Neuropsychologia49 (2011) 3648–3655
ContentslistsavailableatSciVerseScienceDirect
Neuropsychologia
jo u r n al h om ep a ge :w ww . e l s e v i e r . c o m / l o c a t e / n e u r o p s y c h o l o g i a
The
neural
basis
of
the
Enigma
illusion:
A
transcranial
magnetic
stimulation
study
Manuela
Ruzzoli
a,b,∗,1, Simone
Gori
c,d,1, Andrea
Pavan
e,1, Cornelia
Pirulli
b,
Carlo
A.
Marzi
a,
Carlo
Miniussi
b,faDepartmentofNeurological,Neuropsychological,MorphologicalandMotorSciences,UniversityofVerona,Italy bCognitiveNeuroscienceSection,IRCCSSanGiovannidiDioFatebenefratelli,Brescia,Italy
cDepartmentofGeneralPsychology,UniversityofPadua,Italy
dDevelopmentalNeuropsychologyUnit,ScientificInstitute“E.Medea”,BosisioParini,Lecco,Italy eSISSA,CognitiveNeuroscienceSector,ViaBonomea265,34136Trieste,Italy
fDepartmentofBiomedicalSciencesandBiotechnologies,NationalInstituteofNeuroscience,UniversityofBrescia,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received26April2011
Receivedinrevisedform9September2011 Accepted10September2011
Available online 19 September 2011
Keywords:
Enigmafigure Motionillusion
Repetitivetranscranialmagnetic stimulation(rTMS)
V1 V5/MT
a
b
s
t
r
a
c
t
Theaimofthisstudywastotesttheroleofthevisualprimary(V1)andthemiddletemporalarea(V5/MT) intheillusorymotionperceptionevokedbytheEnigmafigure.TheEnigmafigureinducesavisualillusion thatischaracterizedbyapparentrotatorymotioninthepresenceofastaticfigure.Bymeansofrepetitive transcranialmagneticstimulation(rTMS)weshowthatV5/MTiscausallylinkedtotheillusoryperception ofmotion.WhenrTMSwasappliedbilaterallyoverV5/MTjustpriortopresentationoftheEnigmafigure, theperceptionofillusorymotionwasdisruptedforapproximately400msresultinginadelayedillusion onset.Incontrast,rTMSappliedoverV1didnothaveanyeffectontheillusoryperceptionofmotion.These resultsshowthatV5/MT,avisualcorticalareaassociatedwithrealmotionperception,isalsoimportant fortheperceptionofillusorymotion,whileV1appearsnottobefunctionallyinvolvedinillusorymotion perception.
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Enigma,devisedbyLeviant(1981 – Palaisdela Découverte,
Paris)isafigurethatelicitsthespontaneousperceptionofrotary
motionintheabsenceofrealmotion.Theimageconsistsofablack
andwhiteraypatternwithnarrowlyspacedradiallines,ontowhich
threechromaticringsaresuperimposed(Fig.1).Thepresenceof
illusorymotionseenontheringssuggestedthename“TheEnigma”.
Theillusorymotionoftheannulialternatesbetweenclockwiseand
counter-clockwiserotation(Gori,Hamburger,&Spillmann,2006;
Leviant,1996).
Enigmaisa peculiarmotionillusionwhichmakesthisfigure
veryinterestingtostudy.Whilemostothermotionillusions
repre-sentamisperceptionofmotiondirection(Gori&Hamburger,2006;
Gori&Yazdanbakhsh,2008;Gori,Giora,&Stubbs, 2010; Pinna &Brelstaff, 2000; Wallach,1935; Yazdanbakhsh &Gori, 2008),
theillusorymotionintheEnigmafigureis presentunderstatic
viewingconditions;thatis,nomotionoftheobserverorofthe
∗ Correspondingauthorat:CognitiveNeuroscienceSection,IRCCSSanGiovanni diDioFatebenefratelli,viaPilastroni,4,25123Brescia,Italy.
E-mailaddress:manuela.ruzzoli@cognitiveneuroscience.it(M.Ruzzoli).
1 Equallycontributedauthors.
stimulus is necessary to perceive the illusory rotary motion.
Anothermotionillusion,theRotatingSnakes(KitaokaandAshida,
2003),elicitsmotionunderstaticviewingconditions,butitdoes
notshowmotionreversalsastheEnigmafiguredoes.Allofthese
characteristicsmaketheEnigmafigureaveryuniqueandintriguing
motionillusion.
Severalhypotheseshavebeenformulatedonthemechanisms
underlying the illusory motion induced by the Enigma figure.
