Influence of Maternal High Fat Diet, Stress and Cocaine on Neural … · 2017-11-07 · 2.3.5. Gene expression analyses ... Chapter 5: Effects of maternal high fat diet and stress

Influence of Maternal High Fat Diet, Stress and Cocaine on Neural Mechanisms of Reward and

Anxiety in Rat Offspring

by

Aya Sasaki

A thesis submitted in conformity with the requirements for the degree of Doctorate in Philosophy Department of Cell and Systems Biology

University of Toronto

© Copyright by Aya Sasaki 2017

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Influence of Maternal High Fat Diet, Stress and Cocaine on Neural Mechanisms of Reward and Anxiety in Rat Offspring

Aya Sasaki

Doctorate in Philosophy

Department of Cell and Systems Biology

University of Toronto

2017

Abstract Maternalobesityhasimportanthealthconsequencesforthemotherandher

offspring.Experimentspresentedinthisdissertationexploredtheroleofmaternal

overnutritionwithahighfatdiet(HFD)onseveralaspectsofoffspringphenotype:

reward-andstress-relatedbehaviours,theendocrinestressresponse,and

associatedneuralgeneexpression.

First,IexaminedmaternalHFDeffectsonoffspringphenotypeinstress-

relatedbrainregions.MaternalHFDwasassociatedwithalteredexpressionof

stress-relatedgenes,aheightenedendocrinestressresponseandincreasedanxiety

behaviourinadultoffspring.Genescentraltostressanddrugaddiction(THand

CRF)wereupregulatedinHFDoffspring,suggestingthatmaternalHFDaltersneural

systemsunderlyingrelatedprocesses.

Second,IinvestigatedtheroleofmaternalHFDonoffspringphenotype

followingchroniccocaineexposure.MaternalHFDincreasedanxietyinsaline-

treatedcontrolfemales,reducedanxietyincocaine-treatedfemales,butdidnot

interactwithcocaine-primedlocomotoractivityorneuralgeneexpression.These

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findingssuggestthatmaternalHFDmodulatesoffspringanxietybehaviourwith

chroniccocaineexposure.

Third,IinvestigatedtheroleofmaternalHFDandmaternalstresson

offspringphenotypegivenacutecocaineexposure.MaternalHFDdidnotinteract

withcocaineatthelevelofbehaviourorgeneexpression.However,therewasan

increaseinlocomotoractivityinmalesexposedtomaternalHFD,andwithmaternal

stressatahighdoseofcocaine.Thesefindingssuggestthat,overall,maternalHFD

andstressincreasecocaine-inducedlocomotoractivityinoffspringthrough

commonbutnotidenticalneuralmechanisms.

Finally,inparallelIinvestigatedtheroleofpre-gestationalcocaineon

offspringphenotype,anddemonstratedaneffectonthelocomotoractivatingeffects

ofcocaineinadultmaleoffspring,aswellasdopaminereceptor1expressioninthe

medialprefrontalcortex.Thesefindingssuggestincreasedsensitivitytococainein

themaleoffspringofmothersgivenpre-gestationalcocaine.

Thecollectivefindingsarediscussedwithinaframeworkofmaternal

influencesoncocainesensitivityinoffspring,whereinmaternalHFDandpre-

gestationalcocaineconferincreasedsensitivityofstress-andreward-related

responsesinoffspring.

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TableofContents

Chapter1:GeneralIntroduction.........................................................................................................1

1.1Studiesoftheeffectsofmaternaldietonoffspring:Caveatstoconsider...........4

1.2Effectsofmaternalovernutritionontheoffspringdopaminesystem...................5

1.2.1Reward-directedfeedingbehaviourandpsychostimulantinducedlocomotoractivity...........................................................................................................................6

1.2.2Dopamine-relatedneuralgeneexpression...............................................................8

1.3Effectsofmaternalovernutritionontheoffspringstressresponsesystem.......9

1.3.1Anxietybehaviourandstressphysiology................................................................10

1.3.2Stress-relatedneuralgeneexpression......................................................................11

1.3.3Modelsofdiet-inducedobesity....................................................................................12

1.4Relationshipbetweenfoodanddrugaddiction............................................................13

1.5Effectsofmaternalcocaineontheoffspring...................................................................14

1.6Thesisrationaleandobjectives.............................................................................................16

Chapter2:GeneralMethods...............................................................................................................19

2.1.Animals..........................................................................................................................................20

2.2.Proceduralmanipulationsofdams....................................................................................20

2.2.1.Diets........................................................................................................................................20

2.2.2.Assessmentofmaternalbehaviour...........................................................................21

2.3.Proceduralmanipulationsofoffspring.............................................................................22

2.3.1.Subjects..................................................................................................................................22

2.3.2.Cocaine-inducedlocomotoractivity..........................................................................22

2.3.3.Anxietytests........................................................................................................................24

2.3.4.Immobilizationstress-inducedcorticosteroneresponse................................25

2.3.5.Geneexpressionanalyses..............................................................................................26

2.4.StatisticalAnalysis....................................................................................................................28

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Chapter3:Effectsofmaternalhighfatdietonanxietybehaviourandstressanddopaminergicgenesinthelimbicsystem.....................................................................................31

3.1.Introduction.................................................................................................................................32

3.2.MaterialsandMethods............................................................................................................34

3.2.1.Animals.................................................................................................................................34

3.2.2.Proceduralmanipulationsofdams............................................................................34

3.2.3.Proceduralmanipulationsofoffspring....................................................................34

3.2.4.Statisticalanalyses............................................................................................................38

3.3.Results.............................................................................................................................................38

3.3.1.Maternalbodyweightandcaloricintake................................................................38

3.3.2Offspringbodyweight......................................................................................................40

3.3.3Openfield...............................................................................................................................40

3.3.4Elevatedplusmaze............................................................................................................41

3.3.5Light-darktransition.........................................................................................................41

3.3.6Basalandstress-inducedserumcorticosterone...................................................43

3.3.7Corticosteronereceptorgeneexpression................................................................45

3.3.8Pro-inflammatorygeneexpression............................................................................47

3.3.9Anti-inflammatorygeneexpression...........................................................................47

3.3.10.DopaminergicandCRFgeneexpression..............................................................51

3.4.Discussion......................................................................................................................................51

Chapter4:Effectsofmaternalhighfatdietandcocaine-primedlocomotoractivityandgeneexpressioninoffspring......................................................................................................61

4.1Introduction..................................................................................................................................62


4.2.1.Animals.................................................................................................................................63



4.2.4.Statistics.................................................................................................................................68

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4.3Results..............................................................................................................................................68

4.3.1.Maternalbodyweight,offspringbodyweightandoffspringnumbers.....69

4.3.2.Maternalbehaviour..........................................................................................................71

4.3.3.Locomotoractivityduringcocainepre-exposureinoffspring......................71

4.3.4.Locomotoractivityduringtestforconditionedlocomotioninoffspring.74

4.3.5.Locomotoractivityduringtestforcocainesensitizationinoffspring.......74

4.3.6.Elevatedplusmazeinoffspring..................................................................................74

4.3.7.Cocaine-primedgeneexpressioninoffspring......................................................77

4.4Discussion.......................................................................................................................................80

Chapter5:Effectsofmaternalhighfatdietandstressoncocaine-inducedlocomotoractivityandgeneexpressioninoffspring.....................................................................................85

5.1Introduction..................................................................................................................................86


5.2.1.Animals.................................................................................................................................88



5.2.4.Statistics.................................................................................................................................94

5.3Results..............................................................................................................................................94

5.3.1.Maternalbodyweight,offspringbodyweightandoffspringnumbers.....94

5.3.2.Maternalbehaviour..........................................................................................................97

5.3.3.Immobilizationstress-inducedCORTresponsivityinoffspring...................97

5.3.4.Locomotoractivityduringhabituationinoffspring........................................100

5.3.5.Acutelocomotorresponsetococaineinoffspring...........................................100

5.3.6.Anxietytests.....................................................................................................................103

5.3.7.Cocaine-primedgeneexpressioninoffspring...................................................106

5.4.Discussion...................................................................................................................................109

Chapter6:Effectsofpregestationalcocaineoncocaine-inducedlocomotoractivityandgeneexpressioninoffspring...................................................................................................117

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6.1Introduction...............................................................................................................................118

6.2.MaterialsandMethods.........................................................................................................119

6.2.1.Animals..............................................................................................................................119

6.2.2.Proceduralmanipulationsofdams.........................................................................120

6.2.3.Proceduralmanipulationsofmaleoffspring......................................................122

6.2.4.Statistics..............................................................................................................................125

6.3.Results.........................................................................................................................................127

6.3.1.Effectofrepeatedcocaineexposurepriortoconceptiononlocomotoractivityandmaternalbehaviourindams........................................................................127

6.3.2.Effectofrepeatedcocaineexposurepriortopregnancyonbehavioural,physiological,andgeneticresponsesinmaleoffspring.............................................132

6.4.Discussion.................................................................................................................................135

Chapter7:GeneralDiscussion........................................................................................................143

7.1.MaternalHFDresultedinincreasedanxietybehaviourandcorrespondinggeneexpressionchanges..............................................................................................................144

7.2.MaternalHFDandstressincreaselocomotoractivity............................................148

7.3.MaternalHFDhadnoeffectoncocaine-inducedlocomotoractivity...............152

7.4.MaternalHFDincreasedbodyweightsindamsandpre-weaningoffspring155

7.5.Maternalmanipulationsdidnotalterthequalityofmaternalcare..................156

7.6.Limitationsandfuturedirections...................................................................................159

7.6.1.Anxietybehaviour:Drugexposureconditionsandbehaviouralassessmentsofanxiety.............................................................................................................159

7.6.2.Cocaine-inducedlocomotoractivity:self-administrationanddopaminefunction...........................................................................................................................................161

7.6.3.Epigeneticmechanisms...............................................................................................162

7.7.Conclusions................................................................................................................................164

References...............................................................................................................................................169

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ListofTables

Table3.1.Expressionofdopaminereceptorandstress-relatedgenesinoffspring.

Table4.1.Cocaine-primedgeneexpressionanalysesinoffspring.

Table5.1.Expressionofdopaminereceptorandstress-relatedgenesinoffspring.

Table6.1.Expressionofdopaminereceptorandstress-relatedgenesinmale

offspring.

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ListofFigures

Fig.3.1.Experimentaltimelinefordamsandoffspring.

Fig.3.2.Highfatdietaltersmaternalandpre-weaningoffspringbodyweight.

Fig.3.3.Perinatalhighfatdietexposureincreasesanxietybehaviourinadulthood.

Fig.3.4.PerinatalhighfatdietexposurealtersHPAinadulthood.

Fig.3.5.Perinatalhighfatdietexposureincreasescorticosteroidreceptor

expressionintheamygdalainadulthood.

Fig.3.6.Perinatalhighfatdietexposureincreasesproinflammatorygeneexpression

intheamygdalainadulthood.

Fig.3.7.Perinatalhighfatdietexposurealtersanti-inflammatorygeneexpressionin

thehippocampusandamygdalainadulthood.


Fig.4.2.Highfatdietaltersmaternalandpre-weaningoffspringbodyweight.

Fig.4.3.Highfatdietconsumptionindamsdidnotaltermaternalcare.

Fig.4.4.Locomotoractivityduringcocainepre-exposureinoffspring.

Fig.4.5.Locomotoractivityduringthetestforconditionedlocomotioninoffspring.

Fig.4.6.Locomotoractivityduringthetestforcocainesensitizationinoffspring.

Fig.4.7.Anxietybehaviourinoffspring.


Fig.5.2.Highfatdietaltersmaternalbutnotpre-weaningoffspringbodyweight.

Fig.5.3.Highfatdietconsumptionindamsalteredmaternalcare.

Fig.5.4.Immobilizationstress-inducedCORTresponsitivityinoffspring.

Fig.5.5.Locomotoractivityduringthehabituationsessioninoffspring.

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Fig.5.6.Acutelocomotorresponsetococaineinoffspring.

Fig.5.7.Estrusstatusandlocomotoractivityinfemaleoffspring.

Fig.5.8.Anxiety-likebehaviourinmaleandfemaleoffspring.


Fig.6.2.Locomotoractivityduringcocainepre-exposureindams.

Fig.6.3.Locomotoractivityduringtestforcocainesensitizationindams.

Fig.6.4.Maternalbehaviourindams.

Fig.6.5.Acutelocomotorresponsetococaineinmaleoffspring.

Fig.6.6.Immobilizationstress-inducedCORTresponsivityinmaleoffspring.

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ListofAppendices

AppendixTable4.1.Cocaine-primedgeneexpressionanalysesinoffspring.

AppendixTable5.1.Geneexpressionanalysisinoffspringpreviouslyexposedto

acutecocaine.

AppendixTable7.1.Summaryofthesisfindings

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Listofabbreviations

ACTH:adrenocorticotropichormone

AMG:amygdala

CD11b:clusterofdifferentiationmolecule11B

CHD:controlhousechowdiet

CORT:corticosterone

CRF:corticotrophinreleasingfactor

CVS:chronicvariablestress

DARPP-32:dopamine-andcAMP-regulatedneuronalphosphoprotein

DRD1:dopaminereceptorD1

DRD2:dopaminereceptorD2

EPM:elevatedplusmazetask

GR:glucocorticoidreceptor

HFD:highfatdiet

HPA:hypothalamic-pituitary-adrenal

HPC:hippocampus

HYP:hypothalamus

IkBa:i-kappa-B-alpha

IL-1Ra:interleukin-1Receptorantagonist

IL-6:interleukin-6

LD:light-darktransitiontask

LGABN:lickingandgrooming,arched-backnursing

LPS:lipopolysaccharide

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MKP-1:mitogen-activatedproteinKinasePhosphatase-1

mPFC:medialprefrontalcortex

MR:mineralocorticoidreceptor

NAc:nucleusaccumbens

NFkB:nuclearfactorkappaBeta

OF:openfieldtask

PND:postnatalday

TLR4:toll-likereceptor4

TH:tyrosine hydroxylase

VTA:ventraltegmentalarea

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Chapter 1: General Introduction

Thischapterisadaptedfrom:

Sasaki A, Erb S, McGowan PO (2016) The effect of maternal overnutrition on reward and anxiety in offspring. In: Parental obesity: intergenerational programming and consequences (Green LR and Hester RL, eds), pp187-200. New York: Springer-Verlag., with permission from Springer

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Chapter1:GeneralIntroduction

Disorderscharacterizedbyadysregulatedmetabolismhavereachedepidemiclevels

indevelopedcountries.Type2diabeteswasonceadiseaseprimarilyinadults.

Increasingly,however,itispresentinginadolescentsandeveninchildren,asthe

incidenceofobesityincreasesinthesepopulations.Infact,childhoodobesityhas

doubledinchildrenandtripledinadolescentsinthepast30years(Ogdenetal.,

2012)and,accordingly,oneineverythreeAmericanchildrenbornin2000islikely

tobediagnosedwithdiabetesintheirlifetime.Inadditiontobeingathigherriskfor

developingdiabetes,obeseyouthareatgreaterriskforcardiovasculardisease

(Freedmanetal.,2007)andmanyotherdiseases,includingpsychiatricdisorders

laterinlife(Riveraetal.,2015).

Clinicalandanimalstudieshaveattributedthisriseinchildhoodobesityand

diabetestofetalprogrammingbymaternalobesityanddiabetes.Animalstudies

haveshownthatmaternalnutritionhistorypredictsobesityinadultoffspring

independentofpostnataldiet(Howieetal.,2009).Maternalconsumptionofa

palatabledietcanincreasethepreferenceforfatandsugarintakeintheoffspring

(Vuceticetal.,2010).Humanstudiessupportthesefindings,showingthat

preferenceforfatcanbeprogrammedbymaternalfoodintakeduringpregnancy

andprenatallifeindependentofpostnatalmaternalfatintake(Brionetal.,2010).

Palatableorhighfatdietactivatesdopaminergicpathwayswithinthe

mesolimbicrewardsystem,whichhasbeenimplicatedinrewardanddrugaddiction

(KelleyandBerridge,2002;Tobleretal.,2005).Drugaddiction,arelapsingdisorder

characterizedbycompulsiontoseekdrugs,hasbeenlinkedtodysregulationof

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rewardandstress(Koob,2008).Althoughthemechanismsunderlyingnatural

addictions(i.e.,compulsiveconsumptionofnaturalrewardssuchaspalatablefood)

arelesswellunderstood,ithasbeensuggestedthatdrugaddictionandaddictionto

foodmaybemediatedinpartbycommonneuralandmolecularmechanisms

(Nestler,2005).Similartoeffectsofdrugaddictiononneurodevelopment,ithas

beenproposedthatexposuretomaternalhighfatdietmayalterthedevelopmentof

centralrewardcircuitry,increasingthepropensitytooverconsumepalatablefoods

laterinlife(Vuceticetal.,2010).

Todate,themainfocusofstudiesoftheinfluenceofmaternalhighfatdieton

brainfunctionhasbeenonthehypothalamus,whichregulatesthehomeostasisof

energyintake(TaylorandPoston,2007;Ramamoorthy,2015).Recentstudieshave

shownthatmaternalhighfatdietaltersdopaminergicgeneregulation,the

sensitivitytoamphetamine,dopaminergictransmissionintherewardpathwayand

anxietybehaviourinoffspring(Naefetal.,2008;Vuceticetal.,2010;Ongand

Muhlhausler,2011;Sasakietal.,2013;Sasakietal.,2014).Furthermore,ithasbeen

suggestedthatthechangesindopaminergicgeneregulationareduetoepigenetic

mechanismssuchasDNAmethylationofgeneregulatoryelements(e.g.promoters).

Epigeneticmechanismsmayaltergenefunctioninastablemannerthatcanpersist

fromthedevelopmentalperiodthroughadulthood(e.g.(Weaveretal.,2004)).

Thesedatasuggestthatepigeneticmechanismsmayhelpexplaintherapidrisein

metabolicdysfunctioninoffspringasaresultofmaternaldiet.Thisquestionis

particularlyrelevantsinceupto30%ofhumanpregnanciesindevelopedcountries

arenowcomplicatedbymaternalobesity(Catalano,2007).Identifyingthe

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mechanismsthroughwhichmaternalhighfatdietanddrugexposureresultsin

alteredrewardandanxietypathwayslaterinlifewillenabletheunderstandingof

riskfactorsfordisorderscharacterizedbydysregulatedhedonicandnegative

emotionalprocessing,suchasovereatingandanxietyseeninaddiction.

Theoverarchingobjectiveofmydissertationresearchistoexaminethe

effectsofmaternalHFD,stressandpre-gestationalcocaineexposureonrewardand

anxietycircuitriesinthebrainsofadultoffspring,andtheirbehaviouralresponses

tococaine.Indevelopingthetheoreticalandconceptualframeworkformywork,I

willdiscussinthischapterhowmaternaldietduringandaftergestationaffects

behaviourinoffspring.Furthermore,Iwilllinkbehaviouraloutcometoneural

mechanism,andtothepresentationofalteredreward-andstress-related

phenotypebasedonbothdevelopmentalandadulthoodexposuretohighfatdiet.In

sodoing,Iwilldescribeevidenceofsharedneuralcircuitryandbehavioural

outcomesasafunctionofexposuretomaternalovernutritionanddrugsofabuse.

1.1Studiesoftheeffectsofmaternaldietonoffspring:Caveatstoconsider

Beforeproceedingwithadiscussionoftheeffectsofmaternaldietonbehavioural

andneuralphenotypeinoffspring,caveatspertainingtoworkintheareamore

generallyshouldbebrieflyaddressed.First,itshouldbecautionedthatstudiesof

maternalovernutritiontendtobevariableonanumberofcriticalparameters.The

majorityofworkintheareainvolvesamaternaldietmanipulationgivenduring

and/orafterpregnancy,andmoststudiesusedietsthatarehighinfat.However,

studiesdifferintheproportionandqualityoffat(saturated,unsaturated,or

5

transfats)inthediet,thecarbohydratecontent,theuseof‘cafeteria’dietsinsome

cases,andthetimingofexposuretothedietmanipulation(e.g.,before,during,

and/orafterpregnancy).Here,thefocuswillbeondietshighinsaturatedfat,the

mostcommonfatusedtodriveovernutrition,andthetypeofdietarymanipulation

thatIhaveusedinmywork.Thus,Iusetheterm‘overnutrition’interchangeably

with‘highfatdiet’,unlessotherwisespecified.

Second,itisworthnotingthatalthoughthefocushereisontheeffectsof

maternalovernutritiononoffspringbehaviouralphenotype,themajorityofstudies

examiningthebehaviouraleffectsofovernutritionareperformedusingdiet-

inducedobesitymodels,wherethedietisfedcontinuouslyinadulthood.Inthese

diet-inducedobesitystudies,therefore,itisnotalwaysclearwhethertheeffectsof

thedietresultfromcurrentdiet,diethistory,oracombination.Inthepresent

discussion,thediet-inducedobesitystudiesservetoillustrateinstanceswherethe

effectsofhighfatdietindevelopmentdivergefromthoseofchronichighfatdietin

adulthood,aswellaswherecommonbrainmechanismsappeartobealteredbyhigh

fatdietexposure.

1.2Effectsofmaternalovernutritionontheoffspringdopaminesystem

Dopaminecircuitryisassociatedwithneuralrewardmechanismsthatcanserveto

alteranimals’preferenceforenergy-densepalatablefoods.Theregulationoffood

intakebythecentralnervoussysteminvolveshomeostaticmechanismsinthe

hypothalamusandinteractionswiththemesolimbicdopaminepathwaymediating

rewardandmotivation(Narayananetal.,2010).Bothnaturalreinforcers(e.g.

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palatablefoodsuchashighfatdiet)anddrugreinforcersactonthemesolimbic

pathway,whichoriginatesintheventraltegmentalarea(VTA)andprovidesdense

dopamineinnervationofthenucleusaccumbens(NAC)andprefrontalcortex(PFC).

Activationofthispathwaybybothnaturalrewardsanddrugsofabuseresultsin

increaseddopaminergictransmissionwithintheNAC.

Inrodents,thedevelopmentofdopaminergicneuronsisnotfullyestablished

untilthesecondandthirdweeksofpostnatallife,atimeperiodthatoverlapswith

maternallactation(Luedietal.,2005).Thus,ithasbeenconsideredthatthe

maternalnutritionalenvironmentmayalterthefunctionofthemesolimbicpathway

toalterbehaviouralandneuralresponsesinoffspring,includingoverconsumption

ofahighfatdiet.

1.2.1Reward-directedfeedingbehaviourandpsychostimulantinduced

locomotoractivity

Inrodentmodels,maternalovernutritionincreasesthepreferenceforpalatable

foodsinoffspring.Forexample,maternalconsumptionofapalatablehighfatdiet

fourweekspriortoconception,andduringgestationandlactation,increases

preferenceforfatandsugarintakeintheoffspring(Vuceticetal.,2010;Sasakietal.,

2013).Inrodentmodels,it’sknownthatexposureoftheoffspringtoavarietyof

sourcesofovernutrition(maternalHFD,junkfooddiet,additionalmother’smilk,

highproteindiet)increasefuturepreferencefornutritionaloverconsumption,

resultinginobesity(AlfaradhiandOzanne,2011).Humanstudiesareinsupportof

thesefindings,indicatingthatmaternaldietarycontentpredictsadiposityin

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childhood(Okuboetal.,2014),andthatchildfatintakeisassociatedwithprenatal

ratherthanpostnatalmaternalfatintake(Brionetal.,2010).

Appetiteregulationislargelymediatedbyhypothalamicregionsinvolvedin

appetitecontrol,andbyperipheralfactorssuchasleptin,insulin,andghrelinthat

regulateenergybalance(MeierandGressner,2004).Offspringexposedtomaternal

highfatdietappeartohaveanincreasedhungerforfat-richfoodthatoverrides

satietysignalsthatusuallymaintainthebalancebetweenenergyintakeand

expenditureinthebody.Clearly,however,feedingisaboutmorethantheregulation

ofenergyintakeandexpenditure;itproducesapleasurestatethatinvolvesthe

activationofrewardpathwaysinthebrain.

Recentstudieshavesuggestedthatpre-andpost-natalexposuretoadiet

highinfatincreasesthepreferenceforhighfatdiet,andthedrivetoconsume

palatablefoodsinadulthood.Likewise,maternalconsumptionofapalatablediet

increasesthepreferenceandconsumptionoffoodthatishighinfatandsugar,when

comparedtoamicronutrient-balancedcontroldiet(Changetal.,2008;Vuceticetal.,

2010)orfoodrichinproteins(Bayoletal.,2007;OngandMuhlhausler,2011).

