Neural mechanisms underlying heterogeneity in the presentation of anxious temperament

16 Pages·2013·0.99 MB·English

Neural mechanisms underlying heterogeneity in the
presentation of anxious temperament
AlexanderJ.Shackmana,b,c,1,Andrew S.Foxa,b,c,d,e,f,1,JonathanA.Olera,b,c,StevenE. Sheltona,b,
RichardJ.Davidsona,b,d,e,f,andNedH.Kalina,b,c,d,f,2
aDepartmentofPsychiatry,dDepartmentofPsychology,cLaneNeuroimagingLaboratory,eCenterforInvestigatingHealthyMinds,bHealthEmotions
ResearchInstitute,andfWaismanLaboratoryforBrainImagingandBehavior,UniversityofWisconsin,Madison,WI53719
EditedbyBruceS.McEwen,TheRockefellerUniversity,NewYork,NY,andapprovedFebruary22,2013(receivedforreviewAugust18,2012)
Childrenwithananxioustemperament(AT)areatriskfordeveloping To address this question, we used a well-validated nonhuman
psychiatric disorders along the internalizing spectrum, including primate model of early-life AT in combination with high-res-
anxietyanddepression.Likethesedisorders,ATisamultidimen- olution 18fluorodeoxyglucose (FDG)-positron emission tomog-
sionalphenotypeandchildrenwithextremeanxietyshowvarying raphy(FDG-PET)(2,15).
mixturesofphysiological,behavioral,andothersymptoms.Using Youngrhesusmacaques areideal forunderstanding the neu-
a well-validated juvenile monkey model of AT, we addressed the robiology ofdispositional anxietyin humanchildren. Reflecting
degreetowhichthisphenotypicheterogeneityreflectsfundamen- the two species recent evolutionary divergence, the brains of
taldifferencesorsimilaritiesintheunderlyingneurobiology.The monkeysandhumansaregenetically,anatomically,andfunctionally
rhesusmacaqueisoptimalforstudyingATbecausechildrenand similar (16–18). Homologous neurobiological substrates endow
young monkeys express the anxious phenotype in similar ways monkeys and humans with a shared repertoire of complex cog-
andhavesimilarneurobiology.Fluorodeoxyglucose(FDG)-positron nitiveandsocio-emotionalbehaviors(18).Inparticular,juvenile
emission tomography (FDG-PET) in 238 freely behaving monkeys monkeysandyoungchildrenexpressanxietyinsimilarways,and CE
identified brain regions where metabolism predicted variation in inbothspeciesthereareconsiderableindividualdifferencesin CIEN
three dimensions of the AT phenotype: hypothalamic-pituitary- thepresentationoftheanxiousphenotype.Inmonkeys,theAT OS
adrenal (HPA) activity, freezing behavior, and expressive vocal- phenotypecanbeelicitedusingtheNo-EyeContact(NEC)con- UR
E
izations. We distinguished brain regions that predicted all three ditionoftheHumanIntruderParadigm(15).DuringtheNEC N
dimensionsofthephenotypefromthosethatselectivelypredicted challenge,ahuman“intruder”entersthetestroomandpresents
asingledimension.Elevatedactivityinthecentralnucleusofthe hisorherprofiletothemonkeywhileavoidingdirecteyecontact
aaetifrnroelmceegrtievoynhizoasgdeitsnndievmgsiadindlombaduateiaecovahtldrinsiadvidvcawuiotitoaytirethrtlhsd.eienxFwhruatie(grihMntlthaehht1etelli)deraroimviwotnfeefrosalerssrahrbeloeis,epnfaeatpnltHwceottpPceiectvArraieeivitmonsaeyrecpatitnhnuoimvtisaptittyhtphywigeooo,sdaasncesaeanslmpdcawohopdfeimntuenAhssceoiTrsthwett.ayiaegbIapsnnhoseetd-lclslisyoeseeamvnllfceeeottccrliuastvtaiinnivsvottddyeef, (aia(fnr1Hnned5dxeP)iizvo,Abiisunedi)smghua)paaic,lvhlataieiordvnnriitdotfaoyflteepy(irxprniepnoehnrcciwecerbseedeistasruiiesvorieenenadssctisuhpnoesrlmseacesdshmeemidflufod:aunrrhniceyacdonpasarstot(emiito1ssh9nseoai)nnll.()atgr,aUmebdldsieidciushn-picpmagotivisteotiuihontniiirtssosai(noircinaynnh-lscaasarlpdolneeorfaxennistnegtheataedey-l, PSYCHOLOGICALANDCOGNITIVESCIENCES
neousvocalizations).Allthreedimensionsshowrobustchanges
differentexpressionsofanxiety.Thesefindingsprovideaframe-
inresponsetotheNECchallenge(15),parallelingobservations
workforunderstanding the mechanisms thatlead to heteroge-
made in dispositionally anxious and shy children (5).
neityintheclinicalpresentationofinternalizingdisordersand
AkeyadvantageofthejuvenilemonkeyATmodelisthatit
setthestagefordevelopingimprovedinterventions.
permitsconcurrentmeasuresofneuralactivityandnaturalistic
| | responses to an ethologically relevant potential threat, an op-
affectiveneuroscience behavioralinhibition
| | portunitynotaffordedbyresearchinchildren.Here,FDG-PET
developmentalpsychopathology emotion functionalneuroimaging
wasusedtoquantifybrainmetabolicactivityin238freelybehaving
juvenile monkeys. FDG-PET, which provides a measure of re-
There is substantial heterogeneity in the clinical presentation
gionalbrainmetabolismintegratedovertheentire30-minNEC
of anxiety disorders, both within and across diagnostic cate-
challenge,isideallysuitedforassessingsustained,trait-likeneural
gories.Anxietyoftenemergesearlyindevelopmentand,heretoo,
responses(1).
thereisconsiderablevariationinpresentation.Clinicallyrelevant Using these measures, we identified brain regions where me-
anxiety is often accompanied and preceded by an anxious tem-
tabolism predicts variation in one or more of the three AT
perament(AT).ATisatrait-likephenotypethatisevidentearly
dimensions. To understand the degree to which heterogeneity in
in life, stable over time, associated with increased amygdala re- the presentation of AT reflectsinvariant or distinct neural mech-
activitytonoveltyandpotentialthreat,andexpressedsimilarlyin anisms,wedistinguished“common”and“selective”substrates.