Gregory(1993,1995)proposedanexplanationintermsof
fluctua-tionsintheeyeaccommodation(“huntingforaccommodation”)
and fixational eye movements (FEMs) (Gregory, 1994;
Mon-Williams&Wann,1996),suggestingthatthesefactorsmayaccount
forthe illusioninduction. Zeki,Watson, andFrackowiak(1993)
showedbymeansofpositronemissiontopography(PET)thatwhen
participantsperceivedillusorymotion,theregionalcerebralblood
flow (rCBF)increasedin thesame brainareas that wereactive
whenparticipantswerelookingataphysicallymovingstimulus
(V5/MT,anextrastriateareahighlyinvolvedinvisualmotion
pro-cessing(Born&Bradley,2005)andadjacentmotionareas,suchas
V3a,V3bandMST),suggestingthereforethattheillusorymotion
couldbemediatedbythesameneuronsasrealmotion.Moreover,
inaddition,duringtheillusorymotionobservation, theauthors
reportedrCBFincreasesinotherareasoutside thevisualcortex
(rightfrontaloperculumandrightanteriorcingulategyrus),which
0028-3932/$–seefrontmatter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropsychologia.2011.09.020
Fig.1.TheEnigmafiguredrawnbyLeviant(1981).
are notusuallyactivatedwhenparticipantsare exposedtoreal
motion.Therefore,alsothesebrainregionscouldbecandidatesfor
mediatingperceptionoftheEnigmafigure.Basedontheseresults,
Zekietal.(1993)arguedthatthegenerationofillusorymotionmay
dependontheactivationoffrontalareasthatarenotactive
dur-ingexposuretorealmotion,thusexcludingretinalinfluencesinthe
illusionperception.Ontheotherhand,acomputationalmodel
con-sistentwithavailableneurobiologicalevidencewasproposedby
Fermuller,Pless,andAloimonos(1997)thatattemptstomakeboth
FEMsandcorticalactivationnecessaryfortheperceptionofmotion
inducedbytheEnigmafigure.Theirmodelproposesthateye
move-mentsarenecessarytotriggertheillusoryrotationintheEnigma
figurebutthathigh-levelcorticalprocessesaremainlyresponsible
fortheillusion.Recently,Hamburger(2007)andKumarandGlaser
(2006)claimedthattheillusorymotionintheEnigmafigureisnot
theresultofmotionoftheimageontheretinaduetomicrosaccades
but rather hasa strictly cortical origin.Gori et al. (2006)
pro-videddatasupportingthecorticalroleofthisillusoryphenomenon
withoutexcludingthepossibilitythatFEMswereinvolvedinthe
generationoftheillusion.Theseauthorshighlightaclear
regular-ityinthereversalsthatsuggeststhepresenceofaneuralsaturation
mechanismatthecorticallevel.Moreover,theyshowedhowreal
motioncaninfluenceillusorymotion,suggestingthatrealand
illu-sorymotioninteractatsomepointalongthemotion-processing
pathway. On the other hand,Troncoso, Macknik, Otero-Millan,
andMartinez-Conde(2008)convincinglydemonstratedthatsmall
eyemovementsareanecessaryconditiontoperceivetheillusory
motion.Theseauthorssetouttoresolvethelong-standingdebate
astowhethertheinitialneuralprocessesthatultimatelyleadtothe
perceptionoftheEnigmaillusionariseintheeyeorinthebrain.
Becausetheillusionwasfoundtobestronglydependentonsmall
eyemovements,theanswerseemstobethattheillusion
percep-tionstartsintheeye,ratherthaninthebrain,evenifthebrain
isnecessarilyinvolvedintheillusoryperception.Troncosoetal.
(2008)found,indeed,thattherateofmicrosaccadesincreasedjust
beforethereportedperceptionoffasterillusorymotion,compared
withadecrementintherateofmicrosaccadeswhenthe
partic-ipantsreportedslowerornoillusorymotionduringaprolonged
viewingoftheEnigmafigure.Therefore,althoughafull
explana-tionofhowtheillusionarisesisstillacontroversialtopic,itseems
reasonablethat,eventhoughtheFEMsprobablytriggerthe
illu-sion,thecortexplaysapivotalroleintheperceptionoftheillusory
motion.
V5/MTinvisualmotionprocessing(d’Alfonsoetal.,2002;Laycock
etal.,2007;Sacketal.,2006;Stevensetal.,2009):anearly
activa-tionbeginningapproximately60mspriortostimuluspresentation
andalatetemporalwindowofactivationbeginningapproximately
130÷150msafterstimuluspresentation(Stevensetal.,2009).In
thefirstexperimentofthepresentstudy,wevariedthetime
win-dowinwhich repetitiveTMS(rTMS)wasdeliveredoverV5/MT,
eitherbeforeoraftertheappearanceoftheEnigmafigureinorder
todefinethetemporalwindowinwhichperceptionoftheEnigma
illusioncouldbeaffected.Inthesecondandthirdexperiment,in
viewofthenumerousfeedforwardandfeedbackprojections
exist-ingbetweenV1andV5/MT(Born&Bradley,2005)weexploredthe
roleoftheV1cortexintheperceptionoftheillusion.