Importantly,theincreasedpreferenceforpalatablefoodbymaternalovernutrition

appearsnottobeduetoincreasedappetiteperse.Whenanimalsaregivenacontrol

dietinstead,theydonotshowincreasedappetite(i.e.increasedconsumption)for

thecontrolfood(Bayoletal.,2007;Shalevetal.,2010;Sasakietal.,2013).These

studiessuggestthatmaternaleffectsonoffspringdietarypreferencesinvolve

changesinthesalienceofparticularfood-relatedstimuli(i.e.palatablediet),rather

thanmerelyanincreaseinenergyintake.Aswasmentionedabove,studiesin

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humanslikewiseshowthatspecificdietarypreferencesforfatsareassociatedwith

maternalfoodintakeduringpregnancy(Brionetal.,2010;Okuboetal.,2014).

1.2.2Dopamine-relatedneuralgeneexpression

Althoughlittleworkhasbeendonetoexploretheneurobiologicalbasisofthe

effectsofmaternaldietonfoodpreferencesinoffspring,therearedataconsistent

withtheideathatdopamineisinvolved.Indeed,maternalovernutritionhasbeen

showntoaltertheexpressionofmultipledopamine-relatedgenesinthemesolimbic

pathwayofadultoffspring,includingtyrosinehydroxylase(TH),dopamine

receptorsD1andD2(D1R,D2R),anddopaminetransporter(DAT)(Naefetal.,

2008;Naefetal.,2011;OngandMuhlhausler,2011).However,thedirectionof

expressionofthesechangesandthespecificdopaminergicgenesexhibitingchanges

isvariablebetweenstudies,possiblyowingtodifferencesrelatedtothespecificdiet

administered.Ofnote,increasedDATexpressionintheNACisassociatedwithDNA

hypomethylation,suggestingthatthechangeingeneexpressionistransmittedvia

anepigeneticmodification(Vuceticetal.,2010).

Asdiscussed,bothnaturalanddrugreinforcersalterthefunctionofthe

dopaminergicsystem.Thus,aninterestingquestion,andonethatisacentralfocus

ofthisdissertation,iswhethermaternalovernutritionaltersthesensitivityof

offspringtothelocomotor-activatingeffectsofpsychostimulants.Indeed,the

activationaleffectsofdrugssuchasamphetamine,cocaine,andmorphineare

mediatedviadopaminetransmissionintheNAC(Jonesetal.,1998).Inonestudy

thataddressedthisquestion,itwasfoundthatmaternalovernutritionwas

9

associatedwithattenuatedamphetamine-inducedlocomotionandattenuated

expressionofamphetamine-inducedsensitization(Naefetal.,2008).Moreover,the

attenuatedeffectofamphetamineonlocomotoractivitycorrespondedtoblunted

dopaminetransmissionintheNAC(Naefetal.,2011).Inaddition,instudiesofdiet-

inducedobesity,rodentsconsumingahigh-fatdietexhibitincreasedmotivationto

workforsucrosepellets(laFleuretal.,2007),attenuatedamphetamine-induced

locomotorsensitization(Hryhorczuketal.,2015),anddecreaseddopamine

turnoverinthemesolimbicsystem(NAC)(Davisetal.,2008).

ThestudiesbyNaefetal.thussupporttheresultsusingmodelsofdiet-

inducedobesity,however,otherstudiescontradicttheseresults.Forexample,male

adultmiceoffspringofdamsfedHFDshowedincreasedlocomotoractivityin

responsetococaine(GrissomandReyes,2013).Maleadolescentratoffspring

showedpotentiatedlocomotoractivityinresponsetoamphetamine(Brenneman

andRutledge,1982).Maleadultratoffspringofdamsexposedtohighsugardiet

showedincreasedlocomotoractivityinresponsetoamphetamine(Bocarslyetal.,

2012).Thislaststudywasincludedherebecauseovernutritionmanipulationsoften

includehighsugarinthedietcontent.Thesecontradictoryresultswillbediscussed

intermsofthetypeoffatusedintheGeneralDiscussion,section7.2.Overall,there

isaneedforfurtherstudiestoelucidatethedirectionofchangeofdopamine

functioninoffspringwithexposuretomaternalHFD.

1.3Effectsofmaternalovernutritionontheoffspringstressresponsesystem

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Inhumans,exposuretomaternalovernutritionandhighfatdietduring

developmentincreasestheriskinoffspringofdevelopinganxietydisordersand

depression(BaileyandCrawley,2009).Anumberoflinesofevidencesuggestthat

theincreaseinriskmaybedriven,atleastinpart,bydisruptioninthedevelopment

ofneuralpathwaysregulatingresponsestostress(Bersaminetal.,2008;Sullivanet

al.,2010).

1.3.1Anxietybehaviourandstressphysiology

Animalstudieshaveshownthatexposuretomaternalovernutritionimpactsthe

expressionofanxiety-likebehaviouracrossthelifespan.Forexample,inaratmodel,

maternalovernutritionincreasedanxiety-likebehaviourinadultoffspring,as

measuredintheOpenFieldandElevatedPlusMazetasks(Boksa,2004;Bilboand

Tsang,2010;Sasakietal.,2013).Theseresultsaresimilartoprimatestudies

showingthatdevelopmentalexposuretomaternalovernutritionincreasesnovelty-

inducedanxietyinadultoffspring(Brayetal.,2002;Sullivanetal.,2010).Finally,in

humanstudies,resultsgenerallypointtoapositiveassociationbetweentheco-

occurrenceofchildhoodobesityandanxietydisorders(Gariepyetal.,2010).

Theexpressionofanxiety-likebehavioursisknowntobesensitiveto

changesinthefunctionoftheHypothalamic-Pituitary-Adrenal(HPA)axis,which

mediatestheendocrineresponsetostressinpartthroughnegativefeedback

inhibitionofcorticosterone(CORT)release.Likewise,maternalovernutrition

influencesthefunctionoftheHPAaxisofoffspringinalong-termmanner.For

example,maternalovernutritionisassociatedwithlowerlevelsofcirculatingCORT

11

inmaleandfemalerats(Sasakietal.,2013)andmice(Grissometal.,2015),and

femaleratsexposedtomaternalovernutritionexhibitprolongedelevationinCORT

afterphysicalrestraintstress(Sasakietal.,2013).Likewise,neonatalratsexposed

tomaternalovernutritionexhibitanelevatedCORTresponsetoetherstress,

suggestingthattheprogrammingoftheHPAaxisbymaternalhighfatdietoccursin

earlypostnatallife(Brunton,2010).Studiesinhumanshaveshownthatobese

mothersexhibithigherlevelsofcortisol(Aubuchon-Endsleyetal.,2014).Other

evidencehasshownrepeatedlythatmaternalstressorglucocorticoidexposureis

associatedwithelevatedHPAactivityinchildrenandstress-relatedbehavioural

disorders(Lupienetal.,2009b).Likewise,numerousstudieshavereportedthat

maternalobesityisassociatedwithincreasedinternalizingproblemsincluding

anxietydisordersinchildren(PuderandMunsch,2010).Overweightandobese

womenshowlowerratesofbreastfeedingthannormalweightwomen(Amirand

Donath,2007).Therefore,itispossiblethatmaternalovernutritionaffectstheHPA

inoffspringviaalterationsinmaternalcare.

1.3.2Stress-relatedneuralgeneexpression

HPAfunctioncanbealteredbychangesintheexpressionofmineralocorticoid(MR)

andglucocorticoidreceptors(GR)withinlimbicbrainareas,includingtheamygdala

andhippocampus;thesereceptorpopulationsdifferentiallyregulatebasaland

stress-activatedlevelsofCORTincirculation(CordainandHickey,2006;Dantzeret

al.,2008).Ofnote,werecentlyreportedthatMRandGRtranscriptsareelevatedin

theamygdalaofoffspringwhosemotherswerefedahigh-fatdietduringpregnancy

12

andlactation(Sasakietal.,2013).Thesedataareinagreementwithotherstudiesof

maternalstressmanipulationsshowingthatincreasedGRintheamygdalaenhances

theCORT-mediatedresponsetostress(Joelsetal.,2008).Theyarealsoin

agreementwithastudyshowingthattheoffspringofnonhumanprimatesfedwith

highfatdietexhibitincreasedhypothalamicexpressionofproopiomelanocortin

transcript,agenethataffectsHPAfunctionbyalteringlevelsofadrenocorticotropic

hormone(ACTH)and,inturn,cortisolrelease(Graysonetal.,2010).

1.3.3Modelsofdiet-inducedobesity

Thebehaviouraleffectsofchronicexposuretohighfatdietinadultratsaresimilar

tothoseoftheoffspringofmothersfedahighfatdiet,includingasimilarCORT

responsetostress(e.g.(Tannenbaumetal.,1997;Sasakietal.,2013)).Forexample,

after10to12weeksofconsumingahighfatdiet,adultratsexhibitedarelative

increaseinbehaviouralanxietyontheelevatedplusmaze,openfield,andlightdark

task(SharmaandFulton,2013;Sivanathanetal.,2015).Moreover,theseelevated

anxiety-likebehavioursareassociatedwithanalteredHPAaxisresponse,consisting

ofelevatedCORTlevelsafterrestrainstress.Theseresultsagreewithseveralother

studiesshowingthatchronicconsumptionofahighfatdietinadulthoodgenerally

leadstoelevatedcirculatinglevelsofglucocorticoidsandanenhancedCORT

responsetostress(DeSouzaetal.,2005;Ennaceuretal.,2006;Buchenaueretal.,

2009),butsee(Kahnetal.,2006).Finally,chronicconsumptionofahighfatdiet

exacerbatestheeffectsofstressbyimpairingthenegativefeedbackinhibitionofthe

CORTresponsetopsychosocialstress(Kral,2004;SharmaandFulton,2013).

13

Chronicexposuretohighfatdietinadulthoodalsoleadstoalterationsin

stress-relatedgeneexpressionwithinstress-relatedneuralcircuitrythatissimilar,

butnotidentical,tothoseofoffspringexposedtomaternalovernutrition.For

example,animalsconsuminghighfatdietshowreducedtranscriptinthe

hippocampusofbothMRandGR,whencomparedtoanimalsconsumingstandard

housechow(Sivanathanetal.,2015).Theoffspringofanimalsexposedtomaternal

overnutrition,ontheotherhand,exhibitincreasedexpressionoftheGRtranscript

withintheamydgala.Giventheopposingrolesofthehippocampusandamygdalain

theregulationoftheHPAaxis,however,bothfindingsareconsistentwiththeidea

thatthestresssystemisheightenedinresponsetobothdietarymanipulations,and

thatbothmanipulationsleadtoenhancedbehaviouralanxiety.

1.4Relationshipbetweenfoodanddrugaddiction

Basedontherelationshipbetweenmaternalovernutritionandresponsesto

psychostimulantsinoffspring,itisofinterestthatdrugaddiction,likedisorders

involvingfoodconsumption,hasbeenlinkedtodysregulationwithintheprimary

brainpathwaysregulatingrewardandstress.Characterizedbycompulsiontoseek

drugs,drugaddictionconsistsofachroniccycleofdrugintoxicationfollowedby

withdrawalandrelapse(Koob,2008).Thiscyclecorrespondstopowerfulpositive

reinforcement(drugintoxication)and,overtime,theemergenceofanegative

emotionalstate(anxiety)afterwithdrawal.Ithasbeenarguedthatthisnegative

emotionalstatemay,atleastintheshort-term,perpetuatedrugseeking(Koob,

2008).Ithasalsobeenarguedthatdrugaddictionischaracterizedbyashiftinthe

14

motivationalprocessesmediatingongoingconsumptionfrompositive

reinforcementinducedbythedrugtonegativereinforcementresultingfromthe

reliefofnegativeaffectuponresumingdrugtaking(Koob,2008).Andithasrecently

beenproposedthatasimilartransitionmayoccurinthecaseofdisorderedeating

leadingtoobesity(Volkowetal.,2013).Althoughthemotivationaland

correspondingneuralmechanismsinvolvedindrugaddictionareperhapsbetter

understoodthanthoseinvolvedinobesity,ithasbeensuggestedthatcompulsive

drugandfoodconsumptionmayberegulatedbycommonneuralandmolecular

mechanisms,includingthoserelatedtodysregulateddopaminergicandstress-

relatedfunction(Nestler,2005).

1.5Effectsofmaternalcocaineontheoffspring

Amajorfocusofmydissertationisontheeffectsofmaternalovernutritionon

responsestococaineinoffspring,especially,inadulthood.Theeffectofcocaineon

inducinglocomotoractivityhasbeenheavilystudiedinanimalmodelsofdrug

addiction.Cocaine-inducedlocomotoractivityreflectsananimal’svulnerabilityto

behavioursconsistentwithdrugaddiction(e.g.drugsensitization,conditioned

locomotion).Repetitiveuseofcocainecauseslong-lastingchangesinthereward

andstresscircuitryofthebrain.Cocaine-inducedlocomotoractivityinanimals

correlateswithdopaminergicactivityintherewardcircuitry.

Exposuretococaineduringpregnancycanleadtodetrimentaleffectsonthe

physiologyandcentralnervoussystemfunctionofoffspring.Inhumans,themost

commonoutcomesofcocaineuseduringpregnancyincludeprematurebirth,lower

15

thanaveragebirthweight,respiratorydistress,andincreasedriskofseizuresin

offspring(KellerandSnyder-Keller,2000).Prenatalexposuretococainehasalso

beenshowntoaffectthedevelopingnervoussystembydelayingstructuralbrain

maturationofdopamine-richcorticalandsubcorticalbrainstructuressuchasthe

prefrontalcortex(PFC)andbasalganglia,respectively(Deraufetal.,2009).

Cocaineexposureduringgestationleadstodose-dependentincreasesin

maternalbloodpressureanddecreasesinuterinebloodflow,impairingoxygen

transfertothefetus;suchimpairmentmayinturncontributetoobservedincreases

infetallevelsofcocaineandcatecholamines(Woodsetal.,1987).Likewise,prenatal

cocaineexposureleadstoabnormalitiesinfetalbraindevelopment,particularly

withinthedopamine-richneuronsoftheprimaryrewardpathwayprojectingfrom

theVTAtotheNAcandPFC.Prenatalcocaineexposurehasalsobeenlinkedto

alteredcocaine-primeddopaminereleaseintheNAcinadulthood(Malangaetal.,

2009).Takentogether,thesedataindicatethatprenatalexposuretococaineinboth

humansandanimalsleadstophysiologicaldysregulationandneuralalterationin

reward-relatedcircuitryofoffspring.

Cocainecrossestheplacentaandismetabolizedslowlyinthefetus,which

canleadtodirectandprolongedexposuretosignificantlevelsofcocaineinthe

developingfetus(KellerandSnyder-Keller,2000).Mythesisconsistsofparallel

studiestoexaminetheeffectofmaternalexperienceofcocainepriortoconception

inordertoavoiddirectlyexposingfetustococaine.Bystudyingtheeffectsof

cocaineexposurebeforepregnancy,wewereabletodisentanglethepotential

intergenerationaleffectsofcocaineexposureindamsfromthedirecteffectsof

16

cocainetothedevelopingfetus.Thepurposeofthisstudywastocomparethe

effectsofmaternalhighfatdiettotheeffectsofpregestationalcocaineonoffspring

rewardandstressrelatedbehaviour,asbothhighfatdietandcocaineaffectthe

mesolimbicdopaminergicpathway.

1.6Thesisrationaleandobjectives

The literature reviewed in this chapter supports the notion that maternal overnutrition is

linked to increased risk for hedonic and stress dysfunctions in offspring later in life. This

issueisparticularlyrelevantsinceupto30%ofhumanpregnanciesindeveloped

countriesarenowcomplicatedbyfactorsowingtomaternalobesity(Catalano,

2007).Identifyingthemechanismsthroughwhichmaternalovernutritionresultsin

alteredrewardandstresspathwayslaterinlifewillenabletheunderstandingof

riskfactorsfordisorderscharacterizedbydysregulatedhedonicandnegative

emotionalstates.Previous studies in animal models indicate that maternal overnutrition

impacts the function of the mesolimbic pathway leading to dysregulated function of the

reward system and dopamine-related behaviour. Maternal overnutrition also affects the

function of the Hypothalamic-Pituitary-Adrenal axis, leading to activated stress system

and increased anxiety-like behaviour.

Changes in these same neural mechanisms are known to alter the response to

drugs of abuse. Activationofdopaminergicpathwayswithinthemesolimbicreward

systemhasbeenimplicatedinreward-relatedbehaviouranddrugaddiction(Kelley

andBerridge,2002;Tobleretal.,2005).Drugaddiction,arelapsingdisorder

characterizedbycompulsiontoseekdrugs,hasbeenlinkedtodysregulationof

17

rewardandstress(Koob,2008).Asdescribeabove(section1.2),ithasbeen

suggestedthatdrugaddictionandaddictiontofoodmaybemediatedinpartby

commonneuralandmolecularmechanisms(Nestler,2005).Itremainspoorly

understoodwhethermodificationsofthesepathwaysbymaternalexperience

modifytheresponsetostressanddrugsofabusesuchascocaineinoffspring.

Mythesiscomprises4majorstudiesexaminingeffectsofdifferentmaternal

experiencesthatareallknowntoaffectstressandrewardfunction;maternal

exposuretohighfatdiet,chronicstressandpre-gestationalcocaine.The

experimentspresentedinthisdissertationconsistofparallelexplorationsofthese

maternalexposuresonanxietybehaviourandcocaine-inducedlocomotoractivityin

offspring.Inaddition,theexperimentsexploretheeffectsofthedifferentmaternal

exposuresontheexpressionofstress-andreward-relatedgeneswithinlimbicand

mesolimbicbrainregionsofoffspring.Specifically,Ihypothesizedthatmaternal

highfatdiet,chronicstressandpre-gestationalcocainewouldallserveto

increaseanxietybehaviourandcocaine-inducedlocomotoractivityin

offspring,throughcommonbutnotidenticalmechanisms.Ifurther

hypothesizedthattheseeffectswouldbeassociatedwithchangesinthe

expressionofspecificstress-andreward-relatedgeneswithinlimbicand

mesolimbicbrainregions.Thesehypotheseswereaddressedinthefollowing

aims:InAim1,Iexaminedtheeffectsofmaternalhighfatdietonanxietybehaviour

andgeneexpressioninoffspring(Chapter3).InAim2,Iexaminedtheeffectsof

maternalhighfatdietandcocaine-primedlocomotoractivityandgeneexpressionin

offspring(Chapter4).InAim3,Iexaminedtheeffectsofmaternalhighfatdietand

18

stressoncocaine-inducedlocomotoractivityandgeneexpressioninoffspring

(Chapter5).InAim4,Iexaminedtheeffectsofpre-gestationalcocaineoncocaine-

inducedlocomotoractivityandgeneexpressioninoffspring(Chapter6).

19

Chapter 2: General Methods

20

Chapter2:GeneralMethods

Themethodsdescribedinthischapterarecommontothoseusedintheindividual

experimentsdescribedinsubsequentchaptersofthisthesis.Anyvariationsinthese

proceduresaredescribedwithinthemethodssectionsinsubsequentchapters.

2.1.Animals

Breedermalesandfemales:AdultmaleandfemaleLongEvansrats(7weeks)were

obtainedfromCharlesRiverCanada(St.Constant,QC),housedinsame-sexpairs

andmaintainedona12:12-hlight-darkcycle(lightson7:00am-7:00pm)withad

libitumaccesstofoodandwater.Femalebreederswereindividuallyhousedafter

matingandweigheddailyuntilthebirthoftheirpups.

Offspring:OnPostnatalDay1(PND1),alllitterswereweighedandculledtoa

maximumof12,maintainingsimilarnumbersofmalesandfemaleswhenpossible.

Offspringwerecountedandweighedduringweeklycagechanges.Otherwise,the

offspringremainedundisturbeduntilPND21whentheywerehousedinsame-sex

pairsthroughoutadulthood(PND90).

ExperimentalprotocolswereapprovedbytheLocalAnimalCareCommitteeatthe

UniversityofToronto,Scarborough,andwereinaccordancewiththeguidelinesof

theCanadianCouncilonAnimalCare.

2.2.Proceduralmanipulationsofdams

2.2.1.Diets

21

Femalebreederswereplacedononeoftwodiets:ahigh-fatdiet(HFD)oracontrol

housechowdiet(CHD).The5.24-kcal/ghigh-fatdietwasobtainedfromResearch

Diets,Inc.(NewBrunswick,NJ:cat.no.D12492),andcontained(bykcal):20%

protein,60%fat,20%carbohydrate.The3.02-kcal/gCHDwasobtainedfromPurina

LabDiets(St.Louis,MO:cat.no.5001)andcontained28.5%protein,13.5%fat,and

58%carbohydrate.Femalesremainedonthedietfor4weekspriortomatingand

throughoutpregnancyandlactation.UponweaningatPND21,offspringwere

maintainedonCHDthroughoutadulthood.ThisdietregimenofperinatalHFD

exposurehasbeenusedtoexaminetheeffectsofmaternalHFDexposureinseveral

previousstudies(Vuceticetal.,2010;GrissomandReyes,2013).

2.2.2.Assessmentofmaternalbehaviour

(correspondingtoSection4.2.2.2.Chapter4,Section5.2.2.4.Chapter5,Section6.2.2.3.

Chapter6).

ThematernalbehaviourofdamsthathadbeenexposedtoHFD,anddamsthathad

beenexposedtoCHD,wasmonitoredforeitherthefirst6postpartumdays

consecutivelyoreveryotherdaybetweenPND1uptoPND9.Thedatewhenthe

pupswerebornwasregardedasPND0.Thematernalmonitoringprocedureswerea

modifiedversionofmethodsdescribedpreviously(Champagneetal.,2003;

McGowanetal.,2011).Briefly,thebehaviourofeachdamwasvideo-recorded

during4-6sessionsof1-hourobservationperiodsperday.Foreachobservation

period,theindividualvideorecordingsforeachdamwereassessedin3-5min

intervals,usingacomputer-controlledbehaviouralcodingandanalysissystem

22

(Observer4.1orObserverXT,NoldusInformationTechnology,Inc.,Leesburg,VA).

Thefrequencyofmaternalbehavioursoflickingandgroomingofpupsandnursing

pupsinanarched-backposture(LGABN)wasscored.Percentagescoresforeach

behaviourwheregeneratedbydividingthefrequencyscoreforthatbehaviourby

thetotalnumberofobservationsmadeonagivenobservationday.Thecodingof

maternalbehaviourwascarriedoutby5trainedraters.Raterswereblindtothe

subject'smaternalhistory(i.e.,diet,stressorcocainedependingonthestudy),and

weredeterminedtohaveaninter-raterreliabilityofmorethan85%,usingthe

reliabilityanalysistoolinObserver4.1orObserverXT(NoldusInformation

Technology,VA,USA).The5raterswereeachassignedtooneobservationdayper

dam,inordertoavoidobserverbiasforaparticulardam.

2.3.Proceduralmanipulationsofoffspring

2.3.1.Subjects

Anequalnumberofadultoffspring(PND65~90dependingonthestudy)from

damswithahistoryofdifferentexposures(i.e.,dietand/orstressorcocaine,

dependingonthestudies)wereusedforthedifferenttests.

2.3.2.Cocaine-inducedlocomotoractivity

2.3.2.1.Habituationsession

(correspondingtoSection4.2.3.2.Chapter4andSection5.2.3.2.Chapter5).

Onedaypriortothestartofcocaine(orsaline)injections,allratsweregivena

habituationsessiontoacclimatizethemtotheexperimentalapparatusand

23

treatmentprocedures.Duringthissession,ratswereplacedinlocomotoractivity

chambers(26cmx48cmx21cm).Locomotoractivitywasrecordedbyavideo

trackingsystemthatmeasureddistancetraveled(EthovisionorEthovisionXT,

NoldusInformationTechnology,Inc.,Leesburg,VA).

2.3.2.2Cocainepre-exposureinoffspring

(correspondingtoSection4.2.3.2.Chapter4).

Atotalof6drugexposuresessionswereadministeredoverthesubsequentdays.

Ratsweregivenoncedailyinjectionsofcocaine(30mg/kg,i.p.)orsaline(1kg/ml,

i.p.).Duringthesesessions,ratsweregivenaninjectionofcocaineorsalineunder

thesameconditionsdescribedforthehabituationsession.Locomotoractivityafter

theinjectionwasmonitoredasdescribedinthehabituationsessionabove.

2.3.2.3.Testforcocainesensitization

(correspondingtoSection4.2.3.4.Chapter4).

Eighteendaysaftertheterminationofthecocaineexposurephase,alloffspring

weretestedfortheirlocomotorresponsetoachallengeinjectionof10mg/kgof

cocaine(i.p.).Forthistest,ratswereinjectedwithcocaineandplacedinthe

locomotorchambers.Locomotoractivitywasmonitoredasdescribedinthe

habituationsessionabove.