children and young nonhuman primates (1–6). Extreme disposi-
Common neural substrates are those shared by individuals with
tional anxiety and behavioral inhibition in childhood is a well-
varyingexpressionsofanxiety;that is,acoresetofbrainregions
establishedriskfactorfortheinternalizingspectrumofpsychiatric
wheremetabolismpredictsvariationinallthreedimensionsofthe
disorders,includinganxietyandmajordepression(5,7,8).These
disorders are highly prevalent and associated with substantial
morbidityandmortality(9,10).Liketheinternalizingdisorders,
Authorcontributions:A.S.F.andN.H.K.designedresearch;S.E.S.performedresearch;
childhoodATisacomplex,multidimensionalphenotypeandchil- A.S.F.contributednewreagents/analytictools;A.J.S.,A.S.F.,J.A.O.,andN.H.K.analyzed
dren with extreme AT show varying mixtures of peripheral physi- data;andA.J.S.,A.S.F.,J.A.O.,R.J.D.,andN.H.K.wrotethepaper.
ological, behavioral, and other kinds of anxiety-related symptoms Theauthorsdeclarenoconflictofinterest.
(5, 11, 12). This diversity manifests as weak covariation among ThisarticleisaPNASDirectSubmission.
thesefeatures(2,13,14).Fromtheperspectiveofdiagnosisand 1A.J.S.andA.S.F.contributedequallytothiswork.
treatment,animportantunresolvedquestionisthedegreetowhich 2Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected].
heterogeneity in anxious individuals’ symptoms reflects funda-
Thisarticlecontainssupportinginformationonlineatwww.pnas.org/lookup/suppl/doi:10.
mentaldifferencesorsimilaritiesintheunderlyingneurobiology. 1073/pnas.1214364110/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1214364110 PNASEarlyEdition | 1of6
with one another, is important for understanding how phenotypic
variationinATearlyindevelopmentgivesrisetoheterogeneity
intheclinicalpresentationofinternalizingdisorderslaterinlife,
for facilitating the development of improved therapeutic inter-
ventions,andforunderstanding emotionaltraits.
Results
HeterogeneityandContinuityinthePresentationoftheATPhenotype.
Like other emotional traits, individual differences in the pro-
fileofanxiety-relatedsymptomsmanifestsasweakcovariation
amonganxiety-relatedmeasures(2,13,14).Consistentwiththis,
in the present sample a series of robust regressions revealed
that variation in cortisol was largely independent of freezing
andvocalreductions,R2<0.02,P>0.06.AsshowninFig.1,
some individuals are characterized by extreme HPA activity
and little freezing and vocal reductions, some show the op-
posite presentation, and yet others show intermediate pre-
sentations. Regression analyses also showed that, although
individuals who froze more emitted fewer vocalizations, the
two measureswerefarfromredundant,R2= 0.23,P < 0.001.
These results underscore the substantial heterogeneity in the
presentationofAT.Furthermore,allthreedimensionsofthe
anxiousphenotypewerereliable(SIMethods)andcontinuously
distributed (Fig. 1). This continuity suggests that AT, like the
Fig.1. HeterogeneityandcontinuityinthepresentationofAT.Pairwise internalizingdisorders(11,12),representsaspectrumofclosely
partialcorrelationsrevealedthatcortisolwaslargelyindependentoffreezing
related phenotypes rather than a mixture of categorically dis-
andvocalreductions(AandB).Freezingandvocalreductionswerecorrelated
tinct subgroups.
butfarfromredundant(C).Allthreedimensionswerecontinuouslydistrib-
uted,suggestingthatATrepresentsamultidimensionalspectrumofpheno-
types,ratherthanamixtureofdistinctsubgroups.Axislabelsindicatethe Heterogeneity in the Phenotypic Presentation of AT Reflects both
minimum,maximum,andinterquartilerange. CommonandSelectiveSubstrates.Because HPA activity,freezing
behavior,andvocalreductionsarecontinuouslydistributed(Fig.
1),weadoptedadimensionalapproachtoidentifyingtheneural
ATphenotype(HPAactivity,freezingbehavior,andexpressive circuitry underlying different expressions of anxiety. Specifically,
communication).Based onimagingandlesionstudiesinmonkeys we performed a series of voxelwise partial correlations between
andhumans(4,6,20–22),wehypothesizedthatthecentralnucleus metabolism and each AT dimension while accounting for varia-
of the amygdala (Ce) would be among the regions identified as tionin the other two (using robust regression techniques; see
shared substrates. Selective neural substrates are those specifi- SIMethods).Thisprocessrevealedanumberofregionswhere
cally engaged by individuals with high levels of a particular di- metabolismpredictedtheuniquevarianceinoneormoredimen-
mension;thatis,regionswheremetabolismpredictsvariationin sions of the phenotype, including the orbitofrontal cortex (OFC;
only one of the AT dimensions. Mechanistic studies in rodents areas13and47),anteriorinsula(AI),primarymotorcortex(M1),
suggestthattheCecanorchestrateabroadspectrumofresponses amygdala,hippocampus,andperiaqueductalgray(PAG)(Fig.2
to threat via projections to response-specific targets (23, 24). andTablesS1–S3).
Therefore,wefurtherhypothesizedthattheassociationbetween To distinguish common neural substrates, those shared by
commonsubstrates(e.g.,Cemetabolism)andparticulardimensions individualswith differentexpressionsof theanxiousphenotype,
of the anxious phenotype would be mediated by the appropriate weusedathree-wayconjunction(i.e.,logicalAND)ofthethresh-
phenotype-selective substrate. Distinguishing common from se- oldedpartialcorrelationmapsshowninFig.2toidentifyregions
lectivesubstrates,andclarifyingthenatureoftheirrelationships where metabolism predicted variation in HPA activity, freezing
Fig.2. BrainregionswheremetabolicactivitysignificantlypredictedoneATdimensionindependentofvariationintheothertwo(FDRq<0.05).Redcircular
insetshowsmagnifiedview.Coordinatesindicatemillimeterfromtheanteriorcommissure.aHip,anteriorhippocampus;AI,anteriorinsula;L,left;M1,
primarymotorcortex(area4);OFC,orbitofrontalcortex(architectonicareas13and47);PAG,periaqueductalgray;pHip,posteriorhippocampus;R,right;Th,
thalamus.