2. Experiment1:rTMSoverV5/MT
2.1. Materialsandmethods 2.1.1. Participants
Sixteennaïvehealthyparticipants(sevenmales,20÷28yearsold),tookpartin theexperimentaspaidvolunteers.Allofthemhadnormalorcorrectedtonormal visualacuity.Noneofthemhadneurological,psychiatricorotherrelevant medi-calproblemsoranycontraindicationtorTMS(Rossietal.,2009).Allparticipants gaveinformedconsentandtheexperimentalprotocolwasapprovedbytheEthics CommitteeofIRCCSSanGiovannidiDio,Fatebenefratelli,Brescia,Italy.
2.2. Stimuliandapparatus
Stimuliweredisplayedona19-inchmonitorwitharefreshrate
of60HzthatwasgeneratedwithMatlabPsychtoolbox(Brainard,
1997; Pelli,1997). Thescreen resolution was1024×768.Each
pixelsubtended∼1.9arcmin.ThegrayscaleEnigmaillusionused
byHamburger(seeFig.2AinHamburger,2007)servedasstimulus
(Fig.2A).Asacontrolstimulus(Fig.2B),thesameversionofthe
Enigmafigurewasused,exceptthattheradiallineswerereplaced
byablackhomogeneousbackground(seeFig.2Iin Hamburger,
2007). According toHamburger (2007), this stimulus does not
induceillusory motion. Both Enigmaand controlfigures had a
Michelsoncontrastof98%.Thefiguressubtended15.4×18.3◦ of
visualangleand were presentedat thecenter of thescreen. A
blackcentralfixationpoint(0.22◦)overlappedwiththecenterof
thefigures.
2.3. TMSprotocol
TMS was applied simultaneously and bilaterally over
V5/MT using two Magstim Super Rapid Magnetic Stimulators
(50Hz—biphasic, four boosters) and two figure-of-eight coils
(custom double 50mm; Magstim Company Limited, Whitland,
Fig.2. (A)Theexperimentalstimulus(Enigmafigure).(B)Thecontrolstimulus.
positions of severalreference points (e.g., Cz,Oz, O1, O2 from
theInternational10/20EEGsystem)werereproduced.Individual
targetarealocations weredetermined bya functionalmethod,
i.e.,searchingthepositionsforwhichreliablemovingphosphenes
wereinducedbyasingleTMSpulseappliedwithcoilspositioned
onbothhemispheres.Thisprocedurewasperformedstartingfrom
5cmlateraland3cmabovetheinion.Theseareasweremarked
onthecapastheparticipant’sstimulationsitesforthestudy.The
meancoil positionfor left V5/MTstimulation wasidentified at
5.28cm (±0.33) lateralto theinionand 3.57cm (±0.88) above
the inion. The mean coil position for right V5/MT stimulation
wasidentifiedat4.87cm(±0.45)lateraltotheinionand3.30cm
(±0.66)abovetheinion;weplacedthejunctionofthetwocoil
wingsabovetheselocations.Thecoilwaspositionedtangentially
withrespect tothe scalp. The control conditionwas an active
stimulationoverthevertex(Cz,fromtheInternational10/20EEG
system), a location that corresponds to thecaudal part of the
frontalcortex,thatis,abrainareathatisnotinvolvedinvisual
processing. During the experiment, the stimulating coils were
fixed by two articulated mechanical holding arms (Manfrotto
Magicarmswithtwoclampseach)andtwoheavy-dutytripods.
Thepulseintensitywasfixedforalltheparticipantsat50%ofthe
outputofthestimulatorsforbothcoils.DuringtheTMSsessions,
sevenTMSpulsesweredeliveredata frequencyof15Hz.These
parametersareconsistentwithsafetyrecommendationsforrTMS
(Rossietal.,2009).
Weplannedtwoperiodsofstimulationwithinthetargetareas.
InoneTMScondition,thepulsesweredischargedduringfixation,
i.e.,from−400to0msbeforetheappearanceoftheEnigma
fig-ure(conditionTMS-before).Inanothercondition(TMS-during),the
pulsesweredischargedfrom0to400msafterstimulusonset.
TheorderofthefourTMSconditions(TMS-before,TMS-during
bytwolocationsMT/V5and Cz)wasrandomizedacross
partic-ipants. The interval betweenone condition and theother was
approximately 10minutes, during which the participants were
askedtoansweraquestionnaire(Fertonani,Rosini,Cotelli,Rossini,
&Miniussi,2010)aboutpossiblediscomfortsinducedbyTMSin
theprecedingcondition.
