IMPACT OF PROPHYLACTIC INTRANASAL OXYTOCIN …

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IMPACT OF PROPHYLACTIC INTRANASALOXYTOCIN ADMINISTRATION ONSYMPTOMS OF POST-TRAUMATIC STRESSMorgan A. ThomasEastern Washington University

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Recommended CitationThomas, Morgan A., "IMPACT OF PROPHYLACTIC INTRANASAL OXYTOCIN ADMINISTRATION ON SYMPTOMS OFPOST-TRAUMATIC STRESS" (2017). EWU Masters Thesis Collection. 437.http://dc.ewu.edu/theses/437

IMPACTOFPROPHYLACTICINTRANASALOXYTOCINADMINISTRATIONON

SYMPTOMSOFPOST-TRAUMATICSTRESS

__________________________________________________________________________________________________

AThesis

PresentedTo

EasternWashingtonUniversity

Cheney,Washington

__________________________________________________________________________________________________

InPartialFulfillmentoftheRequirements

fortheDegree

MasterofScienceinBiology

__________________________________________________________________________________________________

By

MorganA.Thomas

Spring2017

ii

THESISOFMORGANTHOMASAPPROVEDBY

________________________________________________________________DATE_____________DAVIDDABERKOW,GRADUATECOMMITTEECHAIR_________________________________________________________________DATE_____________JAVIEROCHOA-REPARAZ,GRADUATECOMMITTEEMEMBER_________________________________________________________________DATE_____________ANDREWOSTER,GRADUATECOMMITTEEMEMBER

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Abstract

Post-traumaticstressdisorder(PTSD)isamentalhealthconditionthataffects

peopleafterinstancesofsevereemotionaltrauma.Researchsuggeststhatoxytocin

treatmentdecreasesPTSDsymptoms.Thisstudyservedtoevaluatetheefficacyof

intranasaloxytocinpre-treatmentonsymptomsrelatedtoPTSD.Thehypotheses

arethatoxytocinwilldecreasefearandanxiety,andincreasereward-seeking

behaviors.SpragueDawleyratswereassignedtothreegroups(Control,Stress,

Oxytocin,andOxytocin+Stress;n=6pergroup)toconductthisexperiment.Priorto

footshocktreatment,ratsweretrainedtoexpectafoodreward(Kellogg’sFroot

Loops)inanopenfieldenclosure.Subsequently,theOxytocinandthe

Oxytocin+Stressgroupswerepre-treatedwithintranasaloxytocinandthenthe

StressandOxytocin+Stressgroupswereexposedtoaninescapablefootshock(a

modelPTSDinducingstressor).Afteroxytocinandshocktreatments,rats

underwentvariousbehavioraltests:re-exposuretotheshockchambertoassess

fear,elevatedO-mazetoassessanxiety,andfoodrewardtrialsintheopenfield

enclosuretoassessreward-seekingbehavior.Theoxytocintreatmentdecreased

fearrelatedsymptomsuponre-exposuretothefearconditioningchamber;both

colonicmotilityandfreezingtimewerelowerintheOxytocin+Stressgroup

comparedtotheStressgroup.Thefootshockmodelfailedtoproducesignificant

behavioralchangesrelatedtoanxietyandreward-seekingbehaviorbetweenthe

ControlandStressgroups.

BackgroundandSignificance

PTSD

Post-traumaticstressdisorder(PTSD)isamentalhealthconditionthataffects

peopleafterinstancesofsevereemotionalorphysicaltrauma.Diagnosticcriteria

arecomplexandsymptomsvarybetweenindividuals,butincludeemotional

distress,physicalreactivity,avoidanceoftraumarelatedreminders,decreased

interestinactivities,anddifficultyexperiencingpositiveaffect(American

PsychiatricAssociation,2013).Itisestimatedthatbetween8.3%and9.4%percent

oftheUnitedStatespopulationdevelopsdiagnosablePTSDintheirlifetime

(Kilpatricketal.,2013).

RodentModelofPTSD

Whilepreviousresearchershaveusedcorticosteroneadministrationtoinduce

symptomsofPTSDinrodents(Levyetal.,2001),morerecentlyafear-conditioning

paradigmusingchronicelectricfootshock(describedintheMethodssection)has

beendevelopedtoinducePTSD-likesymptomsinaratmodel(Sahraeietal.,2012;

Yuetal.,2012;Gaoetal.,2014).Thisparadigmproducesbehavioralchangesinrats

suchasfearandanxiety,whichareknowntobeassociatedwithPTSD.