2.3.2.4.Testforacutecocaineexposure


24

Theacutepsychomotorresponseto0,10and30mg/kg(i.p.)ofcocainewas

measuredinoffspring.Onedaybeforethestartoftesting,ratswerehabituatedto

thelocomotorchambers(seeabove).Ratsweregiveni.p.injectionsof0mg/kg(i.e.,

saline),10mg/kg,or30mg/kgofcocaine.Allratsweretestedunderallthreedose

conditionsinacounterbalancedorder.

2.3.3.Anxietytests

2.3.3.1.Elevatedplusmaze

(correspondingtoSection3.2.3.3.Chapter3,Section4.2.3.5.Chapter4,andSection

5.2.3.4.Chapter5).

TheElevatedplusmaze(EPM)containedtwoopenandtwoclosedarms(45x10

cm)andacenterplatform(10x12cm)elevated80cmabovethefloor.Themaze

wasplacedinadimlylitroom(33.7lux).Timespentintheopenarmscomparedto

theclosedarms,numberofheadentriestotheopencomparedtoclosedarms,and

distancetraveledinbotharmswererecordedovera5-10mintrialusingeither

ANY-mazesoftwareorEthoVisionXT.Aftereachtest,themazewascleanedusinga

70%ethanolsolutionandallowedtoairdrytoremoveorhomogenizeodorants.

2.3.3.2.Openfieldtest


TheOpenfield(OF)consistedofanopaquesquarearena(40.3x40.3cm).Thetime

spentinthecenterandthedistancetraveledwererecordedusingeitherANY-maze

softwareorEthoVisionXToverthecourseofa10-15mintrial.Aftereachtest,the

25

mazewascleanedusinga70%ethanolsolutionandallowedtoairdrytoremoveor

homogenizeodorants.

2.3.3.3.Light-darktransitionbox


Thelight-dark(LD)transitionboxconsistedoftwoPlexiglaschambersofequalsize

(30x30cm):oneblack(dark)andonewhite(light).Theboxeswereplacedina

dimlylitroomwithasinglelightbulbcenteredoverthelightportionofthebox.

Eachtrialwas5min.Thewallseparatingthetwochamberscontainedasmall

opening(12x12cm)toallowpassagebetweenthechambers.Thetimespentinthe

lightchamberandthedistancetraveledinthelightchamberwererecordedusing

eitherANY-mazesoftwareorEthoVisionXT.Aftereachtest,themazewascleaned

usinga70%ethanolsolutionandallowedtoairdrytoremoveorhomogenize

odorants.

2.3.4.Immobilizationstress-inducedcorticosteroneresponse


AsubsetofoffspringwasusedtoassessforCORTresponsivityto20minof

immobilizationstress.Ratswerehabituatedtotheprocedureroomandhandledfor

severalminutesperdayfor5consecutivedays,priortothetestday.Onthedayof

testing,ratswerehabituatedtotheprocedureroomfor2h,andthenhand-

restrainedwithalooselyfittingtowel.Bloodwasimmediatelywithdrawnintoa

tubefromasmallnickinthetail,andplacedonice.Ratswerethenplacedinto

26

Plexiglasrestrainersfor20min(6.4cmdiameterx21.6cmlength;Plas-Labs).After

20min,asecondsampleofbloodwaswithdrawnwhileratswerestillinthe

restrainer.Ratswerethenreturnedtotheirhomecagewithouttheirconspecific

partner,andleftundisturbedfor70min.Attheendof70min,ratswerehand-

restrainedagain,andathirdsampleofbloodwaswithdrawn,afterwhichtheywere

returnedtotheirhomecage;atthistime,theywerealsoreturnedtotheircagemate.

Bloodwaskeptoniceforatleast30minbeforebeingcentrifugedat4°C,4000

rpm,for20min.Serumwasthenextractedandstoredat-80°C.Levelsofserum

CORTweredeterminedusingcommerciallyavailableradioimmunoassaykitswith

125I-labeledanti-CORTantibody(MPBiomedicalsInc.,CA,USA).

2.3.5.Geneexpressionanalyses


Chapter5,andSection6.2.3.4.Chapter6).

Differentbrainregionswereanalyzedforgeneexpressiondependingonthe

Chapters.Brainregionsassessedincludedthehippocampus(HPC),amygdala(AMG),

hypothalamus(HYP),medialprefrontalcortex(mPFC),nucleusaccumbens(NAc)

andventraltegmentalarea(VTA).

2.3.5.1.Braindissection

(correspondingtoSection4.2.3.6.Chapter4,Section5.2.3.7.Chapter5andSection

6.2.3.4.Chapter6).

27

Asubsetof6offspringpercondition(dietand/orstressorcocaine)werecollected

forgeneexpressionanalysis.Forbraincollection,ratsweredeeplyanesthetized

withCO2anddecapitated.Brainswererapidlyremovedandflashfrozenin

isopentanechilledwithdryice.Brainswerestoredat-80°Cpriortoslice

preparationandprocessing.Specificbrainregionsweredissectedusingacryostat

andthefollowingcoordinatesrelativetobregma(PaxinosandWatson,1997):

mPFC(+4.2to2.2),NAc(+2.2to1.2),VTA(-5.2to-6.4),AMG(-1.6to-3.4),HYP(-

1.6to-3.6)andHPC(-4.16to-6.3).

2.3.5.2.RNAextractionandquantification


Chapter5,andSection6.2.3.4.Chapter6).

TotalRNAwasisolatedfromthetissuesusingQiazolfollowedbytheRNeasyPlus

kit,accordingtothemanufacturer’sprotocol(QiagenCanada,Toronto,ON,Canada).

ThequalityandquantityofRNAweremeasuredusingaNanoDrop

spectrophotometer(NanodropND-2000C,ThermoScientific,MA,USA).

2.3.5.3.GeneexpressionanalysisbyQuantitativePCR

(correspondingtoSection4.2.3.6.Chapter4,Section5.2.3.7.Chapter5,andSection

6.2.3.4.Chapter6).

EqualamountsofRNApersamplewereconvertedtocDNAusingahigh-capacity

cDNAreversetranscriptionkit(LifeTechnologies,CA,USA).QuantitativePCRwas

thenperformedusingaStepOnePlusrealtimethermocyclerandFastSYBRGreen

28

MasterMix(LifeTechnologies,CA,USA).Astandardcurvewasgeneratedfrom10

serialdilutionsofamixtureofcDNAfromallsubjects,andgeneexpressionwas

quantifiedrelativetoageometricmeanofthreehousekeepinggenes(GAPDH,Actin

B,andUBC),inordertoavoidbiasamonganyhousekeepinggene.Thesamplesfrom

eachratwereanalyzedintriplicate,andameanvalueforthegroupwasgenerated

fromtheaverageofthetriplicates.Thus,asingledatapointfromeachratwas

generated.Therelativetranscriptabundancewascalculatedbydividingthedata

fromthegeneofinterestbythegeometricmeanofthethreehousekeepinggenes.

Eachhousekeepinggenewasexaminedineachbrainregionanalyzed.Theprimers

usedinthisstudyweredesignedusingsequenceinformationfromGenBankatthe

NationalCenterforBiotechnologyInformation(NCBI;www.ncbi.nlm.nih.gov),anda

freelyavailableonlineprimerdesigntool(Primer3;

http://primer3.sourceforge.net).Theefficacyofeachprimersetwasverified

accordingtothemanufacturer’sprotocolforaStepOnePlusrealtimethermocycler

(LifeTechnologies,CA,USA)byensuringefficiencylevelsgreaterthan90%.

2.4.StatisticalAnalysis

AllanalyseswereperformedusingSPSSStatisticsversion21(IBM)orStatview(SAS

institute,CaryNC)forMacOS.Dataarepresentedasmeanvalues±SEMthroughout.

Inallcases,statisticalsignificancewassetatp≤0.05.Incaseswhereinteractions

werestatisticallysignificantorforplannedcomparisons,LSDposthoctestswere

usedtoclarifybetween-groupdifferences.

29

Bodyweightandfoodintake:Dataformaternalandpupweightswereanalyzed

bymixed-modelANOVAwithmaternalhistory(dietand/orstress)asthebetween

subjectfactorandpostnataldays(5to6daysbetweenPND1through9)asthewithin

subjectsfactor.DataforcaloricintakebetweenHFDandCHDdamswereanalyzed

usingastudent-ttest.(correspondingtoSection3.2.4.Chapter3,Section4.2.4

Chapter4.Section5.2.4.Chapter5).

Maternalbehaviour:Thedatafromthematernalcodingwereanalyzedusinga

repeatedmeasuresANOVAwithmaternalhistory(dietand/orstressorcocaine,

dependingontheexperiment)asthebetweensubjectfactorandpostnataldays(5

to6daysbetweenPND1through9)asthewithinsubjectsfactor.(correspondingto

Section4.2.4.Chapter4,Section5.2.4.Chapter5,Section6.2.4.Chapter6).

Anxietymeasures:BehaviouraldataforOF,EPMandLDwereanalyzedbyfactorial

ANOVAwithmaternalhistory(dietand/orstressorcocaine)asthebetweensubject

factor.(correspondingtoSection3.2.4.Chapter3,Section4.2.4Chapter4.Section5.2.4.

Chapter5).

Immobilizationstress-inducedCORTresponse:Thedatafromstress-induced

CORTwereanalyzedusingarepeatedmeasuresANOVAwithmaternalhistory(diet

orcocaine)asthebetweensubjectfactorandtimeafterstressonset(0,20,and70

min)asthewithinsubjectfactor.(correspondingtoSection3.2.4.Chapter3and

Section6.2.4.Chapter6)

Acutecocaineexposure:Thedataexaminingtheoffspring’sacuteresponseto

cocainewereanalyzedusingarepeatedmeasuresANOVAwithmaternalhistory

(dietand/orstressorcocaine)asabetween-subjectsfactorsandcocainedose(0,10,

30

and30mg/kg)asawithinsubjectsfactorinthefirst10minoftesting.

(correspondingtoSection5.2.4.Chapter5andSection6.2.4.Chapter6)

Geneexpression:Thedatafromgeneexpressionassayswereanalyzedusing

factorialANOVAwithmaternalhistory(dietandcocaineorstress)orStudent’st-

testsformaternalcocaineasbetweensubjectsvariables.(correspondingtoSection

4.2.4.Chapter4andSection5.2.4.Chapter5andSection6.2.4.Chapter6)

31

Chapter 3: Effects of maternal high fat diet on anxiety

behaviour and stress and dopaminergic genes in the limbic

system


SasakiA,deVegaWC,St-CyrS,PanP,McGowanPO(2013)Perinatalhighfatdietaltersglucocorticoidsignalingandanxietybehaviorinadulthood.Neuroscience240:1-12.,withpermissionfromElsevier

32

Chapter3:Effectsofmaternalhighfatdietonanxietybehaviourandstressanddopaminergicgenesinthelimbicsystem

3.1.Introduction

Theregulationoffoodintakebythecentralnervoussysteminvolveshomeostatic

mechanismsintheHYP,andinteractionswiththemesolimbicpathwaymediating

rewardandmotivation.Asmentioned(seeChapter1),bothnatural(e.g.palatable

foodsuchasHFD)anddrugreinforcersactonthemesolimbicdopaminepathway.

MidbraindopaminergicneuronsoriginateintheVTAandprojecttotheNAC,PFC

andAMY.TheNACthenextendsitsprojectionstotheHPC.

TheHPCandAMYareknowntobeinvolvedinregulatingthestresssystem

(Meaney,2001)and,itisargued,generatenegativeemotionalstatesthatdevelopin

drugaddiction(Nestler,2005;Koob,2008).Previousstudieshaveshownthatadult

offspringofmotherswhowereexposedtoHFDduringgestationandlactation

preferthisdietandconsumeittoadegreethatexceedsmetabolicdemand;thishas

beenfoundinbothanimalsandhumans(Howieetal.,2009;Brionetal.,2010).One

potentialmechanismforthisoverconsumptionisthoughneuroadaptationsin

rewardandstresscircuitrythatpromoteadditionalconsumption(VolkowandWise,

2005).Theseeffectsarethoughttoleadtoacycleofheightenedreinforcingvalues

ofHFD,andemerginganxietyintheabsenceofsuchdiet,effectssimilartothecycle

ofdrugabuse(VolkowandWise,2005).

Maternalobesitycarriessignificanthealthrisksforoffspringthatmanifest

laterinlife,includingmetabolicsyndrome,cardiovasculardiseaseandaffective

disorders.ProgrammingoftheHPAaxisduringdevelopmentmediatesboth

metabolichomeostasisandtheresponsetopsychosocialstressinoffspring.HFD

33

altersmaternalsystemicCORTlevels,buteffectsinoffspringonlimbicbrainareas

regulatingtheHPAaxisandanxietybehaviourarepoorlyunderstood.Inadditionto

theirroleintheresponsetopsychosocialstress,corticosteroidreceptorsformpart

oftheglucocorticoidsignalingpathwaycomprisingdownstreaminflammatory

processes.IncreasedsystemicinflammationisahallmarkofHFDexposure,though

alteredexpressionofthesegenesinlimbicbrainareashasnotbeenexamined.Also,

effectofmaternalHFDondopaminergicgenesinthesebrainregionswereexamined

asstressisknowntomodulatedopamineactivity.WestudiedtheinfluenceofHFD

exposureduringpre-weaningdevelopmentinratsongeneexpressionintheAMY

andHPCbyquantitativereal-timepolymerasechainreaction(PCR),anxiety

behaviourintheOF,EPMandLDtasks,andCORTlevelsinresponsetostressby

radioimmunoassay.Asadults,offspringexposedtomaternalHFDshowincreased

expressionofCORTreceptorsintheAMYandalteredpro-inflammatoryandanti-

inflammatoryexpressionintheHPCandAMYingenesknowntoberegulatedbyGR.

Thesechangeswereassociatedwithincreasedanxietybehaviour,decreasedbasal

CORTlevelsandaslowerreturntobaselinelevelsfollowingastresschallenge.Also,

tyrosinehydroxylase,arate-limitingenzymeofcatecholaminesynthesiswasfound

increasedintheAMYoftheHFDoffspring.Thedataindicatethatthedietary

environmentduringdevelopmentprogramsglucocorticoidsignalingpathwaysin

limbicareasrelevantfortheregulationofHPAfunctionandanxietybehaviour,

alongwiththealterationofthedopaminergicgene.

34

Intheexperimentspresentedinthischapter,weexaminedtheeffectsof

maternalHFDonoffspringanxietybehaviouranddopaminergicandstress-related

geneexpressioninHPCandAMY.

3.2.MaterialsandMethods

3.2.1.Animals

AdultmaleandfemaleLongEvansrats(7week)weremaintainedwithadlibitum

accesstofoodandwater,asdescribedinChapter2(section2.1).

3.2.2.Proceduralmanipulationsofdams

3.2.2.1.Diets

Femalebreederswereplacedononeoftwodiets:aHFD(n=8)oraCHD(n=8).The

detailofthedietsanddietregimenaredescribedinChapter2(section2.2.1).

3.2.3.Proceduralmanipulationsofoffspring

3.2.3.1.Subjects

Theproceduresforbreedingandbodyweightmeasurementsofdamsandoffspring

aredescribedinChapter2(section2.1).Asubsetofmaleandfemaleadultoffspring

(1-2/sex/litter)wereusedinbehaviouralassays(HFD-Fn=14,CHD-Fn=12,HFD-M

n=14,CHD-Mn=11)andbrainsforasubsetof6animalspersexandperdietgroup

werecollectedatPND110(±10)forgeneexpressionanalysis.Theinfluenceof

maternalHFDonoffspringweightgainwasmeasuredbeforeweaningatPND21and

35

inadulthood(PND90).Bodyweightsinadulthoodweretakenatsacrifice,shortly

aftercompletionofthebehaviouralexperiments.Aseparatesubsetofadultmale

andfemaleoffspringwereusedforbloodcollectionandCORTradioimmunoassays

(HFD-Fn=10,CHD-Fn=10,HFD-Mn=10,CHD-Mn=11).Theexperimentaltimeline

fordamsandoffspringisshowninFig.3.1.

3.2.3.2.Openfield

OpenfieldtaskwasrunasdescribedinChapter2(section2.3.3.2).


Elevatedplus-mazewasrunasdescribedinChapter2(section2.3.3.1).


LightanddarktaskwasrunasdescribedinChapter2(section2.3.3.3).

3.2.3.5.Immobilizationstress-inducedcorticosteroneresponse

36

Fig.3.1

Experimentaltimelinefordamsandoffspring.(A)Damsreceivedeitherhighfatdiet

(HFD)orcontroldiet(CHD)forfourweekspriortopregnancy,duringpregnancy

andduringtheperiodoflactation.Onpostnatalday21,theoffspringwereweaned

fromtheirmothersandgivenCHDdiet.(B)Oncetheoffspringreachedadulthood,a

subsetwastestedontheopenfield(OF),elevatedplusmaze(EPM)andlightand

darktransition(LD)tasksonconsecutivedays.Theresponsetostresswastestedin

anothersetofadultoffspringusingarestraint-inducedcorticosterone(CORT)assay.

Inathirdsetofnaïveadultoffspring,reward-andstress-relatedgeneexpression

wasexaminedinlimbicbrainregions.

37

Immobilizationstress-inducedCORTresponsewasmeasuredasdescribedin

Chapter2(section2.3.4).Theintra-assaycoefficientofvariationwas7.1%.Five

outlierswithCORTvalues±2STDEVfromthemeanwereidentifiedandremoved

fromanalyses(HFD-Fn=1,CHD-Fn=1,HFD-Mn=2,CHD-Mn=1).

3.2.3.6.Geneexpressionanalyses

Thebrainsforasubsetof6animalspersexandperdietgroupwerecollectedat

PND110(±10)forgeneexpressionanalysis,asdescribedinChapter2(section

2.3.5).Theexpressionpatternsof7transcriptswerequantifiedandanalyseswere

performedbyStepOnePlusrealtimePCRusingFastSYBRGreenPCRmastermix

(AppliedBiosystems,LifeTechnologies,Carlsbad,CA,USA)andbyrelative

normalizationagainsttheexpressionofanadditional5houskeepinggenes(Gapdh,

Actb,Ubc,Ywhaz,andrRNA).ThegenesshowingtheleastvariancebetweenHFD

andinCHDgroupswereselectedfortheanalysis(Gapdh,Actb,andYwhaz).A

standardcurvewasgeneratedfrom11serialdilutionsofamixtureofcDNAfrom

offspring,andgeneexpressionwasquantifiedrelativetoageometricmeanofthe3

housekeepinggenes.Allreactionsforallgeneswereperformedintriplicate.

Theprimersusedinthisstudywerepurchasedordesignedusingsequence

informationfromGenBankattheNationalCenterforBiotechnologyInformation

(NCBI;www.ncbi.nlm.nih.gov)asfollows:GR:NR3C1RT2qPCRPrimerAssay

(PPR52805B,SAbiosciences,Qiagen,Valencia,CA,USA),NFkB:RT2qPCRPrimer

Assay(PPR42746A,SAbiosciences,Qiagen,Valencia,CA,USA),IL-6:F:5’–GGCAAA

TTTCCTGGTTATATCC-3’,R:5’-AGAAAAGAGTTGTGCAATGGCA-3’,MR:F:5’–

38

GGCAGCTGCAAAGTCTTCTT-3’,R:5’-GACAGTTCTTTCGCCGAATC–3’,UBC:F:

5’–CACCAAGAAGGTCAAACAGGAA-3’;GAPDH:F:5’–ACATCAAATGGGGTG

ATGCT–3’andR:5’–GTGGTTCACACCCATCACAA–3’,ActinB:F:5’-TTTGAG

ACCTTCAACACCCC–3’andR:5’–ATAGCTCTTCTCCAGGGAGG–3’,18SrRNA:

F:5’–ATGGTAGTCGCCGTGCCTA-3’andR:5’–CTGCTGCCTTCCTTGGATG–3’,

Ywhaz:F:5’–TTGAGCAGAAGACGGAAGGT–3’andR:5’–GAAGCATTGGGG

ATCAAGAA–3’,IL-1Ra:F:5’–CTGGGTACTTACAAGGACCAAATACC–3’andR:

TGGATGCCCAAGAACACATTCCGA–3’.MKP-1:F:5’–GCTCCACTCAAGTCTTCT

TCCTCCAA–3’,IkBa:F:CAGGATTCTGCAGGTCCACT–3’andR:TGGAGCACT

TGGTGACTTTG–3’.

3.2.4.Statisticalanalyses

StatisticalanalyseswererunasdescribedinChapter2(section2.4).Bothmaternal

diethistoryandoffspringsexwereusedasbetweensubjectmeasuresinfactorial

ANOVA.MultipletestingcorrectionswereperformedusingBonferroniforpost-hoc

testsorplannedcomparisons.EffectsconsideredstatisticallysignificantatP≤0.05

andnon-significanttrendsatP≤0.10arereported.

3.3.Results

3.3.1.Maternalbodyweightandcaloricintake

ToensureadequateexposuretoHFD,damsweregivenadlibitumaccesstoHFDor

CHDfor4weekspriortopregnancyandthroughoutgestationandlactation.Dams

39

Fig.3.2

Highfatdietaltersmaternalandpre-weaningoffspringbodyweight.(A)Maternal

bodyweightsduringgestation,(B)maternaldailycaloricintakeduringthelast

weekpriortopupbirth,(C)pre-weaningpupweightsand(D)adultoffspringbody

weights(PND90).HFD=highfatdiet,CHD=chowdiet.**=P<0.01,*=P<0.05

maineffectofdiet.

0

10

20

30

40

50

60

70

1 8 15 21

Pu

p B

od

y W

eig

ht

(g)

Postnatal Day

HFD

CHD

*

*

*

200#220#240#260#280#300#320#340#360#380#400#

1# 2# 3# 4#

Materna

l(Bod

y(Weight((g)(

Weeks(

HFD#

CHD#

*

*

*

*

40

60

80

100

120

Ave

rag

e kc

al/d

ay

HFD

CHD **

A B

C

0

100

200

300

400

500

Females Males

Ad

ult

Bo

dy

Wei

gh

t (g

)

HFD

CHD

D

40

consumingHFDgainedsignificantlymoreweightthanCHDdams[F(1,56)=22.97,P<

0.01;Fig.3.2A].Inaddition,theHFDdams’averagecaloricintakeduringthelast

weekpriortobirthwassignificantlyhigherthanthatofCHDdams[t(1,14)=3.05,P<

0.01;Fig.3.2B].

3.3.2Offspringbodyweight

WenextdeterminedtheinfluenceofmaternalHFDonoffspringweightgainuntil

weaningatPND21andinadulthood(PND90).HFD-exposedoffspringgaining

significantlymoreweightthanCHD-exposedoffspringthroughoutthepre-weaning

period[F(1,15)=15.58,P<0.01;Fig.3.2C].TherateofweightgainamongHFD-

exposedoffspringwasalsosignificantlyhigherthanfortheCHD-exposedoffspring

[daybydietinteraction,F(3,15)=130.72,P<0.01].Uponweaning,boththegroup

developmentallyexposedtoHFDandthegroupexposedtoCHDweremaintained

onCHDuntiladulthood.Inadulthood,maleoffspringweresignificantlyheavierthan

femaleoffspringoverall[F(1,86)=237.29,P<0.01;Fig.3.2D]andtherewereno

significantdifferencesinoffspringweightbetweenHFD-andCHD-exposedmalesor

females.

3.3.3Openfield

IntheOF,entriesintoortimespentinthecenterofthefieldand,conversely,

thigmotaxicbehavioursaretypicallyusedasmeasuresofanxietybehaviour.To

indexpotentialdifferencesinthigmotaxicbehaviour,weexaminedtheproportionof

timespentinthecenteroftheOFrelativetotimespentinboththecenterandarea

41

proximaltothewalls.OffspringexposedtomaternalHFDspentsignificantlyless

timeinthecenteroftheOFasaproportionoftotaltime[F(1,48)=15.73,P=0.01].

ThisdecreaseintherelativetimespentinthecenterportionoftheOFwasmore

pronouncedamongmales[dietbysexinteraction,F(1,48)=3.88,P=0.05;Fig.3.3A].

Therewerenodifferencesbetweendietgroupsorsexesinthemeanspeed,time

spentmobileornumberofentriesintothecenterportionoftheOF.

3.3.4Elevatedplusmaze

MaternalHFD-exposedoffspringgenerallyshowedadecreasedpreferenceforthe

openarmsoftheEPM,suggestingincreasedanxietybehaviour.MaternalHFD-

exposedoffspringdisplayedfewerheadentriesintotheopenarmscomparedto

CHD-exposedoffspring,definedasbehaviouralposturesinwhich60%ofthebody

oftheanimalenteredthearm[F(1,47)=6.14,P=0.01;Fig.3.3B].HFD-exposedfemale

offspringalsomadesignificantlyfewerentriesintotheopenarmsrelativetothe

closedarmsoftheEPMcomparedtoCHD-exposedfemalesorHFDexposedmales

[dietbysexinteraction,F(1,47)=4.68,P=0.03;Fig.3.3C].Therewerenodifferences

betweendietgroupsorsexesinthedistancetraveled,meanspeed,timespent

mobileorthetimespentintheopenversusclosedarmsoftheEPM.

