2.40(0.41) 0.864 ImagerySize 7.25(0.42) 6.95(0.36) 7.05(0.41) 7.10(0.40) 0.718

Easiness:1=veryeasy,9=verydifficult;Details:1=highlydetailed,9=withoutanydetail;Clarity:1=verysharp,9=veryvague;Maintenance:1=veryeasy,9=verydifficult; Changes:1=nothingatall;9=dramaticchange;Informationuse:1=allthescript;9=justasmallportionofthescript,Imagerysize:1=verysmall;9=verylarge.Datarepresent mean(standarderrorofthemean).Boldcharacterssignalstatisticallysignificantdifferences.

Table2

ScoresofVAMSsubjectivefactorsofhealthyvolunteersinresponsetomentalimageryscenarios.

VAMSfactors Scenarios p

Neutral Potential Imminent Pleasant

Cognitiveimpairment Before 49.73(0.91) 46.29(1.25) 46.21(1.27) 45.70(1.26) 0.182

After 45.77(1.97) 42.51(1.77) 41.48(1.81) 47.08(1.57)

Discomfort Before 45.21(1.79) 45.22(1.91) 45.09(1.75) 45.32(1.81) 0.029

After 45.26(1.85) 46.00(1.89) 47.23(1.76) 43.59(2.14)

Sedation Before 45.96(1.43) 46.28(1.68) 46.55(1.51) 46.49(1.58) 0.901

After 46.17(1.55) 46.56(1.54) 45.21(1.69) 45.98(1.65)

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describedinthebehavioral experiment.Eachscenariowas pre-sentedfor 15s(listeningphase), followedby amental imagery phase(30s).Inablockdesignparadigm,thescenarioswere pre-sentedinthefollowingorder:neutral(Meeting)–potential(Noise) –imminent(Elevator)– pleasant(Beach). Thissequenceof pre-sentationofthescenarioswasrepeatedthreetimes.Betweenthe presentationofeachscenariotherewasanintervalconsistingof a30-sofmonotonicauditorystimuli,whenletterswerespoken atrandomorder.Thisrestingblockwasalsosetatthebeginning andattheendofthefunctionalrun.TheentirefMRItasksession lastedapproximately20min.Attheendofscanning,wedebriefed thevolunteeraboutimageryexperienceandhedonicvalenceand arousalrating ofeach scenarioas additionalmeasures.We did notincludesubjectivemeasuresduringthefMRIsessioninorder toavoidincreasingthetimeexpendbytheparticipantinsidethe scanner,whichcouldresultinfatigueandinattentivenessonthe task.

3.2. Neuroimagingdataacquisition

Structuraland functionalimageswere acquiredina 3T MRI scanner(Philips, Achieva, The Netherlands), withan 8-channel headcoil.Ahigh-resolutionT1weightedgradientecho3Dsequence wasacquiredwiththefollowing parameters:180sagittal1mm slices,256×256matrix,TR=5.7ms,TE=2.6ms.ThefMRI acquisi-tionusedanEPIsequencewithsoft-toneparameter,whichallowed forcontinuousacquisitionand12dBnoiseattenuation:32axial slices(4mm),80_×80 matrix, 240_×240mm,TR/TE 2000/30ms, 80◦ﬂipangle.

3.3. Dataanalysis

BehavioraldataatthefMRIexperimentwereanalyzedwithSPSS 17.0.Hedonicvalenceandarousalweresubmittedtorepeated mea-suresANOVAwithGreenhouse-Geisser correctionandposthoc comparisonswithBonferronicorrection.

Functional magnetic resonance imaging data were pre-processed and analyzed using Brain Voyager v. 2.8 (Brain Innovation,Maastricht,Netherlands).Forpre-processing,volumes werecorrectedformotion,slicetime,andspatiallysmoothedwith aGaussianFilter(FWHM=6mm).Threeparticipantsmovedmore than2mmor2◦andwereexcludedfromanalysis.

TheEPIdatasetwasco-registeredwiththeindividual 3DT1 structuralimage,andwerespatiallynormalizedintotheTalairach space[25].Groupanalysisduringtheimageryphasewasperformed

usingrandomeffectsGeneralLinearModel(GLM).Restingblocks

wereusedasbaselinewhensubjectshadjusttolistentolettersin

arandomorder.Thiswassettoavoidprimingandcarry-oneffects

fromonescenariototheother.Also,thiswouldkeeptheauditory

stimulationatbaseline,favoringtheidentiﬁcationofrelevantareas

foremotionalprocessingduringtheimageryphase.

Theeffects ofthe taskswereevaluated usinga wholebrain

approachconsideringsixorthogonalcontrasts:potentialthreatvs.

pleasantscenario;imminentthreatvs.pleasantscenario;neutral

scenariovs. pleasant scenario; potential threatvs. neutral

sce-nario;imminentthreatvs.neutralscenario;andimminentthreat

vs.potential threat.Also, a conjunctionanalysis wascalculated

consideringthe imminentthreat as theﬁrst conditionand the

combinationoftheremainingthreescenariosasthesecond

(immi-nent>potentialthreatplusneutralpluspleasantscenarios).The

statisticalsigniﬁcance ofclustersin each contrastwasassessed

usingarandom-effectsmodel.AplugininBrainVoyagerfor

esti-matingCluster-LevelStatisticalThresholdwasusedtocontrolfor

multiplecomparisons. Using Monte Carlo simulation (500

iter-ations), thepluginestimates the ratefor false-discovery (FDR),

Table3

Scoresinsubjectiveevaluationsofhedonicvalenceandarousalbyhealthy volun-teersinresponsetomentalimageryofscenariosindebrieﬁngafterfMRIsession.

