1,2, Alexander J. Shackman 1,2 - PSYC Home | PSYC...Research Institute, Waisman Laboratory for Brain Imaging and Behavior, and Wisconsin National Primate Center. This work was supported

TheCentralNucleusoftheAmygdalaisaCriticalSubstrateforIndividualDifferencesinAnxiety

JonathanA.Oler1,2,AndrewS.Fox1,2,AlexanderJ.Shackman3andNedH.Kalin1,2

1. DepartmentofPsychiatryUniversityofWisconsinSchoolofMedicineandPublicHealth6001ResearchParkBoulevard,Madison,Wisconsin53719USA

2. HealthEmotionsResearchInstituteUniversityofWisconsin‐MadisonMadison,Wisconsin53719USA

3. DepartmentofPsychologyNeuroscienceandCognitiveScienceProgram,andtheMarylandNeuroimagingCenterUniversityofMaryland,CollegePark3123GBiology‐Psychology,CollegePark,Maryland20742USA

AcknowledgementsWethankthepersonneloftheHarlowCenterforBiologicalPsychology,HealthEmotions

ResearchInstitute,WaismanLaboratoryforBrainImagingandBehavior,andWisconsinNationalPrimateCenter.ThisworkwassupportedbytheNationalInstitutesofHealth(R01‐MH046729,R01‐MH081884,P50‐MH084051,R21‐MH09258),theHealthEmotionsResearchInstituteandtheUniversityofMaryland,CollegePark.

Please cite as Oler, J. A., Fox, A. S., Shackman, A. J. & Kalin, N. H. (in press). The central nucleus of the amygdala is a critical substrate for individual differences in anxiety. Living without an amygdala (D. G. Amaral, M. Bauman, & R. Adolphs, Eds.). Guilford Press.

Researchintothefunctionoftheamygdalabeganwithexperimentsinrhesusmonkeys,

performedbyBrownandSchaefer(BrownandSchafer,1888)andlaterbyKlüverandBucy

(KlüverandBucy,1937;KlüverandBucy,1939).Thesestudiesledtofurthercritical

experimentsinnonhumanprimatesthatcontinuedtospecifytheamygdala’sroleinemotionand

socialbehavior(Weiskrantz,1956;Kling,1968;Kappetal.,1979;Pribrametal.,1979;Aggleton

andPassingham,1981;Rolls,1984;Zola‐Morganetal.,1991).Advancesinlesiontechniquesand

otherinvasiveandnon‐invasivemethodologieshavemotivatedmorenuancedhypotheses

regardingtheadaptiveroleoftheamygdalainfear,dangerdetection,socialbehavior,vigilance,

andtemperament(Kalin,1997;Whalen,1998;LeDoux,2000;Adolphs,2003;Amaral,2003).

Thereisnowgreatinterestinunderstandingalterationsinamygdalafunctioninrelationto

psychopathology,withaparticularemphasisonanxietyandaffectivedisorders.

Understandingtheroleoftheamygdalainanxietyandaffectivedisordersisessentialbecause

thesedisordersareamongthemostcommonpsychiatricillnessesinyouthandadults(33.7%

lifetimeincidenceofanyanxietydisorder;18.3%lifetimeincidenceofmajordepressive

disorder),andtheyarehighlycomorbidandoftenresistanttotreatment(Kessleretal.,2012).

Anxietydisordersfrequentlybeginduringthepreadolescentyearsandinmanycasesare

associatedwiththelateronsetofdepressionduringadolescenceandearlyadulthood.Research

demonstratesthatveryyoungchildrenwithextremeanxiety,asmanifestedbymarkedreactivity

tonoveltyand/orstrangers,areatincreasedrisktodevelopanxietyandaffectivedisorders.For

example,extremetemperamentalchildhoodanxietyisastrongpredictorofthelater

developmentofsocialanxietydisorder(Schwartzetal.,1999;Prioretal.,2000;Biedermanetal.,

2001;Hirshfeld‐Beckeretal.,2007;Chronis‐Tuscanoetal.,2009;Essexetal.,2010),and

depressivedisorders(Caspietal.,1996;GladstoneandParker,2006;Beesdoetal.,2007).A

recentmeta‐analysissupportsthecontentionthatextremechildhoodtemperamentalanxiety

mayrepresentthesinglebestpredictorofthelaterdevelopmentofsocialanxietydisorder

(ClaussandBlackford,2012).Appreciatingwhycertainindividualsarevulnerabletodeveloping

anxietydisordersrequiresanunderstandingoftheneuralmechanismsthatinfluencethe

developmentofadaptiveanxiety,aswellasextremetemperamentalanxiety(Yehudaand

LeDoux,2007;McEwenetal.,2012;Galatzer‐Levyetal.,2013;Goswamietal.,2013;Grupeand

Nitschke,2013;HolmesandSingewald,2013;Shackmanetal.,2013).

Ourultimategoalistoprovideinsightintothedevelopmentalissuesrelatedtotheonset

ofmoodandanxietydisorders.Therefore,wehavefocusedoureffortsonunderstandingthe

developmentalpathophysiologyoftheseillnessesbystudyingtheroleoftheamygdalaearlyin

thelifeofprimatesasitrelatestotheinitialmanifestationsofextremeanxiety.Ourstudiesin

youngrhesusmonkeyssuggestthatthecentralnucleusoftheamygdala(Ce)andthebednucleus

ofthestriaterminalis(BST;partoftheextendedamygdala),arekeysubstratesfortrait‐like

differencesinanxiety.TheCeisoftenconceptualizedasthemajoroutputstructureofthe

amygdalaforprojectionstothebrainstemandhypothalamus,andtheCeisthoughtto

coordinateandgatethephysiologicalandbehavioraleffectsoffear(Davis,2000;Pareetal.,

2004;Ciocchietal.,2010;Haubensaketal.,2010).AdditionalhypothesesofCefunctionhave

beenpostulatedtoaccountforitsroleinappetitivelearningandattention(Kappetal.,1992;

GallagherandHolland,1994;Gallagher,2000;Everittetal.,2003;Gabrieletal.,2003).TheCeis

alsoconceptualizedasthetemporallobecomponentofthe‘centralextendedamygdala’,a

hypothesizedmacrostructualanatomicentitythatextendsintothebasalforebrain(Alheidand

Heimer,1988;deOlmosandHeimer,1999;HeimerandVanHoesen,2006).Thebasalforebrain

isacomplexregionthathasonlyrecentlybecomeaccessibletostudyinthelivingprimate.