3.3.5Light-darktransition

TherewasnosignificantinfluenceofmaternalHFDorsexonthenumberofentries

intothelightedportionoftheLDbox[P’s>0.1;Fig.3.3E].However,thenumberof

fecalboliwasalsorecordedattheendofeachtrial,asanincreasednumberofboliis

42

Fig.3.3

Perinatalhighfatdietexposureincreasesanxietybehaviourinadulthood.(A)Open

field(B-C)Elevatedplusmaze(D-E)Light-darktransitionbox.HFD=highfatdiet;

CHD=chowdiet.**=P<0.01,maineffectofdiet;*=P<0.05post-hoccomparison

withindietorsex.

0

5

10

15

20

25

Females Males

Lig

ht

En

trie

s

LD TASK

0

5

10

15

20

25

30

35

Females Males

Op

en A

rm H

ead

En

trie

s

EPM TASK

**,*

0

0.5

1

1.5

2

2.5

3

3.5

4

Females Males

Nu

mb

er o

f B

oli

LD TASK

** *

0.58

0.6

0.62

0.64

0.66

0.68

0.7

0.72

Females Males

Op

en/C

lose

d A

rm E

ntr

ies

Rat

io

EPM TASK

* *

D E

C

0.58

0.6

0.62

0.64

0.66

0.68

0.7

0.72

HFD CHD

Op

en/C

lose

d A

rm E

ntr

ies

Rat

io

EPM TASK

* *

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

OF TASK

**

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

HFD CHD

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

HFD CHD

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

HFD CHD

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

HFD CHD

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

HFD CHD

,

B

A

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

OF TASK

**

43

sometimesusedasameasureofanxietyinthelight-darktransitiontask(e.g.

(Ennaceuretal.,2006)).AdultoffspringexposedtomaternalHFDshoweda

significantlygreaternumberofbolicomparedtoCHD-exposedoffspring[F(1,47)=

10.92,P=0.01]andtherewasamarginalbutnonsignificantinfluenceofsexonthe

numberofboliobserved,withCHD-exposedfemaleoffspringshowingfewerboli

thanCHD-exposedmales[F(1,47)=2.71,P=0.10;Fig.3.3E].Therewereno

differencesbetweendietgroupsorsexesinthedistancetraveled,speed,timespent

mobileortimespentinthelightedportionoftheLDbox.

3.3.6Basalandstress-inducedserumcorticosterone

CORTlevelswereassessedjustpriortorestraintstresschallenge(0min),attheend

ofthestress(20min)andafter70mintoexaminereturntobaselinelevels.

Repeated-measuresANOVAshowedthatCORTlevelsvariedacrossthetime-points

sampled,withoffspringinbothdietconditionsshowingasignificantincrease

between0minand20minfollowedbyasignificantdecreasebetweenthe20min

and70mintime-point[F(2,74)=125.39,P<0.01;Fig.3.4A].Overall,femalesshowed

higherlevelsofCORTcomparedtomalesateachtime-point[F(1,37)=97.99,P<

0.01].Theinfluenceofmaternaldietexposurevariedovertime,asindicatedby

significantinteractionsbetweensampletimeanddiet,sampletimeandsexand

sampletimebydietbysex[allF’s>3.99,P’s<0.03].BasallevelsofCORTwere

significantlyloweroverallinmalescomparedtofemales[F(1,37)=18.64,P<0.01]

andweresignificantlyloweramongoffspringexposedtomaternalHFD[F(1,37)=

5.08,P<0.05;Fig.3.4B].Withineachsex,CORTlevelstendedtobelowerfor

44

Fig.3.4

PerinatalhighfatdietexposurealtersHPAinadulthood.(A)Stresschallengedlevels

ofCORT.Blacklineshowsdurationofrestraintstressor.Differencesareshown

betweenandwithineachsex.(B)BasallevelsofCORT.HFD=highfatdiet;CHD=

chowdiet.Bar,**=P<0.05,maineffectofsex;**=P<0.01maineffectofdiet;*=P

<0.05,#=P<0.10post-hoccomparisonwithinsex.

0

100

200

300

400

500

600

700

Females Males

Bas

al C

ort

ico

ster

on

e (n

g/m

L)

*

**

**

0

200

400

600

800

1000

1200

1400

0 20 70

Co

rtic

ost

ero

ne

(ng

/mL

)

Time (min)

HFD-F CHD-F HFD-M CHD-M

#

*

*

#

A B

45

maternalHFD-exposedfemaleoffspringrelativetoCHD-exposedfemales(P=0.10).

CORTlevelsweresignificantlylowerinmaternalHFD-exposedmalescomparedto

CHD-exposedmales(P=0.05).Amongfemales,HFD-exposedoffspringalsoshowed

atrendforlowerlevelsofCORTafter20minofstresschallenge(P=0.07),andHFD

-exposedfemaleoffspringhadsignificantlyhigherlevelsofCORTat70min(P=

0.02)comparedtoCHD-exposedfemaleoffspring,demonstratingaslowerreturnto

baselinelevels(Fig.3.4A).Amongmales,CORTlevelsinHFD-exposedoffspring

remainedsignificantlyelevated70minafterstresschallengerelativetothe0min

baseline(P=0.01),whilelevelsinCHD-exposedoffspringwerenodifferentfrom

baselineafter70min(P=0.30),suggestingaslowerreturntobaselineCORTlevels

inHFD-exposedmales.

3.3.7Corticosteronereceptorgeneexpression

Weexaminedrelativeabundanceoftranscriptforthetwomajorcorticosteroid

receptors,MRandGRintheAMYandHPC.MRtranscriptabundanceintheAMY

wassignificantlyhigheroverallamongHFD-exposedoffspringrelativetoCHD-

exposedoffspring[F(1,20)=8.29,P<0.01;Fig.3.5]andamongfemalescomparedto

males[F(1,20)=6.54,P=0.01],withmaternalHFD-exposedfemaleoffspringshowing

greaterexpressionthanCHD-exposedfemaleoffspring(P<0.01)orHFD-exposed

maleoffspring[dietbysexinteraction:F(1,20)=4.76,P=0.04].ForGR,HFD-exposed

offspringshowedhigherabundanceoftranscriptoverallintheAMYcomparedto

CHD-exposedoffspring[F(1,20)=9.90,P<0.01]andmalestendedtoshowhigher

46

Fig.3.5

Perinatalhighfatdietexposureincreasescorticosteroidreceptorexpressioninthe

amygdalainadulthood.HFD=highfatdiet;CHD=chowdiet,Bars,**=P<0.01,#=

P<0.10maineffectofsex;**=P<0.01maineffectofdietandpost-hoccomparison

withinsex.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Females Males

GR

(re

lati

ve a

bu

nd

ance

)

Hippocampus

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1

Females Males

GR

(re

lati

ve a

bu

nd

ance

)

Amygdala

**

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Females Males

MR

(re

lati

ve a

bu

nd

ance

)

Amygdala

**

**

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

HFD CHD

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

HFD CHD

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Females Males

MR

(re

lati

ve a

bu

nd

ance

)

Hippocampus

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

HFD CHD

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Females Males

Cen

ter/

Tota

l Tim

e R

atio

HFD CHD

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Females Males

IkB

a (r

elat

ive

abu

nd

ance

) Hippocampus

*

#

47

levelsthanfemales[F(1,20)=2.87,P=0.10].Post-hoccomparisonsrevealedthat

HFD-exposedfemalesshowedsignificantlymoreGRthanCHD-exposedfemales(P=

0.01).IntheHPC,expressionlevelsofMRandGRdidnotdiffersignificantly

betweendietgroupsorsexes.

3.3.8Pro-inflammatorygeneexpression

Weexaminedtranscriptabundanceofnuclearfactorkappabeta(NFkB),

interleukin-6(IL-6)andclusterofdifferentiationmolecule11B(CD11b),markersof

innateimmuneactivationpreviouslyassociatedwithperinatalHFDexposureinthe

HYPandHPC(BilboandTsang,2010).IntheAMY,HFD-exposedoffspringshowed

increasedtranscriptabundanceofNFkBrelativetoCHD-exposedoffspring[F(1,20)=

4.73,P=0.04;Fig.3.6]andmaternalHFD-exposedfemaleoffspringhadhigher

levelsofNFkBtranscriptcomparedtomaternalHFD-exposedmaleoffspringor

CHD-exposedfemales[dietbysexinteraction:F(1,20)=5.27,P=0.03].HFD-exposed

offspringalsoshowedincreasedabundanceofIL-6[F(1,20)=8.84,P<0.01],with

femalesshowinghigherlevelsoverallthanmales[F(1,20)=16.23,P<0.01;Fig.3.6].

IntheHPC,therewerenosignificantdifferencesbetweendietgroupsorsexesin

NFkBorIL-6expression.Therewerealsonostatisticallysignificantdifferences

betweendietgroupsorbetweensexesintheexpressionofCD11bintheAMYor

HPC.

3.3.9Anti-inflammatorygeneexpression

48

Fig.3.6

Perinatalhighfatdietexposureincreasesproinflammatorygeneexpressioninthe

amygdalainadulthood.HFD=highfatdiet;CHD=chowdiet.Bars,**=P<0.01,*=

P<0.05maineffectofsex;**=P<0.01,*=P<0.05maineffectofdiet;**=P<0.01,*

=P<0.05post-hoccomparisonwithindietandsex.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Females Males

CD

11b

(re

lati

ve a

bu

nd

ance

)

Hippocampus

0 1 2 3 4 5 6 7 8 9

10

Females Males

IL-6

(re

lati

ve a

bu

nd

ance

)

Hippocampus

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Females Males

CD

11b

(re

lati

ve a

bu

nd

ance

)

Amygdala

0 0.5

1 1.5

2 2.5

3 3.5

4 4.5

5

Females Males

IL-6

(re

lati

ve a

bu

nd

ance

)

Amygdala

** **

**

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1

Females Males

NF

kB (

rela

tive

ab

un

dan

ce)

Amygdala *

*

0.00

0.02

0.04

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ter/

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l Tim

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0

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Females Males

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kB (

rela

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ab

un

dan

ce)

Hippocampus

0.00

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Females Males

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ter/

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49

Next,wedeterminedtheimpactofmaternalHFDexposureonexpressionlevelsof

negativeregulatorsofinflammatoryresponse.Proinflammatorystimulibymitogen

activationisknowntoaltertheexpressionofthesignalingmoleculesI-kappa-B-

alpha(IkBa),mitogen-activatedproteinkinasephosphatase-1(MKP-1)and

interleukin-1receptorantagonist(IL-1Ra)viachangesinGR,NFkBandIL-6

signaling(Sorrellsetal.,2009),buttheirregulationinthecontextofthe

inflammatoryresponsetoHFDisunknown.

IntheAMY,HFD-exposedoffspringshowedsignificantlyelevatedabundance

ofIL-1Ra[F(1,20)=15.38,P<0.01;Fig.3.7],andfemalesshowedhigheroverall

levelscomparedtomales[F(1,20)=28.04,P<0.01]aneffectthatwassignificantly

greateramongHFD-exposedfemales[dietbysexinteraction:F(1,20)=6.95,P=

0.01].HFD-exposedoffspringtendedtoshowreducedabundanceofMKP-1

transcript[F(1,20)=2.69,P=0.11],withCHD-exposedmaleoffspringshowinga

trendforhigherlevelsofMKP-1transcriptcomparedtoHFD-exposedmale

offspringorfemaleoffspring[dietbysexinteraction:F(1,20)=2.91,P=0.10].There

werenodifferencesbetweendietgroupsorbetweensexesintheexpressionofIkBa

intheAMY.

IntheHPC,femalesshowedsignificantlyhigherlevelsofIkBaexpression

thanmales[F(1,20)=4.45,P=0.04],therewasatrendforlowerlevelsofexpression

amongHFD-exposedoffspring[F(1,20)=3.37,P=0.08],withCHD-exposedfemale

offspringtendingtoshowhigherlevelsofexpressioncomparedtoeitherHFD-

exposedfemaleormaleoffspring[dietbysexinteraction:F(1,20)=3.43,P=0.07].

PlannedcomparisonsshowedthatCHD-exposedfemaleoffspringhadsignificantly

50

Fig.3.7

Perinatalhighfatdietexposurealtersanti-inflammatorygeneexpressioninthe

hippocampusandamygdalainadulthood.HFD=highfatdiet;CHD=chowdiet.Bars,

**P<0.01,#P<0.10=maineffectofsex;**=P<0.01,*=P<0.05,#=P<0.10main

effectofdietandpost-hoccomparisonwithindietandsex.

0

0.1

0.2

0.3

0.4

0.5

0.6

Females Males

IL-I

Ra

(rel

ativ

e ab

un

dan

ce)

Amygdala

** **

**

0

0.5

1

1.5

2

2.5

3

3.5

Females Males

MK

P-1

(re

lati

ve a

bu

nd

ance

)

Amygdala

#

#

0

0.1

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(rel

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#

0

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ance

)

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ce)

Amygdala

0.00

0.02

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Females Males C

ente

r/To

tal T

ime

Rat

io

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ter/

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ter/

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e R

atio

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0.00

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0.04

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Females Males

Cen

ter/

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l Tim

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atio

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0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Females Males

IkB

a (r

elat

ive

abu

nd

ance

)

Hippocampus

* #

0.00

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Females Males

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51

higherlevelsofIkBa(P=0.04)comparedtoCHD-exposedmaleoffspring.ForIL-

1Ra,therewasatrendamongCHD-exposedfemaleoffspringforincreased

expressionofIL-1RarelativetoHFD-exposedfemaleandCHD-exposedmale

offspring[dietbysexinteraction:F(1,20)=2.96,P=0.10].ExpressionlevelsofMKP-

1didnotdifferbetweendietgroupsorsexesintheHPC.

3.3.10.DopaminergicandCRFgeneexpression

Table3.1showsthemean(±SEM)relativeabundanceoftranscriptforeachofthe

genesthatwereanalyzedinAMY,HYPandHPC.Ofthegenesandregionsassessed,a

significanteffectofmaternalHFDwasfoundfortheTHtranscriptintheAMYfor

maleandfemaleoffspring,CRFinAMYforfemaleoffspring,DARPP-32forfemale

offspringinHPC,andDARPP-32formaleoffspringinHYP(P<.05).

3.4.Discussion

Toourknowledge,thisisthefirststudytoexaminechangesinglucocorticoid

signalinginresponsetoperinatalHFDexposureinlimbicareasknowntoregulate

HPAfunctionandanxietybehaviour.Theresultsindicatethatalteredexpressionof

corticosteroidreceptorsandinflammatorygenesinthebrainofadultoffspring,asa

functionofHFDexposureduringtheperinatalperiodalone,maycontributetoa

heightenedendocrineresponsetostressandincreasedanxietybehaviourin

adulthood.

ThematernalphenotypeofHFDdamsduringgestationandlactation

consistedofincreasedbodyweightandcaloricintake.Thisphenotypewas

52

Table3.1

Mean± SEM relative abundance of transcripts for dopamine receptor D1 (DRD1),

dopamine receptor D2 (DRD2), tyrosine hydroxylase (TH), dopamine-andcAMP-

regulatedneuronalphosphoprotein(DARPP-32), corticotropin releasing factor (CRF)

in hypothalamus (HYP), amygdala (AMY), and hippocampus (HPC). Gene expression

was assessed in male (left) and female (right) offspring that had been pre-exposed to

maternal high fat diet and to cocaine or saline in adulthood. *P < .05 main effect of diet.

Gene Region Maternal diet Gene Region Maternal dietControl chow High fat chow Control chow High fat chow

DRD1 AMY 0.97 ± 0.19 1.39 ± 0.08 DRD1 AMY 1.30 ± 0.32 1.64 ± 0.39HYP 0.56 ± 0.06 0.47 ± 0.04 HYP 0.56 ± 0.05 0.60 ± 0.15HPC 4.57 ± 1.91 8.44 ± 2.37 HPC 12.08 ± 5.95 5.97 ± 2.75

DRD2 AMY 1.03 ± 0.30 1.40 ± 0.25 DRD2 AMY 2.00 ± 0.77 3.68 ± 0.74HYP 0.73 ± 0.05 0.72 ± 0.07 HYP 0.80 ± 0.10 0.71 ± 0.05HPC 3.12 ± 1.62 4.35 ± 1.19 HPC 7.23 ± 3.38 3.62 ± 1.76

TH AMY 0.49 ± 0.05 0.96 ± 0.19* TH AMY 1.31 ± 0.30 2.89 ± 0.33*HYP 0.88 ± 0.03 0.81 ± 0.05 HYP 0.92 ± 0.07 0.89 ± 0.07HPC 4.41 ± 2.05 5.04 ± 0.68 HPC 5.70 ± 1.30 3.02 ± 1.15

DARPP AMY 0.8 ± 0.13 0.74 ± 0.05 DARPP AMY 1.09 ± 0.13 1.07 ± 0.17HYP 0.84 ± 0.05 0.60 ± 0.01* HYP 0.69 ± 0.09 0.85 ± 0.05HPC 0.15 ± 0.02 0.14 ± 0.01 HPC 0.26 ± 0.03 0.10 ± 0.01*

CRF AMY 1.39 ± 0.22 1.81 ± 0.31 CRF AMY 1.58 ± 0.30 3.61 ± 0.80*HYP 1.04 ± 0.08 1.07 ± 0.07 HYP 1.37 ± 0.24 1.09 ± 0.13HPC 3.90 ± 1.69 4.39 ± 0.95 HPC 6.54 ± 3.00 3.03 ± 1.64

53

transmittedtooffspringpriortoweaning,whowereheavierthanCHD-exposed

offspringbeginningatpostnatalday8.However,therewasnodifferenceinbody

weightbetweenmaternalHFD-exposedandCHD-exposedoffspringinadulthood

whenanxietyandphysiologicalmeasuresweretaken,suggestingthattheobserved

phenotypewasassociatedwithperinatalHFDexposureratherthancurrentobesity.

Atpresentwedonotknowwhetherdifferencesinthematernalcarereceived

mayhavecontributedtothedifferencebetweenourHFDandCHD-fedgroups.To

ourknowledge,theonlytworecentstudiesthathaveexaminedthisquestionarrive

atdifferentconclusions.OnestudyfoundthatdamsconsumingHFDfrommating

throughlactationdisplayedincreasedmaternalnursing(i.e.arched-backand

passivenursingpostures)butnochangeinlickingandgroomingbehaviourduring

thefirstweekpostpartumcomparedtoCHD-fedcontrols(Purcelletal.,2011).

AnotherstudyfoundthatdamsconsumingHFDfrommatingthroughlactation

exhibiteddecreasedmaternallickingandgroomingbutnochangeinnursing

behaviourbetweenpostnataldays3and8(Connoretal.,2012).Thesedisparate

resultsindicatethatmorestudyisneededtoelucidatetheroleofmaternal

behaviourinoffspringphenotypeinthecontextofmaternalHFD.

Apotentiallimitationinourstudyisthat,inadditiontorelativelyhighlevels

offat(60%versus13.5%),ourdietarymanipulationalsoconsistedofsomewhat

higherlevelsofrefinedsugars(7%sucrose)comparedtotheCHDcontrol(3.7%

sucrose).Althoughpreviousstudieshaveusedthismodelofdiet-inducedobesity

comparedtoCHDtoexaminethedevelopmentalimpactofovernutrition(El-

Haschimietal.,2000;DeSouzaetal.,2005;DunnandBale,2009;Tamashiroetal.,

54

2009b;Purcelletal.,2011),thepresentdatadonotruleoutthepossibilitythat

increasedexposuretorefinedsugarduringdevelopmentcontributedtothe

observedeffects.Thereisanobvioustradeoffamonglevelsoffats,carbohydrates

andproteinsamongdietsusedtogenerateobesephenotypes.Importantly,

however,whereaslevelsofproteinwerehigherintheCHD-dietcontrolinthisstudy

(28%versus20%),wedidnotobserveadecreasedbodyweightatbirthamongthe

HFDexposedoffspringatbirth,indicativeofinsufficientlevelsofprotein.Thus,

thesedataindicatethatourmodelofdiet-inducedobesitylikelydidnotinvolve

proteinrestrictionduringdevelopment.Futurestudiesareneededtoexaminethe

relativeinfluenceoffatsandcarbohydratesontheobservedphenotype.

Inthisstudy,weusedavarietyofmeasurestoassessanxietybehaviour.Two

previousstudiesinrats(BilboandTsang,2010)andmice(Peleg-Raibsteinetal.,

2012)foundthatoffspringexposedtomaternalHFDshowincreasedanxietyinthe

EPM.OurdataextendthelinkbetweenperinatalHFDexposureandanxiety

behaviourinmalesandfemalesinadulthood;perinatallyexposedanimalsshowed

evidenceofincreasedanxietyinLDtaskaswellasarelativeavoidanceoftheopen

areasonboththeOFandEPM.Importantly,thesedifferencesintheOFandEPM

wereobservedintheabsenceofeffectsonlocomotoractivity,suggestingthat

differencesbetweendietgroupslikelyreflectedananxiety-likephenotyperather

thandifferencesrelatedtometabolicormotivationaleffects.

AheightenedHPAresponsetostresschallengeistypicallyassociatedwith

anxietybehaviour,asbasalCORTlevelssetthethresholdforactivationby

psychosocialstress(Lupienetal.,2009b).Theresultsofalimitednumberof

55

previousstudiesofdevelopmentalHFDexposureonbasalandstresschallenged

levelsofCORThavebeenmixed(Walkeretal.,2008;Shalevetal.,2010;Auvinenet

al.,2011).Differencesinproceduraldetailsincludingdurationofdietary

manipulationandthetimeofdayofCORTsamplingmayaccountinpartforthese

discrepancies(Walkeretal.,2008;Auvinenetal.,2011).Inthisstudy,bothmales

andfemalesshowedasimilarCORTresponsetoHFDexposure.Wefoundthatbasal

CORTwasattenuatedinadultmalesinresponsetoperinatalHFDexposure,and

adultfemalesshowedatrendtowardslowerbasalCORTlevels.HFD-exposed

animalsalsoshowedaheightenedresponsetorestraintstressandaslowerreturn

tobaselineCORTlevels,particularlyamongfemales,indicatinglessefficientCORT

feedback.ThesedataareconsistentwithevidenceofheightenedHPAresponseto

stresschallengeamongfemalescomparedtomalesinbothrodents(McCormicket

al.,1995;Weinstock,2007)andhumans(KudielkaandKirschbaum,2005),and

suggestthatperinatalHFDexposuremayexacerbateHPAreactivitytostressamong

females.

TheHPCandAMYplaykeyrolesinmodulatingtheresponsetostress

throughdirectprojectionstotheparaventricularnucleusoftheHYP.Inturn,the

HYPdirectstheendocrineresponseofCRFfromthePVNoftheHYP,whichthen

activatethereleaseofACTHfromthepituitary,leadingtothereleaseofCORTfrom

theadrenalcortexandexertingfeedbackonlimbicbrainareasviacorticosteroid

receptors.MRisahighaffinityreceptorthatisnormallyboundtoCORTinbasal

conditions,determiningthethresholdofthestressresponse.IntheHPC,increased

CORTasaresultofpsychosocialstressactivatestheloweraffinityGR,inhibitingthe

56

furtherreleaseofCORT,whereasintheAMYGRactivationenhancestheHPA

response(Joelsetal.,2008).Inthismanner,thelimbicsystemhasbeenproposedto

refinetheadaptiveresponsetostress(Groenewegetal.,2011).Wefoundevidence

ofincreasedMRlevelsintheAMYinresponsetomaternalHFDexposure.Increased

MRinAMYhasbeenshowninmaleZuckerdiabeticfattyrats,amodelofdiet-

inducedobesity(Johrenetal.,2007).OurdataindicatethatincreasedMRlevelsin

AMYmayleadtodecreasedbasallevelsofCORTasaresultofmaternalHFD

exposure.WealsofoundevidenceofincreasedGRinAMYandaheightened

responsetostressinoffspringexposedtomaternalHFD.Thesedatasupporta

numberofpreviousobservationsthatincreasedGRintheAMYenhancestheCORT-

mediatedresponsetostress(Joelsetal.,2008).