Scenarios Valence* _Arousal*

Neutral 5.58(0.35) 4.68(0.38) Potential 3.47(0.35) 6.37(0.41) Imminent 2.58(0.51) 7.47(0.43) Pleasant 7.63(0.43) 2.63(0.45)

*_Post_hoc_p_values_<_0.05_to_all_scenarios.

deﬁningaminimumcluster-sizenecessarytoproduceaneffective

alphasmallerthan5%.

Inordertoassesstheinﬂuenceofareaspreviouslyidentiﬁed

asimportantinthemodulationofsceneryperception,fourbrain

areaswere chosen inthe followingvolumes ofinterest (VOIs):

theanterior cingulatecortex (ACC)(x=±9, y=41, z=8),vmPFC

(x=−1,y=51,z=−1),amygdala(x=±23,y=0,z=−15)andPAG

(x=−6, y=−30,z=−5).Thechoicewasbasedonprevious

non-human[26,27]andhumanstudies[13,14]and,whenneeded,MNI

coordinatesweretransformedintotheTalairachspaceusingBrett

algorithm[28].Aboxwasdrawnaroundeachofthesecoordinates,

whosesizevariedfrom125to719,dependingontheanatomical

locationandthespeciﬁcityrequired.Posthocanalysisinspectedfor

changesontheaverage␤-valuesobtainedfromeachoftheseVOIs.

Theanalysiswasseparatelyperformedinthelisteningandimagery

phases,andcomparedeachscenario.Statisticalsigniﬁcance was

setforalphasmallerthan5%,correctedformultiplecomparisons

acrossVOIs.

3.4. Results

3.4.1. Hedonicvalenceandarousal

Inlinewiththebehavioralexperiment,signiﬁcantdifferences

werefoundamongthescenariosforbothvalence[F(2,34)=36.38,

p<0.001] and arousalscales [F(2,36)=38.72,p<0.001](Table3).

Valencescalespointstothepleasantscenarioasthemost

appe-titive(vs.neutral p=0.021;vs.potentialp<0.001;vs.imminent

p<0.001) and imminent threat as the most aversive (vs.

neu-tral p<0.01; vs. potential p=0.053). The scenario neutral was

locatednearthemiddleofthescale,anddifferedfrompotential

(p<0.001).Asforarousal,pleasantwasthelessexciting(vs.

neu-tralp<0.001;vs.potentialp=0.008;vs.imminentp<0.001),and

imminentreceivedthehighestarousalscores(vs.neutralp<0.001;

vs.potentialp=0.032).Neutralwassigniﬁcantlylessexcitingthan

potential(p<0.001)(Table3).

3.4.2. Neuroimagingresults

3.4.2.1. Whole−brain contrasts. Whole brain voxel-wise maps

werecalculatedtofurtherexplorespeciﬁcbrainareasdepending

onthescenario.InregionswheretheBOLDsignalsweremost

dis-paratebetweenscenarios,wecalculatedthemeanBOLDchange

acrossallvoxelsinthesurvivingcluster(cluster-wisecorrection,

p<0.05). See Table 4 for coordinates and values on signiﬁcant

regionsofactivationintheproposedorthogonalcontrasts.

Con-trastsbetweenthreateningscenariosinvolvedwidespreadwhole

brainareas,fromfrontaltosubcorticalregions,includingamygdala

andPAG(Figs.2and3).

Weperformedcontrastsbetweenthemostdisparatescenarios

in terms of arousal and valence. Thus, the three ﬁrst

compar-isonsweremadeagainstproximalthreat(Elevator).Thecontrastof

imminent>potentialthreatrevealedsigniﬁcantclusters(p<0.05,

corrected)intherightmiddletemporalgyrus,whiletheinverse

contrast(potential>imminentthreat)indicatedtosigniﬁcant

clus-tersintherightsuperioroccipitalgyrus,andintraparietalsulcus

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Fig.2.BOLDcontrastdeterminedbyimaginingthreatscenarios.Constrastbetweenimaginationofanimminentthreatandotherscenarios.(A)Contrastimminent>potential threats.Theleftsuperiortemporalgyrus(STG)showsgreateractivationduringtheimaginationofimminentthreatthanpotentialthreat(p<0.05,corrected).(B)Contrast imminentthreat>neutral.TheleftSTGshowsgreateractivationduringtheimaginationofimminentthreatthanmeetingcondition(p<0.05,corrected).Also,abrainstem cluster(whichincludesPAG)showhigherlevelofBOLDsignalduringimminentthreatimagining.(C)Contrastimminent>pleasantscenarios.Temporoparietaljunctionand brainstemshowlargesignalcontrast(p<0.01,clustercorrected).Percentsignalchangewascalculatedfromthemeanvariationofsignalacrossallvoxelsinthecluster. Asterisksshowtheconditionsusedinthecontrast.Errorbarsreﬂectbetween-subjectstandarderror.

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Fig.3.Contrastsdeterminedbyimaginingthreatscenarios,comparinglessthreateningscenarios.(A)Contrastpotential>neutralscenario.Theleftsuperiortemporalgyrus (STG)showsgreateractivationduringtheimaginationofimminentthreatthannoisecondition(p<0.05,corrected).(B)Contrastneutral>pleasant.TheleftSTGshowsgreater activationduringtheimaginationofimminentthreatthanmeetingcondition(p<0.05,corrected).Also,abrainstemcluster(whichincludesPAG)showhigherlevelofBOLD signalduringimminentthreatimagining.(C)Contrastpotentialthreat>pleasant.Temporoparietaljunctionandbrainstemshowlargesignalchange(p<0.01withcluster correction).Percentsignalchangewascalculatedfromthemeanvariationofsignalacrossallvoxelsinthecluster.AsterisksinBOLDsignalchangebargraphsshowthe conditionsbeingcontrasted.Errorbarsreﬂectbetween-subjectstandarderror.