Becauseofitsstrategiclocationandputativefunctions,dysfunctionofthebasalforebrainhas

beenimplicatedinvariousneuropsychiatricdisorders(Heimer,2003).Themajorcomponentsof

thebasalforebrain,includingthecholinergicnucleusbasalisofMynert,theventralstriatopallidal

systemandtheextendedamygdala,arehighlyinterdigitatedmakingitchallengingtoelucidate

selectivefunctionsofthesebasalforebraincomponents(Zaborszkyetal.,2008).Thecentral

extendedamygdalaconceptproposedbyHeimerandcolleaguestodescribethecontinuumof

GABA‐ergicneuronsthatrunfromCe,throughthesubstantiainnominata,toBSTandtheshellof

thenucleusaccumbenscomplementstheothermodelsofCefunctionmentionedabove.In

additiontobeinghighlyinterconnected,theCeandBSTsharemanyofthesameefferenttargets,

reinforcingtheideathatCeandBSTtogetherformacoherentfunctionalunit(deOlmosand

Heimer,1999).Consistentwiththeseanatomicalandneurochemicalfindings,functionalMRI

(fMRI)datafromourlaboratorydemonstratethatinmonkeysandhumanstheCeandBST

displayhighlysignificantfunctionalconnectivityatrestorunderanesthesia,supportingthe

hypothesisthatthesestructuresformadiscretecircuit(Oleretal.,2012).Analternativeview,

however,considerstheCe,sublenticularsubstantiainnominataandBSTcontinuumas

differentiatedcomponentsofastriatopallidalprojectionsystem(Dongetal.,2001;Swanson,

2003).

RodentstudiessuggestanimportantdissociationbetweensubdivisionsoftheCeandtheBST

withrespecttodefensivebehaviors,suchthatthemedialdivisionoftheCe(CeM)isinvolvedin

rapid,phasicfear‐relatedresponding,whereastheBSTviainputsfromthelateraldivisionofthe

Ce(CeL)isthoughttomediateslower,sustainedanxiety‐likeresponsestodiffuseorambiguous

threats(WalkerandDavis,2008).Additionally,recenthumanimagingstudieshaveassociated

theBSTregionwithvigilance,threatmonitoringandanticipatoryanxiety(Straubeetal.,2007;

Alvarezetal.,2010;Mobbsetal.,2010;Somervilleetal.,2010;Choietal.,2013;Grupeetal.,

2013;Averyetal.,2014),andsomeevidenceforaCeandBSTfunctionaldissociation,similarto

thatinrodents,hasbeenreportedinhumans(Davisetal.,2010).

Herewereviewstudiesfromrhesusmonkeysaimedatunderstandingtheroleofthe

amygdalaintemperamentalanxiety,andprovideevidencedemonstratingthatthecentral

extendedamygdalaplaysacriticalroleinearly‐lifeanxiety.Wefirstrecountthedevelopment

andvalidationofthenon‐humanprimatemodelofchildhoodanxiety.Next,wediscuss

neuroimagingandgeneticevidencefromtherhesusmonkeyshowingthattheanxious

phenotype,oranxioustemperament,isheritableandstronglyrelatedtoindividualdifferencesin

Cefunction.Wethendescribeevidencefrommechanisticstudiesdemonstratingthatbehavioral

expressionofprimateanxietycriticallydependsupontheintegrityoftheCe.Weconcludeby

outliningtheimplicationsofthesefindingsforunderstandingtheriskforanxiety‐related

psychopathology,forpotentiallydevelopingmoreeffectiveearly‐lifeinterventions,andfor

understandingnormalvariationinchildhoodtemperament.

DevelopingtheHumanIntruderParadigmandtheConceptofAnxiousTemperament

Fromournonhumanprimatestudies,wedevelopedthetermanxioustemperament(AT)to

describeanindividual’sunderlyingpredispositiontodisplayextremeanxiety‐relatedbehavioral

andphysiologicalresponsesearlyinlife.Thereisconsiderableevidencethattheamygdalaplays

acriticalroleinnormalfearandemotionalprocessing(Aggleton,1992;Aggleton,2000;

Shinnick‐Gallagheretal.,2003),alteredamygdalafunctionhasbeenreportedinadultswith

anxietydisorders(EtkinandWager,2007)andadministrationofclinicallyeffectiveanxiolytics

reducesamygdalaactivationinadose‐dependentmanner(Paulusetal.,2005).Inaddition,

adultswithahistoryofchildhoodATdisplayincreasedamygdalareactivitytonovelor

potentiallyfearfulstimuli(Schwartzetal.,2003;BlackfordandPine,2012).However,the

amygdala’scontributiontoearlylifepresentationoftrait‐likeindividualdifferencesinchildhood

anxietyremainsunclear.Specifyingtheprocesseswithintheamygdalathatunderliethe

developmentofnormalandabnormalanxietywillbeessentialfordevelopingnovel

neuroscientifically‐groundedinterventionsfortreatingandpreventinganxiety‐related

psychopathology.

ThebehavioralassayformonkeysdevelopedbyKalinandSheltontermed‘thehuman

intruderparadigm’wasconceptualized,inpart,tomapontostudiescharacterizingbehavioral

inhibitioninhumanchildren.Thehumanintruderparadigmconsistsofthreedifferent

consecutivelypresentedconditions(Alone,No‐Eye‐Contact,andStare)thatelicitdifferent,

contextuallyappropriate,anxiety‐relateddefensiveresponses(KalinandShelton,1989);see

Figure1).Inthe‘Alone’condition,animalsareseparatedfromtheircage‐matesandplacedby

themselvesinanoveltestcage.Duringthe‘No‐Eye‐Contact’(NEC)condition,whichfollowsthe

Alonecondition,ahumanintruderenterstheroomandat2.5metersfromthecagepresents

his/herprofiletothemonkey.Thecriticalcomponentofthisconditionisthelackofeyecontact

betweenthehumanintruderandthetestmonkey.Whileeyecontactsignalsadirectthreat,the

avoidanceofeyecontactprovidesadifferentpotentiallythreateningcontext.Theintruderthen

leavestheroomforabriefperiod.Uponreentering,the‘Stare’conditionensues,duringwhich

theintrudercontinuouslystaresatthemonkeywithaneutralfacialexpression(Kalin,1997).

[insertFigure1here]

Anumberofstandardizedbehavioralparadigmsexisttomeasurechildhoodbehavioral

inhibition(Foxetal.,2005).Theseparadigmsincludetheintroductionofastrangertotheroom

withayoungchild(Bussetal.,2004),andexposureofachildtonovelobjectsandsocial

situations(Kaganetal.,1988).Individualdifferencesinphysiologicalresponsestostresshave

alsobeenexaminedinrelationtobehavioralinhibition.Manyofthesestudieshavefocusedon

pituitary‐adrenalactivityandreportmixedresults.Initialstudiesdemonstratedassociations

betweencortisolandbehavioralinhibitioninchildren,orbetweencortisolandATinmonkeys

(Kalinetal.,1998;Essexetal.,2002),howeverlaterstudiesdidnotconsistentlyreplicatethese

findings(Shackmanetal.,2013).Whilenotasextensivelystudied,evidencepointstoan

associationbetweenheartrateandrightfrontalEEGasymmetrywithextremechildhoodBIand

monkeyAT(Davidsonetal.,1992;Davidsonetal.,1993;Kalinetal.,1998;Foxetal.,2005).