2.4. Procedure
Theparticipantswereseated57cm awayfromthescreen.A
chinrestwasusedtostabilizethehead.Theyfirstfamiliarized
themselveswiththestimuliandwiththescalelevelsforranking
thestrengthoftheillusion.Subsequently,theyperformeda
train-ingblocktopracticethetask.Eachtrialstartedwithapuretone
of500ms.Afixationpointthenappearedandlasteduntiltheend
ofthetrial.Theparticipantswereinstructedtomaintaintheir
fix-ationatthecenterofthescreen.Theywererequiredtopressthe
spacebaronakeyboardassoonastheyperceivedthemotion
illu-sion(Gorietal.,2006).Ifnoresponsewasprovided,thestimulus
automaticallydisappearedafterfiveseconds.Afterresponse,the
participantswereaskedtorankthestrengthoftheillusionona
five-levelscale(Kumar&Glaser,2006)bypressingoneoftheindicated
buttons(0=noillusoryperception,1=weakperception,2=average
perception,3=clearperception,and4=vividperception).The
fol-lowingtrialstartedtensecondsaftertheresponserelatedtothe
strengthoftheillusorymotionwasgiven.
Afterthefirsttrainingblock,inwhichparticipantsperformed
thetaskwithoutanystimulation(i.e.,No-TMScondition),the
tar-getareas(bilateralV5/MTandCzconditions)wereidentified(as
previouslydescribed)andmarkedontheskullcap.Thepositionsof
thecoilsweremonitoredbytheexperimentersduringtask
execu-tion.Eachexperimentalblockconsistedof40trials.Thirtyofthese
consistedofEnigmafiguredisplays,whereastheremainingten
tri-alsconsistedofcontrolstimulusdisplays(catchtrials),asshownin
Fig.2B.
2.5. Results
Theresultsofthereactiontimes(RTs)areshown inFig.3A.
Becausethecatchtrialsneverproducedillusorymotion,the
anal-yseswereconductedexclusivelyonthetrialsinwhichtheEnigma
figurewasdisplayed.Apaired-sample t-testdidnotrevealany
significantdifference betweenthe two Cz TMS conditions (i.e.,
CzTMS-beforevs. CzTMS-during)(t7=−0.69, p=0.52).For this
reason, in the other analysis, we considered the Cz TMS
con-ditions as a single control condition (i.e., with no distinction
betweenCzTMS-beforeandCzTMS-during).ThemeanRTswere
as follows: No-TMS condition,2.50s (SE:0.24); Cz TMS
condi-tion,2.52s(SE: 0.25); V5/MT TMS-before condition,2.93s (SE:
0.25);andV5/MTTMS-duringcondition,2.46s(SE:0.25).A
one-wayrepeated-measuresanalysesofvariance(ANOVA)performed
on the RTs and considering the following conditions—Cz TMS,
V5/MTTMS-beforeandV5/MTTMS-during—revealedasignificant
Fig.3.(A)ResultsofExperiment1.RTsareplottedasafunctionoftheTMScondition.WhenrTMSwasdeliveredoverV5/MT-beforetheappearanceofthestimulus,theRTs weresignificantlylongerwithrespecttotheCzTMSconditionandV5/MT-duringTMScondition.(B)ThestrengthoftheillusionisplottedasafunctionoftheTMScondition. rTMSoverV5/MT-beforethestimulusappearanceslightlyreducedtheperceivedstrengthoftheillusionwhencomparedwiththeCzTMSconditionbutthedifferencewas notsignificant.Errorbarsindicatestandarderrors.
Pairwisecomparisons(Bonferronicorrected)revealedasignificant
differencebetweentheV5/MTTMS-beforeconditionand theCz
TMScondition(t15=3.83,p=0.004)andbetweentheV5/MT
TMS-beforeconditionandtheV5/MTTMS-duringcondition(t15=3.56,
p=0.008).In summary,theRTs weredelayedbyapproximately
400mswhenTMSwasappliedbilaterallyoverV5/MTbeforethe
appearanceoftheEnigmafigure,comparedwithbothCzTMSand
V5/MTTMS-duringconditions.
Fig.3Bshowstheresultsoftheperceivedstrengthofthe
illu-sion.Apaired-samplet-testdidnotrevealanysignificantdifference
betweenCzTMS-beforeandCzTMS-during(t7=−0.54,p=0.60).As
forRTs,weconsideredtheCzTMSconditionsasasinglecontrol
condition.Inordertoprovideaparallelwiththeresultsobtained
with theRTs,we conducted a seriesof t-tests(Bonferroni
cor-rected)consideringtheCzTMSandV5/MTTMS-beforeconditions.