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Table4

BOLDsignalchange(%)andt-valuesdependingonthecontrastbetweenscenarios.Positivet-valuesindicatetheclusterissigniﬁcantfortheﬁrstscenariointhecomparison. SignalBOLDchangeinpercentagewascalculatedfromthemeanofallvoxelssurvivingcluster-wisecorrection(p<0.05.corrected).

Contrast Brainarea R/L %BOLDchange t-valuecontrast Voxels Talairachcoordinates

Condition1 Condition2 x y z

Imminentvs.potential Superioroccipital gyrus(BA7)

R −0.107 0.030 −3.21 2677 22 −72 31

L Intraparietalsulcus(BA 40)

R −0.105 0.035 −3.23 3624 38 −35 39

L Middletemporalgyrus (BA22)

R 0.292 0.113 3.46 5959 43 −28 2

L Imminentvs.neutral Intraparietalsulcus(BA

39) R −0.139 0.027 −3.48 3050 37 −63 37 L Superiortemporal gyrus(BA22) R 0.268 0.075 3.70 9001 44 −22 −3 L 0.394 0.164 3.52 9541 −57 −33 6 Cerebellum R L 0.086 −0.036 3.34 2341 −18 −43 −20 Occipital(BA18) R L 0.126 −0.047 3.69 3338 −4 −77 4

Brainstem(PAG) 0.122 −0.031 3.53 23,507 −3 −33 −9

Imminentvs.pleasant Inferiorparietallobe (BA40) R −0.136 0 −3.18 2412 44 −33 38 L 0.139 −0.098 3.32 2859 −59 −46 31 Brainstem 0.123 −0.031 3.49 8770 0 −22 −2 Cerebellum R 0.157 0.027 3.47 4946 20 −65 −32 L Middletemporalgyrus (BA21)

R 0.381 0.198 3.57 3653 47 −25 −1

L

Potentialvs.neutral Parahippocampal(BA

19) R L 0.079 −0.049 3.91 6442 −32 −47 −6 Posteriorcingulate gyrus(BA30) R 0.142 −0.049 3.84 3363 17 −59 13 L 0.158 −0.007 3.45 2479 −18 −58 11 0.088 −0.045 3.43 2201 −14 −42 42 Parahippocampal (BA20) R 0.127 −0.023 3.57 1634 24 −40 −16 L

Mid-insula(BA13) R 0.073 −0.061 3.25 1804 37 2 4

L

Neutralvs.pleasant Hippocampus(BA37) R

L −0.027 0.117 −3.64 6314 −30 −35 −12

Lateralinferiorparietal (BA39)

R

L 0.114 −0.041 3.32 1293 −52 −58 29

Inferiorparietallobe (BA40)

R 0.003 −0.159 3.42 3371 48 −56 39

L Potentialvs.pleasant Inferiorparietallobe

(BA40) R L 0.032 −0.115 3.33 2517 −56 −45 34 Imminentvs.other scenarios Brainstem/cerebellum R 0.689 −0.241 2.52 7774 4 −43 −20 L

Medialfrontal(BA32) −0.006 −0.468 2.57 13,908 −13 23 22

PCC(BA29) 0.290 0.704 −2.56 4316 0 −54 11

Temporal(BA21) L 1.243 0.882 −3.50 24,121 −57 −22 −2

R 1.406 1.101 −2.79 15,977 54 −10 −4

L:lefthemisphere;R:righthemisphere;BA:Brodmannarea;Condition1:ﬁrstconditioninthecontrast;Condition2:secondconditioninthecontrast.

differentialBOLDsignalactivationinaregionintheleftsuperior temporalgyrus,leftcerebellum,leftoccipital,rightsuperior tem-poralgyrusandbrainstem(PAG/thalamus)(p<0.01,corrected). Theinversecontrast(neutral>imminentthreat) showedgreater activationintherightintraparietalsulcus(Fig.2B).Thethird

con-trastinvolvingproximalthreatcomparedittothemostpleasant

one. Resultingsigniﬁcantclusters for imminent>pleasant were

foundinleftinferiorparietallobe,rightmiddletemporal gyrus,

right cerebellum, and brain stem. The inverse contrast

(pleas-ant>imminent threat) showed onecluster in the rightinferior

parietallobe(Fig.2C).Allmapswerecorrectedattheclusterlevel

withtheBrainVoyagerplugin(p<0.05,corrected).

Otherthreecontrastswereperformedtocomparespeciﬁc

acti-vationsbetweenless arousingscenarios. Theﬁrstwaspotential

threat>neutral scenario Signiﬁcant clusters were found in the

parahippocampalgyrusand inposteriorcingulategyrus

bilater-allyandintherightmid-insula(Fig.3A).Noclusterwasfoundfor

theinversecontrast(neutral>potentialthreat).

The contrast neutral>pleasant revealed clusters in left

lat-eral inferior parietal cortex and in the right inferior parietal

lobe(p<0.05,corrected).Theinversecontrast(pleasant>neutral)

showedasigniﬁcantclusterinthelefthippocampus(Fig.3B).

Finally,thecontrast potentialthreat>pleasant scenario

indi-cated one signiﬁcant cluster in the left inferior parietal lobe

(p<0.05,corrected).

Aconjunctioncontrastwasperformedtotestforregionsthat

weremorestronglyactiveduringimminentthreatthanallother

scenarios(imminent>distalplusneutralpluspleasant).This

con-junctioncontrastshowedsigniﬁcantlygreateractivationinbrain

stem, cerebellum and medial frontal (p<0.05, corrected). The

inverse contrastshowed signiﬁcantclusters in bilateralmiddle

temporal and posterior cingulate cortex. Detailed information

abouttheclustersfoundinthecontrastsandconjunctioncontrast

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Table5

tValuesofcontrastsbetweenscenariopairsduringtheimageryphase.