OurdefinitionofATparallelstheconstructofbehavioralinhibitionusedbyKaganand

colleaguesintheirdescriptionofextremelyshytoddlersthatwereobservedtobecomeimmobile

andhesitanttovocalizeinthefaceofpotentialthreat(Kaganetal.,1988).Freezingbehaviorin

responsetotheNECconditionofthehumanintruderparadigm,becauseofitsobvioussimilarity

tohumanbehavioralinhibition,wastheinitialmetricusedtoassessthreat‐relatedanxietyin

youngmonkeys(KalinandShelton,1989;KalinandShelton,2000;KalinandShelton,2003;

Kalinetal.,2005).Asavalidationofitsrelevancetoanxiety,wedemonstratedthatNEC‐induced

freezinginmonkeyscanbereducedbyadministrationofthebenzodiazepine,diazepam,a

commonpharmacologicaltreatmentforclinicallysignificantanxiety(KalinandShelton,1989;

Davidsonetal.,1993;Kalin,2003),andincreasedwithadministrationofß‐carboline,an

anxiogenicbenzodiazepineinverseagonist(Kalinetal.,1992).Welaterexpandedthe

assessmentofmonkeyanxietytomovebeyondjustasinglebehavioralmeasure(i.e.,freezing)to

acompositemeasure,byincludingdecreasesinspontaneouscoo‐calls(Foxetal.,2005)aswell

asindividualdifferencesinthreat‐inducedcortisollevels(Jahnetal.,2010).Thiswas,inpart,

basedontheobservationthatanimalswithelevatedfreezinginresponsetotheNECcondition

concomitantlyemittedfewervocalizations(KalinandShelton,1989).Threat‐inducedcortisol

wasaddedtogaugeindividualdifferencesinpituitary‐adrenalreactivity(KalinandShelton,

1989;Kalinetal.,1998).Itisimportanttonote,thatwhenexaminingtherelationsamongthe

threecomponentsofAT(freezing,reducedcooingandcortisollevels)inalargesample,

individualdifferencesincortisollevelsdonotsignificantlycorrelatewitheitherbehavioral

metric,whereasfreezingandcooingaremoderatelyinverselycorrelated(Shackmanetal.,

2013).TheinclusionofcortisolinthecompositemeasureofATisintendedtocapturethe

heterogeneityinindividualdifferencesinthephysiologicalresponsetofearandanxietyeliciting

stimuli.Interestingly,theATcompositebetterpredictsindividualdifferencesinamygdala

metabolismthananyoneofitsthreecomponents(Foxetal.,2008;Shackmanetal.,2013).

Tobeclear,wespecificallyusethetermATtooperationalizethetheoreticalconstruct

representinganindividual’sdispositiontobehavewithreticenceandrespondtopotentialthreat

withextremebehavioralandphysiologicalreactivity.OurdefinitionofATincludesbehavioral

inhibition(i.e.,freezinganddecreasedspontaneousvocalization),butalsotakesintoaccountthe

degreeofpituitary‐adrenalstress‐responsivenessoftheindividual(seeFigure2a).

Table1demonstratesthetranslationalutilityofATasamodelforchildhoodbehavioral

inhibitionortheearlychildhoodriskfordevelopingsocialanxiety.Asmentionedabove,itiswell

documentedthathighlyanxiouschildrenareatsubstantialriskforsocialanxietydisorder(SAD).

TheDiagnosticandStatisticalManualofMentalDisorders(DSM‐V)liststhefollowingascriteria

forSADdiagnosis,manyfeaturesofwhicharesharedbybothchildhoodbehavioralinhibition

andmonkeyAT(italicsadded):SADCriterionA:Markedfearoranxietyaboutoneormoresocial

situationsinwhichtheindividualisexposedtopossiblescrutinybyothers.Examplesinclude

socialinteractions(e.g.,meetingunfamiliarpeople),beingobserved,andperforminginfrontof

others.CriterionB:Theindividualfearsthatheorshewillactinawayorshowanxiety

symptomsthatwillbenegativelyevaluated.CriterionC:Thesocialsituationsalmostalways

provokefearoranxiety.Note:Inchildren,thefearoranxietymaybeexpressedbycrying,

tantrums,freezing,clinging,shrinking,orfailingtospeakinsocialsituations.CriterionD:The

socialsituationsareavoidedorenduredwithintensefearoranxiety.CriterionE:Thefearor

anxietyisoutofproportiontotheactualthreatposedbythesocialsituationandtothe

socioculturalcontext.CriterionF:Thefear,anxiety,oravoidanceispersistent,typicallylasting

for6monthsormore.CriterionG:Thefear,anxiety,oravoidancecausesclinicallysignificant

distressorimpairmentinsocial,occupational,orotherimportantareasoffunctioning.Criterion

H:Thefear,anxiety,oravoidanceisnotattributabletothephysiologicaleffectsofasubstanceor

anothermedicalcondition.CriterionI:Thefear,anxiety,oravoidanceisnotbetterexplainedby

thesymptomsofanothermentaldisorder.CriterionJ:Ifanothermedicalconditionispresent,the

fear,anxiety,oravoidanceisclearlyunrelatedorisexcessive(AmericanPsychiatricAssociation,

2013).AsshowninTable1,theATphenotypeinyoungmonkeysandthebehaviorallyinhibited

phenotypeinyoungchildrenshareanumberofcommonfeatures.Manyofthesecommon

featuresareantecedentsofSAD.WebelievethatextremeATinchildren,whenstableandtrait‐

like,hasthehallmarksofsub‐thresholdSADbutisnotsevereenoughtosatisfythefunctional

impairmentcriterion.

[inserttable1here]

NeuroimagingstudieslinkindividualdifferencesinCefunctiontoanxiety

Ourinitial18F‐fluorodeoxyglucose(FDG)‐positronemissiontomography(PET)imagingstudies

demonstratedthatmonkeyATwascorrelatedwithmetabolismintheamygdalaandthe

extendedamygdala(i.e.,BST),aswellasanteriorhippocampus,anteriortemporallobe,and

periaqueductalgrey(PAG)(Foxetal.,2005;Kalinetal.,2005).FDGisaradiolabeledglucose

analogwithahalf‐lifeof~110minutesthatdoesnotgetmetabolizedandremainstrappedin

metabolicallyactivecells(Sokoloffetal.,1977).BecausethetimecourseofFDGuptakereflects

brainactivityoveranapproximate30‐minuteperiod,andremainsstablydetectableinthebrain,

itisanidealradiotracertosimultaneouslystudybehaviorandbrainactivityelicitedbyexposure

toethologically‐relevantsituations(seeFigure2b).FDG‐PETisthereforeparticularlyusefulin

understandingthesustainedbrainresponsesassociatedwithtemperament,whichbydefinition

isapersistentandrelativelycontext‐independentemotionaldisposition.

[insertFigure2here]

WeperformedFDG‐PETscansonanimalsexposedto4differentconditions,2ofwhichwere

stressful(NECandAlone‐separationfromcage‐mateintoatest‐cage)and2ofwhichwere

nonstressful(inhome‐cagewithoutcage‐mate,andinhome‐cagewithcage‐mate).Ourfindings

revealedconsistentpositivecorrelationsbetweenindividualdifferencesinNEC‐elicitedATwith

metabolismintheamygdala,hippocampus,anteriortemporalpole,andPAGregardlessofthe

stressfulornonstressfulconditioninwhichbrainmetabolicactivitywasassessed(Foxetal.,

2008).Remarkably,theATbrainmetabolismphenotypewasdiscernibleintheabsenceof

provocation,whenmonkeyswereathomewiththeircage‐mate,somethingthatisvirtually

impossibletomeasureinhumans.TheseresultssuggestthattheneuralcorrelatesofATare

stableacrosscontextsandnotascontext‐dependentastheobservablebehavioralandpituitary‐

adrenalresponsesassociatedwithAT.Similarly,weexaminedthestabilityofAT’sneural

substratesacrosstimebyassessingFDG‐PETandATinresponsetoNECin24animals3‐times

overthecourseof6‐18months(Foxetal.,2012).Resultsdemonstratedthatbrainmetabolism

withinAT‐relatedregionswasstableovertime,andmeanbrainmetabolism(acrossthe3

assessments)predictedmeanAT(Foxetal.,2012).Collectively,thesedataindicatethatthetrait‐

likenatureofATisreflectedbycontext‐independentandtemporallystableneuralsubstrates

thatareinstantiatedintheinherentactivityofanindividual’sbrain.