Fearinratsiscommonlyassessedbyincreasedfreezingbehavior(Fanselow,

1994;Sahraeietal.,2012;Yuetal.,2012;Gaoetal.,2014).Freezingisdefinedas

motionlessperiodsuponre-exposuretothepreviouslytraumatizingsourceof

stimuli,thefootshockchamber(Yuetal.,2012;Gaoetal.,2014),andisahallmark

ofstress-inducedbehavioralchangesinrodents.Anotherassessmentoffearin

rodentsisincreaseddefecationwhenre-exposedtofearrelatedstimuli(Lesteretal.,

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1968;Gaoetal.,2011).Increaseddefecation,oftenreferredtoas“colonicmotility”,

isameasurehighlysensitivetopsychologicalstress(Stametal.,1995).Colonic

motilityisthoughttoberegulatedbythereleaseofcorticotropin-releasinghormone

causedbystressfulevents(Verleye&Gillardin,2004).

Anxietyiscommonlymanifestedandmeasuredinrodentsasdecreased

locomotion(Fernandesetal.,1999;Prut&Belzung,2002)andincreasedtimein

enclosedspaces(Pellowetal.,1985;Shepherdetal.,1994;Ennaceuretal.,2006).

Manydifferentmethodshavebeenusedtoassessanxietyinrodentsandincludethe

open-fieldtest,elevatedplus-maze,andelevatedO-maze(reviewedinSestakovaet

al.,2013).Theopen-fieldtestisthesimplestandlongestusedanxietytestforrats,

datingbackto1930’s(HallandBallachey,1932).Whilespecificrequirementsofthe

open-fieldarenotstandardized,ingeneralitconsistsofanopenareainwhich

ambulationcanbeobserved(WalshandCummings,1976).Thetestmeasures

locomotionandpropensitytoexplore;loweramountsoflocomotionand

explorationcorrelatetohigherlevelsofanxiety(Peralsetal.,2017;Prutand

Belzung2003).Theelevatedplus-mazeisafour-armedmazethatisraisedoffthe

groundbylegs.Eacharmisconnectedatacenterplatform.Twoarmsoftheplus-

mazehavehighwallsandtheothertwoarmsdonothavewalls.Thetimespentin

thewalledareasisassociatedwithanxiousbehavior(Pellowetal.,1984;Rodgers

andDalvi,1997)astheratisthoughttobeavoidingthenoveltyoftheopenarea

(Dawson&Tricklebank,1995).TheelevatedO-maze(orelevatedzero-maze)was

developedasanimprovementtotheelevatedplus-maze.Theconceptsoftheplus-

maze,suchasheightoffthegroundandtheopenversusclosedareas,remainthe

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samebuttheO-mazeformsacontinuouscircle(orzero)withalternatingquadrants

ofopen(nowalls)andclosed(walledoff)areas.Thisconfigurationallowsforeasier

explorationbetweenmazesectionssincetherearenocornersandtheratsdonot

havetoturnaroundwhilecontinuingtomoveaboutthemaze(Shepherdetal.,

1994).IthasbeensuggestedthattheelevatedO-mazeproducesmoreconsistent

resultsovertime(Tucker&McCabe,2017);however,theresultsoftheelevatedO-

mazearediminishedbydailyre-exposure(Cooketal.,2001).

Anhedonia(thedecreaseinthecapacitytofeelpleasure)hasrelatively

recentlybeenincorporatedintothediagnosticcriteriaofPTSD(Steinetal.,2014).It

isthoughtthatPTSDmaybeassociatedwithreward-seekingimpairments.While

theresponseisvaried,thereseemstobeanoveralldecreaseinrewardanticipation,

approach(orwanting),andhedonicresponsestoreward(Nawijnetal.,2015).This

symptomofPTSD,observedinhumans,hasnotyetbeeninvestigatedintherodent

model.Rewardseekinginrodentsismostcommonlyassessedbytheuseofan

operantbox(Dingessetal.,2017;Piantadosietal.,2017).Sinceoperantboxesare

currentlynotavailableinourlab,oneofthegoalsofthisstudywastoinvestigate

theimpactofstressonthereward-seekingbehaviorinanopenfieldcircular

enclosure(describedintheMethodssection).Furthermore,arecentstudy

conductedbyNawijnetal.(2016)suggeststhatoxytocintreatmentinhumanswith

PTSDappearstoimpactthebrainregionsinvolvedinrewardprocessing.Therefore,

anothergoalofthisstudywastoinvestigatetheimpactofoxytocinonPTSD-related

behaviors(i.e.,fear,anxiety,andreward-seeking).