Glucocorticoidshavewellknownanti-inflammatoryactivitiesinthebody,

andhavebeenrecentlybeenshowntohavebothinflammatoryandanti-

inflammatoryrolesinthebrain(Sorrellsetal.,2009).IncreasedGRexpression

underconditionsofchronicstressoralteredlevelsofglucocorticoidsgenerally

enhancescentralinflammatoryresponses.Indeed,underconditionsofchronic

stress,GRactivationisrequiredforinflammatoryresponsesasaresultofincreased

NFkB(Sorrellsetal.,2009).AlteredinflammatorygeneexpressionintheHYPisa

well-knownconsequenceofdiet-inducedobesity,anddietinducedobesityis

associatedwithincreasedIL-6expressionintheHYP(DeSouzaetal.,2005)andin

thecortex(Whiteetal.,2009)inadiet-doseresponsivemanner.Thatis,increased

IL-6transcriptisonlyobservedwithprolongedHFDexposure.Inthisstudy,we

foundthattranscriptabundanceofthemajorpro-inflammatorygenesNFkBandIL-

57

6wasalsoincreasedintheAMYamonganimalsexposedtoHFDonlyduring

perinatallife.WedidnotfindachangeinIL-6intheHPC,supportingpreviouswork

(BilboandTsang,2010).ThepresentdatashowingparallelincreasesinGR,NFkB

andIL-6suggestthat,inadditiontochangesinducedbyHFDconsumption,changes

intheregulationofthesegenesoccurswithHFDexposureduringdevelopment,

independentofcurrentconsumption.WedidnotdetectaninfluenceofHFDonIkBa

expression,aknownnegativeregulatorofNFkB(Munhozetal.,2010),suggestinga

disruptioninthenegativeinhibitionofNFkBnormallymediatedbyIkBa.Amongthe

othernegativeregulatorsofinflammationexaminedinthisstudyandknowntobe

activatedbyincreasedGR,wefoundaHFD-dependentincreaseintheexpressionof

IL-1Ra,aninhibitorofIL-1,andatrendfordecreasedexpressionofMKP-1

particularlyamongfemales.Abalancebetweeninflammatoryresponsesisacritical

mechanismtomaintainanadequateresponsetopathologicalchallengessuchasin

diseaseandinfection(Spulberetal.,2009).Inperipheralmonocytes,analysisby

microarrayhasshownthatsyntheticglucocorticoidincreasestheexpressionofa

numberofbothpro-andanti-inflammatorygenes(Galonetal.,2002).Seeninthis

context,ourdataindicatethatthecentralhomeostaticequilibriumbetween

inflammatoryandanti-inflammatoryresponsesmaybedisruptedbyHFDexposure

duringdevelopment.

Anumberofpreviousstudiesoftheinteractionbetweenglucocorticoids,GR

andinflammatoryprocesseshaveexaminedtheeffectsofmitogenchallengeby

lipopolysaccharide(LPS)onglucocorticoidsignaling.LPSactivatessignal

transductionthroughtheToll-likereceptor4(TLR4)pathway,leadingtoincreased

58

NFkBandMAPkinaseactivation.Studiesoftheeffectofstressandsustained

alterationsinglucocorticoidshavedemonstratedthatGRsmodulatetheabilityof

LPStodirectapro-inflammatoryresponseinpartbymodulatingtheactivityof

MKP-1,IkBa,IL-1RaandsubunitsofNFkB(Madrigaletal.,2001;Sorrellsetal.,

2009;Franketal.,2010;Franketal.,2012).ThesedataindicatethattheTLR4

inflammatorypathwayissensitivetoalteredglucocorticoidsignaling.HFDexposure

isknowntoinduceinflammationthroughtheTLR4pathwayintheHYP,whichleads

toendoplasmicreticulumstress,theexpressionofinflammatorycytokinesand

eventuallyapoptosisofneurons,contributingtothedysregulationofenergy

homeostasis(Zhangetal.,2008;Milanskietal.,2009;Moraesetal.,2009).The

effectsofglucocorticoidsonsignalingviathispathwaymay,tosomeextent,be

brain-region(Munhozetal.,2010)andcell-typespecific(Sorrellsetal.,2009).

Therefore,somedegreeofcautioniswarrantedinextrapolatingsuchfindingsto

limbicbrainareas.However,ourdataimplicateglucocorticoidmodulationof

inflammatorysignalinginlimbicareasinanxietybehaviourasafunctionofHFD

exposure.

Anxietybehaviourhasbeenlinkedtochangesininflammatorygene

expressioninseveralstudies(Dantzeretal.,2008).Inturn,mitogen-activated

inflammationinlimbicareasofthebrain,includingAMYandtheHPC,islinkedto

anxietybehaviour,indicatingadirecteffectofinflammationonanxiety(Rodgerset

al.,2012).Futurestudiesareneededtoidentifyinteractionsamongkeyregulators

ofglucocorticoidsignalingsuchasGR,NFkBandotherdownstreaminflammatory

signalingmoleculesandtheircontributiontoanxietybehaviour.

59

Femaleratsshowedthelargestchangesingeneexpressionasafunctionofthe

maternaldietinthisstudy.Fewstudieshaveexaminedsexdifferencesingene

expressionprogrammingbydiet.However,similarresultshavebeenreportedin

studiesofmaternalHFDontranscriptionalregulationduringplacental

development.MaternalHFDhasamorepronouncedinfluenceongeneexpressionin

femaleplacentainmice,withagreaternumberofgenesshowingincreased

expressioninfemalescomparedtomales(Maoetal.,2010).Arecentstudyusinga

genome-widetranscriptomicapproachreportedaselectiveincreaseinthe

expressionofanumberofgenesinvolvedintheinflammatoryresponsesinfemales

comparedtomales(Gaboryetal.,2012).Thesedatamayindicateasexually

dimorphicdevelopmentaltrajectoryofimmunesignalingthroughadaptationsatthe

fetal-maternalinterface.Inhumans,incidencesofanxietydisordersaswellas

obesityaremuchmorecommoninfemales(Desaietal.,2009;Rofeyetal.,2009).

Thereisalsoevidencethatfemalesmaybemoresensitivetotheeffectsof

alterationsinimmunefunctiononanxietybehaviour(Bouret,2009;Schwarzand

Bilbo,2012).Ourdatatodateareconsistentwiththehypothesisthattheresponse

toperinatalHFDobservedinthisstudymayariseasafunctionofbothincreased

HPAreactivityandincreasedinflammatorygeneexpression,particularlyamong

females.

Aclearmajorityofadultsindevelopedcountriesarenoweitheroverweight

orobese(68%intheUS(Flegaletal.,2010;Sullivanetal.,2010)),aconditionlinked

toimportanthealthrisks.Whenexperiencedduringdevelopment,aHFDmayhave

long-termimpactsonmentaldisordersassociatedwithanxiety.Itwillbeimportant

60

toelucidatechangesinsignalingpathwaysincriticalbrainareasinoffspringsothat

targetedinterventionsmaybeeffectivelyaimedatmitigatingdeleteriouseffectsof

overnutritionondevelopmentalprogramming.

61

Chapter 4: Effects of maternal high fat diet and cocaine-

primed locomotor activity and gene expression in offspring

62

Chapter4:Effectsofmaternalhighfatdietandcocaine-primedlocomotoractivityandgeneexpressioninoffspring

4.1Introduction

InChapter3,wefoundthatmaternalconsumptionofHFDalteredoffspring’sstress

systemandanxietybehaviour.Wealsofoundthattyrosinehydroxylase,arate-

limitingenzymeofcatecholaminesynthesiswasupregulatedintheamygdalaofthe

HFDoffspring.Additionallycorticotropin-releasingfactor(CRF),aneuropeptide

centraltothestresssysteminvolvedindrugaddiction,wasfoundupregulatedin

theamygdalaoffemaleHFDoffspring.Thedopaminergicinputfromtheventral

tegmentalareaconnectstothemesolimbicpathway,whichthenextendstothe

amygdala.Palatablediet,includingHFD,activatesdopaminergicpathwayswithin

themesolimbicrewardsystem,whichhasbeenimplicatedinrewardandaddiction

tococaine(KelleyandBerridge,2002;Tobleretal.,2005).Inthischapter,weused

cocaine-inducedlocomotoractivitytodetectalterationsindopaminergicfunction.

TherehavebeenonlyafewstudiesthathavelookedattheeffectsofmaternalHFD

ondopaminergicfunction(Naefetal.,2008;Vuceticetal.,2010;Ongand

Muhlhausler,2011).Howevercocaine-inducedlocomotoractivityhasneverbeen

examinedinHFDoffspring.

WeexaminedtheeffectofmaternalHFDintakeonthemesolimbicsystemof

offspringbyexaminingtheirsensitivitytococaine.Wehypothesizedthatexposure

tomaternalHFDincreasescocaine-sensitivebehaviour,increasesanxietybehaviour

andalterscocaine-primedregulationofdopamine-andstress-relatedgene

transcriptioninthebrainsofadultoffspring.Specifically,wehypothesizethat

increasedcocaine-sensitivebehaviourwillbeaccompaniedbyincreasedexpression

63

ofdopaminergicgenesinthemesolimbicsystem.Wealsohypothesizethatchronic

cocaineexposurewillbeaccompaniedbychangesintheexpressionofstress-related

genesinamannerconsistentwithheightenedanxiety.WeusedtheHFDregimen

appliedinChapter3(Sasakietal.,2013).Wealsousedachroniccocaineexposure

drugregimenthatisknowntoinduceconditionedlocomotoractivity(Johnsonetal.,

2012),followedbyanelevatedplusmazetestandcocainesensitizationtest.Overall,

weobservedincreasedlocomotoractivitywithcocainetreatmentinthedrug

exposurephase,conditionedlocomotion,andsensitizationtestinbothsexes.


4.2.1.Animals

AdultmaleandfemaleLongEvansrats(7weeks)wereobtainedandprovidedad

libitumaccesstofoodandwater,asdescribedinChapter2(section2.1).


4.2.2.1.Diets

Femalebreederswereplacedononeoftwodiets:ahighfatdiet(n=16,HFD)ora

housechowdiet(n=14,CHD).Thedetailofthedietsanddietregimenwere

describedinChapter2(section2.2.1).OneHFDdamwasremovedfromthestudy

duetodifficultyduringbirth.

4.2.2.2.Assessmentofmaternalbehaviour

64

ThematernalbehaviourofdamsthatwereexposedtoHFD(n=15)priortoand

duringpregnancy,anddamsthatwereexposedtoCHD(n=14),wasmonitoredon

eachof6consecutivedaysbetweenPND1andPND6.Briefly,thebehaviourofeach

damwasvideo-recordedduringsix1-hourobservationperiods(i.e.,0100h-0200h,

05000h-06000h,1000h-1100h,1300h–1400h,1700h–1800h,2100h–2200h).

Foreachobservationperiod,theindividualvideorecordingsforeachdamwere

assessedin3minintervals(i.e.,20Observations/periodx6periodsperday=120

observations/dam/day),usingacomputer-controlledbehaviouralcodingand

analysissystem.Additionaldetailsregardingtheassessmentofmaternalbehaviour

aredescribedinChapter2(section2.2.2).


4.2.3.1.Subjects

AnequalnumberofoffspringfromdamswithahistoryofHFDandCHDcontrol

wereusedforthedifferenttestsdescribedbelow.Asubsetofmale(CHD;n=24,

HFD;n=24)andfemaleadultoffspring(CHD;n=24,HFD;n=24)wereusedin

behaviouralassays.Asubsetofanimalsfromeachgroupwasselectedforanalyses

ingeneexpression(CHDsalinen=6,CHDcocainen=6,HFDsalinen=6,HFD

cocainen=6persex).Theexperimentaltimelinefordamsandoffspringisshownin

Fig.4.1.

4.2.3.2.HabituationandCocainepre-exposureinoffspring

65

Thecocainepre-exposureregimenbeganwhenoffspringreachedadulthood,at

approximately65daysofage.Onedaypriortothestartofcocaineorsaline

injections,allratsweregivenahabituationsession.Duringthissession,ratswere

givenasalineinjection(1kg/ml,i.p.),afterwhichtheywereplacedinlocomotor

activitychambersforaperiodof30min.Locomotoractivityaftertheinjectionwas

monitored,asdescribedinChapter2(section2.3.1.1.1).

Atotalof6drugexposuresessionswereadministeredoverthesubsequent7

days.Ratsweregivenoncedailyinjectionsofcocaine(30mg/kg,i.p.)orsaline

(1kg/ml,i.p.).Halfofeachdietgroupwasexposedtococainewhiletheotherhalf

receivedsalineinjections(CHDsalinen=12,CHDcocainen=12,HFDsalinen=12,

HFDcocainen=12persex).Duringthesesessions,ratsweregivenaninjectionof

cocaineorsaline,afterwhichtheywereplacedinlocomotoractivitychambersfora

periodof30min,underthesameconditionsdescribedforthehabituationsession.

Locomotoractivityaftertheinjectionwasmonitored,asdescribedinChapter2

(section2.3.1.1.2).

4.2.3.3.Testforconditionedlocomotion

Twenty-fourhoursaftercocainepre-exposurewascomplete,animalsweretested

forconditionedlocomotionwhereanimalswerereturnedtoanenvironment

66

Fig.4.1



andduringtheperiodoflactation.Onpostnatalday21,theoffspringwereweaned

fromtheirmothersandgivenCHDdiet.(B)Oncetheoffspringreachedadulthood

theyweregivenahabituationsession(HAB)andatotalof6drugpre-exposure

sessions(DE1-DE6)toeithersaline(1mL/kg.,i.p.)orcocaine(30mg/kg.,i.p.).The

nextday,theoffspringweretestedforconditionedlocomotion(CL)followedby

elevatedplusmaze(EPM).EighteendaysafterDE6,alltheoffspringweregivena

cocainechallenge(10mg/kg.,i.p.).Twenty-fourhoursafterthecocainechallenge,

reward-andstress-relatedgeneexpressionwasexaminedinmesolimbicandlimbic

brainregions.

67

wheretheyhadpreviouslyreceiveddruginjectionsandlocomotoractivitywas

measuredintheabsenceofdrug.Testingprocedureswereidenticaltodrug

exposureproceduresexceptthatallratsweregivenvehicleinjections(0.9%saline,

1mL/kg)priortoplacementintheactivitymonitoringchambersfor30min.


Eighteendaysaftertheterminationofthecocaineexposurephase,alloffspring

weretestedfortheirlocomotorresponsetoachallengeinjectionof10mg/kgof

cocaine(i.p.).Forthistest,ratswereinjectedwithcocaineandplacedinthe

locomotorchambersfor30minperiod.

4.2.3.5.Elevatedplus-maze

Twenty-fourhoursafterthetestforconditionedlocomotion,thatis,48hafterthe

lastexposuretorepeatedinjectionsofcocaineorsaline,theEPMwasusedto

measureanxietybehaviour.Anxiety-likebehavioursemergewithrepeated

exposuretococaineasafunctionoftheanxiogeniceffectsofcocainewithdrawal

(Sarnyaietal.,1995).Theelevatedplus-mazewasrunasdescribedinChapter2

(section2.3.1.2.1).

4.2.3.6.Cocaine-primedgeneexpressionanalysesinoffspring

Asubsetofanimalswasselectedforanalysesingeneexpression(CHDsalinen=6,

CHDcocainen=6,HFDsalinen=6,HFDcocainen=6persex).24hrsafterthelast

injectionofcocaine,thebrainsoftheseanimalswerecollectedasdescribedin

68

Chapter2(section2.3.1.4.1).Thisprocedureallowedfortheanalysesofcocaine-

activatedgeneexpressionlevelsratherthansteady-statemRNAlevels(Renthalet

al.,2009).Theexpressionfor4dopamine-related(DRD1,DRD2,TH,DARPP-32)and

2HPA-related(CRF,GR)genesofinterestwascomparedtothegeometricmeanof

threehousekeepinggenes(GAPDH,ACTb,UBC)in5brainregions(AMY,HPC,PFC,

VTA,NAC)foreachsex,dietanddrugcondition(seeChapter2,section2.3.1.4.1for

coordinatesandsection2.3.1.4.2-3forgeneralprocedures).Analysesofdopamine-

relatedgeneswererestrictedtorewardpathwaysandanalysesofHPA-related

geneswererestrictedtolimbicregions,exceptforGR,whichisknowntointeract

withdopaminergictransmissioninthePFCandNAC.

4.2.4.Statistics

Thedatafromthedrugpre-exposurephasewereanalyzedusingtwo-wayrepeated

ANOVAwithmaternaldiet(HFDorCHD)asthebetweensubjectfactoranddrug

exposuredays(1through6)asthewithinsubjectsfactor.Thedatafromthedrug

exposureday1,conditionedlocomotion,sensitizationtestandelevatedplusmaze

wereallanalyzedinthesamewayusing2x2factorialANOVAwithmaternaldiet

(HFDorCHD)anddrugpre-exposure(salineorcocaine)asbetween-subjects

factors.ThedatafromthematernalcodingwereanalyzedasdescribedinChapter2

(section2.4).

4.3Results

69

4.3.1.Maternalbodyweight,offspringbodyweightandoffspringnumbers

DamsweregivenadlibitumaccesstoHFDorCHDcontrolfor4weekspriorto

pregnancyandthroughoutgestationandlactation.DamsconsumingHFDgained

significantlymoreweightthanCHDdams[F(1,20)=3858.68,P<0.001;Fig.4.2A].

WenextdeterminedtheinfluenceofmaternalHFDonoffspringweightgain

untilweaningatPND21andinadulthood(PND65).HFD-exposedoffspringgained

significantlymoreweightthanCHD-exposedoffspringthroughoutthepre-weaning

period[F(1,23)=6.258,P=0.02;Fig.4.2B].TherateofweightagainamongHFD-

exposedoffspringasalsosignificantlyhigherthanfortheCHD-exposedoffspring

[daybydietinteraction,F(3,69)=11.903,P<0.01].Uponweaning,HFD-exposed

offspringreceivedCHDandCHD-exposedoffspringweremaintainedonthesame

dietuntiladulthood.Inadulthood,therewerenosignificantdifferencesinoffspring

weightbetweenHFDandCHD-exposedmalesorfemales(datanotshown).

WealsodeterminedtheinfluenceofmaternalHFDonoffspringnumbers

duringthepre-weaningperiodaftercullinganimalstoequalnumbersacross

conditionsatbirth.Thenumberofoffspringatbirthwasnotsignificantlydifferent

acrossthedietconditions(P>.05).Fig.4.2Cshowsthemean(±SEM)numberof

offspringforthedifferentmaternaldiet(CHD,HFD)conditionsonPND1,11,17and

21.Therewasasignificantdaybymaternaldietinteraction[F(3,69)=3.84,P<.05].

70

Fig.4.2

Highfatdietaltersmaternalandpre-weaningoffspringbodyweight.(A)Maternal

bodyweightspriortopupbirth(B)pre-weaningpupweights(C)numberof

offspringduringthepre-weaningperiodaftercullingatbirth.HFD=highfatdiet,

CHD=chowdiet,CON=stresscontrol,CVS=stress.Bars,*P<.05maineffectofdiet,

Bar,#P<.05maineffectofstress,*P<.05posthoccomparison.

0

2

4

6

8

10

12

14

1 11 17 21

Pup

num

ber p

rior t

o w

eani

ng

Postnatal Day

CHDHFD

*C

A

050100150200250300350400450500

1 2 3 4 5

Mat

erna

l Bod

y W

eigh

t (g)

Number of weeks prior to birth

HFDCHDPupbirth

*

0

10

20

30

40

50

60

70

1 11 17 21

Bod

y W

eigh

t (g)

Postnatal Day

CHDHFD

*

*

*

*

B

71

Post-hoctestsrevealedthatthenumberofHFDoffspringwasloweratPND21

comparedtotheCHDoffspring.

4.3.2.Maternalbehaviour

Themeanpercentageofmaternalbehaviouroversixobservationsdaysisshownin

Fig.4.3.Dam’slickingandgroomingofpupand/ornursingpupinanarched-back

posturewasmeasured.Therewasnoeffectofmaternaldietonmaternalbehaviour.

4.3.3.Locomotoractivityduringcocainepre-exposureinoffspring

Fig.4.4showsthemeandistancetraveled(cm)byoffspringbetweenDays1and6

ofthepre-exposurephase.Overall,repeatedmeasuresANOVArevealedasignificant

maineffectofdrugpre-exposureinmale[F(1,43)=133,673,P<0.001]aswellasin

femaleoffspring[F(1,44)=172.64,P<0.001].Inaddition,inthefemaleoffspring,

therewasasignificantinteractionofdrugpre-exposurexday[F(5,220)=3.448,P<

0.01]thatcanbeattributedtoanincreaseinactivityincocaine-treatedratsduring

theearlyexposuredays.Onpre-exposureday1,anANOVArevealedmaineffectsof

drug[F(1,44)=65.347,P<0.001],aswellasdiet[F(1,44)=4.133,P<0.05],

howeverthemaineffectofdietdisappearedbypre-exposureday6.Although

cocainetreatmentdidnotinteractwithmaternaldietonday1(P=.14),basedon

ourpriorihypothesis,weperformedaplannedcomparisononcocaine-treated

femalesonly,andfoundthatlocomotoractivityincreasedspecificallyamong

cocaine-treatedHFDfemalescomparedtococaine-treatedCHDfemales(P<.05).

72

Fig.4.3

Highfatdietconsumptionindamsdidnotaltermaternalcare.Mean± SEM

percentage of licking/grooming and arched back nursing (ABN) by dams during postnatal

days 1 though 6. HFD=highfatdiet,CHD=chowdiet.

73

Fig.4.4

Locomotoractivityduringcocainepre-exposureinoffspring.(A)Mean± SEM

distance traveled (cm) in 10 min during Day 1 through 6 of the cocaine pre-exposure

phase by male (left) and female offspring (right). (B) Mean± SEM distance traveled (cm)

in 10 min during Day 1 of the cocaine pre-exposure phase by male (left) and female

offspring (right). HFD=highfatdiet,CHD=chowdiet.SAL=saline,COC=cocaine.*P

<.05maineffectofdrugexposure,Bars,*P<.05maineffectofdiet,(*)P<.05

plannedcomparison

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6

Dis

tanc

e Tr

avel

ed (c

m)

Drug Exposure Day

CHD SalineHFD SalineCHD CocaineHFD Cocaine*

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6D

ista

nce

Trav

eled

(cm

)Drug Exposure Day

Saline CHDSaline HFDCocaine CHDCocaine HFD

*

*

0

1000

2000

3000

4000

5000

6000

7000

1 2 3 4 5 6

Dis

tanc

e Tr

avel

ed (c

m)

Drug Exposure Day

Saline CHDSaline HFDCocaine CHDCocaine HFD

0

1000

2000

3000

4000

5000

6000

CHD HFD

Dis

tanc

e Tr

avel

ed (c

m)

SalineCocaine

0

1000

2000

3000

4000

5000

6000

CHD HFD

Dis

tanc

e Tr

avel

ed (c

m)

SalineCocaine

* **

**

(*)

A

B

74

4.3.4.Locomotoractivityduringtestforconditionedlocomotioninoffspring

Fig.4.5showsthemeandistancetraveled(cm)byoffspringduringtestfor

conditionedlocomotion.Inthistest,allratsreceivedsalineinjectionsintheactivity

chambers,wheredrugpre-exposuresessionshadbeencarriedout.Overall,there

wasasignificantmaineffectofdrugpre-exposureinbothmales[F(1,44)=8.864,P

<0.01]andfemales[F(1,44)=37.579,P<0.001].Infemales,therewasasignificant

maineffectofmaternaldiet[F(1,44)=8.333,P<0.01],withHFDexposedfemales

travelingagreaterdistancethanCHDfemales.Notably,thiseffectwassimilarto

thatobservedonday1ofthepre-exposuresessions.

4.3.5.Locomotoractivityduringtestforcocainesensitizationinoffspring

Approximately16daysafterthelastpre-exposure,alloffspringweregivenatestfor

locomotorsensitizationinresponsetoacocainechallenge(10mg/kg.,i.p.).Fig.4.6

showsthatoffspringpre-exposedtococaineexhibitedasignificantlyhigherlevelof

locomotoractivityinresponsetoanacutecocainechallengerelativetooffspring

pre-exposedtosaline,bothinmale[F(1,44)=12.935,P=0.001]andfemale

offspring[F(1,44)=19.622,P<0.001].Thus,theeffectsofrepeatedcocaine

exposureonlocomotoractivitypersistedoveratleasta16-dayperiod.Therewas

nosignificantinteractionbetweencocainepre-exposureandmaternaldiet.

4.3.6.Elevatedplusmazeinoffspring

HFDexposedmaleoffspringdidnotshowanydifferenceinthetimespentinthe

openarmscomparedtoCHDoffspring.However,infemaleoffspring,ANOVA

75

Fig.4.5

Locomotoractivityduringthetestforconditionedlocomotioninoffspring.Mean±

SEM distance traveled (cm) in 10 min following an injection of saline (1mL/kg, i.p.) in

the same room where the animals received drug pre-exposure (saline or cocaine)

previouslyinmale(left)andfemaleoffspring(right).HFD=highfatdiet,CHD=

chowdiet.*P<.05maineffectofdrugexposure,Bar,*P<.05maineffectofdiet.