TofurtherexploretheneuralsubstrateunderlyingATandtoelucidatetheheritablebasisof

AT,weperformedanexperimentexaminingFDG‐PETandATinresponsetotheNECcontextina

largesample(n=238)ofyoungrhesusmonkeys(Oleretal.,2010).Becauseofthestatistical

poweraffordedbythelargesamplesize,weusedextremelystringentstatisticalthresholds

(Šidákcorrected),whichincreasesconfidenceinthefindings.Consistentwithearlierfindings,

theimagingdatademonstratedthatmetabolisminanteriortemporallobestructuresincluding

theCeregion,anteriorhippocampusandanteriortemporalcortexpredictedindividual

differencesinAT(Figure3).

[insertFigure3here]

AttheŠidákthreshold(p=0.00000005875),largebilateralanteriortemporallobeclusters

thatcorrelatedpositivelywithATwereobserved(Oleretal.,2010).Theanteriortemporallobe

clusterscontainedmultiplespatialpeaks,eachofwhichcorrelatedwithAT.Therefore,we

furtherresolvedthelocationofthepeakcorrelationswithintheanteriortemporallobeclusters

bycalculatingthespatialconfidenceintervalsrepresentingvolumesthatwith95%certainty

containedthepeakcorrelationsbetweenmetabolicactivityandAT(seeOleretal.,2010for

details).Tofurtherdemarcateanddefinethelocationofthesepeaks,weusedinvivo

chemoarchitectonictechniquestodemonstratethatthisfunctionally‐definedregioncorresponds

totheCe,adegreeofprecisionthatisdifficulttoachieveusingconventionalimagingtechniques

inhumans.Thevolumescontainedwithinthe95%confidenceintervalsweresuperimposedona

voxelwisemapofserotonintransporter(5‐HTT)bindingcreatedfromanindependentsampleof

rhesusmonkeysassessedwith11C‐DASBPET(Christianetal.,2009;Oleretal.,2009).This5‐

HTTmap(seeFigure4)canbeusedtolocalizetheCeanddifferentiateitfromtheanterior

hippocampus,sincecomparedtosurroundingregionsthelateraldivisionoftheCe(CeL)hasthe

highestdensityof5‐HTTbinding(O'RourkeandFudge,2006).

[insertFigure4here]

DemonstratingheritabilityofATandinitialstudiesofthegeneticbasisofAT

ToascertainwhetherindividualdifferencesinATareheritable,wetookadvantageofthefact

thattheyoungrhesusmonkeysinthestudyallbelongtoasinglemultigenerationalpedigreeof

morethan1,800individuals.Thepoweroftheextendedpedigreeapproachtoquantitative

geneticanalysisstemsfromthemanycloselyrelated,distantlyrelatedandunrelatedpairsof

individualsthatallcontributeinformationabouttheeffectsofsharedgenesonphenotypic

similarity.Specifically,amongthemonkeyswithphenotypedataandconfirmedlineage,there

werethreefull‐siblingpairs,189half‐siblingpairs,128third‐degreerelativepairs,372fourth‐

degreerelativepairs,andmuchlargernumbersofmoredistantlyrelatedandunrelatedpairs.

Usingageneralvariancecomponentsmethod(AlmasyandBlangero,1998),weestimatedthe

heritabilityofATwhileincludingcovariatessuchassex,age,andtheirinteractionsinthemean

effectsmodeltocontrolforextraneoussourcesofvariance(formethodologicaldetailssee

supplementalmaterialsfromOleretal.,2010).Consistentwithpreviousreportsinrhesus

monkeys(Williamsonetal.,2003;Rogersetal.,2008),andthegeneticepidemiologyofhuman

anxietydisorders(Hettemaetal.,2001),approximately36%ofthevariabilityinATwas

accountedforbythepair‐wiserelationshipsamongtheanimals.

Weusedthissamequantitativegeneticapproachtoestimatetheheritabilityofmetabolic

activityateachvoxelwhereFDGmetabolismsignificantlypredicteddifferencesintheanxious

phenotype(seeFigure5).Remarkably,althoughglucosemetabolismintheCeandanterior

hippocampalpeakregionsweresimilarlypredicativeofAT,theseregionsweredifferentially

heritable.UnlikeCemetabolism,anteriorhippocampalmetabolismwassignificantlyheritable

andthislevelofheritabilitywassignificantlygreaterthantheheritabilityestimatefortheCe

(Oleretal.,2010).Weinterpretedthesefindingscautiouslyaseventhislargesamplesizeis

relativelymodestfortestsofadditivegeneticeffects,buttheresultssuggestthattheCemaybe

particularlyinfluencedbytheenvironmentandexperience,andsetthestageforfurther

experimentsaimedatunderstandingtheneurodevelopmentaloriginsorAT.Theseresultsalso

highlighttheimportantobservationthatitispossibletodissociateheritablefromnon‐heritable

neuralsubstrates‐somethingthat,toourknowledge,hasneverbeenshowninpriorwork.

[insertFigure5here]

Atamorespecificlevel,weexaminedDNAvariationincandidategenesastheyrelatetoAT

anditsunderlyingamygdalarandhippocampalmetabolism.Weselectedtheserotonin

transporter‐linkedpolymorphicregion(5‐HTTLPR)becausevariationinthisgenewasshownby

numerousgroupstopredictfear‐relatedbehaviorsandtheriskforaffectivedisorders(Hariri

andHolmes,2006).Theeffectsofthe5‐HTTLPRgenotypeontherisktodevelopanxietyarenot

straightforward,andmayonlyberevealedwhenexaminingbrainreactivity,forexamplewhen

comparingstressfulandnon‐stressfulconditionsor,asisrequiredintheanalysisoffMRIdata,a

changefrombaseline.Weobservednoeffectofthe5‐HTTLPRpromoterrepeatlength

polymorphismonATorAT‐relatedglucosemetabolism(Oleretal.,2010).Thiswasnot

surprisingconsideringthat1)alargeimagingstudyusingarterialspinlabelingfoundnoeffectof

5‐HTTLPRgenotypeonbaselineamygdalabloodflow(Vivianietal.,2010),and2)aprevious

studyinasmallersampleofmonkeysfailedtoobserve5‐HTTLPRgenotype‐relateddifferences

inNEC‐inducedFDG(Kalinetal.,2008).Kalinetal.,(2008)did,however,find5‐HTTLPR

genotype‐relatedalterationswhencomparingthedifferenceinmetabolismbetweentheNEC

conditionanda“safe”condition,wheretheanimalswereadministeredFDGintheirhomecages.