Oxytocin

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Oxytocinisaneurohormoneproducedbyneuronsoftheparaventricularand

supraopticnucleiinthehypothalamus.Itisassociatedwithmanyfunctionsofthe

bodyincludinghumanemotionandmotivation(Love,2014).Administeredafter

traumatization,oxytocinhasbeenshowntoreducesymptomsassociatedwithPTSD

(i.e.,fearandanxiety)inbothrodent(Missigetal.,2010;Ayersetal.,2011;Zoicaset

al.,2014;Janezicetal.,2016;Sacketal.,2017)andhumanclinicaltrials

(Bakermans-Kranenburg&VanIJzendoorn,2009;Achesonetal.,2013;Frijilinget

al.,2014).

Oxytocinisaneuropeptidethatdirectlyinteractswithoxytocinreceptorsin

specificpartsofthecentralnervoussystem,assuchitisconsidereda

neuromodulatorinbrainregionsassociatedwithfear,aggressionandsocial

behaviors(Febo&Ferris,2014;Heinrichs&Domes,2008).Oxytocinreceptorsare

expressedintheamygdala,whichisanareaofthebrainintimatelyinvolvedin

processingofemotionandcognition(reviewedinPhelps,2006).Furthermore,

oxytocinreceptorsarealsofoundinrewardprocessingareasofthebrain(Feboand

Ferris,2014)includingtheventraltegmentalareaandthenucleusaccumbens(Wise

andBozarth,1987;Nicola,2016).Hypothalamicoxytocinneuronshavedirect

axonalconnectionstotheamygdala,ventraltegmentalarea,andnucleusaccumbens

andarethoughttodirectlymodulatetheactivityofthesebrainregions(Bethlehem

etal.,2012).

Inadditiontoitsdirecteffectsonbrainregionsassociatedwiththe

processingofemotionsandmotivation,oxytocinalsointeractswiththe

hypothalamic-pituitary-adrenal(HPA)axis.Thisendocrinefeedbacksystemis

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implicatedinstressreactionsaswellasregulatingmanybodyprocesses(Bhatnagar

etal.,2006;Stranahanetal.,2008;Halletal.,2012;Daskalakisetal.,2013).TheHPA

axiscanbemodifiedpermanentlybyearlychildhoodtrauma,renderingithyper

reactive(vanBodegometal.,2017).Oxytocinhasbeenshowntoinhibitstress

responsesassociatedwiththeHPAaxissuchascorticosteronerelease(Windleetal.,

1997;Heinrichsetal.,2003;DeKloetetal.,2006).Asanaturalmechanism,oxytocin

offersprotectiontostressassociatedwiththeHPAaxisinpostpartum,

breastfeedingmothers(Coxetal.,2014).Oxytocinlevelsriseperipherallyfollowing

stressfulincidentsandhigheroxytocinlevelscorrespondtofasterrecoveryfrom

stressrelatedsymptoms(Engertetal.,2016).Therefore,oxytocintreatmentcould

potentiallyprovideneuroprotectionduringstressfulevents.

Prophylaxis

Variouspharmaceuticalandpsychosocialinterventionshavebeenstudiedas

potentialpreventativetreatmentsforPTSD(reviewedinBakeretal.,2009;

Daskalakisetal.,2013).Mostpreventativemeasuresthathavebeenexploredfall

intothecategoryof“earlyintervention”inwhichtreatmentisgivenafterthe

traumaticevent,butpriortodevelopmentofPTSDrelatedsymptoms(reviewedin

Biruretal.,2017).Multipledrugshavebeenstudiedaspotentialpreventative

treatmentsforPTSD(Vaivaetal.,2003;Bakeretal.,2009;Daskalakisetal.,2013,

Morenaetal.,2017).However,theefficacyofoxytocinasaprophylactic,or

preventative,treatmenthasbeenminimallyexploredinhumans(Frijlingetal.,

2014;vanZuidenetal.,2017)andthereisadearthofresearchwithrodents

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(Renickeretal.,2015).Oxytocinmayofferthesameneuroprotectivebenefitsof

othertreatments(Vaivaetal.,2003;Bakeretal.,2009;Daskalakisetal.,2013,

Morenaetal.,2017)withouttherisksassociatedwithlong-termtreatment,

includingantidiuresisandhyponatremia(Bakeretal.,2009).