Paired-samplet-testsrevealedanon-significanteffectbetweenthe
V5/MTTMS-beforeconditionandtheCzTMScondition(t15=−1.55,
p=0.14).Alltheotherpaired-samplet-testsdidnotreachthe
sig-nificancelevel.
Theresultsofthediscomfortquestionnaireshowedthatcoil
pressureandnoisewerethemostdisturbingeffectswithno
dif-ferenceforthelocationofthecoil(V5/MTorCz).
3. Experiment2:rTMSoverV1
Inthepreviousexperiment,wefoundthatTMSapplied
bilat-erally overV5/MTfrom−400msto0msbeforetheappearance
oftheEnigmafiguredelayedthetimenecessarytoreliably
per-ceivetheillusorymotion.Someauthors(Laycocketal.,2007;Sack
etal.,2006;Stevensetal.,2009)foundthatthedisruptionprofile
ofV5/MTTMSforglobalmotionperceptionconsistsoftwo
dis-tinctepochs(i.e.,anearlyactivationandalateractivation)during
whichglobaldirectionjudgmentsareimpaired.Whilethelate
acti-vationdoesnotappearcontroversial,differentexplanationshave
beenproposedtoexplaintheroleoftheearlyactivation.Sacketal.
(2006)referredtoitasaTMS-inducedartifact,suggestingthe
possi-bilitythatTMSmightinduceeyeblink,asalsoreportedbyCorthout,
HallettandCowey(2003).Onthecontrary,Laycocketal.(2007)
proposedtwoalternativehypotheses.Inparticular,they
hypothe-sizedthatTMScoulddisruptmotionprocessingdirectlythroughan
antidromicinterferencewiththelateralgeniculatenucleusofthe
thalamus(LGN)(deLabraetal.,2007),bypassingcorticalactivation.
Alternatively,theyhypothesizedthatthedeficitinmotion
percep-tioninducedbyV5/MTTMSpriortostimulusappearancemightbe
aconsequenceofadisruptedattentionalmechanismor
expecta-tion(Laycocketal.,2007).Finally,Stevensetal.(2009)suggested
afeed–forward–feedbackmodelinwhichtheearlyimpairmentof
performanceissupposedtobeduetoanindirect(backward)effect
ofTMSonV1.
Inthelightofthesecontroversies,inthisexperiment,we
inves-tigatedwhetherV1 isinvolvedin theperceptionoftheEnigma
illusionandwhetherthereisatemporalwindowofactivationthat
iscomplementarytothatofV5/MT.
3.1. Materialsandmethods
Thesameparticipantsfromthepreviousexperimentparticipatedinthesecond experimentafteratleastonemonthhadpassed.Thestimuli,apparatusand proce-duresofthesecondexperimentwerethesameasinthefirstexperiment,withthe exceptionofthestimulationsite,whichchangedfromV5/MTtoV1.
3.2. TMSprotocol
TMSwasappliedoverV1usingaMagstimSuperRapidMagnetic
Stimulatorand one figure-of-eight coil (customdouble 70mm;
MagstimCompanyLimited,Whitland,UK).Becausethemagnetic
fieldsfromtwocoilsinteractwithoneanotherifthecoilsareplaced
tooclosetogether,wedecidedtouseasinglebiggercoil,insteadof
two50mmcoils,aswasdoneinthepreviousexperiment.Before
theexperiment,individualtargetarealocationsweredetermined
bysearchingthepositionatwhichreliablestaticphospheneswere
inducedbyasingleTMSpulse.Thisprocedurewasperformed
start-ingfrom1cmabovetheinionandmovingslightlyinalldirections.
Thetargetareawasmarkedonthecapastheparticipant’s
stimula-tionsiteforthestudy.ThemeancoilpositionforV1stimulation
wasidentified at2.15cm (±0.55)abovetheinion. Alltheother
conditionswerethesameasinExperiment1.
3.3. Results
Fig.4AshowstheRTresultsforExperiment2.Becausewetested
thesame participantsof Experiment1, we usedthe datafrom
thesamecontrolconditions(No-TMSandCz-TMS)oftheprevious
experiment.InExperiment2,themeanRTfortheV1TMS-before
Fig.4.(A)ResultsofExperiment2.MeanRTsforeachTMScondition.NoeffectoftheTMSconditionwasobserved.(B)Thestrengthoftheillusionisplottedasafunctionof theTMScondition.rTMSoverV1didnotreducetheperceivedstrengthoftheillusionwithrespecttotheCzTMScondition.Errorbarsindicatestandarderrors.
was2.62s(SE:0.25).Aone-wayrepeated-measuresANOVA
con-sideringthefollowingconditions—CzTMS,V1TMS-beforeandV1
TMS-during—didnotrevealanysignificanteffectoftheTMS
con-dition(F2,30=0.13,p=0.87,2=0.009).Fig.4Bshowstheresultsof
theperceivedstrengthoftheillusion.AsinExperiment1,we
con-ductedat-test(Bonferronicorrected)consideringtheCzTMSand
V1TMS-beforeconditions.Apaired-samplet-testrevealedno
sta-tisticaldifferencebetweentheV1TMS-beforeconditionandthe
CzTMScondition(t15=1.3,p=0.20).Alloftheotherpaired-sample
t-testsperformeddidnotreachthesignificancelevel.