Brainarea Neutralvs. Pleasant Potentialvs. Pleasant Imminentvs. Pleasant Imminentvs. Neutral Imminentvs. Potential Potentialvs. Neutral

Voxels Talairachcoordinates

x y z ACC L −1.42 0.01 −2.34 −0.85 −2.34 1.35 512 −9 41 8 R −0.42 0.91 −1.54 −1.44 −2.45 0.89 512 9 41 8 vmPFC −2.02 −1.76 −3.35 −1.21 −1.59 0.26 512 −1 51 −1 AMY L −2.51 −0.95 −0.80 1.70 0.17 1.54 125 −23 0 −15 R −3.03 −0.07 −0.77 2.24 −0.68 2.91 125 23 0 −15 PAG 0.29 1.22 4.83 4.37 3.60 0.90 729 −6 −30 −5

L:lefthemisphere;R:righthemisphere;ACC:anteriorcingulatecortex;AMY:amygdala;PAG:periaqueductalgraymatter.Boldcharactersindicatestatisticallysignificant differences(p<0.05uncorrectedorp<0.0013,correctedformultiplecomparisonsacrossVOIs).Thesignalinthistableindicateswhichscenariocontributestothesignificance oftheactivity.Positivesignalsindicatethefirstexperimentalcondition,andnegativevaluestothesecond.

Fig.4.BOLDcontrastdeterminedbyimaginingthreatscenarios.(A)AttenuationofthevmPFCresponseinimminentvs.pleasantcontrast(Talairachx=4,y=51,z=−3). (B)Activationoftherightamygdalaresponseinpotentialvs.neutralcontrast(Talairachx=24,y=−1,z=−22).(C)ActivationofthePAGresponseinimminentvs.pleasant contrast(Talairachx=−6,y=−30,z=−5).(D)IllustrationofPAGtimecourseofpercentageofBOLDsignalchangeasresultoflinearregressionanalysisoflisteningphase (untiltime=0)andimageryphase(aftertime=0).

3.4.2.2. VOIanalysis. VOIanalysisshowedthatimaginationofthe scenariosdidnotchangesigniﬁcantlyintheACC,bilaterally,forall contrasts(Table5).SigniﬁcantchangeswereobservedinthevmPFC

whenimminentthreatwascontrastedwithapleasantsituation

(Fig.4A;Table5),andalsointherightamygdala,whenpleasant

wascontrastedwithneutralscenario(Table5).

Additionally,therightamygdalashowedagreatersignal

dur-ingtheimaginationofthepotentialthreatscenariothanduring

theneutralscenario(Table2,Fig.4B).Asigniﬁcantlygreatersignal

wasobservedinthePAGduringtheimaginationoftheimminent

threatsituationthanduringpleasant(Fig.4C),neutralandpotential

(Table5).

Fig.5showsthebetas(columns)±standarderror(bars)ofthe

VOI-GLManalysesforbothlisteningandimageryphases.

Signiﬁcantsignalchanges werefoundinACC bilaterally[left

ACC F(23,50)=2.56, p<0.001; right ACC F(23,50)=4.28, p<0.001].

Increasedsignalwasobservedduring listeningtothescenarios

neutralandimminentinbilateralACC(p<0.001).Decreasedsignal

wasfoundduringtheimaginationofbothscenarios(leftACC:

neu-tralp=0.030andimminentp<0.001;rightACC:neutralp=0.017

andimminentp<0.001).

Ventromedial prefrontal cortex [F(23,50)=2.98, p<0.001]

showedapatternsimilartothatofACCregardingactivationswith

thelistening epochfor thescenarios neutral (p=0.002) and of

imminentthreat(p<0.001).Theperiod ofimagininga pleasant

situationalsoelicitedincreasedsignalinthevmPFC(p<0.001).

Atrendtoward signiﬁcancebetweenthepresentationofthe

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Fig.5.BetasandstandarderrorsoftheGLManalysesovervariationsoftheBOLDsignaldependingonstimulationphasesineachscenariocondition(neutral,potential, imminentthreatandpleasant)inthelisteningandimageryphases.VOIsselectedwereleftandrightamygdala,ventromedialprefrontalcortex(vmPFC),leftandrightanterior cingulatecortex(ACC)andperiaqueductalgraymatter(PAG).

rightamygdala[F(23,50)=1.51,p=0.055].Posthocanalysesshowed

asigniﬁcantincreaseduringthelisteningphaseoftheimminent

threat(p<0.001)andtheimageryphasesofpotential(p<0.001),

imminentthreats(p=0.016)andpleasant(p<0.001).

ChangesinPAGactivitywereobserved[F(23,50)=3,21,p<0.001]

duringthelisteningphasesofthepleasantscenario(p<0.001)and

imminentthreat(p<0.001)andtheimageryphasesofthe

scenar-iospotential(p=0.023)andimminentthreats(p<0.001).Posthoc

analysesshowedasigniﬁcantsignalincreaseinthePAGduringthe

imageryphaseoftheimminentthreat,whencomparedtoallother

scenarios(FisherLSDtvalues=2.54,2.04and2.58,p<0.05).

4. Discussion

Whole-brainanalysisshowedthatthemostarousingscenario

(imminent threat) had higher BOLD signal in areas related to

secondaryauditoryprocessing(middletemporalgyrus)when

con-trastedwiththethreeremainingscenarios.Brainstem,cerebellum,

occipitalandinferiorparietalgyriwerespeciﬁcallyactivatedfor

proximalthreatscenarioonlywhenitwascontrastedwithneutral

orpleasant.Thisresultseemsplausibleinawaythatonlythese

twoscenariosaredifferentenoughfromproximalthreattoresult

inclustersinthebrainstem(PAGandthalamus)andcerebellum.