IntranasalAdministration

Intranasaladministrationofoxytocinisaneffectivetherapeuticdelivery

methodinhumans(Fischer-Shoftyetal.,2010;Guastellaetal.,2010;Achesonetal.,

2013;Nawijnetal.,2016).Whilethemechanismsarenotclearlyunderstood,

intranasaloxytocinadministrationproduces“clearandspecificchangesinneural

activation”(Veening&Olivier2013)andhasbeenshowntoincreaselevelsof

oxytocinincerebralspinalfluid(Stevensetal.2013,Streipensetal.,2013).

Intranasaloxytocinadministrationhasalsobeenshowntoimpactspecificbrain

regionsconsidered‘social’regions(Bethlehemetal.,2012).Oxytocinisaverysmall

peptideofnineaminoacids(anonapeptide)andisbelievedtoatleastpartiallypass

throughthebloodbrainbarrier(Ermischetal.,1985).Oxytocinadministrated

intranasallyisthoughtbypassthebloodbrainbarrier(Talegaonkar&Mishra2004)

andhasbeenshowntoproducehigherlevelsofoxytocininthebrainthan

peripheraladministration(Neumannetal.,2013).Additionally,peripheraloxytocin

mayhavedifferenteffectsonstressthanoxytocindelivereddirectlyintothecentral

nervoussystemviaintranasaladministration.Onestudyfoundthatincreased

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plasmaoxytocinlevelscorrelatetoincreasedcortisollevels(Tayloretal.,2006),

althoughcausationwasnotimplied.Researchsuggestingthatintranasal

administrationofoxytocindirectlyandeffectivelyimpactsbrainfunctionwarrants

investigationintoitspotentialtherapeuticeffectsonconditionssuchasPTSD.

Thepurposeofthisstudywastotestthehypothesisthatprophylactic

oxytocintreatmentwithdecreasePTSDrelatedsymptomsinaratmodel,

specificallydecreasingfearandanxietyrelatedbehaviorsandincreasingreward-

seekingbehaviors.

Methods

Twenty-fourmaleSpragueDawleyratswererandomlyassignedtofour

groups(n=6,pergroup):1.Controlgroup(noshockandnooxytocintreatment),2.

Stressgroup(exposedtoshockandnooxytocintreatment),3.Oxytocingroup(no

shockandtreatedwithoxytocin),and4.Oxytocin+Stressgroup(treatedwith

oxytocinandexposedtoshock).Forpracticalpurposestheratsweresplitinto3

cohorts,testingn=2ratsfromeachtreatmentgroupatatime(2-3month

timeframe).

Generalhousing

Ratswerehousedindividuallytomosteffectivelymonitorfoodconsumption

duringbehavioraltasks.Ratswerehousedinapolysulfonefiltertopcage.Cages

containedcorncobbedding,PVCpipe,andwatersuppliedadlibitum.“Harlan2018”

foodwasprovidedadlibitum,exceptduringtheweeksoffooddeprivation

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(describedbelow).Allratswerehousedinthesameroomkeptatatemperatureof

22+1°C,andahumidityof23-33%witha12hourlight/darkcycle.

RewardTraining

Priortooxytocintreatmentandfearconditioning(footshock),allratswere

pre-trainedtoexpectandretrieveafoodreward(Kellogg’sFrootLoop)inacircular

openfieldenclosure(3-ftx3-ft,1-ftwalls)witharewarddeliverytubeatoneend

(Fig.1).Foroneweek,ratswerehabituatedtotheenclosurebyallowingthemto

roamfreelyforaminimumof5-10minutesaday(Monday-Friday),threeFroot

Loopsweredeliveredintothechamberduringthistimetofamiliarizethemtothe

rewarddeliveryprocedureandassesstheirinterestinthefoodreward.Forthenext

2-3weeksratswerefoodrestricted,placedinastartboxintheopenfieldenclosure

and3FrootLoopsweredeliveredindependentlyintothereward-seekingarea(Fig.

1).Foreachreward-seekingtrial,theratwasplacedinthestartbox,theFrootLoop

wasdeliveredandthestartboxdoorwasopenedmanuallybytheexperimenter.

Thetimefortherattoretrievethereward(withinaminuteofrewarddelivery)was

monitoredandrecordedasanassessmentoftheir“reward-seeking”behavior.