2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1214364110 Shackmanetal.
behavior,andvocalreductions[false-discoveryrate(FDR)q<
0.05].AsshowninFig.3,thisanalysisrevealedaclusterinthe
right dorsal amygdala and bilateral clusters in the hippocampi
(Table S4) (hemispheric asymmetryanalyseswerenot significant
for the amygdala cluster; see SI Methods). Using a probabilistic
chemoarchitectonic map (SI Methods), we localized the dorsal
amygdala cluster to the lateral division of the central nucleus
(CeL)(Fig.S1).Themagnitudeofthethreepartialcorrelations
wassimilarintheamygdalaandanteriorhippocampus(P>0.19),
reinforcingtheideathattheseregionsmakesimilarcontributions
tothethreedimensionsoftheanxiousphenotype.Takentogether,
these results demonstrate that elevated metabolism in the CeL
and anterior hippocampus is a consistent feature of individuals
withdivergentphenotypicexpressionsofanxiety.Thisimplication
reflectsthefactthatthepartialcorrelationsestimatetheunique
relationshipbetweeneachphenotypedimensionandmetabolism
at a fixed level of the other two. For example, the partial cor-
relationforcortisolindicatesthatindividualswithhighlevelsof
HPAactivity(andintermediatelevelsoffreezingbehaviorand
vocalizations on average) show more activity in the CeL and
anteriorhippocampuswhencomparedwithindividualswithlow Fig. 4. Selective substrates: Regions where one AT dimension predicted
levelsofHPAactivity(andintermediatelevelsoffreezingbehavior significantvarianceinbrainmetabolism(partialcorrelation:FDRq<0.05)and
andvocalizationsonaverage).Forillustrativepurposes,these explainedmorevariancethantheothertwodimensions(differenceincorre-
effects are shown in Fig. S2. [Note: The results of the con- lations:FDRq<0.05),identifiedusingatwo-wayconjunction(logicalAND). CE
N
junction analysis suggest, but do not demonstrate, that aggre- ConventionsaredescribedinFig.2. CIE
gatingthethreedimensions—cortisol,freezing,andvocalization OS
R
reductions—into a composite would provide a more sensitive theothertwo.Thisprocessrevealedanumberofregions(Tables NEU
index of AT-related variation in metabolic activity. Confirmatory S1–S3).Inparticular,asshowninFig.4,individualscharacterized
analysesdemonstratedthatthiswasindeedthecase(SIMethods).] by high levels of HPA activity were distinguished by elevated
To distinguish selective neural substrates, regions that are metabolicactivityinthelateralanteriorhippocampus,whereas DS
specificallyengagedbyindividualswithhighlevelsofaparticular thosewhopresentedwithhighlevelsoffreezingbehaviorshowed ANNCE
AwsihoTendrseiamfmetenerstiacobonon,ltwirsoemlul:isn(eigd)faporrtewvdoai-rcwitaeatydiocononnienjuotnhfcettihooenthtteohrrietdweeonAt(iTfFyDdreiRmgiqoenn<s- acpotrtemefnrmuouantnteaidclaacticootrintveistxyho(invwlePthdFeCiMn)c.1rTe,aahsneeddlathateocrtsiaevliwtayinthitnelrotihowerlehvveiepnlpstrooocflaavmtoepcraaalll OLOGICALTIVESCIE
c0o.0m5)paarneddw(iitih) tehxepolathineerdtwmoo(aressevsasreidanucseinginavtohxaetlwdiismeetnesstioonf cfrluosmterththeantewaarsesstelcelcutsitveertothcaotrtpirseodliwctaesdloaclaltethdreseeveArTalmdiimlliemnseitoenrss PSYCHCOGNI
the difference in correlations; FDR q < 0.05). That is, we dis- (Fig. 3 and Table S4). These results clarify the neural systems
tinguishedregionswhereoneof thethreepartialcorrelations underlyingheterogeneity intheexpressionofanxiety.
showninFig.2wasbothsignificantandsignificantlystrongerthan We identified the CeL and anterior hippocampus as shared
substrates,regionswheremetabolismpredictsallthreedimensions
oftheanxiousphenotype.Toassesswhetherthosebrain-phenotype
relationships are explained by activity in phenotype-selective
regions,weusedaseriesofmultivariatemediationmodels(SI
Methods) to test whether the partial correlation between the
shared substrates (Fig. 3) and a particular dimension of the
AT phenotype depends on the relevant selective substrate
(Fig.4)(e.g.,CeL→ M1→freezing).AsshowninFig.5and
detailedinTableS5,thisprocessdemonstratedthatrelations
betweenthesharedsubstratesandHPAactivityweremediated
by activity in the cortisol-selective anterior hippocampal region,
P<0.005(uncorrected).Similarly,relationsbetweentheshared
substratesandfreezingbehaviorweremediatedbyactivityinM1,
P < 0.005 (uncorrected). Significant mediation was not found
for vocal reductions. To assess the specificity of the cortisol
and freezing results, we recomputed the mediation models
after reversing the candidate mediating regions (e.g., CeL →
lateralanteriorhippocampus→freezing).Noneofthesecontrol
models was significant, indicating a double dissociation (Table
S5). Specifically, metabolism in the lateral anterior hippo-
campus explains relations between the shared substrates and
HPA activity, but not freezing behavior, whereas M1 metabo-
lism explains relations between the shared substrates and
Fig.3. Sharedneuralsubstrates:Amygdalarandhippocampalmetabolism
freezing,butnotHPAactivity.Collectively,theseresultsclarify
pwraeydimctisnaimlluthmreceodnijmunecntsioionns(loofgitchaelAATNDph)eonfotthyepes.igFnigifiurceandteppiacrttsiathlecotrhrreelae-- howsharedsubstratescaninfluencemultipledimensionsofthe
anxious phenotype. In particular, these results indicate that
tions shown in Fig. 2. Regions shown in cyan/gold predicted the unique
varianceinallthreedimensionsoftheATphenotype.Circularinsetsshow relations between thecore set of shared substrates,such as the
magnifiedviews.Forotherconventions,seeFig.2. CeL, and the endocrine and behavioral dimensions of the
Shackmanetal. PNASEarlyEdition | 3of6
reflects the activity of a neurobiological system comprised of
bothsharedandphenotype-selectivecomponents.Asdescribed
below, these findings have mechanistic, translational, and theo-
reticalimplications.