0

500

1000

1500

2000

2500

3000

3500

4000

CHD HFD

Dis

tanc

e Tr

avel

ed (c

m)

SalineCocaine

0

500

1000

1500

2000

2500

3000

3500

4000

CHD HFD

Dis

tanc

e Tr

avel

ed (c

m)

SalineCocaine

* **

**

76

Fig.4.6

Locomotoractivityduringthetestforcocainesensitizationinoffspring.Mean±

SEM distance traveled (cm) in 10 min following a challenge injection of cocaine (10

mg/kg, i.p.)inmale(left)andfemaleoffspring(right).HFD=highfatdiet,CHD=

chowdiet.*P<.05maineffectofdrugexposure.

0

1000

2000

3000

4000

5000

6000

CHD HFD

Dis

tanc

e Tr

avel

ed (c

m)

SalineCocaine

0

1000

2000

3000

4000

5000

6000

CHD HFD

Dis

tanc

e Tr

avel

ed (c

m)

SalineCocaine

* *

**

77

showedthattherewasasignificantmaineffectofcocainepre-exposure[F(1,43)=

4.644,P<0.05]andasignificantinteractionbetweencocainepre-exposureand

maternaldiet[F(1,43)=3.865,P=0.05;Fig.4.7].Posthoctestsrevealedthat

saline-treatedHFDfemalesreducedtimespentinopenarmscomparedtosaline-

treatedCHDfemales(P<.05),whichisconsistentwithpreviousfindings(Chapter

3).Surprisingly,repeatedexposureofcocainedidnotleadtoincreasedanxietyas

cocaine-treatedHFDfemalesshowedincreasedtimespentintheopenarms

comparedtosaline-treatedHFDfemales(P<.05).

4.3.7.Cocaine-primedgeneexpressioninoffspring

Cocaine-primedgeneexpressionanalyseswerecarriedoutinmesolimbicregions

(PFC,NACandVTA)aswellasinthelimbicregions(HPC,AMYandHYP)forreward

andstress-relatedgeneexpression(Table4.1).MaleoffspringofHFDdamsshowed

increasedCRFintheHPCandincreasedDRD2intheHYPcomparedtomale

offspringofCHDcontroldams.Cocaine-treatedmaleoffspringshowedincreasedTH

intheAMYcomparedtosaline-treatedmaleoffspring.FemaleoffspringfromHFD

damsshoweddecreasedDARPPintheVTAandAMYandasignificantdrugbydiet

interactionforCRFintheHPC.Cocaine-treatedfemaleoffspringshowedreducedGR

intheHPC,PFCandNACandincreasedTHinthePFCcomparedtosaline-treated

femaleoffspring.Forthecompletetableofresultspertainingtoallthegenes

examined,seeAppendixTable4.1.PleaseseetheGeneralDiscussionforanalysis

ofgenesshowingsimilareffectsacrossstudies.

78

Fig.4.7

Anxietybehaviourinoffspring.Mean± SEMratiooftimespentintheopenarms

overtimespentintheopenarmsandclosedarmsmeasuredintheelevatedplus

mazeinmale(left)andfemaleoffspring(right).HFD=highfatdiet,CHD=chowdiet.

SAL=saline,COC=cocaine.*P<0.05posthoccomparisons.

00.050.10.150.20.250.30.350.4

CHD HFD

OA

time/

tota

l tim

e ra

tio

SALCOC

00.050.10.150.20.250.30.350.4

CHD HFD

OA

time/

tota

l tim

e ra

tio

SALCOC

**

79

Table4.1

Cocaine-primedgeneexpressionanalysesinoffspring.Twenty-fourhoursaftera

challengeinjectionofcocaine(10mg/kg,i.p.),thebrainswerecollectedforanalyses

ofcocaine-activatedgeneexpressionlevels.Mean± SEM relative abundance of

transcripts for dopamine receptor D1 (DRD1) and dopamine receptor D2 (DRD2) in

medial prefrontal cortex (mPFC), nucleus accumbens (NAC) and ventral tegmental area

(VTA) and for glucocorticoid receptor (GR) and corticotropin releasing factor (CRF) in

hypothalamus (HYP), amygdala (AMY), and hippocampus (HPC). Gene expression was

assessed in male (left) and female offspring (right) that had been pre-exposed to maternal

high fat diet and to repeated injections of cocaine or saline in adulthood. *P < .05 main

effect of diet, #P < .05 main effect of drug treatment, &P < .05 diet by drug interaction.

Gene Region Gene RegionControl chow High fat chow Control chow High fat chow

Saline Cocaine Saline Cocaine Saline Cocaine Saline CocaineDRD1 mPFC 0.95 ± 0.15 0.88 ± 0.11 0.83 ± 0.08 0.85 ± 0.08 DRD1 mPFC 1.46 ± 0.14 1.48 ± 0.13 1.52 ± 0.17 1.51 ± 0.13

NAC 1.77 ± 0.22 1.22 ± 0.17 1.31 ± 0.15 1.25 ± 0.20 NAC 1.16 ± 0.01 1.04 ± 0.04 1.17 ± 0.09 1.07 ± 0.04VTA 1.23 ± 0.14 1.23 ± 0.06 1.24 ± 0.06 1.26 ± 0.12 VTA 0.89 ± 0.05 0.93 ± 0.05 0.97 ± 0.05 1.04 ± 0.10

DRD2 mPFC 1.07 ± 0.09 1.09 ± 0.08 1.05 ± 0.06 1.06 ± 0.03 DRD2 mPFC 1.74 ± 0.11 1.70 ± 0.11 1.95 ± 0.06 1.82 ± 0.07NAC 1.64 ± 0.19 1.75 ± 0.24 1.72 ± 0.40 1.11 ± 0.14 NAC 0.84 ± 0.05 0.82 ± 0.02 0.89 ± 0.04 0.74 ± 0.04VTA 1.23 ± 0.16 1.16 ± 0.08 1.19 ± 0.10 1.22 ± 0.08 VTA 0.96 ± 0.11 0.95 ± 0.07 1.07 ± 0.08 0.96 ± 0.11

GR AMY 1.26 ± 0.04 1.26 ± 0.01 1.22 ± 0.03 1.15 ± 0.06 GR AMY 1.12 ± 0.03 1.11 ± 0.04 1.11 ± 0.05 0.97 ± 0.05HYP 1.03 ± 0.11 0.82 ± 0.06 0.94 ± 0.06 0.85 ± 0.05 HYP 0.76 ± 0.06 0.71 ± 0.04 0.71 ± 0.06 0.69 ± 0.03HPC 1.19 ± 0.10 1.31 ± 0.08 1.33 ± 0.06 1.40 ± 0.08 HPC 1.28 ± 0.05 1.10 ± 0.03 1.10 ± 0.04 1.04 ± 0.02#

CRF AMY 1.49 ± 0.14 1.43 ± 0.14 1.41 ± 0.13 1.29 ± 0.09 CRF AMY 1.33 ± 0.08 1.37 ± 0.08 1.34 ± 0.19 1.11 ± 0.05HYP 1.26 ± 0.11 1.09 ± 0.21 1.32 ± 0.22 1.30 ± 0.11 HYP 1.02 ± 0.20 0.93 ± 0.13 1.22 ± 0.18 1.00 ± 0.13HPC 1.30 ± 0.07 1.40 ± 0.15 1.65 ± 0.19 1.85 ± 0.09* HPC 1.12 ± 0.07 1.33 ± 0.08 1.31 ± 0.05 1.15 ± 0.06&

Maternal diet

Offspring drug treatment

Maternal diet

Offspring drug treatment

80

4.4Discussion

Thecurrentinvestigationprovidesthefirstanalysisoftheinfluenceofmaternal

HFDonoffspringbehaviouralresponsestococainesensitization.Overall,cocaine

treatmentwasassociatedwithincreasedlocomotoractivityinthedrugexposure

phase,conditionedlocomotion,andsensitizationtestsinbothsexes,whichprovided

evidencethatthedrugtreatmentwassuccessfullyexecuted.Asexpected,maternal

HFDleadtoincreasedbodyweightinoffspringduringthepre-weaningperiod.On

drugexposureday1,locomotoractivitywasincreasedspecificallyamongcocaine-

treatedHFDfemalescomparedtococaine-treatedCHDfemales.Withrepeated

exposures,however,HFDandCHDfemalesshowedasimilarmagnitudeofresponse

tococaine.Thesefindingssuggestthatonlytheinitialpsychomotorresponseto

cocaineisdifferentbetweenHFDfemalescomparedtoCHDfemales.Ithasbeen

proposedthattheacutelocomotorresponsetococainereflectsthereleaseof

dopamineinthemesocorticolimbicsystem(KoobandNestler,1997).Withrepeated

exposure,dopaminereleaseandanassociatedincreaseinglutamatergictoneoccur,

leadingtobehaviouralsensitization(BrennemanandRutledge,1982).

Thus,thefindingsinthischapterimplicateachangeindopaminergicactivity

infemaleoffspringexposedtomaternalHFD.Importantly,however,similar

behaviouraldifferenceswerenotobservedinmaleoffspring,althoughmalesdid

showchangesinstress-relatedgeneexpression.Similarly,therewereno

differencesinmaternalcarebetweenthedietgroups,ineithermaleorfemale

offspring.

81

Repeatedexposuretococaineisknowntoproducelong-lastingbehavioural

changes.,includingincreasedlocomotoractivityinanenvironmentpreviously

associatedwithcocaine(i.e.,conditionedlocomotion;(Johnsonetal.,2012)),and

increasedreactivitytoachallengeinjectionofcocaine(i.e.,cocainesensitization;

(KalivasandStewart,1991)).Inthisstudy,bothCHDandHFDoffspringdisplayeda

conditionedlocomotionandasensitizedresponsetococaine.Likewise,bothCHD

andHFDoffspringrespondedtothedrugtreatmentinasimilarmanner.HFD

femaleoffspringshowedenhancedconditionedlocomotionoverall,indicating

greatercontextualmemoryintheabsenceofdrug,butthiseffectofdietdidnot

interactwithdrugtreatment.

Duringthecocainepre-exposurephase,bothmaleandfemaleoffspring

showedenhancedlocomotoractivityinresponsetococainetreatment.However,

onlyonDay1ofthepre-exposurephase,andonlyinthefemaleoffspring,wasthere

adifferenceinresponsetococainebasedonmaternaldiet.Specifically,inresponse

tothefirstacuteinjectionofcocaineonDay1,HFDfemalesshowedenhanced

locomotoractivityrelativetoCHDfemales.ThisresultonDay1isinterestinginthat

locomotorresponsivitytoacutecocaineissuggestedtoreflectinnatesensitivityto

thedrug(KoobandNestler,1997),whereaschroniccocaine-inducedsensitization

oflocomotoractivity(wheredifferencesbasedonmaternaldietwerenotobserved)

hasbeenproposedtoreflectenhancementoftherewardingpropertiesofcocaine

(RobinsonandBerridge,1993).

Onepotentialmechanismfortheenhancedlocomotoractivitytococaine

observedinfemaleHFDoffspringonDay1ofthepre-exposurephasemaybean

82

alteredHPAsystem.ThefindingsinChapter3showedalterationsintheCORT

negativefeedbacksysteminfemaleoffspringfromHFDdams,indicatinganeffectof

maternaldietontheincreasedresponsetostressinoffspring(Sasakietal.,2013).In

addition,previousstudieshaveshownthatlevelsofthestresshormoneCORTare

closelyrelatedtotheindividual’sresponsivenesstococaine.HighCORTlevelsdue

tomaternalstressexperienceleadtoanincreaseinacutecocaine-induced

locomotion(Kippinetal.,2008).ThesefindingssuggestthatmaternalHFDexposure

mayalterlocomotoractivityinpartthroughchangesintheHPAfunctionthatare

exacerbatedbycocaineexposure.

ThefindingsinChapter3alsoshowedthatmaternalHFDexposurewas

associatedwithincreasedanxietybehaviourintheEPM(Sasakietal.,2013).This

findingisconsistentwiththefindingsfortheEPMinthischapter,wheresaline-

treatedfemaleoffspringfromHFDdamsshowedincreasedanxietycomparedto

femaleoffspringfromCHDdams.Theparadigmofrepeatedcocaineexposureused

inthischapterisknowntoinduceanxietybehaviourinrodents.However,an

increaseinanxietyincocaine-treatedfemaleCHDoffspringwasnotobserved.The

cocainepre-exposureregimentdid,however,reversetheeffectofmaternalHFDon

anxiety.Itisknownthatcuesassociatedwithdrugadministrationenhanceanxiety

duringthewithdrawalperiod(Erbetal.,2006).Futurestudiesusingpre-exposure

tocuesassociatedwiththecontextinwhichcocainewasadministeredmayenhance

anxietyintheCHDandHFDgroupsexposedtococaine.

Theanalysisofgeneexpressionrevealedchangesindopamine-andstress-

relatedgenesinseveralrelevantbrainregions,includingmPFC,NAc,VTA,AMG,

83

HYPandHPCamongHFDoffspringcomparedtocontrols(seeAppendixTable4.1

forfulldescriptionofgeneexpressionchanges).Withoneexception,however,these

changesdidnotcorrespondtothebehaviouraleffectsweobserved.Female

offspringfromHFDdamsshowedasignificantdrugbydietinteractionforCRFgene

expressionintheHPC.Therewasatrendindicatingthatsaline-injectedHFDfemale

offspringmayshowincreasedCRFcomparedtosaline-injectedCHDfemale

offspring(p=.07).Therewasalsoatrendindicatingthatcocaine-injectedHFD

femaleoffspringmayshowreducedCRFcomparedtosaline-injectedHFDfemale

offspring(p=.08).Likewise,intheanxietytesting,therewasasignificant

interactionbetweendruganddietinfemaleoffspring.Consistentwiththeresultsof

Chapter3,posthoctestsrevealedthatsaline-treatedHFDfemalesreducedtime

spentinopenarmscomparedtosaline-treatedCHDfemales,whichwasconsistent

withthedirectionofchangeinCRFgeneexpression.Contrarytothehypothesis,

repeatedexposuretococainedidnotleadtoincreasedanxiety,ascocaine-treated

HFDfemalesshowedincreasedtimespentintheopenarmsoftheEPMcomparedto

saline-treatedHFDfemales,whichwasalsoconsistentwiththedirectionofchange

inCRFgeneexpression.However,therewasnocorrelationbetweentheCRFgene

expressionandtheratiooftimespentintheopenarmsoftheEPM.

Overall,theseresultsareconsistentwithpastreportsofchangesinother

dopamineandstress-relatedgenesinducedbysimilartypesofmanipulations(Naef

etal.,2008;Vuceticetal.,2010;OngandMuhlhausler,2011).Aswillbediscussedin

furtherdetailbelow(seeGeneralDiscussion),itispossiblethatprocedural

modificationsmayrevealchangesintheexpressionofadditionalgenesthathave

84

beenpreviouslyassociatedwithdrugadministration(Nestler,2001).Analternative

proceduremayalsohelptoincreasethesensitivityofthebehaviouralmeasures.For

instance,theoveralllackofmaternaldieteffectonlocomotoractivityinmalesmay

beduetosexdifferencesinresponsivitytococainethatvaryasafunctionofdrug

dosage.Comparedtotherelativelyhighdosageselectedforthecocainepre-

exposurephase(30mg/kg),apsychomotortestingparadigmusingacute

administrationoflowerdosesofcocainemayaidinincreasingthesensitivityand

thelikelihoodofdetectingadifferenceinmales,andstrengtheningtheeffect

observedinfemales.

85

Chapter 5: Effects of maternal high fat diet and stress on

cocaine-induced locomotor activity and gene expression in

offspring

86

Chapter5:Effectsofmaternalhighfatdietandstressoncocaine-inducedlocomotoractivityandgeneexpressioninoffspring

5.1Introduction

ThefindingsinChapter3showedalterationsintheCORTnegativefeedbacksystem

inoffspringfromHFDdams,indicatinganeffectofdietontheresponsetostress,

andincreasedanxietybehaviour(Sasakietal.,2013).InChapter4,wefoundthat

therewasincreasedlocomotoractivityspecificallyamongcocaine-treatedfemales

fromHFDdamscomparedtococaine-exposedCHDfemalesondrugexposureday1

(i.e.withacutecocaineexposure).Inaddition,andsimilartotheresultsinChapter3,

saline-treatedfemaleoffspringfromHFDdamsshowedincreasedanxietyonthe

elevatedplusmaze(EPM)taskcomparedtofemaleoffspringfromcontrolCHD

dams.PreviousstudieshavefoundthatlevelsofthestresshormoneCORTare

closelyrelatedtotheindividual’sresponsivenesstococaine.HighCORTlevelsdue

tomaternalstressexperienceorpharmacologicalmanipulationsofCORTleadtoan

increaseinlocomotioninanoveltestingenvironmentaswellastoincreasedacute

cocaine-inducedlocomotion(Kippinetal.,2008).Ontheotherhand,reductionor

removalofCORTreversestheseeffects(Aubuchon-Endsleyetal.,2014).These

findingsimplythatmaternalHFDexposurealterslocomotoractivityinpartthrough

changesinHPAfunctionthatareexacerbatedbycocaineexposure.

Stressduringthegestationalperiodisassociatedwithbehaviouraland

endocrineabnormalitiesinoffspringthataresimilartothoseofmaternalHFD

exposure.Stressexposureindamsduringthelasthalfofpregnancyleadstodirect

exposureofoffspringtoglucocorticoids,whichmayresultinsmallerlittersizes

(Ryanetal.,2014).Maternalbehaviourisalsodisruptedbymaternalstress,leading

87

toincreaseddepressive-likebehavioursindamsandincreasingthelikelihoodof

cannibalismofpups(Perez-Lasoetal.,2013).Offspringexposedtomaternalstress

showheightenedHPAreactivityandincreasedanxiety(Piazzaetal.,1991;Amirand

Donath,2007;GrissomandReyes,2013).Maternalstressalsoaltersdopamine

activityandthedensityofDRD2intheNAC(Henryetal.,1995;Alfaradhiand

Ozanne,2011),andenhancesthelocomotorresponsivenesstococaine(Kippinetal.,

2008).ThesedataindicatethattheremaybesynergisticeffectsofmaternalHFD

andmaternalstressexposureonneuralsystemsunderlyingreward-andstress-

relatedbehaviour,thoughfewstudieshaveexaminedtheinteractionbetweenthe

two.

Inthischapter,weinvestigatedtheinteractionbetweenmaternalstress

duringgestation(i.e.chronicvariablestress)andmaternalHFDonsensitivityto

acutecocainetreatmentinadulthood.Wehypothesizedthatexposuretomaternal

HFDenhancestheeffectsofprenatalstressoncocaine-sensitivebehaviourand

anxietybehaviour.WefurtherhypothesizedthatmaternalHFDaswellasmaternal

stressincreaselocomotoractivityinoffspring,includinginthepresenceofcocaine.

Wehypothesizethatincreasedcocaine-sensitivebehaviourwillbeaccompaniedby

increasedexpressionofdopaminergicgenesinthemesolimbicsystem.Wealso

hypothesizethatmaternalstresswillexacerbatetheeffectsofmaternalHFDon

stress-relatedgeneexpressioninamannerconsistentwithheightenedanxiety.

Cocainewasadministeredtoeachoffspringacutelyatoneofthreedifferentdoses(0,

10and30mg/kg)everyotherdayforatotalofthreeinjectiondays.Threedifferent

anxietymeasureswereemployedafterthedrugtreatment:theEPMtask,openfield

88

task,lightanddarktransitionbox.HalfofeachgroupofHFDandCHDdamswere

exposedtoachronicvariablestress(CVS)duringthe3rdweekofgestation,

whereasnon-stressedcontroldamswereleftundisturbed(CON).Maternalcarewas

assessedasapossibleinterveningvariable.Cocaine-inducedlocomotoractivitywas

measuredinadultoffspring.Inaddition,dopamine-andstress-relatedgene

transcriptlevelswereexaminedinthemesolimbicpathway.


5.2.1.Animals

AdultmaleandfemaleLongEvansrats(7week)usedwereobtainedandprovided

adlibitumaccesstofoodandwater,asdescribedinChapter2(section2.1).


5.2.2.1.Diets

Femalebreederswereplacedononeoftwodiets:ahighfatdiet(n=19,HFD)ora

housechowdiet(n=17,CHD).Thedetailofthedietsanddietregimenwere

describedinChapter2(section2.2.1).OneHFDdamwasremovedfromthestudy

duetodifficultyduringbirth.

5.2.2.2.Chronicvariablestress(CVS)

Beginningongestationday14,asubsetofdamsfromeachdietgroup(CHD-CVS;

n=9,andHFD-CVS;n=11)wassubjectedtoan8-dayscheduleofvariablestress,as

previouslydescribed(AmirandDonath,2007;Hellemansetal.,2010;Purcelletal.,

89

2011;St-CyrandMcGowan,2015).Thestressesconsistedof:(1)afifteen-minute

forcedswimminginroomtemperaturewater(21-24°C),(2)aone-hourrestraint

challengeincylindricalrestraintchambers,(3)atwelve-hourexposuretonovel

objects(marbles),(4)anexposuretowhitenoiseandtiltedcageovernight,(5)a

twelve-hourexposuretobeddingsubmergedin450mLofwater,(6)afive-minute

exposureonanelevatedplatform,(7)lightsonovernight(8)athirty-minute

exposuretothepredatorodour2,3,5-trimethyl-3–thiazoline(TMT).Stresswas

presentedinanunpredictableorderandrestrictedtothe3rdweekofgestation,asit

isthecriticalperiodwhenmaternalstressaffectsthedevelopmentofoffspring

stresssystem(Matthews,2002).Theremainingcontroldamswereleftundisturbed

(CHD-CON;n=8andHFD-CON;n=8).


ThematernalbehaviourofdamsthathadbeenexposedtoHFDpriorandduring

pregnancywithorwithoutCVSanddamsthathadbeenexposedtoCHDwithor

withoutCVS,wasmonitoredoneachof6daysbetweenPND1andPND6(CHDCON;

n=8,CHDCVS;n=8,HFDCON;n=8,HFDCVS;n=11).Briefly,thebehaviourofeach

damwasvideo-recordedduringsix1-hourobservationperiods(i.e.,0100h-0200h,

05000h-06000h,1000h-1100h,1300h–1400h,1700h–1800h,2100h–2200h).

Foreachobservationperiod,theindividualvideorecordingsforeachdamwere

assessedin3minintervals(i.e.,20Observations/periodx6periodsperday=120


analysissystem.Additionaldetailsregardingtheassessmentofmaternalbehaviour

90

aredescribedinChapter2(section2.2.2).Duetoanunexpectedpowerinterruption,

videofilesforsomesubjectswerelost.Consequently,dependingonthepostnatal

day,thenumberofsubjectsineachconditionvaried(CHDCON;n=6-8,CHDCVS;n

=7-9,HFDCON;n=6-8,HFDCVS;n=10-11).Inaddition,oneHFDCVSdamwas

removedfromthestudyduetocannibalism.


5.2.3.1.Subjects

Asimilarnumberofoffspringfromdamswithamaternaldiet(HFDorCHD)and/or

maternalstress(CVSorCON)wereusedforthedifferenttestsdescribedbelow.A

selectionofmaleoffspring(CHD-CON;n=12,CHD-CVS;n=12,HFD-CON;n=12,

HFD-CVS;n=12)andasubsetoffemaleoffspring(CHD-CON;n=16,CHD-CVS;n=

15,HFD-CON;n=18,HFD-CVS;n=13)wereusedinbehaviouralassays.Amongthe

samesubjects,asubsetofanimalswereselectedforanalysesingeneexpression

(CHDsalinen=6,CHDcocainen=6,HFDsalinen=6,HFDcocainen=6persex).

TheexperimentaltimelinefordamsandoffspringisshowninFig.5.1.

5.2.3.2.Testsforcocaine-inducedlocomotoractivityinoffspring

91

Fig.5.1



andduringtheperiodoflactation.Onpostnatalday17,thedamsandoffspringwere

allgivenCHD.Onpostnatalday21,theoffspringwereweanedfromtheirmothers.

(B)Oncetheoffspringreachedadulthood,theyweregivenahabituationsession

(HAB)and,everyotherday,theoffspringweregiven0(i.e.,saline),10,or30mg/kg

ofcocaine.Alloffspringweretestedunderallthreedoseconditionsina

counterbalancedorder.Twodaysafterthelastexposuretoadrugsession,the

offspringweretestedintheelevatedplusmaze(EPM),openfield(OF)andlightand

darktransition(LD)taskseveryotherday.Inasubsetofoffspring,reward-and

stress-relatedgeneexpressionwasexaminedinmesolimbicandlimbicbrain

regions.Theresponsetostresswastestedinanothersetofadultoffspringusinga

restraint-inducedcorticosterone(CORT)assay.