FoodDeprivation

Afterthefirstweekofhabituation,ratswerefoodrestrictedonMonday-

Fridayandfedadlibitumontheweekends.Ratswereweigheddailywhilefood

restrictedandgivenanamountoffoodthatcorrelatedtotheirweightchange.Rats

wholost0-5grams,orgainedweightwerefedonehalfofafoodpellet,thosewho

lost6-10gramswerefed1pellet,ratswholost11-15gramswerefed1.5pelletsand

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thosewholost15gramsormorewerefed2pellets.Ratswhodropbelow80%of

theirstartingweightduringfoodrestrictionwouldhavebeenfedadlibitum,though

therewerenoinstancesofthisinourstudy.

OxytocinAdministration

Ratswerelightlyanesthetizedwithisofluranetocalmthemsufficientlyto

allowforintranasaladministration.TheOxytocin+StressandtheOxytocingroups

weretreatedwithintranasaloxytocinat0.1µL/kg,30minutespriortofear

conditioning(describedbelow)basedonAyersetal.(2011)procedure.TheControl

andStressgroupswereadministeredanequivalentamountofsaline.Theratswere

thenassessedforfear,anxietyandreward-seekingbehaviors(describedbelow).

FearConditioning

TheStressandOxytocin+Stressgroupswereexposedtoanelectricfoot-shock

paradigm(usedasamodelforaPTSDinducingstressor;Gaoetal,2014).Overa

periodofthreedays,ratsinthesegroupswereexposedtofootshocktwicedaily.

Theywereplacedintothefear-conditioningchambertwicedailyandgiven20

inescapablefootshocks(8mAintensity,3secondduration,10secondintervals

betweenshocks;similartoGaoetal.,2014).

BehavioralAssessments

Afteroxytocinandshocktreatments,allratswerereintroducedtotheshock

chambertoassessbehaviorsrelatedtofear(i.e.,timemotionlessanddefecation),

runonanelevatedO-maze(Fig.2)toassessbehaviorsrelatedtoincreasedanxiety

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(i.e.,decreaseintimespentintheopensegmentsofthemaze),andgiventhree

rewarddeliverytrialsintheopenfieldenclosuretoassessreward-seekingbehavior

(i.e.,timetoretrievereward).

Ratswereplacedintheshockingchamberforfiveminutes,butnotshocked,

toobservefearrelatedbehaviors.Fearisoftenexpressedandmeasuredbyan

increaseinfreezingtime(definedastheabsenceofallmovementsexceptforthose

relatedtorespiration;Gaoetal.2014)andbyanincreaseinfecalproduction(Gaoet

al.,2011)whenreintroducedintothefearconditioning(shocking)chamber.

Therefore,theratswereputbackintotheshockingchamberfor5minutesandwere

observedforfreezingtimeandtheamountoffecalproduction.

Toassessanxiety,theratswereplacedinanelevatedO-mazeforfive

minutes,measuringtheamountoftimespentintheopensegmentswhichis

inverselycorrelatedtoanxietyleveloftheanimalasproposedbyShepherdetal.

(1994).TheelevatedO-maze(Fig.2)consistedofanannularplatformelevated65

cmabovethefloor(105cmindiameter,10cminwidth),theplatformofthe

elevatedO-mazewasdividedinto4segments:2opposingopensegmentswithno

walls,and2opposingclosedsegmentswithwallsextending27cmabovethe

platformsurface.TheO-mazewaslocatedinanotherwiseemptyroomandthe

researchersteppedoutofroomandclosedthedooronceratwasplacedintheopen

segmentoftheO-mazeastonotdistractfromtheratsnormalexploratorybehavior.

Behaviorwasrecordedonvideoforlateranalysis.

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Toassessreward-seekingbehavior,theratswerethenplacedintheopen

fieldenclosure(Fig.1)for3consecutive1-minutetrials,basedonthereward

trainingprocedure.Atthebeginningofeachtrial,theratwasenclosedinastartbox.

ThestartboxdoorwasopenedandaFrootLoop(foodreward)deliveredthrough

therewarddeliverytube.ThetimetoretrievetheFrootLoopwasmeasured.

Behaviorwasrecordedonvideoforlateranalysis.

Allbehavioraltestsweredonetwice:week1(anxietytested1dayafter

shocktreatment,fearandreward-seekingtested2daysaftershocktreatment)and

againinweek2(anxietytested8daysaftershocktreatment,fearandreward-

seekingtested9daysaftershocktreatment).