Withrespecttomechanism,ourresultsshowthattheCeLand
anterior hippocampus are consistently engaged by individuals
withdivergentpresentationsofanxiety(Fig.3andFig.S2).This
findingisinaccordwithevidencethattheamygdalaandanterior
hippocampus show exaggerated activation to potentially threat-
relevantcuesinindividualswithavarietyofanxietydisordersor
a childhood history of extreme AT (6, 21). Similarly, lesions of
either region attenuate many signs of anxiety (20, 22, 23, 25).
Interestingly,recentworkinrodentssuggeststhattheCeLplays
a key role in gating the output of the amygdala (26, 27). In
particular, the CeL is poised to modulate both acute fear and
sustained anxiety via inhibitory projections to the two major
outputstationsoftheextendedamygdala:themedialdivisionof
the Ce and the lateral division of the bed nucleus of the stria
terminalis(26,27).
Ourresultsindicatethatindividualdifferencesintheexpression
ofanxietyreflecttheproximalcontributionofphenotype-selective
regions. In particular, we observed a double dissociation: M1
mediatedfreezingbehavior,butnotcortisol,whereasthelateral
Fig.5. Linkingcommonandselectivesubstrates:Brain-phenotyperelations anteriorhippocampusshowedtheoppositeprofile.Theselective
areselectivelymediated.(A)Metabolisminthecortisol-selectiveregionof
lateral anterior hippocampus mediates relations between the common role of the lateral anterior hippocampus in the endocrine dimen-
substrates(CeLandaHip)(Fig.3)andHPAactivity,P<0.005(uncorrected). sionofdispositionalanxietyisconsistentwithmechanisticevidence
(B)Metabolisminthefreezing-selectiveregionofM1mediatesrelations thatthehippocampusregulatestheHPAaxis(28).Thisresultmay
between the common substrates and freezing behavior, P < 0.005 (un- reflectthedensedistributionofmineralocorticoidreceptorsinthe
corrected).Theplusandminussymbolsindicatethesignofthepartialcor- primatehippocampus(29),whichareinvolvedinmoretrait-likeor
relation(seeTableS5). basalaspectsofHPAactivity(28).Theinvolvementof M1inthe
behavioral dimension of AT is consistent with its well-established
roleinvoluntaryaction.Weobtainedmorelimitedevidencethat
anxious phenotype strongly depend upon regions that are di-
mension-specific. thevlPFCisselectivelyinvolvedinthereductionofexpressivecoo
vocalizations,consistentwithworkimplicatingventralpremotor
Discussion areasinvocalizations andotherorofacial behaviors(30).
Althoughnoninvasivetechniques,suchasFDG-PET,cannot
Liketheinternalizingdisorders,thereismarkedvariationinthe
establish causation, our results are in accord with mechanistic
presentationofATduringearlydevelopment.Ourobservations
research demonstrating that the Ce orchestrates many of the
provide compelling evidence that this heterogeneity reflects the
peripheral physiological, behavioral, and expressive dimensions
jointcontributionofcommonsubstrates,acoresetofbrainregions
ofanxietyandthattheseeffectsaremediatedbyfunctionalinter-
that are shared by individuals with different manifestations of actionswithresponse-specifictargets(22–24).Ourresultsaddress
extreme anxiety, and selective substrates, regions that are spe-
the proximal substrates of individual differences in the presen-
cifically associated with particular expressions. Consistent with
tation of anxiety. Individuals characterized by high levels of
priorwork(2,13, 14),theendocrine, behavioral,and communi-
freezing,for example, are distinguished by attenuated activity
cative dimensions of the anxious phenotype were weakly corre-
inM1.However,thedistaldeterminantsofthisheterogeneity
latedandcontinuouslydistributed,suggestingthatATrepresents remain unclear; it may reflect variation in the strength of
a multidimensional spectrum of closely related phenotypes (Fig.
functional connectivity between the CeL and anterior hippo-
1).Usingadimensionalanalyticapproachthatcircumventedthe campus and particular phenotype-specific regions. Another pos-
need to impose artificial categorical boundaries on the data, we sibilityis thatitreflectsindividual differencesinsubpopulations
identified a number of regions where metabolic activity predicts of phenotype-specific neurons that are intermingled at a level
oneormoredimensionsoftheAT phenotype(e.g.,amygdala, beyond the resolution of conventional imaging techniques (31).
hippocampus, PAG, AI, and OFC) (Fig. 2). We demonstrated Indeed, evidence for distinct subpopulations of freezing- and
that variation in each dimension of the phenotype—increased cardiovascular-specific neurons in the Ce has led some inves-
HPA activity, more freezing behavior, and fewer expressive tigators to suggest the possibility of developing therapeuticinter-
vocalizations—wasindependentlypredictedbyactivityintheCeL ventions targeting disorder-specific or patient-specific differences
and anterior hippocampus (Fig. 3). Elevated activity in this core insymptomprofiles(31),consistentwithearliersuggestionsinthe
set of brain regions was consistently found in individuals who translationalliterature(32).
displayedhighlevelsofanyofthesedimensions(Fig.S2).We Between one-third and two-thirds of anxiety patients are
identifiedasecondsetofregionsthatspecificallypredictparticular treatment-resistantorrefractory(33),underscoringtheneedto
dimensions of the anxious phenotype, including the lateral an- developmoreefficaciousinterventions.Thepresentresultshigh-
teriorhippocampusandM1,andvlPFC(Fig.4).Activityinthese light the potential utility of broad-spectrum(i.e., multisymptom)
phenotype-selective regions distinguished individuals with high approaches.Inparticular,ourfindingssuggestthattherapeutics
levels of HPA activity, freezing behavior, and vocal reductions, aimed at molecular targets within the CeL and anterior hippo-
respectively. Finally, we demonstrated that these regions selec- campus, particularly when administeredearly inlife, could ame-
tively mediate the association between the shared substrates, liorateavarietyofmaladaptiveorexcessiveresponsestopotential
suchastheCeL,andtheendocrineandbehavioraldimensions threat. Overtime, suchresponses likelypromote morecomplex
of the AT phenotype (Fig. 5). In sum, these observations sug- and chronic symptoms (e.g., avoidance, anticipatory worry) and
gest that variation in the expression of dispositional anxiety neurobiologicalalterations(34,35).Thissuggestionisreinforced
4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1214364110 Shackmanetal.