Theresultsofthediscomfortquestionnaireshowedthatcoil
pressureandnoisewerethemostdisturbingeffectswithno
dif-ferenceamongcoillocations(i.e.,V1,CzorV5/MTofExperiment
1).
4. Experiment3:rTMSoverV1withsmallerstimuli
InthepreviousexperimentwefoundthatrTMSappliedoverV1
didnotdelaytheperceptionoftheillusorymotionincomparison
totheCzTMScondition.However,itcouldbepossiblethatthe
stimuluswastoolargetobeaffectedbyrTMS(asitcoveredan
areaof15.4×18.3◦).Thatis,rTMSoverV1mayhavenotaffected
asufficientportionofV1tointerferewiththewholeextentofthe
stimulus.Inordertocontrolforthispossibilityweperformedan
additionalexperimenthalvingthesizeofthestimuli.
4.1. Materialsandmethods 4.1.1. Participants
Anewsampleofsixnaïvehealthyparticipants(threemales,25÷37yearsold), participatedintheexperimentaspaidvolunteers.Theyallmetthesamecriteriaas theparticipantsofthepreviousexperiments.
4.2. Stimuli,apparatus,TMSprotocolandprocedure
Thestimuliwerethesameofthepreviousexperimentsbutthe
figuressubtended7.7×9.15◦ofvisualangle.Thestimulisizewas
chosenbasedonthesmalleststimulusstillconveyingavivid
illu-soryeffect.Apparatus,TMSprotocolandprocedurewerethesame
ofExperiment2.
4.3. Results
Fig.5AshowstheRTresultsforExperiment3:meanRTforthe
V1TMS-beforeconditionwas2.20s(SE:0.23),fortheV1
TMS-duringconditionwas2.18s(SE:0.24)andforCz(averagedbetween
beforeandduring)conditionwas2.64(SE:0.22).TheCzTMS
con-ditionshowedthelargestdelayfortheillusorymotiononset:there
wasnodisruptiveeffectduetorTMSonV1.Aone-way
repeated-measuresANOVAconsideringthefollowingconditions–CzTMS,
V1TMS-beforeandV1TMS-during– revealedasignificanteffectof
TMS(F2,10=5.38,p=0.026,2=0.52).Howevernoneofthe
paired-samplet-tests(Bonferronicorrected)reachedthesignificancelevel.
Fig.5Bshowstheresultsoftheperceivedstrengthofthe
illu-sion.AsinExperiment1and2,weconductedat-test(Bonferroni
corrected)consideringtheCzTMSandV1TMS-beforeconditions.
Apaired-samplet-testrevealednostatisticaldifferencebetween
theV1TMS-beforeconditionandtheCzTMScondition(t5=0.63,
p=0.9).Alloftheotherpaired-samplet-testsperformeddidnot
reachthesignificancelevel.
Theresultsof thediscomfort questionnaireshowedthat coil
pressureandnoisewerethemostdisturbingeffectswithno
dif-ferenceamongthelocationsofthecoil(i.e.,V1,CzorV5/MTofthe
Experiment1).
5. Discussion
Inthepresentstudy,weaimedatassessingaselectivecortical
involvementintheperceptionoftheEnigmaillusionbyapplying
rTMSovervisualareasV1andV5/MT.TheresultsshowedthatrTMS
deliveredoverV5/MTbeforetheonsetoftheEnigmafigure
dis-ruptedtheillusorymotionperceptionforapproximately400ms,
comparedwiththeconditioninwhich rTMSwasappliedatthe
onsetoftheEnigmafigureorwhenrTMSwasdeliveredoverCz.In
addition,wefoundthatrTMSappliedoverV5/MTbeforethe
stim-ulusonsetslightly reducedtheperceivedillusorystrength with
respecttotheCzTMScondition,eventhoughthatdifferencedid
notreachsignificancelevel.Thus,itseemsthatV5/MTisdirectly
involvedintheperceptionoftheEnigmaillusion.Thisresultis
con-sistentwithpreviousfindingsreportedbyZekietal.(1993)who,
usingPET,foundanactivationofV5/MTandrightfrontalregions
duringthepresentationoftheillusion.WhileZekietal.(1993)
pro-posedthattheillusorymotionarisesanewinthebrain,weretain
thattheperceivedmotionistriggeredbytheFEMs(Troncosoetal.,
2008)butthesameareathatprocessestherealmotion(V5/MT)is
alsocrucialfortheillusorymotionoftheEnigmafigure.