Statisticalanalysis

Todeterminesignificantdifferencesinbehavioral(dependent)measures,we

firstcompareddatafromallfourgroupsusingaone-wayANOVA(VassarStats).If

overallsignificance(p≤0.05)wasfound,asubsequentTukey’spost-hocanalysis

wasusedtodeterminesignificantdifferencesbetweenindividualgroups.Standard

two-tailedt-tests(VassarStats)wereusedtoanalyzechangeswithingroups

betweenweekoneandtwo.Wewereparticularlyinterestedinthedifferences

betweentheStressandStress+Oxytocingroupstomostdirectlyaddressthe

objectivesofthisstudy.

Theproceduresdescribedinthisproposalhavebeenreviewedandapprovedbythe

EasternWashingtonUniversityInstitutionalAnimalCareandUseCommittee(effective

June7,2016).

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Results

Forbehavioralmeasuresassessingfear,wefoundoverallsignificanceinboth

measures(freezinganddefecation),duringbothweek1andweek2(Fig.3,

p<0.0001;Fig.4,p<0.001;Fig.7,p<0.0005;Fig.8,p<0.05).Freezingtimeinseconds

wassignificantlylowerforControl,Oxytocin,andOxytocin+Stressgroupswhen

comparedtotheStressgroup(Fig.3,p<0.01)duringweek1.Inweek2,the

decreasedfreezingtimebetweentheStressgroupandtheOxytocinandControl

groupsremainedsignificant(Fig7,p<0.01)buttheOxytocin+Stressgroupwasno

longersignificantlylowerthantheStressgroup,orsignificantlyhigherthanthe

Controlgroup(Fig7).Whencomparingweek1toweek2,thefreezingtimesforthe

Stressgroupshowsasignificantdecreaseovertime(Fig.11,p<0.05)whilethis

decreasefortheOxytocin+Stressgroupwasnotsignificantacrossthetwoweeks(Fig.

11).

FecalProduction(measuredingramsoffeces)wassignificantlylowerfor

Control,Oxytocin,andOxytocin+StressgroupswhencomparedtotheStressgroup

(Fig.4,p<0.05)inweek1afterfearconditioning.Byweek2theStressgroupwas

stillsignificantlyhigherthantheOxytocinandControlgroups(Fig8,p<0.01)butthe

Oxytocin+StressgroupwasnolongersignificantlylowerthantheStressgroup,or

significantlyhigherthantheControlgroup(Fig8).

Testsforanxietyandrewardseekingbehaviorfailedtoproducesignificant

resultsineitherweek1orweek2(overallone-wayANOVA;Fig.5,p=0.15;Fig.6,

p=0.47;Fig.9,p=0.68;Fig.10,p=0.55).

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Discussion

ItisclearthatthePTSDparadigmworkedtoinducefearintheratsbasedon

thedifferencesbetweentheControlandStressgroups(Fig.3-4,Fig.7-8).These

resultsalsosupportedourhypothesisthatprophylacticoxytocintreatmentwould

decreasefearrelatedbehaviorsafteraPTSD-likestressor.WhiletheOxytocin+Stress

grouphadincreasedfearrelatedbehaviorscomparedtotheControlgroup,boththe

timespentfreezing(Fig.3)andtheamountoffecalproduction(Fig.4)were

decreasedsignificantlycomparedtotheStressgroup.However,thesignificant

differenceinfearrelatedbehaviorsbetweentheStressandOxytocin+Stressgroups

doesnotcontinueinthesecondweekpoststressor(Fig.7-8).Thislackof

significancecouldbeduetotheattenuationoffearresponsesovertimeintheStress

groupwhichdecreasedfromweekonetoweektwo,asopposedtodiminished

efficacyoftheprophylactictreatmentintheOxytocin+Stressgroup,whichalso

decreasedinfearrelatedmeasuresbutwerenotsignificant(Fig.11).

AstherewasnosignificanceinanxietyrelatedbehaviorsbetweentheControl

andtheStressgroups(Fig.5andFig.9),itappearsourparadigmdidnotworkfor

testingorinvokingthesesymptoms.ItisunclearwhytheelevatedO-mazedidnot

produceresultsasithasshowntobereliableinothersimilartestsofthePTSD-like

model(Gaoetal.,2014;Renickeretal.,2015).Gaoetal.(2014)whodevelopedthe

PTSD-likemodelinratsusedanelevatedplus-mazetotestanxietysotheirresults

cannotbecompareddirectlytoourO-mazeresults,althoughasimilareffectwould

beexpected.Onepossibleexplanationisthetemporalsequenceoftesting,Gaoetal.