IncontrasttotheATfindings,thesedatademonstrateanassociationbetweencontext‐

dependentmetabolicchangesandthe5‐HTTLPRgenotype.Interestingly,inthesamesampleof

monkeysthe5‐HTTLPRgenotypewasnotsignificantlyassociatedwith11C‐DASBbinding,a

measureof5‐HTtransporteravailability(Christianetal.,2009).Collectively,thesefindings

highlightthecomplexityoftheinfluencethatthe5‐HTTLPR,andotherfunctional

polymorphisms,haveonbehaviorandtheriskforpsychopathology,andsupporttheideathat

neurogeneticsresearchshouldfocusongene environmentinteractions(Caspietal.,2010;

Hydeetal.,2011;Bogdanetal.,2013).

Incontrasttotheshortandlongallelicvariationinthe5‐HTTLPR,singlenucleotide

polymorphisms(SNPs)inthecorticotropinreleasinghormonereceptor1(CRHR1)gene,which

hasbeenassociatedwithriskforthedevelopmentofanxiety‐relateddisorders(Bradleyetal.,

2008),weresignificantlyassociatedwithbothATandAT‐relatedglucosemetabolism.

Specifically,SNPsinexon6oftherhesusCRHR1geneappeartoconferanincreasedlikelihoodof

elevatedATandgreaterNEC‐relatedmetabolismintheCeandanteriorhippocampus(Rogerset

al.,2013).Thisfindingisparticularlyinterestingbecauseexon6isfoundprimarilyinanthropoid

primates.MuchofthehumanCRHR1geneticdatareportgene environmentinteractions,

especiallyinteractionswithearlychildhoodtrauma(Bradleyetal.,2008).Thus,thesefindings

suggestthattheearly‐lifeeffectsofCRHR1geneticvariationmaybetosupportthedevelopment

ofadiathesisthatinteractswithearlyadversitytoincreasethelikelihoodofdeveloping

pathologicalanxiety.

MolecularsubstrateswithintheCerelevanttoAT

Asanxietyandaffectivedisorderscanberesistanttocurrenttreatments,andthese

treatmentsarecommonlyassociatedwithsignificantadverseeffects(Bystritsky,2006;Cloosand

Ferreira,2009;Kessleretal.,2012)thereisgreatneedforidentifyingnewanxiolyticand

antidepressantmoleculartargets.Furthermore,becauseoftheearly‐lifeonsetofanxiety,

establishingnovelearly‐lifeinterventionsaimedatpreventingchronicanddebilitatingoutcomes

wouldbeanidealtreatmentapproach.Todevelopnovelinterventionsforanxietydisorders,itis

necessarytoidentifypotentialtreatmenttargetsandtotesttheirtherapeuticfeasibilityina

speciesthatexpressesanxiety‐relatedpsychopathologythatissimilartohuman

symptomatology.Inthisregard,quantitativemRNAapproachesareparticularlyusefulbecause

theycapturethecombinedimpactofgeneticandenvironmentalepigeneticregulation(Jaenisch

andBird,2003).WithmicroarrayordeepRNAsequencingdatawecanidentifyindividual

differencesinmRNAexpressionlevelsofspecificgenesthatpredictATandalteredmetabolism

withintheATneuralcircuit(Foxetal.,2012;Roseboometal.,2013).

ThemonkeymodelofchildhoodATallowsustodovetailthesamemultimodalimaging

methodsroutinelyusedinhumanswithindepthpost‐mortembrainmolecularanalyses.Our

initialapproachhasbeentocollectbraintissuepunchesfromasubsetofmonkeysphenotyped

forAT.Usingtheimagingdataasaguide,fromthebrainsof24malemonkeysweselectively

biopsiedtheregionofthedorsalamygdalawhereitsmetabolismwasmostpredictiveofAT

(Figure6).AffymetrixrhesusmicroarraychipswereusedtoassessmRNAexpressionthatwas

analyzedinrelationtoindividualdifferencesinATandCemetabolism(seeFigure6).Analyses

controllingforhousingdifferences,hemispheresampled,andagerevealedthatATwas

associatedwithanumberofmRNAsthathadatleastmoderateexpressionlevels[>log2(100)],

andremainedsignificantlycorrelatedwithATaftercorrectingformultiplecomparisons(FDRq

<0.05,two‐tailed;seeFoxetal2012fordetailedmethods).Ageneontologyenrichmentanalysis

ofallthesignificantAT‐relatedmRNAsrevealedthatexpressionlevelsofgenefamilies

associatedwithneuroplasticityandneurodevelopmentsignificantlypredicteddifferencesinAT

(Foxetal.,2012).Specifically,thistranscriptome‐wideanalysisrevealedthatATandincreased

CemetabolismwasassociatedwithdecreasedexpressionlevelsofseveralgenesintheNTF‐3

(neurotrophin‐3)‐NTRK3pathway(seeFigure6).NTRK3(neurotrophictyrosinekinase

receptor‐3,alsotermedTrkC)isofconsiderableinterestbecauseitsactivationcaninitiate

synaptogenesisandneurogenesis(Bernd,2008).Inaddition,NTRK3geneticvariationhasbeen

linkedtohumanpsychopathology(Otnaessetal.,2009)andbecausetheNTRK3proteinisacell

surfacereceptor,NTRK3mayprovideanaccessibledrugtarget.Theseuniquefindingsina

primatespeciessuggestthattheexpressionandmaintenanceofATandthesubsequent

increasedrisktodevelopanxietyanddepressionmaybeduetoearlymaladaptive

neurodevelopmentalprocesses(Foxetal.,2012).

[insertFigure6here]

ThefindingsfromthemicroarrayexperimentalsodemonstratedthatCemetabolismandAT

wereassociatedwithalteredexpressionofsomeexpectedcandidatesgenes(e.g.,5HT2Cand

NPY1R).LevelsofmRNAsforbothofthesegeneswerenegativelycorrelatedwithAT,suchthat

individualswiththelowestexpressionlevelsofNPY1RmRNA,forexample,werethosewiththe

mostextremeAT(Roseboometal.,2013).NYP1Risofinterestbecauseofthenumerousreports

linkingdecreasedNPYsystemactivitytodepression.WhileCeNYP1RmRNAlevelsdidnot

predictCemetabolism,awhole‐brainvoxelwiseanalysisrevealedseveralotherregionswhere

CeNYP1RmRNAexpressiondidpredictmetabolism.Theseregionsincludedthedorsolateral

prefrontalcortex(dlPFC)andperigenualanteriorcingulatecortex,corticalregionsknowntobe

partofthecircuitthatregulatesamygdalaractivity(Davidson,2002;Etkinetal.,2006;Buhleet

al.,2013;Shackmanetal.,2013).ThesedatasuggestthatNPY1RmRNAlevelsintheCemaybe

regulatedbyprefrontalcorticalinputstoNPY1R‐expressingCeneurons.Alternatively,NPY1R‐

expressingCeneuronscouldmodulatemetabolisminthesedistalbrainregionsviadirector

indirectmechanisms.