by evidence that anxiety disorders with distinct presentations— Insummary,usingawell-validatednonhumanprimatemodel
includingposttraumaticstressdisorder,socialanxietydisorder, ofATandhigh-resolutionfunctionalimaging,thepresentstudy
specific phobias, and generalized anxiety disorder—are all char- demonstratesthatstrikingdiversityinthepresentationofanxiety
acterizedbyelevatedamygdalareactivitytoaversiveorpotentially reflectsthedistinctcontributionsofbothsharedandphenotype-
threatening stimuli (21, 36). Targeting upstream regions that are selectivesubstrates.Individualscharacterizedbyhighlevelsof
poised to regulate the CeL and anterior hippocampus (37), such HPAactivity,highlevelsoffreezingbehavior,orlowlevelsof
as the OFC, represents another approach to broad-spectrum expressive vocalizations all exhibit elevated metabolic activity
treatment.Inthisregard,cognitive-behavioraltherapy,whichis intheCeLandanteriorhippocampus.Thesebrain-phenotype
thought to be mediated by emotion regulatory processes imple- associations were dependent upon a second set of regions, in-
mentedintheprefrontalcortex(38,39),orcognitive-behavioral cluding the lateral anterior hippocampus and M1, which selec-
therapy combined with novel pharmacological interventions tivelymediateparticulardimensionsoftheanxiousphenotype.
thatincreaseneuroplasticity,maybeparticularlyeffective(40). Importantly,theseresultswereobtainedusingarelativelylarge
unselectedsampleandrobustanalyticprocedures,increasingthe
Developingmoreeffectiveearly-lifeinterventionsisparticularly
likelihood of replication. More broadly, these observations pro-
importantforminimizingthecumulativesocialandinterpersonal
videaframeworkforunderstandingtheneurobiologyofearly-life
damageassociatedwithextremeanxietyandbehavioralinhibition
anxiety and other emotional traits and set the stage for mecha-
duringearlydevelopment(5).
nisticstudiesaimedatidentifyingmoreeffectiveinterventionsfor
Thesedataalsohaveimplicationsforgeneraltheoriesofemo-
theinternalizingspectrumofdisorders.
tion.Theperipheralphysiologicalandbehavioralfeaturesthatare
thehallmarksofemotionhavetraditionallybeencastastightly
Methods
synchronized(41,42).However,facedwithgrowingevidenceof
Subjects received FDG before testing. The AT phenotype was elicited
weakresponsecoupling,theoristshavespeculatedthatnosingle bythepresentationofahumanintruder’sprofile(30-min).Anobserver
brainregioncouldorchestratealloftheseresponses—thatthe quantified freezing behavior and expressive vocalizations. Following
alterations in the face, voice, body, and mental experience testing,plasmawascollectedandsubjectswerescanned.HigherFDG-PET
characteristic of emotional statesand traits reflect the activity of signalsindicategreatermetabolismduringtesting.Plasmacortisolwas NCE
segregatedneuralcircuits(14,43).Thispossibilityisnotaddressed quantitatedbyradioimmunoassay.MRIandPETimageswereprocessed CIE
bypriorimagingstudies,whichhavetypicallymeasuredonlyone using standard methods and normalized to a stereotactic template OS
ortwoconcurrentresponsesorcompositeanxietymeasures.Nor (0.625 mm3). Robust regressions identified regions where activity pre- EUR
dicted the unique variance in cortisol, freezing, and vocal reductions. N
has it been directly addressed by lesion studies, which lack the
Analyses controlled for nuisance variation in mean-centered age, sex,
statistical power required to investigate phenotypic heterogene-
andvoxelwisegraymatterprobability.Commonandphenotype-selective
ity.Therefore,thepresentobservationthatactivityintheCeL substrates were identified using criteria described in the text. Mediation DS
and anterior hippocampus explains independent variation in analysesusedstandardanalytictechniques. ANNCE
eNclnaEdiCmocsrcihonafel,nleebnuegrheaalvpsiroeorgavrield,geaastniodunn.ciqoMumoemregurgnoeiucnnaetdriasvlelfyo,rrethsrpiesojefincnstedinsingtgositnrtdihciet- AB.CKCNhrOisWtiaLEnD,GAM. CENonTSv.eWrsee,thL.anFkrieE.dAmhalenr,s,MA..AJelessxoann,deEr.,LVa.rBsaolnch,eKn.,MR.aByiernr,, OLOGICALTIVESCIE
T.Oakes,P.Roseboom,N.Vack,H.VanValkenberg,L.Williams,thestaffs HNI
csiaotnesstohfaatnaxliaectykdoofesstrnoontgpcroevcaluridaetitohneaemxiostnegncdeifofefraenrtesdpiomnesne-- oInfsttihtuetHe,aarlnodwWCeisncotenrsifnorNBaitoiolongailcParliPmsaytcehoCleongtye,rH,aenaldthtEwmooatnioonnsyRmeosuesarrceh- PSYCCOG
independentsubstrate(44);individualscanvaryinthestrength viewers for their assistance. This work was supported by the National
InstitutesofHealth(NIH)(GrantsHD003352,HD008352,MH018931,MH046729,
andpredominanceofdifferentanxietydimensions,yetrelyon
MH081884,MH084051,MH091550,OD011106,andRR000167),theHealth-
the same core set of shared substrates. EmotionsResearchInstitute,andMeriterHospital.
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Supporting Information
Shackman et al. 10.1073/pnas.1214364110
SIMethods in age and, for cortisol, time-of-day using SPSS (v20.0.0; IBM).