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(2014)performedtheelevatedplus-mazetestafterre-exposuretothefear

conditioningchamberwhileourelevatedO-mazetestsweredonethedaybeforere-

exposure.Renickeretal.(2015)alsotestedtheelevatedO-mazeafterre-exposure

tothefearconditioningchamber,althoughtherewasagapoftime(5days)between

thetests.Previousresearchhassuggestedthatareminderofthestressexperience

caninfluencememoryandbehavior(Zoladzetal.,2010;Burkeetal.,2013).Assuch,

itispossiblethatthere-exposuretothefear-conditioningchamberenhancedthe

anxietyresponseseeninthepreviousstudiesofGaoetal.(2014)andRenickeretal.

(2015).

Therewasalsoalackofsignificanceinreward-seekingbehaviorsbetween

theControlandtheStressgroups(Fig.6andFig.10).Sincereward-seeking

behaviorshavenotbeenanalyzedintherodentmodelofPTSD,itispossiblethat

thesebehaviorsarenotaffectedinrodentsasisfoundinhumancasesofPTSD.

Alternatively,sincethereward-seekingparadigmusedinthisstudywasdeveloped

inourlabasacosteffectivesubstitutetotheclassicaloperantbox,itispossiblethat

thesereward-seekingbehaviorsareimpactedintheratmodelofPTSDbutour

dependentmeasuresdidnotexposetheseeffects.Ifthisexperimentweretobe

repeated,itmightbenefitfromtheuseofoperantboxesashasbeenusedin

previousstudiestoassessreward-seekingbehaviors(Dingessetal.,2017;

Piantadosietal.,2017).

Animportantaspecttoconsideristhetimingof“prophylaxis”.Whileour

studydefinedprophylaxisastreatmentpriortothetraumaticstressor,otherstudies

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definepreventativetreatmentasinterventionsperformedsoonafterthetraumatic

eventbeforesymptomsofPTSDmanifest,alsocalled“earlyintervention”(Vaivaet

al.,2003;Bakeretal.,2009;Daskalakisetal.,2013;Rothbaumetal.,2014;Frijlinget

al.,2014;Renickeretal.,2015).Earlyinterventionorprophylactictreatmentscould

alsobeaffectingdifferentmechanismsthantraditionaltreatments,suchasmemory

consolidation(Morenaetal.,2017).Futurestudiesshouldevaluatetheprosand

consofeachmethod.Treatmentafterthetraumaticeventwouldbemostpractical

asthevastmajorityoftraumaticeventscannotbepredicted;however,iftreatment

beforeatraumaticstressorofferssignificantprotectionitcouldbebeneficialin

settingssuchascombatwarfare,lawenforcementoperations,andsurgicalsettings.

Whetheroxytocintreatmentisdeliveredsoonafterthetraumaticevent

(Frijlingetal.,2014;Renickeretal.,2015;vanZuidenetal.,2017)orbefore,aswas

doneinthisstudy,researchsuggeststhatintranasaloxytocinadministration

providesneuroprotectionandattenuatesfearrelatedbehaviorsinhumans(Frijling

etal.,2014;vanZuidenetal.,2017)androdents(Renickeretal.,2015).Thedirect

effectsofoxytocinonbrainregionsthatcontainoxytocinreceptorsandprocess

emotion(e.g.,amygdala)arelikelyimplicatedinthebehavioralchangeswe

observedinthemeasuresoffear.Furthermore,PTSDhasbeenshowntoalter

endocrineoutputrelatedtotheHPAaxis(reviewedinDaskalakisetal.,2013).

IntranasaloxytocinadministrationislikelyimpactingtheHPAaxisbuthow

oxytocinalterstheHPAaxisispoorlyunderstood(reviewedinStockhorstand

Antov,2016).

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Certainfactorsshouldbeconsideredwhendesigningfuturestudies.Genetics

andepigeneticsbothplayaroleinthelikelihoodofdevelopingPTSD(Yehuda&

Bierer2009;Gerritsenetal.,2017)aswellasthepotentialfortreatmentsuccess

(Yehudaetal.,2013).Additionally,sexdifferencesarefoundtobeverypronounced

intheinstancesofPTSD(2:1femaletomaleratio)(Kilpatricketal.,2013)and

oxytocinefficacy(Sacketal.,2017;Smith&Wang2013).Asourstudypopulation

consistedofonlymaleratswewerenotbeabletoaddressthesedifferences.Many

factorsinfluencetheprobabilityofdevelopingPTSD.Inhumans,while

approximately80%ofthepopulationisexposedtotraumaticstressors,onlyasmall

percentage(~11%)ofthemgoontodevelopthedisorder(Kilpatricketal.,2013).