Livingwithoutanamygdala

Lesionstudiesinhumanandnon‐humanprimatessuggestacausalrolefortheamygdalain

AT.InitialstudiesbyBrown&Schaferdemonstrateddecreasedfearfulnessinmonkeyswith

amygdaladamage(BrownandSchafer,1888).Specificexperimentallesionstotheamygdala

havebeenshowntodecreasethereticencetoactinpotentiallythreateningsituations(Kalinet

al.,2001;MurrayandIzquierdo,2007;MachadoandBachevalier,2008;Chudasamaetal.,2009)

andalterstress‐inducedcortisolrelease(MachadoandBachevalier,2008).Importantly,

amygdalalesionsalsoresultedinlessanxietyinsocialsituationswherehumanATismost

commonlyobserved(Emeryetal.,2001;Machadoetal.,2008).Wenotethatotherstudieshave

usedthehumanintruderparadigmtoassesstheeffectsofamygdalalesionsonbehavior;these

studiesarereviewedinotherchaptersinthisvolume.Alsoreviewedelsewhereinthisvolume

aretheseminalstudiesofpatientS.M.,awomanwithcalcificationoftheamygdalaasaresultof

Urbach‐Wiethedisease.YearsofclinicalandexperimentalassessmenthavefoundthatS.M.is

moretrustingofandmorelikelytoapproachstrangers(Adolphsetal.,1998),doesnotrecognize

fearinothers(Adolphsetal.,1994),showsa“blindness”forsociallyacceptablephysicalspace

(Kennedyetal.,2009),doesnotreadilylearnnovelPavlovianfearassociations(Becharaetal.,

1995),anddoesnotshowtypicalsignsofanxiety(Feinsteinetal.,2011).Takentogether,these

datasuggestthatSMdisplayslessanxietyinsocialandotherthreateningsituations,andfitwith

datafromadultrhesusmonkeyswithamygdalarlesionsthatdisplayalteredsocialbehavior

(Emeryetal.,2001;Amaral,2002;Machadoetal.,2008).Seealso(Terburgetal.,2012),and

Chapter12inthisvolume,foradifferentinterpretationofthedeficitsassociatedwithhuman

amygdalalesionsresultingfromUrbach‐Wiethedisease.

InaninitialstudyaimedatunderstandingtheroleofamygdalainmonkeyAT,we

lesionedtheentireamygdalawiththeneurotoxinibotenicacid(Kalinetal.,2001).Lesioned

animalsdisplayedlessfear‐relatedbehaviorinthepresenceofalivesnakeornoveladult

conspecific.However,noreductioninfreezingbehaviorwasobservedinresponsetothehuman

intruder.Inhindsight,webelievethatthisnullresultreflectsanunintendedconsequenceofthe

factthatthelesionedmonkeysinthisstudywererepeatedlyexposedtothehumanintruder

paradigmpriortosurgery.Otherworkbyourgroup(Foxetal.,2012)indicatesthatalthough

individualdifferencesinfreezingaremoderatelystable,absolutelevelsoffreezingtendto

decreasewithrepeatedexposuretothehumanintruderparadigm.Thus,itispossiblethatthe

apparentlackofeffectofthelesionsonfreezinginthisexperimentwasduetorepeatedexposure

associatedhabituation.

Alterationsinsleepwerealsoobservedinthemonkeyswithlargeamygdalalesions

(Bencaetal.,2000).Specifically,lesionedandcontrolmonkeyswereadaptedtoEEGrecording

duringtheirnocturnalsleepperiod.Despiteapparentadaptation,thesleeppatternsofcontrol

animalswerepunctuatedbyfrequentarousals.Monkeyswithlargebilaterallesionsofthe

amygdalahadmoresleepandahigherproportionofREMsleepcomparedtocontrolanimals,

suggestingthattheamygdalamaybeimportantinmediatingtheeffectsofstressonsleep.Thisis

interestingconsideringthatanxietyisthepsychiatricsymptommostoftenassociatedwith

insomnia,andthegrowingrecognitionofthatsleepdisturbancesaccompanyalmostallformsof

psychopathology(Bencaetal.,1992).

Inafollow‐uplesionstudywefocusedmorespecificallyontheCe.Inthatstudy,the

monkeyswereintentionallykeptnaïvetothehumanintruderparadigmandwereexposedtoit

onlyonce,followingrecoveryfromthelesionsurgery(Kalinetal.,2004).Smallselectivelesions

intheCeregionwereproducedtoexaminetheextenttowhichtheCemediatesunconditioned

fear,AT‐relatedbehavioralresponses,andstress‐inducedpituitary‐adrenalactivity(Figure7).

Thereweretwoexperimentalgroups[bilaterallesion(n=9)andunilateral(n=5)Celesions)and

anage‐matchedunoperatedcontrolgroup(n=16).

[insertFigure7here]

TheCelesionssignificantlyaffectedcoovocalizationsandfreezingduration,thetwo

behavioralcomponentsofAT.Comparedwiththeage‐matchedcontrols,cooingwasincreasedin

thebilateral‐lesionandunilateral‐lesiongroups(p<0.04).Thebilateral‐lesiongroupshowed

significantlylessfreezingbehaviorcomparedtotheothergroups(p<0.023).Thebilateral

lesionedanimalsalsodisplayedlessfearwhenexposedtoalivesnake,suggestingthatthese

effectsgeneralizebeyondthehumanintruderparadigm.Decreasesinadrenocorticotropin

releasinghormone(ACTH)andcerebrospinalfluidlevelsofcorticotropinreleasinghormone

(CRH),thetwokeyupstreammediatorsofcortisolreleasewereobserved,andindividual

differencesintheextentofthelesionsignificantlypredictedstress‐relatedcortisollevels(Kalin

etal.,2004).InconjunctionwiththeFDGimagingresults,thesefindingsindicateamechanistic

rolefortheCeinmediatingthebehavioralandpituitary‐adrenalcomponentsofAT,aswellas

otherfear‐relatedbehaviors,earlyinlife.

CorticalandsubcorticalsystemsinteractingwithCeinrelationtoAT

Psychiatricdisorderslikelyreflectalterationsinthecoordinatedactivityofdistributed

functionalcircuits.WhiletheresultsofourFDGandlesionstudiessuggestthattheCeisakey

substrateforstableindividualdifferencesinAT,theydonotdirectlyaddressthelarger

functionalnetworkinwhichtheCeisembedded.Tounderstandthelong‐rangeneuralnetworks

thatmayinteractwiththeCeinrelationtoAT,weusedfMRItoassessfunctionalconnectivityof

theCeregion.Basedonworkdemonstratingtheabilitytoreliablyassessfunctionalconnectivity

inanesthetizedrhesusmonkeys(Vincentetal.,2007),weusedtheCeasaseedregionto

examinetemporalcorrelationsoftheBOLDsignalinasubsetofthemonkeysfromthelarge‐

sampledescribedabove(Oleretal.,2010).Bycombiningdatafrommultiplemodalities(FDG‐

PETandfMRI)wefoundthatgreaterCeglucosemetabolismwasassociatedwithdecreased

functionalcouplingbetweentheCeanddlPFC,andthatdecreasedfunctionalcouplingbetween

theCeanddlPFCwasalsoassociatedwithhigherlevelsofAT(Birnetal.,2014).DecreasedCe‐

dlPFCconnectivitywasalsoobservedinasampleofpre‐adolescentchildren(ages8‐12)with

anxietydisorders,furthervalidatingthemonkeyATmodel,suggestingaroleforaltereddlPFC‐

