impact of prophylactic intranasal oxytocin …
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Eastern Washington UniversityEWU Digital Commons
EWU Masters Thesis Collection Student Research and Creative Works
Spring 2017
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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Control Oxytocin +Stress
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+
020406080100120140160180
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Freezing
time(s)
29
+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