On theotherhand,we foundnoeffect ofrTMS onthe
per-ceptionoftheillusionwhenitwasappliedoverV1,regardlessof
stimulationtimewindowandstimulussize.ThelackofanyrTMS
Fig.5. (A)ResultsofExperiment3.MeanRTsforeachTMScondition.rTMSoverV1didnotdelaytheillusorymotiononsetwithrespecttotheCzTMScondition.(B)The strengthoftheillusionisplottedasafunctionoftheTMScondition.rTMSoverV1didnotreducetheperceivedstrengthoftheillusionwithrespecttotheCzTMScondition. Errorbarsindicatestandarderrors.
inprocessingtheillusorymotioninductedbytheEnigmafigure.
However,inprinciple,ourresultscannotruleoutaV1
involve-mentintheEnigmaillusion.ApossibleinvolvementofV1inthe
early motionprocessingis suggested by physiological evidence
(Maunsell&vanEssen,1983;Movshon&Newsome,1996;Pack, Conway,Born,&Livingstone,2006)showingthatneuronsinarea
V5/MTaretunedforthedirectionofstimulusmotion,primarilyas
aconsequenceofastrongprojectionfromdirection-selectiveV1
neurons.However,V1direction-selectiveneuronsarethoughtto
sensethemotionoflocalcontours,whileV5/MTneuronsintegrate
this localmotioninformationtoextract globalmotion(Mather,
2011). It could bepossible that the illusory motion of Enigma
depends mainlyonhighlevelofmotionprocessing wherelocal
motionsignalsareintegratedtoextractglobalmotion(i.e.,V5/MT).
Inthiscase,rTMSwouldinterferewiththeillusorymotiononly
whendeliveredovertheextrastriateareaV5/MT.
Analternativeexplanationaboutthelackoftheeffectwhen
stimulating V1 couldbe that Enigma elicits a rotatory illusory
motion. Electrophysiological, human neuroimaging and
psy-chophysical studieshave shown that complex moving patterns
likeradialmovingpatterns(i.e.,contracting/expanding),spiraland
rotationalmovingpatternsareselectivelyprocessedbyhigh-level
visualareassuchasV5/MTandMST(Morroneetal.,2000;Smith,
Wall, Williams, &Singh, 2006;Wall, Lingnau,Ashida, &Smith,
2008).Theseauthors,usingfMRI,foundthatthecorticalareasthat
respondtocomplexmotionandtranslationalmotionwereclearly
distinctwithintheconfinesoftheMT+complex.Thus,itislikely
thatEnigmaelicitsakindofillusorymotionthatisselectively
pro-cessedbyhigh-levelvisualareas.
However,inprincipleonecouldarguethatrTMSisnotableto
selectivelyaffectthemotiondirection-selectiveV1neuronsorat
leastthatitisnotaffectingthemuptoalevelthatproducesa
per-ceptualeffect.Basedonthatpossibilitywecanonlysafelyclaim
that,withourexperimentalsetting,V1seemsnottobeinvolvedin
theillusorymotionperceptionevocatedbyEnigmaandthata
dif-ferentapproachmightbemoreeffective.Thisisforfurtherstudies
toascertain.
Furthermore,inthepresentstudy,a potentialconfoundthat
could lead toa differencebetween theeffect of stimulation of
V5/MT and V1 is the useoftwo coils for theV5/MT condition
andone,bigger,coilfortheV1condition.PotentiallytheV5/MT
condition couldbemore distracting and uncomfortable for the
participants.However,theresultsofthediscomfortquestionnaire
showednodifferencesamongthecoillocations(V5/MT,V1orCZ).
Mostimportantly,ifthedelayobservedintheV5/MT-before
con-ditionwithrespecttotheV1TMSandtheCzTMSconditionswas
merelyduetothediscomfort givenbythetwo coils,thiseffect
shouldbepresentalsointheV5/MTTMS-duringandnotonlyin
theV5/MTTMS-beforecondition,butthiswasnotthecase.Thus,
thispotentialconfoundseemstoberuledout.
Anothercharacteristicofthisstudythatisimportantto
high-lightistheuseofdifferenttimewindowsofrTMS.Theabsenceof
therTMSeffectduringtheEnigmaobservationwasanunexpected
resultthatcouldleadtofuturestudiestoabetterunderstanding
ofthephenomenon.Basedonourresultsitseemsthatthe
pre-activationofV5/MTismoreimportantthantheactivationduring
theEnigmaobservation,atleastforthespecifictaskandstimulus
weemployed.TheactivationofV5/MTduringtheEnigma
observa-tioncouldbemorerelevantinsessionswithlongerexposuretime,
i.e.,whensomespontaneousreversalsarepresent.