Hypothesistesting usedthesampledescribedinref. 1, whichfo- Priorworkindicatesthatcortisol,freezing,andcoovocalizations
cused onthe heritability ofmetabolicactivity inthe medial tem- consistently show robust changes in response to the NEC chal-
porallobe.Methodsfortheelicitationandassessmentofanxious lenge(4,5,11–23).Brain-behavioranalysesusedreverse-scored
temperament(AT)andthequantificationof18fluorodeoxyglucose vocalizations(“vocalreduction”=−1×coo-frequency1/2)toensure
(FDG)-positronemissiontomography(PET)aredetailedinrefs1 thateffectswereconsistentlysignedacrossthethreedimensions
and2andsummarizedhere. ofthephenotype(i.e.,highervaluesindicatemoreintensereactions
tothe phenotype-eliciting NECchallenge).
Subjects.Briefly, 240 prepubescent monkeys [Macaca mulatta;
mean (SD) age = 2.41 (0.92) years; 51.3% female] from the FDG-PETandMRI.FDGandattenuationscanswereacquiredusing
Harlow Primate Laboratory or Wisconsin National Primate Re- a Siemens/Concorde microPET P4 scanner (24). Images were
searchCenterunderwentbehavioraltestingandFDG-PETaspart reconstructed using standard filtered-backprojection techniques
ofalargerinvestigationofthegeneticunderpinningsofAT(1,3). with attenuation- and scatter-correction. MRI were collected
PETdata from two individuals proved unusable. Housing and underanesthesia(seeabove)usingaGeneralElectricDiscovery
experimental procedures were performed in accord with guide- 3Tscanner(GE)andstandardquadratureextremitycoil.Scans
linessetforthbytheUniversityofWisconsin–MadisonInstitutional used a 3D T1-weighted inversion-recovery fast gradient echo
AnimalCareandUseCommittee. prescription(TR/TE/Flip/NEX/FOV/Matrix:9.4ms/2.1ms/10°/2/
140mm/512×512;248×1-mmaxialslices;gap:−0.05mm).
Overview. Subjects received intravenous FDG immediately before
the30-minNo-EyeContact(NEC)challenge.Individualdifferences ProcessingPipelineforImagingData.Beforespatialnormalization,
inbehavioralresponses(freezingandvocalizations)werequantified brainsweremanuallyextractedfromT1imagesusingSPAMALIZE
byanexperiencedobserver.Followingtesting,plasmawascollected (http://psyphz.psych.wisc.edu/~oakes/spam/spam_frames.htm).
for quantifying cortisol and subjects were deeply anesthetized Brain-extractedT1imageswerelinearlyregistered(12df)toa
(15 mg/kg ketamine), intubated, and positioned in a stereotactic preexisting in-house macaque template (2) in the stereotactic
devicewithinthePETscanner.MetabolicactivityduringthePET spaceofPaxinosetal.(25)usingFLIRT(http://fsl.fmrib.ox.ac.
scan reflects the amount of FDG uptake during the preceding uk/fsl/flirt).Imageswereinspectedandaveragedtocreateanage-
behavioralparadigm;regionsthatweremoremetabolicallyactive appropriate,study-specificlineartemplate(0.625mm×0.625mm×
during the NEC challenge took up more radio-labeled glucose. 0.625 mm = 0.244 mm3). Native-space, brain-extracted T1 images
Anesthesia was maintained using 1–2% (vol/vol) isoflurane gas. werethennonlinearlyregisteredtothetemplate usingFNIRT
MRIwerecollectedduringaseparatesession.Themedian(SD) (http://www.fmrib.ox.ac.uk/fsl/fnirt). Normalized brains were seg-
timebetweentheFDG-PETandMRIsessionswas37.0(37.5)d. mented into gray matter (GM), white matter, and cerebrospinal
fluidprobabilitymapsusingFAST(www.fmrib.ox.ac.uk/fsl/fast4).
NECChallenge.Individual differences in the three dimensions of Single-subject PET images were linearly registered to the cor-
theATphenotypewereelicitedusingtheNECcomponentofthe responding native-space T1 images (6 df). The resulting trans-
HumanIntruderParadigm(HIP)(4).TheHIPisamongthemost formation matrices were concatenated with those defining the
commonlyusedproceduresformeasuringdispositionalanxietyin nonlineartransformationtothestudy-specificstandardtemplate
nonhuman primates (5). Subjects were placed in a testing cage. and then used to normalize the PET images. Normalized and
SimilartolaboratoryproceduresusedforassessingATinchildren interpolatedPETimages(0.625mm×0.625mm×0.625mm=
(e.g.,strangerapproach)(6–8),potentialthreattooktheformof 0.244 mm3) were global-mean scaled within the brain using
amalehumanexperimenter(“intruder”)whoenteredtheroom SPAMALIZE. Scaled PET and GM probability maps were spa-
andstood motionless∼2.5mwhile presentinghis profiletothe tially smoothed (4-mm FWHM Gaussian). Some figures were
subject(30 min). created using MRIcron (http://www.mccauslandcenter.sc.edu/
mricro/mricron).
QuantifyingIndividualDifferencesintheThreeDimensionsoftheAT
Phenotype. NEC-elicited behavior was unobtrusively quantified HypothesisTestingStrategy.Thecentralaimofthepresentstudy
byawell-trainedraterusingaclosed-circuit audiovisualsystem. was to distinguish common and selective neural substrates. Ac-
Freezingwasdefinedasaperiodof>3scharacterizedbyatense cordingly, we first identified regions where metabolic activity
bodypostureandtheabsenceofvocalizationsormovementsother predicted varianceineachofthethreedimensionsof theAT
thanslowheadmovementsoreye-blinks.“Coo”callsarecontact phenotype (cortisol, freezing, and vocal reduction) while con-
or separation vocalizations that are elicited by exposure to the trolling for the other two. Specifically, a series of whole-brain
testcage(i.e.,the“alone”conditionoftheHIP)andsuppressed robustregressionanalyseswereperformedusingMULTISTATIC
byexposuretotheNECchallenge(i.e.,humanintruder’sprofile) (26), an extension of FMRISTAT (http://www.math.mcgill.ca/
(9–11). Coo vocalizations were defined as audible calls charac- keith/fmristat).Consistentwithrecentrecommendations(27,28),
terized by an increase then decrease in frequency and intensity analysesusedrobustprocedures,whichminimizetheinfluenceof
madebyroundingandpursingthe lips.Meanfreezingduration outlyingobservations.Similartoothertoolboxesforimagingdata
and cooing frequency were log and square-root transformed, (29), robustregression inMULTISTATIC isimplemented using
e
respectively.Plasmacortisol(μg/dL)wasquantifiedinduplicate the robustfit function in MATLAB (http://www.mathworks.com).