Iffromonesidecerebellumisresponsibleformotorplanningand

preparedness,PAGandmorerostralsubcorticalstructures

(tha-lamus) maybe related withresponses toemotionally arousing

stimuli.Asdescribedpreviously,theresultsherecorroboratethe

viewthatinformationtowardsactivationofthePAGoccursasa

functionofthreatproximity[29].Interestingly,inversecontrasts

againstproximalthreatresultedinclustersintherightmultimodal

parietallobesofthebrain,whatcouldsuggesttop-down

modula-tionofintegrativebrainareas.

Assumingthat mentalimageryandactualsituationsactivate

thesamebrainregions[16,30],wepredictedthatimaginationof

aproximalthreatscenariowouldactivatethePAG,whilethatof

potentialthreatwouldactivateforebrainstructures,aspreviously

reportedwithactualthreatsituations[13–15].Ourresultsmeet

thesepredictions.ThePAGshowedhigherBOLDsignalduringthe

processingofasituationrelatedtoimminentandintensethreat

whencomparedwithsituationsrepresentingpotentialthreat,

neu-tralorpleasantcontent.Ontheotherhand,theBOLDresponsein

thevmPFCduringtheimaginationoftheimminentthreatscenario

wassigniﬁcantlyreducedincomparisonwiththeBOLDresponse

duringtheimaginationofthepleasantscenario.Theactivityofthe

rightamygdalaalsoincreasedduringtheprocessingofapotential

threatincomparisontoaneutralsituation.

Theabove resultsare inagreementwiththose describedby

Mobbsandco-workersusingactualthreatsituations.Intheirﬁrst

study,inwhichavirtualpredatorcapableofchasing,capturing,

andinﬂictingpainpursuedavirtualpreythroughamaze,there

wasanincrease of brainactivityin thevmPFCduring the

pro-cessingofapotentialordistantthreat,butasthethreatbecame

closer,thebrainactivityshiftedtothePAG.Moreover,the

magni-tudeofPAGactivationpositivelycorrelatedwithsubjectivefeelings

ofdreadandnegativelycorrelatedwithfeelingsofconﬁdence[13].

In a furtherexperiment, it was shown that the anticipationof

anaversiveevent(potentialthreat)increasedtheactivityof

fore-brainstructures,mainlythevmPFC,hippocampus,hypothalamus

andamygdala,whereasimminentthreatelicitedactivitymainly

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aphylogeneticthreat,theresultsshowedthatasatarantulagrew

nearertooneofthesubject’sbarefoot,themagnitudeofsubjective

fearcorrelatedwithincreasedactivityinthePAG,amygdala,and

bednucleusofthestriaterminalis,whereasthevmPFCwas

acti-vatedasthetarantulawasmovedaway[15].However,wedidnot

ﬁndahigheractivationofthevmPFCduringthepotentialthreat

scenario,probablybecauseourparadigmwasnotstrongenough

toelicittheactivationpatternobservedinthestudiesdescribed

above,withactualthreatscenarios.

Thepresentlinearmodelpointedtoapositiveresponseofthe

BOLDsignalduringthelisteningphaseoftheimminentthreat

sce-nario,whenthevolunteershadtoplanwhattodotoaddressthe

situationpicturedintheinstructions.Thisoutcomeisinaccordance

withtheroleattributedtothevmPFCofgatheringcontingencyand

contextualinformationand,indirectly,viatheamygdala,of

mod-ulatingthedefensivebehaviorandtheshiftfromforebrainareas

tothemidbrainPAGactivationinmoreintensethreatsituations

[9,14].

Inthesamedirection,duringthelisteningphaseoftheimminent

threatthere wasanincreaseintheBOLDsignalofACC,

bilater-ally,whichmayberelatedtoitsproposedfunctionofmonitoring

goal-directedbehavior,processingstimulusvalence,and

engag-ingattentiontorelevantstimuli[31,32].Duringtheimageryphase,

therewasanegativeBOLDsignalinACC;bilaterally,duringthe

imagerycondition.Thisresultcanbeassociatedtotheproposition

thattheACCismainlyactivatedattheearlystagesofdemanding

tasks,whenitisintegratingsensorialandautonomicinformation

[33].It is interestingtonotethat this patternof hemodynamic

activityintheACCduringtheprocessingofanimminentthreat

wasnotobservedduringtheprocessingofalessintenseandmore

ambiguoussituation.However,asituationsupposedtobeneutral

presentedanalternativepatternofassociation−apositivesignal

duringthelisteningphaseandnegativesignalduringtheimagery

phase.Apossibleexplanationforthisphenomenonisthat even

thoughthesituationisneutral,thescenariorequiredactionsas

answeringthephone andmakingpreparationsfor the

appoint-ment.Incontrast,thewakingupwithavagueanddubiousdistal

noiseinthesecondscenariomaylackthiscalltoactiononspeciﬁc

objectsorpeople.

We alsofoundanincreased BOLDsignalin theright

amyg-daladuringtheimaginationphaseofthescenarios,notonlywith

aversivecontent,butalsowithappetitivecontent.Theamygdala

isassociatedwithhedonicvalenceattribution,morespeciﬁcally

reward processing [34,35] and also intensity-related activation

[36].Nevertheless,wewerenotabletodiscriminatewhich

amyg-dalarnucleiwouldbeinvolvedinpositiveornegativeemotional

processing due to thelow spatial resolution of the fMRI

tech-niqueused.Inthepresentexperiment,thecommonfactorwasthe

salienceoftheemotionalcontent[37,38]ofthethreescenariosthat

activatedtheamygdala.Accordingly,thebrainactivityoftheright

amygdaladuringtheneutralscenariowassigniﬁcantlylowerthan

duringthepleasantscenario.