92

Theacutepsychomotorresponsetothreedosesofcocainewasmeasuredinmale

andfemaleoffspringfrommaternalHFDandormaternalCVSdamsortheircontrol

groups(seesection5.2.3.1above)inadulthoodatapproximately65daysofage.

Onedaypriortothestartofcocaine(orsaline)injections,allratsweregivena

habituationsessionasdescribedinChapter2(section2.3.1.1.1).Duringthissession,

ratsweregivenasaline(1kg/ml,i.p.)injection,afterwhichtheywereplacedin

locomotoractivitychambersforaperiodof30min.

Beginningthedayafterthehabituationsessionandeveryotherday,rats

weregiveni.p.injectionsof0mg/kg(i.e.,saline),10mg/kgor30mg/kgofcocaine

asdescribedinChapter2(section2.3.1.1.4).Duringthissession,ratsweregivenan

injection,afterwhichtheywereplacedinlocomotoractivitychambersforaperiod

of30min.

5.2.3.3.Estruscyclemonitoringinfemaleoffspring

Attheendofeachdayofcocaine-inducedlocomotoractivitytestinginfemale

offspring,thestateofestrusornotestruswasdeterminedbyvaginalsmear.Estrus

wascharacterizedbythepresenceofnon-nucleated,cornifiedepithelialcells.Asthe

effectsofcocaineareknowntointeractwiththestateofestrusinfemalerats(Van

Swearingenetal.,2013),thestateofestruswasdeterminedtoexcludethe

possibilityoftheinfluencebyestrogenlevels.

5.2.3.4.Elevatedplus-maze

Elevatedplus-mazewasrunasdescribedinChapter2(section2.3.1.2.1).

93

5.2.3.5.Openfieldtask

OpenfieldtaskwasrunasdescribedinChapter2(section2.3.1.2.2).


LightanddarktaskwasrunasdescribedinChapter2(section2.3.1.2.3).

5.2.3.7.Immobilizationstress-inducedcorticosteroneresponse

Immobilizationstress-inducedCORTresponsewasmeasuredasdescribedin

Chapter2(section2.3.4).Theintra-assaycoefficientofvariationwas7.1%.

5.2.3.8.Geneexpressionanalysis

Asubsetofanimalswasselectedforanalysesingeneexpression(CHDCONn=6,

CHDCVSn=6,HFDCONn=6,HFDCVSn=6persex).10daysafterthelast

injectionofcocaine,thebrainsoftheseanimalswerecollectedasdescribedin

Chapter2(section2.3.1.4.1).Geneexpressionfor4dopamine-related(DRD1,DRD2,

TH,DARPP-32)and2HPA-related(CRF,GR)genesofinterestwascomparedtothe

geometricmeanofthreehousekeepinggenes(GAPDH,ACTb,UBC)in5brain

regions(AMY,HPC,PFC,VTA,NAC)foreachsex,dietanddrugconditionas

describedinChapter2(section2.3.1.4.3;seeChapter2section2.3.1.4.1for

coordinatesofeachbrainregion).Analysisofdopamine-relatedgeneswas

restrictedtorewardpathwaysandanalysisofHPA-relatedgeneswasrestrictedto

94

limbicregions,exceptforGR,whichisknowntointeractwithdopaminergic

transmissioninthePFCandNAC.

5.2.4.Statistics

Thedataexaminingtheoffspring’sacuteresponsetococainewereanalyzedas

describedinChapter2(section2.4).Thedataforeachcocainedosewereanalyzed

using2x2ANOVAswithmaternaldiet(HFDorCHD)andmaternalstress(CVSor

CON)asabetween-subjectsfactors.Anxietymeasureswereanalyzedinthesame

wayusing2x2ANOVASwithmaternaldiet(HFDorCHD)andmaternalstress(CVS

orCON)asbetween-subjectsfactors.Thedatafromthematernalcodingwere

analyzedasdescribedinChapter2(section2.4)usingathree-wayrepeated

measuresANOVA.

5.3Results

5.3.1.Maternalbodyweight,offspringbodyweightandoffspringnumbers

DamsweregivenadlibitumaccesstoHFDorCHDcontrolfor4weekspriorto

pregnancyandthroughoutgestationandlactation.DamsconsumingHFDgained

significantlymoreeightthanCHDdams[F(1,31)=27.298,P<0.001;Fig.5.2A].

WenextmeasuredoffspringweightgainuntilweaningatPND21andin

adulthood(PND65).Surprisingly,unlikeourpreviousstudies(Chapters3&4),

HFD-exposedoffspringdidnotgainmoreweightthanCHD-exposedoffspring

duringthepre-weaningperiod(Fig.5.2B).Uponweaning,HFD-exposedoffspring

receivedCHD,andCHD-exposedoffspringweremaintainedonthesamedietuntil

95

adulthood.Inadulthood,therewerenosignificantdifferencesinoffspringweight

betweenHFD-andCHD-exposedmaleorfemaleoffspring(datanotshown).

WedeterminedtheinfluenceofmaternalHFDonoffspringnumbersduringthepre-

weaningperiodaftercullinganimalstoequalnumbersacrossconditionsatbirth.

Thenumberofoffspringatbirthwasnotsignificantlydifferent(p>.05).Fig.5.2C

showsthemean(±SEM)numberofoffspringforthedifferentmaternaldiet(CHD,

HFD)andstress(CON,HFD)conditionsonPND1,10and21.Therewasasignificant

differencewithinsubjectsacrosstime[F(2,66)=5.214,p<0.01],asoffspring

numberdecreasedwithprogressivepost-nataldays.Therewasalsoasignificant

timebydietbystressinteractionthatwasobserved[F(2,66)=4.222,p<0.05].The

interactioncanbeattributedtogreateroffspringnumberforHFDCONdamsthan

HFDCVSdams,acrossPND1,10and21andgreateroffspringnumberforCHDCON

thanHFDCVSdamsonPND10and21(p<0.05).Lastly,therewasamaineffectof

stressbetweensubjects[F(1,35)=8.470,p<0.01],withCONdamshavingalarger

averagenumberofoffspringthanCVSdams.

96

Fig.5.2

Highfatdietaltersmaternalbutnotpre-weaningoffspringbodyweight.(A)

Maternalbodyweightspriortopupbirth,(B)pre-weaningpupweights(C)number

ofoffspringduringthepre-weaningperiodaftercullingatbirth.HFD=highfatdiet,

CHD=chowdiet,CON=non-stressedcontrol,CVS=chronicvariablestress.Bar,*P

<.05maineffectofdiet,Bar,#P<.05maineffectofstress,@P<.05posthoc

comparison:HFDCVS<HFDCON,&P<.05posthoccomparisons:HFDCVS<CHD

CONandHFDCON.

0

100

200

300

400

500

1 2 3 4

Bod

y W

eigh

t (g)

Weeks prior to birth

CHDHFDPupbirth

02468

101214

1 10 21

Num

ber o

f pup

s/da

m

Postnatal day

CHD CONCHD CVSHFD CONHFD CVS

0

10

20

30

40

50

60

1 10 21

Offs

prin

g Bo

dy W

eigh

t (g)

Postnatal day


#

@& &

* B

C

A

97

5.3.2.Maternalbehaviour

Themeanpercentageofmaternalbehaviouroverthesixobservationsdaysis

showninFig.5.3.Thedam’slickingandgroomingofpups(LG)andnursingofpups

inanarched-backposture(ABN)wasmeasuredasacompositescoreand

designatedLGABN.TherewasasignificantincreaseinLGABNwithexposureto

maternalHFD[F(1,20)=4.4,P=.047].Therewasnoeffectofmaternalstress.

5.3.3.Immobilizationstress-inducedCORTresponsivityinoffspring

Fig.5.4showsthemean(+/-SEM)levelsofplasmacorticosterone(ng/ml)inthe

maleoffspringjustpriortotheonsetofimmobilizationstress(0min),justafterthe

terminationofthestress(20min),andagain60minlater(80min).TheANOVA

revealedonlyasignificantmaineffectoftime[F(2,52)=185.564,p<.0001]forthe

maleand[F(2,42)=73.197,p<.0001]forfemaleoffspring.Asexpected,therewasa

significantincreaseinplasmacorticosteronelevelsbetween0ad20min(p<.0001),

andasignificantdecreasebetween20-and80-min(p<.0001).Therewas,however,

noeffectofmaternalhistory.

98

Fig.5.3

Highfatdietconsumptionindamsalteredmaternalcare.Mean± SEM percentage of

licking/grooming and arched back nursing (ABN) by dams during postnatal days 1

though 6. HFD=highfatdiet,CHD=chowdiet.CON=non-stressedcontrol,CVS=

chronicvariablestress.Bar,*P<.05maineffectofdiet.

*

99

Fig.5.4

Immobilizationstress-inducedCORTresponsivityinmale(A)andfemale(B)

offspring.Mean+/-SEMcorticosterone(ng/ml)inoffspringbefore(Time0),20and

80minaftertheonsetofimmobilizationstress.StresswasappliedfromTime0to

Time20.*Time20mindifferentfromTime0and70,p<.001.CHDCON:n=7,CHD

CVS:n=7,HFDCON:n=8,HFDCVS:n=8forthemalesandCHDCON:n=7,CHD

CVS:n=7,HFDCON:n=8,HFDCVS:n=8forthefemales.Thesedatawere

providedbySameeraAbuaish.

A B

0

200

400

600

800

1000

1200

0 20 80

Cor

ticos

tero

ne (n

g/m

L)

(min after stress onset)


0

200

400

600

800

1000

1200

0 20 80C

ortic

oste

rone

(ng/

mL)

( min after stress onset)


** *

*

100

5.3.4.Locomotoractivityduringhabituationinoffspring

Fig.5.5showsthemeandistancetraveled(cm)byoffspringduringthehabituation

session,whereanimalswereputinthelocomotorchamberafterasalineinjection

forthefirsttime.Therewasasignificanteffectofmaternaldietinmaleoffspring

[F(1,44)=10.85,P<0.01].Infemaleoffspring,therewasasignificantinteraction

betweenmaternaldietandmaternalstress[F(1,58)=4.3,P<0.05].Posthoctests

didn’trevealanydifferencesacrossconditions.

5.3.5.Acutelocomotorresponsetococaineinoffspring

Themeandistancetraveled(cm)byoffspringduringacutelocomotortestateach

doseofcocaine(0,10,30mg/kg,i.p.)isshowninFig.5.6Aformale(leftpanel)and

femaleoffspring(rightpanel).Thereweresignificanteffectsofcocainedoseinboth

males[F(2,88)=55.785,P<0.001]andfemalesF(2,116)=72.172,P<0.001].In

maleoffspring,therewasasignificantmaineffectofmaternalHFD[F(1,44)=15.57,

P<0.001],andanearlysignificant3-wayinteractionbetweencocainedose,

maternaldietandmaternalstress[F(2,88)=2.847,P=0.06]and,infemale

offspring,asignificantinteractionbetweenmaternaldietbymaternalstressF(1,58)

=7.656,P<0.01].Malesshowedsignificanteffectsofmaternaldietwithineach

doseofcocaine[0mg/kg;F(1,44)=17.637,P<0.001],[10mg/kg;F(1,44)=9.265,

101

Fig.5.5

Locomotoractivityduringthehabituationsessioninoffspring.Mean± SEM distance

traveled (cm) in 10 min during the exposure to the locomotor activity chambers for the

first time inmale(left)andfemaleoffspring(right).HFD=highfatdiet,CHD=chow

diet,CON=non-stressedcontrol,CVS=chronicvariablestress.Bar,*P<.05main

effectofdiet.

0

1000

2000

3000

4000

5000

6000

CHD HFD

Dis

tanc

e Tr

avel

ed (c

m)

ControlCVS

0

1000

2000

3000

4000

5000

6000

CHD HFD

Dis

tanc

e Tr

avel

ed (c

m)

ControlCVS

*

102

Fig.5.6

Acutelocomotorresponsetococaineinoffspring.(A)Mean± SEM distance traveled

(cm) in 10 min following an acute injection of cocaine (0, 10, and 30 mg/kg, i.p.) inmale

(left)andfemaleoffspring(right).(B)Mean± SEM distance traveled (cm) in 10 min

following an acute injection of cocaine(30mg/kg,i.p.)inmale(left)andfemale

offspring(right).HFD=highfatdiet,CHD=chowdiet,CON=non-stressedcontrol,

CVS=chronicvariablestress.Bar,*P<.05maineffectofdiet,*P<.05posthoctests,

(*)P<.05plannedcomparison.

0

1000

2000

3000

4000

5000

6000

0 10 30

Dis

tanc

e Tr

avel

ed (c

m)

Cocaine dosage in mg/kg

CHD-CONCHD-CVSHFD-CONHFD-CVS

0

1000

2000

3000

4000

5000

6000

0 10 30

Dis

tanc

e Tr

avel

ed (c

m)

Cocaine Dosage in mg/kg

CHD-CONCHD-CVSHFD-CONHFD-CVS

*A

0

1000

2000

3000

4000

5000

6000

CHD HFD

Dis

tan

ce T

rave

led

(cm

)

CONCVS

***

*

*(*)B

103

P<0.01],[30mg/kg;F(1,44)=13.801,P<0.01].Malesalsoshowedasignificant

effectofmaternalstressinresponsetothehighdoseofcocaine(30mg/kg)[F(1,44)

=3.82,P=0.05](Fig.5.6B).Infemaleoffspring,thereweresignificantinteractions

betweenmaternaldietbymaternalstressinthesaline(0mg/kg)andhighcocaine

doseconditions(30mg/kg)[0mg/kg:F(1,58)=6.231,P<0.05],[30mg/kg;F(1,58)

=4.939,P<0.05].Basedonliteratureshowingthatmaternalstresspotentiatesthe

psychomotorresponsetococaine,weexaminedthepossibilityofadifferentialeffect

ofmaternaldietonthestress-potentiatedlocomotorresponsetococaine.Inboth

malesandfemales,posthoctestswithineachdietgrouprevealedthat,atthe

30mg/kgdoseofcocaine,CVSpotentiatedthelocomotorresponsetococaineinthe

HFDgroupbutnotamongCHDcontrols(Ps<.05:Fig.5.6B).Theobserved

locomotorresponsetococaineinthefemaleoffspringwasunlikelyduetodifference

inestruscycleatthetimeoftesting,asavisualinspectionofthelocomotoractivity

dataateachdrugdosageamongestrusvsnon-estrusanimalsshowedsimilar

locomotoractivity(Fig.5.7).

5.3.6.Anxietytests


HFD-exposedoffspringspentmoretimeintheopenarmsovertotaltimecompared

toCHD-exposedoffspring;thiswasthecaseforbothmale[F(1,43)=11.242,P<

0.01]andfemaleoffspring[F(1,56)=8.527,P<0.01](Fig.5.8A).Inthemale

offspring,asinthelocomoteractivitytests,HFD-exposedoffspringtraveleda

greaterdistanceduringthetest[F(1,43)=5.031,P<0.05].

104

Fig.5.7

Estrusstatusandlocomotoractivityinfemaleoffspringforeachcocainedosage.Thenumberofsubjectsisshownatthebottomofeachbar.HFD=highfatdiet,CHD=chowdiet,CON=non-stressedcontrol,CVS=chronicvariablestress.

0mg

10mg

30mg

10 6 9 5 10 8 8 5

13 3 9 6 12 5 7 6

12 4 13 2 13 2 7 6

105

Fig.5.8

Anxiety-likebehaviourinmale(left)andfemale(right)offspring.(A)Mean± SEM

ratio time spent in open arms over total time measured in the elevated plus maze, (B)

Mean± SEM ratio time spent in the center over total time measured in the open field test

and (C) Mean± SEM ratio time spent in the light chamber over total time measured in

the light-dark transition box. HFD=highfatdiet,CHD=chowdiet,CON=non-

stressedcontrol,CVS=chronicvariablestress.Bar,*P<.05maineffectofdiet.

00.050.10.150.20.250.30.350.40.45

CHD HFD

OA

time/

tota

l tim

e ra

tio

CONCVS

00.050.10.150.20.250.30.350.40.45

CHD HFD

OA

time

/tota

l tim

e ra

tio

CONCVS

0

0.2

0.4

0.6

0.8

1

1.2

1.4

CHD HFD

Larg

eCen

ter/E

dge

time

ratio

CONCVS

0

0.2

0.4

0.6

0.8

1

1.2

1.4

CHD HFD

Larg

eCen

ter/E

dge

time

ratio

CONCVS

00.10.20.30.40.50.60.70.80.91

CHD HFD

Ligh

t/Tot

al ti

me

ratio

CONCVS

00.10.20.30.40.50.60.70.80.91

CHD HFD

Ligh

t/tot

al ti

me

ratio

CONCVS

* *

106

5.3.6.2.Openfieldtask

HFDormaternalstressexposedoffspringspentasimilaramountoftimeinthe

centerrelativetotimespentontheedgesoftheopenfieldcomparedtoCHDor

non-stressedcontrols(Fig.5.8B).Therewasnodifferenceindistancetraveledby

maleorfemaleoffspringduringthetest.

5.3.6.3.Lightdarktransitionbox

HFDormaternalstressexposedoffspringdidnotshowanydifferenceinthetime

spentinthelightcompartmentrelativetotimespentinthedarkcompartmentin

thelight-darktransitionbox(Fig.5.8C).Therewasnodifferenceindistancetraveled

bymaleorfemaleoffspringduringthetest.

5.3.7.Cocaine-primedgeneexpressioninoffspring

Geneexpressionanalyseswerecarriedoutinmesolimbicregions(PFC,NACand

VTA)aswellasinthelimbicregions(HPC,AMYandHYP)forrewardandstress-

relatedgeneexpression(Table5.1).MaleoffspringofHFDdamsshowedreduced

DRD1,DRD2andGRintheNAC,increasedCRFintheHPCandincreasedTHinthe

AMY.MalesalsoshowedadietbystressinteractioninDARPPintheVTA.Maternal

stresswasassociatedwithreducedCRFintheAMYinmaleoffspring.Overall,

femaleoffspringofHFDdamsshowedincreasedGRinPFC,whereasfemales

offspringexposedtomaternalstressshowedreducedGRinthePFC.Forthe

completetableofresultspertainingtoallthegenesexamined,seeAppendixTable

107

5.1.PleaseseetheGeneralDiscussionforananalysisofgenesshowingsimilar

effectsacrossstudies.

108

Table5.1

Mean±SEMrelativeabundanceoftranscriptsfordopaminereceptorD1(DRD1)

anddopaminereceptorD2(DRD2)inthemedialprefrontalcortex(mPFC),nucleus

accumbens(NAC)andventraltegmentalarea(VTA),andforglucocorticoidreceptor

(GR)andcorticotropinreleasingfactor(CRF)inthehypothalamus(HYP),amygdala

(AMY),andhippocampus(HPC).Geneexpressionwasassessedinmale(left)and

femaleoffspring(right)thathadbeenpre-exposedtomaternalhighfatdietandto

acuteinjectionsofcocaineinadulthood.*P<.05maineffectofdiet,#P<.05main

effectofmaternalstress.


Control Stress Control Stress Control Stress Control StressDRD1 mPFC 1.05 ± 0.09 1.14 ± 0.08 1.04 ± 0.13 1.00 ± 0.08 DRD1 mPFC 1.03 ± 0.07 1.20 ± 0.09 1.29 ± 0.13 1.15 ± 0.08

NAC 1.27 ± 0.09 1.18 ± 0.10 1.03 ± 0.06 0.80 ± 0.12* NAC 0.75 ± 0.13 0.80 ± 0.05 0.72 ± 0.17 0.85 ± 0.07VTA 1.51 ± 0.57 2.57 ± 0.86 0.74 ± 0.05 1.56 ± 0.80 VTA 1.01 ± 0.03 0.99 ± 0.07 1.02 ± 0.08 1.10 ± 0.06

DRD2 mPFC 1.29 ± 0.16 1.38 ± 0.13 1.06 ± 0.06 1.23 ± 0.07 DRD2 mPFC 0.90 ± 0.08 0.93 ± 0.06 0.77 ± 0.08 0.93 ± 0.07NAC 0.96 ± 0.11 0.90 ± 0.07 0.80 ± 0.06 0.64 ± 0.10* NAC 0.60 ± 0.10 0.75 ± 0.08 0.58 ± 0.16 0.78 ± 0.11VTA 0.89 ± 0.06 0.87 ± 0.06 0.85 ± 0.03 0.96 ± 0.07 VTA 0.75 ± 0.07 0.85 ± 0.05 0.90 ± 0.08 0.81 ± 0.06

GR AMY 1.09 ± 0.07 0.93 ± 0.06 1.06 ± 0.09 1.03 ± 0.07 GR AMY 1.20 ± 0.05 1.23 ± 0.04 1.43 ± 0.09 1.28 ± 0.08HYP 1.05 ± 0.02 0.91 ± 0.04 1.06 ± 0.04 1.04 ± 0.03 HYP 1.15 ± 0.07 1.09 ± 0.04 1.73 ± 0.66 1.08 ± 0.08HPC 2.0 ± 1.04 0.81 ± 0.09 4.03 ± 1.61 2.05 ± 1.29 HPC 1.20 ± 0.23 1.39 ± 0.37 8.29 ± 5.11 1.78 ± 0.72

CRF AMY 1.13 ± 0.10 0.78 ± 0.05 1.12 ± 0.10 1.08 ± 0.07# CRF AMY 1.36 ± 0.09 1.28 ± 0.07 1.24 ± 0.14 1.17 ± 0.06HYP 1.42 ± 0.25 1.02 ± 0.32 1.64 ± 0.31 0.99 ± 0.21 HYP 1.31 ± 0.24 1.44 ± 0.23 1.46 ± 0.30 1.82 ± 0.25HPC 1.14 ± 0.03 1.09 ± 0.02 1.24 ± 0.11 1.28 ± 0.05* HPC 1.17 ± 0.12 1.27 ±0.06 1.19 ± 0.10 1.28 ± 0.05

Maternal diet Maternal diet

Maternal stress Maternal stress

109

5.4.Discussion

Thecurrentinvestigationprovidesthefirstanalysisoftheinfluenceofthecombined

effectsofmaternalHFDandmaternalstressonbehaviouralresponsestoacute

cocaineinadultoffspring.Overall,maleHFDoffspringshowedincreasedlocomotor

activityuponfirstexposuretothelocomotoractivitybox,aswellaswithineach

drugtreatmentcondition.Inresponsetothehighdoseofcocaine(30mg/kg),male

offspringfromtheHFDCVSgroupshowedgreaterlocomotoractivitycomparedto

maleoffspringfromtheHFDCONgroup.Infemaleoffspring,maternalHFDintake

didnotaffectlocomotoractivityoverall.However,inresponsetothehighdoseof

cocaine(30mg/kg),femaleoffspringfromtheHFDCVSgroupshowedgreater

locomotoractivitycomparedtofemaleoffspringfromtheHFDCONgroup.

MaternalstresspotentiatedlocomotoractivityamongHFDoffspringbutnot

amongCHDcontrolsinbothmaleandfemaleoffspringatthehighdoseofcocaine

(30mg/kg).Similarly,inChapter4,HFDfemalesshowedenhancedlocomotor

activityrelativetoCHDfemalesontoanacuteinjectionof30mg/kgcocaineonDay

1ofthepre-exposurephase.Maternalstressisknowntoincreasecocaine-induced

locomotoractivity(Kippinetal.,2008),possiblyduetochangesinCORTlevels

(Lupienetal.,2009b).InChapter3,HFDoffspringexhibitadysregulatedCORT

negativefeedbacksystem(Sasakietal.,2013).However,inthisstudy,stress-

inducedCORTwasunaltered,andtherewasnoincreasedanxietybehaviouror

changegeneexpressionpatternwithmaternalstressexposure.Thesedatasuggest

thattheeffectofmaternalstressobservedamongHFDoffspringatthehighdoseof

cocainemaybeassociatedwithanothermechanism.

110

Onepotentialmechanismthatinteractswithmaternalstressandmaternal

HFDinresponsetococainemaybeperipheralfactorssuchasleptinthatregulate

energybalance.Forexample,leptinhasbeenshowntoregulatedopaminergicstate,

viaactionsatleptinreceptorsintheVTA,modulatingthereleaseofdopamineinthe

NACreflectiveofpsychostimulant-inducedlocomotoractivity(Hommeletal.,2006).