amygdalafunctionalcouplinginthepathogenesisofchildhoodanxietydisordersand

demonstratingthatthemodulatoryinfluenceofdlPFConamygdalafunctionisevolutionarily

conserved(Birnetal.,2014).Importantly,themonkeyFDG‐PETdataprovidedevidencethat

elevatedCemetabolismstatisticallymediatestheassociationbetweenCe‐dlPFCconnectivityand

elevatedAT(Birnetal.,2014).Thus,thesefunctionalconnectivitydatasuggestthatcoordinated

activitybetweendlPFCandCeisanimportantmodulatorofindividualdifferencesinthe

expressionofAT.Thishighlightsanimportantbenefitofassessingfunctionalconnectivity,as

findingsarenotconstrainedbydirectneuroanatomicalconnections.Futurestudiesaimedat

directlymodulatingdlPFC‐Cefunctionalconnectivitywouldhelpinfurtherunderstandingthe

roleofdlPFCinregulatingamygdalafunctionandATaswellasinchildrenwithanxiety

disorders.Inthisregard,transcranialmagneticstimulationisanoninvasivestrategythatcould

beusedinbothhumanandnon‐humanprimatestostimulatethedlPFCandexamine

downstreameffectsonamygdalafunctionaswellasonaffectingdlPFC‐amygdalaconnectivity.

Inadditiontotheamygdala,FDG‐PETimagingstudiessuggestthatATreflectsindividual

differencesinanumberofregionsthatincludetheanteriorhippocampus,BST,anterior

temporalcortexandPAG.Thecaudalorbitofrontalcortex(OFC)alsoappearstoplayarole(FDR

q<.05,corrected;unpublishedanalysesofthen=238sampledescribedbyOleretal2010).

Furthermore,aspirationlesionsoftheOFCreducefreezinginresponsetotheNECchallenge

(Kalinetal.,2007).Importantly,whole‐brainFDG‐PETimagingprovidedevidencesuggesting

thatthereductioninfreezingobservedinOFC‐lesionedanimalsreflectsanindirectconsequence

oflesion‐inducedalterationsintheextendedamygdala.Specifically,OFClesionsreducedNEC‐

relatedmetabolismintheBST(Foxetal.,2010).ItisimportanttoemphasizethatwhileOFC

lesionsattenuatefreezinganddecreaseBSTmetabolism,thecorrelationbetweenBSTactivity

andfreezingbehavior,evidentpriortothelesions,remainedsignificantafterthelesions(Foxet

al.,2010).ThissuggeststhatdecreasedfreezingbehaviorinOFClesionedanimalswasdirectly

relatedtodecreasedactivityintheBST,andsupportspreviouslyreportedfindingsthat

individualdifferencesinBSTmetabolicactivityarepredictiveofindividualdifferencesin

freezingand/orATinyoungmonkeys(Kalinetal.,2005;Foxetal.,2008).Thus,futurestudies

examiningthemechanisticroleofBSTinprimateanxietyshouldemployselectiveBSTlesion

techniquessimilartothosedescribedabove,andinotherchaptersinthisvolume,todissociate

theselectiverolethatthiscomponentoftheextendedamygdalamayplayinnormaland

pathologicalanxiety.

ConcludingRemarksandFutureDirections

ThefunctionalneuroimagingdatainintactanimalsandbehavioraldatafromCe‐lesioned

animalsreviewedaboveextendpriorstudiesonthefunctionoftheCe.First,wedemonstrateda

mechanisticrolefortheCeinthebehavioralandpituitary‐adrenalcomponentsofATusing

selectiveibotenicacidlesions.Then,buildingonearlierstudies,wedemonstratedthatCe

metabolismstronglypredictsindividualdifferencesinAT.Inthislargesample,wedemonstrated

thatpolymorphismsintheCRHreceptorsystemareassociatedwithheightenedanxietyand

elevatedmetabolicactivityintheCeinresponsetopotentialthreat.Inasubsample,wefound

thatmRNAexpressionofneurodevelopment‐relatedgenesisdecreasedintheCeofanxious

monkeys,whichsuggeststhatlearning‐relatedneuroplasticityphenomenaintheamygdalamay

becompromisedinindividualswithextremeanxiousphenotypes.Additionally,weuncovered

evidencesuggestingthatdorsolateralandorbitalregionsofthePFCinfluenceAT‐relatedactivity

withintheextendedamygdala.Takentogether,thesedataindicatearoleforacircuitcenteredon

theextendedamygdala,encompassingtheCeandBST,intheestablishingandmaintaining

normativeandextremeanxietyearlyinlife.

Futurestudiesemployinglesionorreversibleinactivationtechniquesthattargetspecific

neuronalsub‐populationswilllikelydeepenourunderstandingoftheamygdalarmicrocircuits

thatunderlieprimateAT.Furthermore,rapidimmunohistochemicalstainingtoidentifyspecific

cellpopulationsformicro‐dissectionandsubsequentdeepRNAsequencingisapromising

methodforunderstandingthecell‐specificmolecularmechanismsrelatedtoAT.Genedelivery

withviralvectorstoinduceorsuppressexpressionofspecificmoleculesisanothertechnique

withthepotentialtoenrichourunderstandingofprimateamygdalarmicrocircuitfunctionand

theroleoftheextendedamygdalaintemperamentalanxiety.Withtheultimateaimof

developingmoreeffectiveearly‐lifeinterventionstotreatandpreventanxiety‐related

psychopathology,itisourhopethatsuchstudieswillshedlightontheriskforanxiety‐related

psychopathologyaswellasdeepenourunderstandingofamygdalafunctionandnormal

variationintemperament.

Table1.Parallelsbetweenmonkeyanxioustemperament(AT)andchildhoodbehavioralinhibition(BI).

Phenotypic features  AT in Juvenile Monkeys  BI in Children 

Increased freezing/reduced motor activity/passive 

avoidance in the presence of adult strangers 

YES (Kalin and Shelton, 1989; Kalin et al., 1998; Fox et al., 2008; Oler et al., 2010; Fox et 

al., 2012; Shackman et al., 2013)

YES (Fox et al., 2005; Hirshfeld‐Becker et al., 2008; Degnan et 

al., 2010) 

 Less frequent vocal communication 

YES (Kalin and Shelton, 1989; Fox et al., 2008; Oler et al., 2010; Fox et al., 2012; Shackman et al., 2013)

YES (Fox et al., 2005; Hirshfeld‐Becker et al., 2008; Degnan et 

al., 2010) 

 Moderate stability across 

time and context 

YES (Fox et al., 2008; Fox et al., 2012; Shackman et al., 2013) 

YES (Pfeifer et al., 2002; Fox et al., 2005; Hirshfeld‐Becker et al., 2008; Degnan et al., 2010; 

Brooker et al., 2013)  

Significant functional impairment or distress 

 Unknown 

Variable (Fox et al., 2005; Hirshfeld‐Becker et al., 2008; 

Degnan et al., 2010)  

Heritable YES (Williamson et al., 2003; 

Oler et al., 2010) YES (Rickman and Davidson, 1994; Hirshfeld‐Becker et 

al., 2008)  