ThemeaningofthepresentfMRIanalysisisunderscoredbythe

presentassessmentofbehavioralresponsesassociatedwiththe

dif-ferentimaginedscenarios.Supportingthehypothesisthatmental

imageryisasuitableparadigmfortheinvestigationofdefensive

neuralprocessinginhumans,theobtainedresultsshowedthat

dif-ferentnarrativescenariosprovokedexpectedchangesinsubjective

feelingstothescenario’sfeatures, fromneutraland pleasantto

potentialandimminentdanger.

Theratingofqualitiesofthementalimagerytaskshowedthat,

ingeneral,theimageswereeasyandclearlyimagined,visualizedas

mediumsizedpictures.Moreover,thetaskofassumingactionsto

speciﬁcsituationswasevaluatedaseasytoperform,with

preserva-tionofthecontentofthenarratives.Theseresultsarecomparableto

previouslyreportedobservationswithasimilarparadigmof

con-speciﬁcconfrontationscenariosshowingareductionin cerebral

bloodﬂowinvmPFCduringtheimaginationofadefensiveattack

behavior[19].Interestingly,thescenariosselectedtorepresentthe

extremesofthehedonicvalencecontinuum(pleasantscenariovs.

imminentthreat) weretheonesimaginedwithbestclarity.The

engagementofthevolunteerswiththetaskwasalsosuccessfully

evaluated,basedonthereportedreductionincognitiveimpairment

aftertheimageryphase,independentlyofthescenario,andwith

noincreaseinsedation.

AccordingtoLang etal.[6],thescenarios wereevaluatedin

termsofhedonicvalence,andarousal.Overall,althoughthe

differ-encesinthesubjectiveresponsesforthedifferentscenarioswere

mild,ourﬁndingsdrawa gradientofthreat,froma neutral

sit-uationtoanintensethreat,withamoderatethreatplacedatan

intermediate position;totheopposite directionwasa pleasant

and calmingscenario.Thisgradient wassupportedbythedata

regardingsubjectiveanxiety.Boththreateningsituationsstudied

–thedistal/potentialscenarioand theproximaland high

mag-nitudethreatscenario–wereabletoincreasesubjectiveanxiety.

However,complaintsaboutdiscomfortandsomaticsymptomsof

anxietyweresigniﬁcantlyincreasedonlyaftertheimminentthreat,

mostlikely becauseitrepresentsa threatthatdemands urgent

responses.Inthepreviousvalidationstudiesoftheseaversive

sce-narios[4,5,39],theﬁrstchoiceresponseforthisscenariowasto

attack,coherentwiththeevaluationofthehighestmagnitudeof

threat,lowestpossibilityofescapeandlowestpossibilityof

ﬁnd-ingahidingplace.Ontheotherhand,theﬁrstchoiceresponsefor

themostambiguousscenariowastocheckoutthesituation,which

correspondstotheriskassessmentbehaviorobservedinrodents.

Thisstudyhasseverallimitations:thesamplesizeofboth

exper-imentswassmall,andwomenwerethemostwhovolunteeredto

participate,moreover,withoutcontrolforthephaseofthe

men-strualcycle;thenwemustbecarefulingeneralizingtheresultsto

otherpopulations.TheBlanchards’scenariosareatoolresulting

froma composite ofﬁve dimensions inﬂuencingthreat

evalua-tion,notbeingourintent,inordertomaintainitsconstructand

itsecologicalvalidity,toisolateonlyoneofthem[40].However,

weintroducedanewfactor,theprosodyintheauditorystimuli,

intentionallychosentoincreaseemotionalengagementofsubjects.

Thechoiceofsingleprototypicalscenariosofproximaland

dis-talthreatimpliesrestrictiontothegeneralization,butitwasdone

becauseofthecomplexityofthefMRIexam.Wetriedtoavoidvery

longdatacollectionthatincreasesthechanceofheadmovements

andfatigue.Wealsodidnotcheckforordereffectofscenario

pre-sentationinthebehavioralandfMRIparadigms.Asanexploratory

study,ordertesting wouldextendexcessivelytheexperimental

sessionordemandabiggersample.Oncethedecisionofnot

pro-longingthefMRItaskwastaken,thescenarioswerepresentedin

aﬁxedsequence.Wehopedthattheintervalbetweentwo

scenar-iosusedwassufﬁcienttodisengagethevolunteerandpreventa

carry-overeffectfromonesituationtothenext,especiallyfrom

potentialtoimminentthreat.Evenso,werecognizetheneedto

testordereffectinfuturestudies.Finally,thefMRItechniqueused

hasrelativelylowspatialresolution,whatcouldraisesome

criti-cismonproblemsofVOIsizes,spatialﬁlterrangeandanatomical

location.ThispointcouldbeespeciallyrelevantintheePAG.

Nev-ertheless,eventhoughsomeresultsintheVOIsanalysisarenot

coincidentwithpreviousresults[13,41],ourwhole-braincontrast

mapsindicatethattheclustersshowingdifferentialactivitiesare

inPAG,chieﬂyintheproximalthreatcondition.

4.1. Conclusion

Inspiteoftheseshortcomings,thepresentresultsshow,toour

knowledgefortheﬁrsttime, thatimaginaryscenariosof

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fearandanxietynotasstrongasbutinthesamewayasactual

threatsituations[13–15],and,inbothcases,theactivationpattern

wasconsistentwiththatreportedinnon-humanspecies.