using the DPC Coat-a-count radioimmunoassay (Siemens). As- Ineachanalysis,oneofthedimensionsservedastheexplanatory
saying procedures were highly reliable (interassay CV = 6.6%; variable and the other two served as covariates of no interest.
intra-assayCV=4.0%)andsensitive(lowerdetectionlimit= Analysesalsocontrolledfornuisancevariationinmean-centered
1 μg/dL). Standardized cortisol, freezing, and vocalization re- age, sex, and voxelwise GM probability, an indirect measure of
sponseswerecreated(1,2)bylinearlyremovingnuisancevariance differencesinspatialnormalizationandgrossanatomy(26).This
Shackmanetal.www.pnas.org/cgi/content/short/1214364110 1of10
analytic procedure is formally equivalent to computing three recomputed each mediation model using another candidate me-
voxelwise partial correlation maps. diatorregionasacontrol(e.g.,usingthecortisol-selectiveregion
Commonsubstrates.Commonneuralsubstratesarethosesharedby forthe freezing mediationmodel:CeL→ lateralanteriorhippo-
individualswithvaryingexpressionsofextremeAT,acoresetof campus→freezing).Forthesecondmodel,werecomputedeach
brainregionswheremetabolismpredictsvariationinallthreeAT modelusinganotherATdimensionasacontrol(e.g.,cortisolfor
dimensions (cortisol levels, freezing behavior, and vocal reduc- themediationmodelincorporatingthefreezing-selectiveregion:
tions).Toidentifyregionswhereindividualdifferencesinregional CeL→M1→cortisol).Controlanalyseswereonlycomputedfor
brainmetabolismsignificantlypredictedallthreeATdimensions, regionswhere significant mediation effects wereobtained.
the three partial correlation maps were thresholded using the
false-discoveryrate(FDRq=0.05;whole-braincorrectedacross PhenotypeReliabilityAnalyses.Wecomputedtest-retestreliability
maps)(30,31)andthencombinedusingathree-wayminimum ofindividualdifferencesincortisol,freezingbehavior,andvocal
conjunction test (i.e., logical AND) (32). The conjunction test reductions for a subset of individuals exposed to the NEC chal-
yielded a “t-minimum” map containing voxels that were signifi- lengeonthreeoccasionsover1.21y(SD=0.27;n=63).Thefirst
cantinallthreeparentmaps;voxelssatisfyingthiscriterionwere assessmentoccurredatthetimeofFDG-PETsessionfeaturedin
assignedthevaluecorrespondingtothet-statisticfromtheleast- themaintext.Therank-orderofindividualdifferencesincortisol,
significantparentmap,otherwisesetto0(www.math.mcgill.ca/ freezing, and vocal reduction were reliable over the three occa-
keith/fmristat/#conjunctions). sions(intraclasscorrelation=0.66–0.88;meansingle-response
Selectivesubstrates.Selectiveneuralsubstratesarethosespecifically correlationbetweenadjacentsessions=0.46–0.79).Theselevels
engagedbyindividualswithhighlevelsofaparticulardimension aresimilartothoseobtainedforself-reportmeasuresofaffective
oftheATphenotype(e.g.,freezing),regionswheremetabolism traits (e.g., negative affect) in human adults over comparable
significantlyandstronglypredictsvariationinonlyoneoftheAT spans(41).Thetruepsychometricreliabilityofthesemeasures
dimensions.Selectiveregionsweredefinedasthosethat:(i)were
(i.e.,intheabsenceofgenuinechange)islikelytobesomewhat
significantly correlated with one of the three dimensions of the higherbecauseofthelengthyperiodfromthefirsttothethird
AT phenotype (cortisol levels, freezing behavior, or vocal re-
sessions(42)andthefactthatdatawereacquiredduringtheperi-
ductions;FDRq<0.05),and(ii)explainedsignificantlymore
adolescentperiod,aperiodofsubstantialneuralandpsychological
varianceinthatdimensioncomparedwiththeothertwo(FDR
maturation(43).ThecompositeindexofAT(2)wasalsoreliable
q<0.05).Effectively,thisprocessidentifiedpartialcorrelations (intraclasscorrelation=0.83;meancorrelationbetweenadjacent
thatwerebothsignificantandsignificantlydifferentfromtheother sessions= 0.71).
two (e.g., individual differences infreezing explained more var-
ianceinbrainmetabolicactivitythancortisolandvocalreductions).
HemisphericAsymmetryAnalysesfortheRightDorsalAmygdala.To
Differences in correlations correlations were assessed using the test whether the right dorsal amygdala cluster identified by the
Hotelling–Williamstest (33, 34).
three-wayvoxelwiseconjunction(seeFig.3andTableS4)showed
Mediationanalyses.Weidentifiedthelateraldivisionofthecentral asignificanthemisphericasymmetry,wecomputedthedifference
nucleus of the amygdale (CeL) and anterior hippocampus as
incorrelations separately for eachdimension of the AT pheno-
commonsubstrates,regionswheremetabolismpredictseachone
type. Regressions were conducted in SPSS using data extracted
of three dimensions constituting the AT phenotype (Fig. 3 and
fromtherightdorsalamygdalaclusterandthehomologousregion
TableS4).Toassesswhetherthosebrain-phenotyperelationships
in the left hemisphere, and controlling for nuisance variance in
areexplainedbyactivityinregionsidentifiedasselective,weused
mean-centeredage,sex,andGMprobability.Becausenoneofthe
a series of multivariate mediation models to test whether the
tests approached significance, t < 1.46, P > 0.14 (uncorrected),
partial correlation betweenthecommonsubstratesandapar-
werefrainfrominterpretingtheapparentlateralityofthiseffect.