Reduced by anxiolytic administration 

YES (Kalin and Shelton, 1989; Davidson et al., 1992; Davidson 

et al., 1993)

 Unknown 

 Increased pituitary‐adrenal 

activity (cortisol) 

Not consistently observed (Kalin et al., 1998; Fox et al., 2008; Oler et al., 2010; Fox et 

al., 2012; Shackman et al., 2013)

Not consistently observed (Schmidt et al., 1997; de Haan et al., 1998; Fox et al., 2005) 

Right‐lateralized frontal EEG activity 

YES (Davidson et al., 1993; Kalin et al., 1998) 

YES (Davidson and Rickman, 1999; Buss et al., 2003; Fox et 

al., 2005) Increased or sustained 

amygdala activity to novelty and potential threat 

YES (Fox et al., 2008; Oler et al., 2010; Fox et al., 2012; Shackman et al., 2013) 

YES (some data are from retrospective studies in adults) 

(Schwartz et al., 2003; Blackford et al., 2011) 

Altered functional connectivity between the amygdala and prefrontal 

cortex 

 YES (Birn et al., 2014) 

 YES (Hardee et al., 2013) 

Figure1

Figure 1. The three experimental conditions of the human intruder paradigm elicit distinct fear‐relatedbehaviors in young rhesus monkeys. When alone and separated from their cagemate (left), youngmonkeysactivelyexplorethetestcageandspontaneouslyemit“coo”calls,thoughttoreflectanattempttoattracthelpfromtheirmothersorotherconspecifics.Inthenextconditionahumanintruderpresentshisor her profile while avoiding direct eye contact with the monkey (NEC, center). In this situation themonkeystypicallyorienttheir focusonthe intruder, tryingtoevadediscoverybyremainingcompletelystill(freezing)orhidingbehindtheirfoodbin(opaqueboxinthecenterpanel).Inthethirdcondition,thehumanintruderenterstheroomandstaresattheanimal(right).Thisdirectthreatconditionoftenelicitsaggressive behaviors (e.g., barking, threatening gestures, cage rattling). This figure was reprintedwithpermission(Kalin,1997).

Figure2

A. B.

Figure2. (A)AT is calculatedas themeanz‐scoresofNEC‐induced freezing, coovocalizations [reverse‐scored], and plasma cortisol levels. (B) To measure NEC‐induced regional brain metabolism, monkeyswere injectedwith a radiotracer (18‐FDG) immediately prior to exposure of the 30‐minNEC challengedepicted in Figure 1. FollowingNEC exposure themonkeyswere anesthetized, bloodwas collected forcortisol, and the animals were placed in a high‐resolution microPET scanner to measure FDG uptake,integratedacrossthe30‐minNECchallenge.

Figure3

Figure3.TounderstandtherelationbetweenindividualdifferencesinregionalbrainmetabolismandAT,whole‐brainvoxelwiseregressionanalysiswasperformedin238youngmonkeyswhilecontrollingfornuisanceeffectsofage,sexandvoxelwisegray‐matterprobability.ResultsrevealedapeakFDG‐ATcorrelation in theregionof theCe(significanceofcorrelations:yellow,p<0.05; lightorange,p<0.01;darkorange,P<0.001,adjustedformultiplecomparisonsusingtheŠidákcorrection.)Theareainpinkrepresentsthe95%spatialconfidenceintervalofthepeakFDG‐ATcorrelationintheamygdala.ThisfigurewasadaptedwithpermissionfromOleretal.,2010).

Figure4

Figure 4.Invivo serotonin transporter (5‐HTT) binding localized the dorsal amygdalacluster to the Ce. (Top) A low‐power photomicrograph of ex vivo 5‐HTTimmunohistochemistry showing substantial immunoreactivity in the lateraldivisionofCe[adaptedwithpermissionfromO'RourkeandFudge(2006)CopyrightElsevier].Highlevels of 5‐HTT are a chemoarchitectonichallmarkof the lateral subdivisionof the Ce(CeL). (Middle)Overlapbetween the amygdala 95%spatial confidence interval of thepeak FDG‐AT correlation (pink) and in vivo 5‐HTT availability (dark blue = 250Xbackground 5‐HTT binding). High 5‐HTT availability was also observed within thesubstantia innominata,which canbe seen just below the anterior commissure,medialanddorsaltotheCeandintheregionofthedorsalraphenucleus(notshown).(Bottom)Magnified coronal view of the overlap between 5‐HTT binding and the FDG‐PETcorrelationasshowninthemiddlepanel.

Figure5

Figure5.OverlapbetweenregionalmetabolicactivitypredictiveofAT (yellow)andregionsthatare significantly heritable. No significantly heritable voxels were observed in the dorsalamygdalaregion(top),althoughwithinthesameslicesignificantheritabilitywasdetectedinthesuperiortemporalsulcus.(Bottom),Glucosemetabolismwassignificantlyheritableinboththerightandleftanteriorhippocampus,whereitoverlapswiththeleftanteriorhippocampalregionthat correlated with AT (yellow, regions predictive of AT; dark green to light green, falsediscoveryrate:q=0.05,q=0.01,q=0.001).ThisfigurewasadaptedwithpermissionfromOleretal.,(2010).

Figure6

Figure6.MicroarraydatademonstratedthatindividualswithhigherlevelsofCeNTRK3mRNAexpressionexhibitedlowerAT.(Top)CeregionspredictiveofdispositionalATwereusedtoguideamygdalabiopsyforanalysisofAT‐relatedRNAexpression.Aslicethroughthefunctionallydefinedamygdalaregionjuxtaposedwitharepresentativesingle‐subjectslabinwhichthedorsalamygdalawasbiopsied.(Middle)NTRK3expressionnegativelypredictsCemetabolism.IndividualsshowinghigherlevelsofNTRK3mRNAexpression,indexedbyqRT‐PCR,showreducedCemetabolisminvivo(green)[FDR‐correctedwithinthestableAT‐relatedregion(pink)].(Bottom)Portrayaloftheneuroplasticity‐associated,NTRK3(Trkreceptor,green)pathway.AsimilarpatterninrelationtoATwasfoundforIRS2(orange)andRPS6KA3(pink),twodownstreamtargetsofNTRK3.OthermoleculesintheNTRK3pathwayaredepictedingray.FigurewasadaptedfromFoxetal.,(2012)andreprintedwithpermission.

Figure7

Example CeA Lesion

Ce LesionGroup

ControlGroup

Stress‐induced plasm

a ACTH

(+/‐ SEM

)*

Cooing Frequency 

square‐root transform

ed (+/‐ SEM)

*

Freezing Duratio

n 

log transform

ed (+/‐ SEM)

*

Figure7.TheeffectsofCelesionsoncomponentsof AT.Left,arepresentativelesionisdisplayedonfourcoronalsectionsthroughtheanterior–posterior(toptobottom)extentofCe.TheintactCeisdepictedinblue,theareaofthetotallesionisdisplayedinyellow,andtheCeregionthatislesionedisdepictedbytheoverlapingreen.Right,monkeyswithCelesionsdisplayedlessfreezing(top),emittedmorecoocalls(middle),andreleasedlessACTH(bottom)duringexposuretothehumanintruderparadigm.FiguremodifiedfromKalinetal.,(2004)andreprintedwithpermission.

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