There-fore,thepresentresultsindicatethatneuralprocessingofrelevant

behavioralprocessescanbestudiedinhumansubjectsusing

men-talimagery,obviatingtheethicalandpracticallimitationsofreal

threatsituations. Onceit is knownthat differentmental

disor-dersembodydisturbanceinthedefensivesystem,futureresearch

investmentsmightdisentanglethedimensionsinﬂuencingofthe

agonistencounterthatcomposethethreatintensity,asdistance,

andshedlightonspeciﬁccontributiontothedefensiveprocessing.

Becauseanxietyandfeardisordersarelikelytoinvolve

dysfunc-tionofbrainstructuresthatcontroldifferentdefensestrategies,

suchstudiesarelikelytoenhanceourunderstandingofthe

patho-physiologyofthesedisorders.

Acknowledgements

ThisstudywassupportedbyFundac¸ãodeAmparoàPesquisa

do EstadodeSãoPaulo(FAPESP-2007/03685-3).DBA,ACS,FGG

andCMDBaresupportedbyresearchfellowshipsfromConselho

Nacional de Desenvolvimento Cientíﬁco e Tecnológico. RS and

CRaresupportedbyFAPESP.Theauthorsdeclarenocompeting

ﬁnancialinterests.Thefundershadnoroleinstudydesign,data

collectionandanalysis,decisiontopublish,orpreparationofthe

manuscript.

References

[1]D.C.Blanchard,G.Griebel,R.J.Blanchard,Mousedefensivebehaviors: pharmacologicalandbehavioralassaysforanxietyandpanic,Neurosci. Biobehav.Rev.25(May(3))(2001)205–218.

[2]R.J.Blanchard,K.J.Flannelly,D.C.Blanchard,Defensivebehavioroflaboratory andwildRattusnorvegicus,J.Comp.Psychol.100(June(2))(1986)101–107.

[3]R.J.Blanchard,M.A.Hebert,P.F.Ferrari,P.Ferrari,P.Palanza,R.Figueira,etal., Defensivebehaviorsinwildandlaboratory(Swiss)mice:themousedefense testbattery,Physiol.Behav.65(November(2))(1998)201–209.

[4]D.C.Blanchard,A.L.Hynd,K.A.Minke,T.Minemoto,R.J.Blanchard,Human defensivebehaviorstothreatscenariosshowparallelstofear-and anxiety-relateddefensepatternsofnon-humanmammals,Neurosci. Biobehav.Rev.25(December(7–8))(2001)761–770.

[5]R.Shuhama,C.Del-Ben,S.Loureiro,F.Graeff,Defensiveresponsestothreat scenariosinBraziliansreproducethepatternofHawaiianAmericansand non-humanmammals,Braz.J.Med.Biol.Res.41(April(4))(2008)324–332.

[6]P.J.Lang,M.M.Bradley,B.N.Cuthbert,Emotion,motivation,andanxiety:brain mechanismsandpsychophysiology,Biol.Psychiatry44(December(12)) (1998)1248–1263.

[7]A.M.Perkins,P.J.Corr,Reactionstothreatandpersonality:psychometric differentiationofintensityanddirectiondimensionsofhumandefensive behaviour,Behav.BrainRes.169(April(1))(2006)21–28.

[8]A.P.Carobrez,K.V.Teixeira,F.G.Graeff,Modulationofdefensivebehaviorby periaqueductalgrayNMDA/glycine-Breceptor,Neurosci.Biobehav.Rev.25 (December(7–8))(2001)697–709.

[9]J.F.Deakin,F.G.Graeff,5-HTandmechanismsofdefence,J.Psychopharmacol. 5(January(4))(1991)305–315.

[10]N.McNaughton,P.J.Corr,Atwo-dimensionalneuropsychologyofdefense: fear/anxietyanddefensivedistance,Neurosci.Biobehav.Rev.28(May(3)) (2004)285–305.

[11]V.Magierek,P.L.Ramos,N.G.daSilveira-Filho,R.L.Nogueira,J.

Landeira-Fernandez,Contextfearconditioninginhibitspanic-likebehavior elicitedbyelectricalstimulationofdorsalperiaqueductalgray,NeuroReport 14(August(12))(2003)1641–1644.

[12]L.J.Bertoglio,H.Zangrossi,Involvementofdorsolateralperiaqueductalgray N-methyl-d-asparticacidglutamatereceptorsintheregulationofrisk assessmentandinhibitoryavoidancebehaviorsintheratelevatedT-maze, Behav.Pharmacol.17(November(7))(2006)589–596.

[13]D.Mobbs,P.Petrovic,J.L.Marchant,D.Hassabis,N.Weiskopf,B.Seymour, etal.,Whenfearisnear:threatimminenceelicitsprefrontal-periaqueductal grayshiftsinhumans,Science317(August(5841))(2007)1079–1083.

[14]D.Mobbs,J.L.Marchant,D.Hassabis,B.Seymour,G.Tan,M.Gray,etal.,From threattofear:theneuralorganizationofdefensivefearsystemsinhumans,J. Neurosci.29(September(39))(2009)12236–12243.

[15]D.Mobbs,R.Yu,J.B.Rowe,H.Eich,O.FeldmanHall,T.Dalgleish,Neural activityassociatedwithmonitoringtheoscillatingthreatvalueofatarantula, Proc.Natl.Acad.Sci.U.S.A.107(November(47))(2010)20582–20586.

[16]D.Sabatinelli,P.J.Lang,M.M.Bradley,T.Flaisch,Theneuralbasisofnarrative imagery:emotionandaction,Prog.BrainRes.156(2006)93–103.