ticular dimension of the phenotype depends on the relevant se-
lectivesubstrate(e.g.,CeL→M1→freezing)(Fig.4andTables
S1–S3). Weuseda standard ordinary least squares(OLS)multi- ProbabilisticChemoarchitectonicMapoftheCeL.Theamygdalaisa
complexstructure,comprisedofnumerousanatomicallyandphys-
variateanalyticframework(35,36)(forrecentapplicationsto
iologicallydistinctnuclei(44).Hereweusedpreviouslypublishedin
neuroimaging data, see refs. 37 and 38). Fully satisfying the
criteria of this framework would demonstrate that a significant vivoserotonintransporter(5-HTT)bindingdata(45,46)tolocalize
thedorsalamygdalaclustertotheCeLoftheamygdala.Exvivo
proportion of the association (i.e., partial correlation) between
researchdemonstratesthatthelateraldivisionoftheprimate
metabolicactivityinoneofthethreecommonregions(e.g.,CeL)
centralnucleusoftheamygdala(CeL)expressesmuchhigher
andaparticularATdimension(e.g.,freezingbehavior)ispredicted
5-HTT levelscompared with neighboring regions (47–50). Cap-
bymetabolisminoneofthecandidatemediatingregion(e.g.,M1,
italizing on this chemoarchitectonic signature, we used the dis-
or the primary motor cortex). Operationally, this framework
requiredfoursignificanttests:(i)acommonsubstratepredicts tributionof11C-DASB(ahigh-affinityradiolabeled5-HTTligand)
fromanindependentsampleofyoungmonkeystodefineaprob-
a particular dimension of AT, (ii) a selective substrate pre-
abilisticCeLregionofinterest(Fig.S1).Fordetailedmethods,see
dictsaparticulardimensionofAT,(iii)thecommonsubstrate
predictstheselective substrate,and(iv)controllingforvariance ref. 45; for a similar mapping application, see ref. 51. Briefly,
intheselectivesubstrateweakensthepartialcorrelationbetween 5-HTTavailability was assayedusing11C-DASB,aradiolabeled
the common substrate and the relevant dimension of the AT high-affinity 5-HTTligand. Dynamic PET time series were trans-
phenotype. Consistent with our prior work (39), the final crite- formedintovoxelwisedistributionvolumeratio[DVR;anindexof
rion was assessed using Clogg’stest (35, 40). This test was con- binding(52)]mapsnormalizedtoactivityinacerebellarreference
servativelythresholdedatanominalP<0.0057(Sidak-corrected region.Single-subjectDVRmapswerenormalizedtothestudy-
for the nine tests-of-interest, one-tailed given the directional hy- specific template and averaged. The resulting probabilistic (i.e.,
pothesis). Because FDG-PET lacks the temporal resolution nec- mean)5-HTTbindingmapwasthresholded(250×cerebellum).
essarytodeterminewhetheractivitywithinthecommonsubstrates We then assessed the degree of overlap with the conjunction-
(e.g.,CeL)temporallyprecedesdifferencesinactivitywithinthe defined cluster. This process revealed that the peak amygdala
selectivesubstrates(e.g.,M1),thistestdoesnotprovideevidence voxelandmostofthedorsalamygdalaclusteroverlappedwith
ofcausalmediation. the ROI (Sørensen-Dice similarity coefficient = 0.86), indi-
Toassessthespecificityofthemediationfindings,wecomputed cating that individuals with different presentations of extreme
twokindsofcontrolmodels(TableS5).Forthefirstmodel,we AT commonly engage the CeL (Fig. S1).
Shackmanetal.www.pnas.org/cgi/content/short/1214364110 2of10
ATCompositeIsaMoreSensitiveIndexofMetabolicActivitythanany findingindicatesthatindividualswithastrongaverageresponse
One of Its Constituents. The results of the conjunction analysis tothe NEC—high levelsofcortisol, long bouts offreezing, and
suggest, but do not demonstrate, that aggregating the three few vocalizations—tended to show the highest levels of activity
dimensions—cortisol, freezing, and reduced vocalizations—into in the CeL and anterior hippocampus; conversely, individuals
acompositewouldprovideamoresensitiveindexofAT-related
withaweakaverageresponsetendedtoshowthelowestactivity.
variation in core network activity. Confirmatory analyses dem-
Whole-brain analyses yieldedsimilar conclusions.
onstrated that this was the case. Confirmatory analyses used
Theseresultsalsoprovideuniqueempiricalsupportfortheuse
techniquessimilartothosedescribedin HypothesisTestingStrategy
of composite measures of AT and other dimensions of temper-
(see above), but did not partial covariation across the three di-
ament. Often, such composites are derived using statistical cri-
mensions.ThisprocessrevealedthattheATcomposite,computed
teriathatmandatestrongcovariationamongconstituents(e.g.,
asthearithmeticmeanofthethreestandardizeddimensions(2),
explained significantly more variance in CeL and anterior hip- factoranalysis).Ourresultsempiricallydemonstratetheutility
ofmultidimensional composites constructedfromanxiety-related
pocampal metabolic activity than any one of the constituent di-
mensionsin the clusters identified by the voxelwise three-way measures that are theoretically or clinically related,but not nec-
conjunctionanalysis,t>2.18,P<0.03.Furthermore,planned essarilysignificantlyintercorrelated.Infact,ourcompositeAT
contrastsrevealedthattheslopeoftherobustregressionline indexshowedstrongpredictivevaliditydespiteshowingrelatively
fit to neural activity was significantly steeper for the AT com- weak covariation among its constituent dimensions, consistent
positecomparedwithitsconstituents(P<0.04,one-tailed).This withempirical work byKagan etal. andothers (7, 53–55).
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Fig.S1. Invivoserotonintransporter(5-HTT)bindinglocalizedthedorsalamygdalaclustertotheCeL.Highlevelsof5-HTTbindingareahallmarkofthe
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(magenta).High5-HTTavailabilitywasalsoobservedalongthemidline(substantialinnominataandraphe),butnotintheanteriorhippocampalclusters
shownintheaxialview(goldboxes).(B)Comparisonwithexvivo5-HTTbinding.Fromlefttoright,magnifiedcoronalviewsoftheoverlapshowninA,
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photomicrographof5-HTTimmunohistochemistry[adaptedwithpermissionfromref.47,CopyrightElsevier(2006)].
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