Ourresultsshowthatthecriticalperiodofactivationwasbefore
theappearanceofthefigure.Laycocketal.(2007)suggestedthat
TMSdeliveredprior tothestimulusappearance mighthave an
effectby interfering withthesubject’sexpectation or withthe
attentionalprocessing.Moreover,theearlyV5/MTTMSdisruptive
effecthasbeenlinkedtoanindirectnoise inductionintheLGN
(Laycocketal.,2007)orinV1(Stevensetal.,2009)throughfeedback
connectionfromV5/MT.AnotherexplanationcouldbethatTMS
induceseyeblinkingwhenappliedbeforethestimulusappearance
(Corthoutetal.,2003;Sacketal.,2006).However,ifTMSincreased
theRTtoperceivetheillusioninourparticipantsbecauseofeye
blinks,onewouldhaveexpectedastrongeroratleastasimilar
effectwhenstimulatingacorticalareadifferentfromV5/MT(e.g.,
V1inourexperiment).Furthermore,Stevensetal.(2009)directly
addressedthepossibilitythatTMSinduceseyeblinksby
stimulat-ingtheprimarymotorcortexwhiletheparticipantswereengaged
inamotion–directiondiscriminationtaskandconcurrently
record-ingtheeyeblinksthroughavideocameraintwoparticipants.Their
resultsclearlyrefutedtheeyeblinkhypothesis.Itseemslikelythat
theperformanceimpairmentintheearlyperiodofV5/MT
stimula-tionmightbeexplainedbyaninterferingmechanismviafeedback
connectionsfromV5/MTtoV1(Stevensetal.,2009).Incontrastto
thispossibility,inthepresentstudyrTMSoverV1didnotyielda
delayintheperceptionofillusorymotion.
AnotherpossibleexplanationfortheRTdelayfoundwhen
brainoscillationsplayinvisualperception(i.e.,alpha-band)and
thepossibilityofentrainingaparticularoscillationfrequencyby
brainstimulation(Thut&Miniussi,2009).Romeietal.(2008)found
thatthe restingalpha-band (8–15Hz) powerover the
parietal-occipitalcortexisinverselycorrelatedtotheperceptualoutcomeof
asingle-pulseTMStoinducephosphenes.Moreover,Romei,Gross,
andThut(2010)showedthatshorttrainsofrTMSatalpha
fre-quencyoverthevisualareascausallydeterminedtheperceptual
outcomeofforthcomingstimulusdetection.Inthepresentstudy
weusedastimulationfrequencyof15Hzwhichcanbeconsidered
tobewithintherangeofthealpha-band.Onepossibilityisthat
TMSdeliveredatthistemporalfrequencybeforetheappearance
oftheEnigmafiguremight“entrain”thebrainoscillationwithin
thealpha-band,leadingtoanimpairmentoftheperceptionofthe
illusoryrotatorymotion.However,theroleofbrainoscillationsin
illusoryvisualmotionprocessingwasoutofthescopeofthepresent
studyandfurtherTMS/EEGorMEGstudiesarenecessarytocast
lightonthispoint.
6. Conclusions
Eventhoughfixationeyemovementscanprobablytriggerthe
Enigma illusion(Gregory, 1994; Mon-Williams& Wann, 1996;
Troncosoetal.,2008),clearlycorticalactivityplaysacrucialrole
intheperceptionof illusorymotionevoked bytheEnigma
fig-ure.Ourresultsareinagreementwiththehypothesissuggested
byTroncosoetal.(2008)thatFEMswouldtriggerneural
process-inginmotion-sensitiveareasofthebrain.V5/MT,anareathatis
normallyassociatedwiththeperceptionofrealmotion,appears
tobeimportantalsofortheperceptionofillusorymotion,while
V1doesnotappear tobecausallyinvolved.Theseresultsinvite
futurestudiesaimedatunderstandingwhetherandwhereillusory
andrealmotionsdifferintheircorticalprocessingpathwaysthus
contributingtosolveanotheraspectofthepuzzlenamedEnigma.
Disclosurestatement
Noneoftheauthorshasanypersonalorinstitutionalconflictof
interest.
Thispaperisnotunderconsiderationbyanyotherjournaland
ithasnotpreviouslypublished.
Theprotocolhasreceivedprior approval bythe appropriate
Ethicscommitteeandinformedconsentwasobtainedfromeach
subject.
Allco-authorshaveagreedwiththecontentsofthemanuscript.
Acknowledgement
ThisresearchworkwassupportedbytheItalian Ministryof
UniversityandResearchandbytheMinistryofHealth.
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