[17]E.P.Vianna,N.Naqvi,A.Bechara,D.Tranel,Doesvividemotionalimagery dependonbodysignals?Int.J.Psychophysiol.72(April(1))(2009)46–50.

[18]P.J.Lang,M.M.Bradley,B.N.Cuthbert,Internationalaffectivepicturesystem (IAPS):affectiveratingsofpicturesandinstructionmanual,Gainesville, TechnicalReport,A-6(2005).

[19]P.Pietrini,M.Guazzelli,G.Basso,K.Jaffe,J.Grafman,Neuralcorrelatesof imaginalaggressivebehaviorassessedbypositronemissiontomographyin healthysubjects,Am.J.Psychiatry157(November(11))(2000)1772–1781.

[20]H.Norris,Theactionofsedativesonbrainstemoculomotorsystemsinman, Neuropharmacology10(March(21))(1971)181–191.

[21]A.W.Zuardi,I.G.Karniol,Estudotransculturaldeumaescaladeauto-avaliac¸ão paraestadossubjetivos,J.Bras.Psiquiatr.31(5)(1981)403–496.

[22]A.W.Zuardi,F.S.Guimarães,A.C.Moreira,Effectofcannabidiolonplasma prolactin,growthhormoneandcortisolinhumanvolunteers,Braz.J.Med. Biol.Res.26(February(2))(1993)213–217.

[23]A.C.Parente,C.Garcia-Leal,C.M.Del-Ben,F.S.Guimarães,F.G.Graeff, Subjectiveandneurovegetativechangesinhealthyvolunteersandpanic patientsperformingsimulatedpublicspeaking,Eur.Neuropsychopharmacol. 15(December(6))(2005)663–671.

[24]W.Schneider,A.Eschman,A.Zuccolotto,E-PrimeUser’sGuide,Psychology SoftwareToolsInc.,Pittsburcg,2002.

[25]J.Talairach,P.Tournoux,Co-PlanarStereotaxicAtlasoftheHumanBrain, 3-DimensionalProportionalSystem:AnApproachtoCerebralImaging, ThiemeMedical,NewYork,1988.

[26]E.F.Carvalho-Netto,C.Markham,D.C.Blanchard,R.L.Nunes-de-Souza,R.J. Blanchard,Physicalenvironmentmodulatesthebehavioralresponses inducedbychemicalstimulationofdorsalperiaqueductalgrayinmice, Pharmacol.Biochem.Behav.85(September(1))(2006)140–147.

[27]R.K.Butler,A.C.Sharko,E.M.Oliver,P.Brito-Vargas,K.F.Kaigler,J.R.Fadel, etal.,Activationofphenotypically-distinctneuronalsubpopulationsoftherat amygdalafollowingexposuretopredatorodor,Neuroscience175(February) (2011)133–144.

[28]M.Brett,K.Christoff,R.Cusack,J.Lancaster,UsingtheTalairachatlaswiththe MNItemplate,Neuroimage1(2001).

[29]E.J.Hermans,M.J.Henckens,K.Roelofs,G.Fernández,Fearbradycardiaand activationofthehumanperiaqueductalgrey,Neuroimage66(February) (2013)278–287.

[30]E.A.Holmes,A.Mathews,Mentalimageryinemotionandemotional disorders,Clin.Psychol.Rev.30(April(3))(2010)349–362.

[31]P.Kanske,S.A.Kotz,Effortfulcontrol,depression,andanxietycorrelatewith theinﬂuenceofemotiononexecutiveattentionalcontrol,Biol.Psychol.91 (September(1))(2012)88–95.

[32]P.G.Gasquoine,Localizationoffunctioninanteriorcingulatecortex:from psychosurgerytofunctionalneuroimaging,Neurosci.Biobehav.Rev.37 (March(3))(2013)340–348.

[33]C.S.Weston,Anothermajorfunctionoftheanteriorcingulatecortex:the representationofrequirements,Neurosci.Biobehav.Rev.36(January(1)) (2012)90–110.

[34]M.G.Baxter,E.A.Murray,Theamygdalaandreward,Nat.Rev.Neurosci.3 (July(7))(2002)563–573.

[35]P.H.Janak,K.M.Tye,Fromcircuitstobehaviourintheamygdala,Nature517 (January(7534))(2015)284–292.

[36]L.Bonnet,A.Comte,L.Tatu,J.L.Millot,T.Moulin,E.MedeirosdeBustos,The roleoftheamygdalaintheperceptionofpositiveemotions:anintensity detector,Front.Behav.Neurosci.9(2015)178.

[37]A.B.Fernando,J.E.Murray,A.L.Milton,Theamygdala:securingpleasureand avoidingpain,Front.Behav.Neurosci.7(2013)190.

[38]D.J.Hayes,N.W.Duncan,J.Xu,G.Northoff,Acomparisonofneuralresponses toappetitiveandaversivestimuliinhumansandothermammals,Neurosci. Biobehav.Rev.45C(September)(2014)350–368.

[39]D.C.Blanchard,G.Griebel,R.Pobbe,R.J.Blanchard,Riskassessmentasan evolvedthreatdetectionandanalysisprocess,Neurosci.Biobehav.Rev.35 (March(4))(2011)991–998.

[40]D.C.Blanchard,G.Griebel,R.Pobbe,R.J.Blanchard,Riskassessmentasan evolvedthreatdetectionandanalysisprocess,Neurosci.Biobehav.Rev.35 (March(4))(2011)991–998.

[41]J.T.Buhle,H.Kober,K.N.Ochsner,P.Mende-Siedlecki,J.Weber,B.L.Hughes, etal.,Commonrepresentationofpainandnegativeemotioninthemidbrain periaqueductalgray,Soc.Cogn.Affect.Neurosci.8(August(6))(2013) 609–616.