Therefore,arelativelysmallstudysuchasoursmaynotseealargeenoughsample

ofaffectedindividualstogainanaccuraterepresentationoftheeffects.

Theresultsofthisstudysuggestthatfurthertestingiswarrantedtoascertain

whetherornotprophylacticoxytocintreatmentisaneffectiveandpractical

preventionmethodforPTSD.OurfearresultscorroboratethefindingsofvanZuiden

etal.(2017)Friljingetal.(2014)andRenickeretal.(2015);however,theimpactof

prophylacticoxytocintreatmentonanxietyandrewardseekingneedfurther

investigation.

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Figures

Figure 1. Schematic diagram of open field enclosure and reward delivery area.

reward delivery tube

26

Figure2.Ratintheenclosedareaoftheelevatedzeromaze.

Figure3.Timespentmotionless(freezing)during5minutere-exposuretothefootshockchamberfollowingoxytocinpretreatment(n=6,pergroup)weekonepoststress.OverallsignificantdifferencebyANOVAp<0.0001*significantlydifferentthanOxytocin+Stressp<0.05;+significantlydifferentthanControlandOxytocinp<0.05

+*

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Figure4.Fecalproductionduring5minutere-exposuretothefootshockchamberfollowingoxytocinpretreatment(n=6,pergroup)weekonepoststress.OverallsignificancebyANOVAp<0.001;*significantlydifferentthanOxytocin+Stressp<0.05;+significantlydifferentthanControlandOxytocinp<0.05

Figure5.Timespentintheopenarmduring5-minuteexposuretotheelevatedzeromazefollowingoxytocinpretreatment(n=6,pergroup)weekonepoststress.Therewasnosignificantdifferencebetweenthegroupsp=0.15

+*

+

00.20.40.60.81

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Figure6.Averagetimeacross3trialstoretrievefoodreward(FrootLoop)inopenfieldenclosurefollowingoxytocinpretreatment(n=6,pergroup)weekonepoststress.Therewasnosignificantdifferencebetweengroupsp=0.47

Figure7.Timespentmotionless(freezing)during5-minutere-exposuretothefootshockchamberfollowingoxytocinpretreatment(n=6,pergroup),weektwopoststress.OverallsignificancefromANOVAp<0.0005

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020406080100120140160180

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+significantlydifferentthanControlandOxytocinp<0.05

Figure8.Fecalproductionduring5-minutere-exposuretothefootshockchamberfollowingoxytocinpretreatment(n=6,pergroup),weektwopoststress.Overall,p=0.016;+significantlydifferentthanControlandOxytocinp<0.05

Figure9.Timespentintheopenarmduring5minuteexposuretotheelevatedzeromazefollowingoxytocinpretreatment(n=6,pergroup)twoweekspoststress.Therewasnooverallsignificancebetweengroupsp=0.68.

+

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Figure10.Timetoretrievefoodreward(FrootLoop)inopenfieldenclosurefollowingoxytocinposttreatmentpretreatment(n=6,pergroup)twoweekspoststress.p=0.55

Figure11.Comparisonoffreezingtimesbetweenweek1andweek2inStressandOxytocin+Stressgroups.

*p<0.05two-tailedt-test

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31

VITA

Author:MorganA.Thomas,néePowell

PlaceofBirth:Spokane,Washington

UndergraduateSchoolsAttended:UniversityofWashington

DegreesAwarded:BachelorofScience,2013,UniversityofWashington

HonorsandAwards:GraduateFellowship,BiologyDepartment,2015-2017,EasternWashingtonUniversity

BiologyDepartmentMiniGrant,2017,EasternWashingtonUniversity

ProfessionalExperience:

AnatomyandPhysiologyTeachingAssistant,EasternWashingtonUniversityBiologyDepartment,2015-2017

SeniorCapstoneTeachingAssistant,EasternWashingtonUniversityBiologyDepartment,2016

DataCoordinator,FredHutchinsonCancerResearchCenter,2013-2015

SportsMedicineIntern,UniversityofWashingtonAthletics,2010-2013