Ob/obmice,wherethegenescodingforleptinarelacking,showedreduced

amphetamine-inducedlocomotoractivity,whichwasrescuedbyinfusionofleptin

intotheVTA(Fultonetal.,2006).Nevertheless,maternalconsumptionofHFDis

knowntoincreaseleptinlevelsinoffspring(AlfaradhiandOzanne,2011).Similarly,

maternalstresshasbeenshowntoincreaseleptinlevelswhenCVSoffspringwere

givenHFDinadulthoodcomparedtocontroloffspring,suggestinganinteraction

betweenmaternalstressandHFDonleptinlevels(Tamashiroetal.,2009a).Thus,it

couldbethattheincreasedleptinlevelswithmaternalstressandHFDleadto

enhancedlocomotoractivitywithadministrationofthehighdoseofcocaine.

Overall,therewasasignificanteffectofacutecocaineexposureonthe

locomotorresponseregardlessofmaternaldietormaternalstressexposure.Acute

exposuretococaineproducesheightenedlocomotoractivityinrodents,andthe

degreeoflocomotoractivityhasbeenproposedtoreflecttheanimal’ssensitivityto

cocaine(KoobandNestler,1997).Whenlowtomediumcocainedosesare

administered,asinthisstudy,adoseresponsecurveininducedlocomotoractivity

isexpected.Thus,thesefindingsprovideevidencethatdrugtreatmentwas

successfullycarriedout.Contrarytoourhypothesis,however,theeffectofHFDor

maternalstressexposuredidnotvaryasafunctionofthedoseofcocainegiven.

111

MaternalHFDhadaconsistenteffectonlocomotoractivityacrosslocomotor

activitytestsessions.Maleoffspringshowedincreasedlocomotoractivityinthe

habituationsession,wheretheywereexposedtothelocomotoractivitychambers

forthefirsttime,andthroughoutthedrugsessions.Femalesshowedadietbystress

interactioninthehabituationsessionandinthe0mg/kgand30mg/kgdrugsessions.

Itispossiblethattherewasacarry-overeffectofbasallocomotoractivityon

thetestforcocaine-inducedlocomotoractivity.Otherstudiesofgroupsthatdifferin

baselinelocomotoractivity[forexample:(Kippinetal.,2008)]useself-

administrationparadigmstoassesscocainesensitivity,astheoutputmeasureis

independentoflocomotoractivity.Infuturestudies,self-administrationtaskscould

beusefultodisambiguatethepossiblecontributionofbasaldifferencesinlocomtor

activitytotheeffectsofHFDoncocaine-inducedlocomotoractivity,becausethe

self-administrationparadigmmeasuresmotivationtoseekcocaine(Robinsonand

Berridge,1993).Wewillreturntoadiscussionofthepotentialbehaviouraland

neuralmechanismsinvolvedinthemotivationalcomponentassessedbytheself-

administrationtaskintheGeneralDiscussion(section7.4.1).

Theoffspringweretestedforanxietybehaviourtwodaysafterthelast

exposuretodrugtreatment.Therewere,however,nodifferencesinanxietyinthe

OFtaskortheLDtask.Contrarytoourhypothesis,exposuretomaternalHFDwas

associatedwithreducedanxiety-likebehaviourintheEPMtaskinmaleandfemale

offspringcomparedtoCHDcontrols.Theseresultsareconsistentwiththoseof

Chapter4,whereanxietylevelsinHFDfemaleoffspringexposedtorepeatedcocaine

weresimilartothoseofCHDcontrols,andwerereducedcomparedtosaline-

112

injectedHFDrats.Acuteexposuretococainedoesnotgenerallyalteranxiety

behaviour;however,atleastonepreviousstudyhasreportedreducedanxietyin

cocaine-exposedanimals(VanSwearingenetal.,2013).Infuturestudies,the

additionofasubgroupofdrugnaïveanimals,aswasdonepreviouslyinChapter3,

couldbeusefulindeterminingtheeffectofmaternalHFDonanxietybehaviourin

theabsenceofcocaineexposure.

ItisimportanttonotethatwedidnotobservebodyweightgaininHFD

offspringduringthepre-weaningperiod,whichhasbeenshownrepeatedlytobe

oneoftheeffectsofmaternalHFDexposureinoffspring(Vuceticetal.,2010;Purcell

etal.,2011;Sasakietal.,2013).ThelackofweightgainamongHFDoffspringduring

thepre-weaningperiodcouldindicatethatmaternalHFDinthisstudydidnothave

thesameeffectsonoffspringphenotypeasinChapter3and4.Infact,maternalHFD

didnotaffectoffspring’sCORTresponsetostressinadulthoodcontrarytothe

resultsinChapter3.

Inaddition,andincontrasttotheresultsofChapter4,HFDincreased

maternalcare.Visualexaminationofthedatashowedthattheincreaseinmaternal

carewaslikelyduetotheincreasedmaternalcareprovidedbytheHFDCVSdams.

Thisresultiscontrarytotheliteratureshowingmaternalstressalonedecreases

maternalcare(ChampagneandMeaney,2006).Furtherinvestigationconcerning

theinteractionbetweenmaternalHFDandmaternalstressiswarrantedtoelucidate

potentialmechanisms.

Althoughmaternalstressalonedidnotaffectmaternalcare,offspringanxiety

behaviouroroffspringCORTresponsetostress,itaffectedoffspringsurvivalduring

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thepre-weaningperiod.Therewasasignificant3-waypostnataldaybymaternal

dietbymaternalstressinteraction.Thisanalysisrevealedthatagreaternumberof

HFDCVSoffspringthanHFDCONandCHDCONdiedduringthepre-weaningperiod.

HFDdamsareknowntoengageinahigherfrequencyofcannibalismthanCHD

dams(Bellisarioetal.,2015).Anumberofstudiesalsoshowthatprenatalstresscan

haveprofoundeffectsonneonatalbehaviour,whichinturnhasdirectconsequences

forthesurvivalofoffspring.Forexample,offspringborntostress-exposeddams

experienceadecreasedrateofultrasonicvocalization(USV)andmyoclonic

twitching(anindicatorofactivesleepinrats)(PuderandMunsch,2010).Decreased

ratesofUSVandmyoclonictwitchingwillinterferewiththepup'sabilitytocalland

communicatewithitsmother,whichinturnpreventsitsretrievaltothenest.This

maybeapotentialmechanismtoexplainwhydamsintheCVSconditionhadfewer

offspringonaveragethanCONdams.Itispossiblethatadecliningnumberofpups

duringthepre-weaningperiodleadtoacompensatoryincreaseinmaternal

behaviouramongHFDCVSdams.

Although,contrarytoourhypothesis,evidenceofincreasedsensitivityto

cocaineamongHFDexposedoffspringwasnotobserved,maleoffspringofHFD

damsneverthelessshowedreducedDRD1andDRD2intheNAC.Thesedataparallel

apreviousstudywithasimilarmaternaldietmanipulationinmalemicethatwas

associatedwithincreasedconsumptionofpalatablediet(Vuceticetal.,2010).It

shouldbenotedthatanotherpreviousstudyofmaternalHFDexposurefoundno

changeinreceptorexpressionintheNAC(Naefetal.,2008)butdecreased

locomotorsensitivitytoamphetamine.Thus,theresultsofthepresentstudy

114

indicatethatDRD1andDRD2expressionarenotlikelytobedirectlyrelatedto

locomotorsensitivitytopsychostimulants.

Infuturestudies,itwouldbeimportanttodeterminewhetherthechangein

DRD1andDRD2intheNACwithmaternalHFDarerelatedtochangesinmotivation

relatedtodrugseekingbehaviour.Infact,apreviousstudyshowedthatmice

lackingDRD2self-administeredcocaineathigherratesthanheterozygousor

wildtypelittermates(Caineetal.,2002).Theseresultsareconsistentwiththe

propositionthatdecreasedsignalingthroughdopaminereceptorsmayleadto

increasedconsumptionofrewardingstimuliinordertorestorehomeostasis(Koob

andLeMoal,2008).Aself-administrationtaskcouldbeusedtoquantifythis

relationshipinmaternalHFDanimals.

WeexpectedthatmaternalHFDwouldbeassociatedwithincreasedanxiety

behaviour,basedonourpreviousfinding(Chapter3)thatmaternalHFDleadto

increasedanxietybehaviourtogetherwithdysregulatedCORTresponsetostress

andgeneexpressionintheAMY.However,itisimportanttonotethattheseeffects

weremoreprominentlyseeninthefemaleoffspring(CORTandgeneexpression).

Basedonanumberofpreviousstudies,wealsoexpectedthatmaternalCVSwould

beassociatedwithincreasedanxietyinoffspring(Lupienetal.,2009a).TheHPA

axisisregulatedbytheHPCandtheAMYinanopposingway,wherethe

hippocampalneuronsinhibitandAMYneuronsactivatetheHPAaxis.Ingeneral,an

increaseinCRFgeneexpressionhasbeenassociatedwithanxietybehaviourinthe

AMYbutalsointhebrainglobally(Lupienetal.,2009a).Also,reducingCRFlevels

115

throughtheapplicationofantisenseoligonucleotidesresultsininhibitsanxiety

behaviour(Heinrichs,1999).

Contrarytoourhypothesis,maleHFDoffspringdidnotshowincreased

anxietybehaviour.Intheabsenceofincreasedanxietybehaviour,HFDmale

offspringstillshowedincreasedCRFintheHPCcomparedtoCHDmaleoffspring,

whichhasbeenassociatedwithincreasedHPAreactivityintheliterature(Meaney,

2001).However,increasedCRFintheHPCwasnotassociatedwithdifferences

betweenthedietgroupsintheCORTresponsetorestraintstress.Intermsofthe

relationshipbetweenmaternalstressandgeneexpression,CVSmaleoffspring

showedreducedCRFintheAMY,comparedtoCONmaleoffspring.Maternalstress

hasbeenshowntoincreaseCRFexpressionintheAMY(AmirandDonath,2007;

GrissomandReyes,2013),accompaniedbyoveractiveCORTinresponsetostress.

Thesegeneexpressiondifferencesareconsistentwiththeobservedlackof

differenceseeninanxietybehaviourandCORT.TherewerenodifferencesinCRF

expressioninfemaleoffspringbetweenmaternaldietormaternalstressconditions.

Insum,thefindingsinthischapterdidnotsupportaneffectofmaternalHFD

exposureontheresponseinoffspringtoacuteadministrationofcocaine.The

resultsoftheexaminationoflocomotoractivitysupportourpreviousobservations

implicatingtheinvolvementofchangesintheHPAinanxietybehaviourinHFD

offspring,andsuggestthepossibleinvolvementoftheHPAindifferential

modulationoflocomotoractivityinHFDoffspringwithexposuretococaine.

However,therewerenodifferencesinCORToranxietybehaviourreflecting

dysregulationoftheHPA,suggestingthatthedifferenceinlocomotoractivitymay

116

occurviaanothermechanism.Thedataongeneexpressionlevelsexaminedinthis

Chapterwillbediscussedrelativetothoseobservedwithexposuretochronic

cocaine(Chapter4)intheGeneralDiscussion.

117

Chapter 6: Effects of pregestational cocaine on cocaine-

induced locomotor activity and gene expression in offspring


SasakiA,ConstantinofA,PanP,KupferschmidtDA,McGowanPO,ErbS(2014)Cocaineexposurepriortopregnancyaltersthepsychomotorresponsetococaineandtranscriptionalregulationofthedopamined1receptorinadultmaleoffspring.Behaviouralbrainresearch265:163-170.,withpermissionfromElsevier.

118

Chapter6:Effectsofpregestationalcocaineoncocaine-inducedlocomotoractivityandgeneexpressioninoffspring

6.1Introduction

Wehypothesizedthatpalatabilityindietcontainingfatmodifiestheoffspring

dopaminergicbrainsystembyindirectexposurethroughthemothers.As

mentioned(seeChapter1),thishypothesiswasbasedonliteraturedemonstrating

thatbothnaturalanddrugreinforcerssuchasHFDandcocaineimpactsimilar

neuralmechanisms,andthatoverconsumptionofeitherleadstosimilarbehavioural

effects.Inthisstudy,weaskedwhethermaternalcocaineexposurecouldalter

offspring’ssensitivitytococaine.

Thereisevidencethatmaternalexperiencepriortopregnancycanplayan

importantroleinbehavioural,physiological,andgeneticprogrammingofoffspring.

Likewise,exposuretococaineinuterocanresultinmarkedchangesincentral

nervoussystemfunctionofoffspring.Inthisstudy,weexaminedwhetherexposure

ofratdamstococainepriortopregnancysubsequentlyaltersindicesofbehaviour,

physiology,andgeneexpressioninoffspring.Multipleoutcomemeasureswere

examinedinadultmaleoffspring:(1)behaviouralexpressionofcocaine-induced

psychomotoractivation;(2)levelsofCORTinresponsetoimmobilizationstress;

and(3)expressionofmultiplegenes,includingdopaminereceptorD1(DRD1)and

D2(DRD2),glucocorticoidreceptor(GR),andcorticotropin-releasingfactor(CRF),

infunctionallyrelevantbrainregions.AdultSprague-Dawleyfemaleswereexposed

tococaine(15-30mg/kg,i.p.)orsalinefor10days,andwerethenmatedtodrug

naïvemalesofthesamestrain.Separategroupsofadultmaleoffspringweretested

119

fortheiracutepsychomotorresponsetococaine(0,15,30mg/kg,i.p.),CORT

responsivityto20minofimmobilizationstress,andexpressionofmultiplegenes

usingquantitativePCR.Offspringofdamsexposedtococainepriortoconception

exhibitedincreasedpsychomotorsensitivitytococaine,andupregulatedgene

expressionofDRD1inthemedialprefrontalcortex(mPFC).Neitherstress-induced

CORTlevelsnorgeneexpressionofGRorCRFgeneswerealtered.Thesedata

suggestthatcocaineexposurebeforepregnancycanservetoenhancepsychomotor

sensitivitytococaineinoffspring,possiblyviaalterationsindopaminefunctionthat

includeupregulationoftheDRD1.


6.2.1.Animals

AdultmaleandfemaleSprague-Dawleyrats(7week)usedwereobtainedand

maintainedwithadlibitumaccesstofoodandwater,asdescribedinChapter2

(section2.1).Allratswereallowedatleast1weekofacclimatizationtothevivarium

beforethestartofthecocainepre-exposureregimen,whichconsistedofoncedaily

injectionsofcocainefor10days(seebelow).Afterthecocainepre-exposure

regimen,ratswereleftundisturbedfor5days,afterwhichtimetheywerehoused

withsexuallyexperiencedmaleSprague-Dawleyrats.Onemalewashousedwith

eachpairoffemalesfor7days.Afterthismatingperiod,maleswerereturnedto

theirhomecagesandfemalesweresinglyhoused.

Oftheoriginal20femalesubjects,18becamepregnant:9cocainepre-

exposedand9salinepre-exposed.Theoffspringof6damsfromeachpre-exposure

120

conditionwererandomlyselectedfortheexperiments.OnPostnatalDay1(PND1),

alllitterswereweighedandculledtosixmalesandsixfemales.Afterweaningon

PND21,offspringwerepair-housedwithalittermateofthesamesex.


6.2.2.1.Cocainepre-exposure

Thecocainepre-exposureregimenwasstartedwhentheratdamswere

approximately65daysofage.Onedaypriortothestartofcocaine(orsaline)

injections,allratsweregivenahabituationsession,toacclimatizethemtothe

experimentalapparatusandtreatmentprocedures.Duringthissession,ratswere

placedinlocomotoractivitychambers(26cmx48cmx21cm)foraperiodof30

minutes.Thentheyweregivenasaline(1kg/mli.p.)injection,afterwhichtheywere

replacedintheactivitychambersforanadditional60min.Locomotoractivityboth

beforeandaftertheinjectionwasmonitoredandrecordedbyavideotracking

systemthatmeasureddistancetraveledduringeachminofthesession(Ethovision,

NoldusInformationTechnology,Inc.,Leesburg,VA).Inordertoequatebaseline

levelsofactivityinthecocaineandsalinepre-exposureconditions,animalswere

assignedtotheconditionsbasedonactivityduringthehabituationsession.

Overasubsequentandconsecutive10-dayperiod,ratsweregivenoncedaily

injectionsofcocaineorsaline.OnDays1and10,cocaine(15mg/kg,i.p.)orsaline

injectionsweregiveninthelocomotoractivitychambers,underthesameconditions

describedforthehabituationsession.Thus,ratswereplacedinthechambersfor30

min,injectedwithcocaineorsaline,andplacedbackinthechambersforan

121

additional60-minrecordingperiod.OnDays2-9,cocaine(30mg/kg,i.p.)orsaline

injectionsweregiveninthehomecages.Thisisacocainedosingregimenthatwe

andothershavefoundpreviouslytoproducerobustbehaviouralsensitizationto

cocaine(Churchilletal.,1999;Erbetal.,2003).


Approximately8weeksaftertheterminationofthecocaineexposurephase,and

withinoneweekofweaning(whichoccurredonPND21),alldamsweretestedfor

theirlocomotorresponsetoachallengeinjectionof15mg/kgofcocaine(i.p.).For

thistest,ratswereplacedinthelocomotorchambersfor30min,injectedwith

cocaine,andplacedbackinthelocomotorchambersforanadditional60minperiod.


Thematernalbehaviourof9damsthathadbeenexposedtococainebefore

pregnancy,and9damsthathadbeenexposedtosaline,wasmonitoredoneachof5

daysbetweenPND1andPND9(i.e.,PNDs1,2,5,7,and9).Thebehaviourofeach

damwasvideo-recordedduring4consecutive1-hourobservationperiodsthat

occurredeitherinthemorning(i.e.,0900h–1000h,1000h-1100h,1100h-1200h,

and1200h–1300h)orafternoon(1300h–1400h,1400h–1500h,1500h–1600h,

and1600h–1700h).Themorningandafternoonobservationperiodsalternated

betweenobservationdays,andwerecounterbalancedbetweenconditions.Foreach

observationperiod,theindividualvideorecordingsforeachdamwereassessedin5

minintervals(i.e.,12Observations/periodx4periodsperday=48

122


analysissystem,asdescribedinChapter2(section2.2.2).Thecodingofmaternal

behaviourwascarriedoutby5trainedraters.Raterswereblindtothesubject's

maternalhistoryofcocaineexposure,andweredeterminedtohaveaninter-rater

reliabilityofmorethan85%,usingthereliabilityanalysistoolinObserver4.1

(NoldusInformationTechnology,VA,USA).

6.2.3.Proceduralmanipulationsofmaleoffspring

6.2.3.1.Subjects

An equal number of male offspring (1-2 per litter) from mothers with a history of cocaine

and saline exposure, were used for the different tests described below. Each group of rats

was selected from 6 out of 9 litters per maternal condition (saline or cocaine). The

experimentaltimelinefordamsandoffspringisshowninFig.6.1.Thischapterwas

basedonmypublishedworkonmaleoffspringonly.Althoughwedidexaminethe

femaleoffspringaswell,theyshowedhighlyanomalousanxiety-likebehaviour

(regardlessofmaternalcocaineexperience)thatwewereunabletoaccountforand

thatprecludedreasonablestatisticalanalysis.Therefore,furtheranalysesonthe

femaleoffspringbrainswerenotpursuedandare,therefore,notincludedinmy

thesis.

6.2.3.2.Testsforcocaine-inducedlocomotoractivity

Theacutepsychomotorresponseto0,10and30mg/kg(i.p.)ofcocainewas

measuredin12maleoffspringofcocaine-exposeddams,and12maleoffspringof

123

saline-exposeddams,startingonapproximatelyPND75.Onedaybeforethestartof

testing,ratswerehabituatedtothelocomotorchambers.Duringthishabituation

session,ratswereplacedindividuallyinthelocomotoractivitychambersfor30min,

afterwhichtheyweregivenani.p.injectionofsalineandplacedbackinthe

chambersforanadditional60min.Oneachof3subsequentandconsecutivedays,

ratsweregiveni.p.injectionsof0mg/kg(i.e.,saline),10mg/kg,or30mg/kgof

cocainebetweenthe30minpre-injectionand60-minpost-injectionsessions.All

ratsweretestedunderallthreedoseconditions,inacounterbalancedorder.Two

maleoffspringofcocainepre-exposeddamswereconsideredasoutliers,andwere

excludedfromtheanalyses;theiractivityscoresinresponsetoasalineinjection

weregreaterthantwostandarddeviationsfromthegroupmedian.Theseratsalso

exhibitedareducedlocomotorresponsetococaine(10mg/kgand30mg/kg,

respectively)comparedtosaline.

6.2.3.3.Immobilizationstress-inducedcorticosteroneresponsivity

Aseparatesubsetof24maleoffspring(12ofcocaine-and12ofsaline-exposed

dams)wasusedtoassessforCORTresponsivityto20minof

immobilizationstress;theseassessmentsweremadeonapproximatelyPND90,as

describedinChapter2(section2.3.4).Theintra-assaycoefficientofvariationwas

5.4%.Thedatafromonemaleoffspringofasalineexposeddamwasexcludedfrom

theanalysis,duetoprematureinterruptionofthestressregimenandresulting

failuretocollectallsamples.

124

Fig6.1

Experimentaltimelinefordamsandoffspring.(A)Damsreceivedeithercocaineor

salineeverydayfor10days.Duringthedrugpre-exposuredays(DE)1and2,the

cocainegroupreceived15mg/kgofcocaine(i.p.)andfromDE3-10,30mg/kg(i.p.)

ofcocaine(i.p.).Aftermating,pupbirthandaweekafterpupweaningatpostnatal

day21,alldamsweretestedwithachallengeinjectionof15mg/kgofcocaine(SEN).

(B)Oncetheoffspringreachedadulthood,asubsetofmaleoffspringweretestedfor

inahabituationsession(HAB)and,oneachofthreesubsequentdays,weregiven0

(i.e.,saline),10,or30mg/kgofcocaine.Alltheoffspringweretestedunderallthree

doseconditionsinacounterbalancedorder.Theresponsetostresswastestedin

anothersetofadultmaleoffspringusingarestraint-inducedcorticosterone(CORT)

assay.Inathirdsetofadultmaleoffspring,reward-andstress-relatedgene

expressionwasexaminedinmesolimbicandlimbicbrainregions.

125

6.2.3.4.Geneexpressionanalysesinmaleoffspring

Thebrainsofaseparatesubsetof12maleoffspring(6ofcocaine-and6ofsaline-

exposeddams),atapproximatelyPND130,werecollectedandprocessedfor

expressionofthefollowinggenes:DRD1,DRD2,GRandCRF,asdescribedinChapter

2(section2.3.5).Theprimersusedinthisstudyweredesignedusingsequence

informationfromGenBankattheNationalCenterforBiotechnologyInformation

(NCBI;www.ncbi.nlm.nih.gov),andafreelyavailableonlineprimerdesigntool

(Primer3;http://primer3.sourceforge.net).Theefficacyofeachprimersetwas

verifiedaccordingtothemanufacturer’sprotocolforaStepOnePlusrealtime

thermocycler(LifeTechnologies,CA,USA)byensuringefficiencylevelsgreaterthan

90%.UBC:F:5’-CACCAAGAAGGTCAAACAGGAA-3’;R:5’-

AAGACACCTCCCCATCAAACC-3’,GAPDH:F:5’-ACATCAAATGGGGTGATGCT-3’and

R:5’-GTGGTTCACACCCATCACAA-3,’ActinB:F:5’-TTTGAGACCTTCAACACCCC-3’and

R:5’-ATAGCTCTTCTCCAGGGAGG-3’,DRD1:F:5’-TCCACTCTCCTGGGCAATAC-3’and

R:5’-CAGGACAGCCACCAAGAGAT-3’,DRD2:F:5’-TCCCAGCAGAAGGAGAAGAA-3’and

R:5’-GTGGGATGTTGCAATCACAG-3’,GR:F:5’-CTCGAAAGGCTCCACAAGCAATGT-3’

andR:5’-GCAATGCTTTCTTCCAGAAGCCGA-3’,CRF:F:5’-TTCCTGTTGCTGTGAGCTTG-

3’andR:5’-TCACCTTCCACCTTCTGAGG-3’.

6.2.4.Statistics

Thedatafromthecocainepre-exposurephasewereanalyzedusingatwo-way

repeatedmeasuresANOVAwithpre-exposure(cocaineorsaline)asthebetween

126

subjectsfactorandpre-exposureday(1and10)asthewithinsubjectsfactor.

Student’st-testswereusedforcomparisonsbetweenDays1and10.

Thedatafromthetestforcocainesensitizationwereanalyzedusing

student’st-testforthefactorofpre-exposurecondition(cocaineorsaline)inthe

first30minoftesting.Althoughalltestsessionswere60mininduration,careful

scrutinyofthebehaviouraldataindicatedthatbetween-groupdifferenceswere

revealedonlyinthefirst30minofeachsession,afterwhichtimeactivitylevels

returnedtobaselineinbothconditions;therestorationofbaselineactivitywas,

however,accompaniedbyahighlevelofvariabilitythatinterferedintheexpression

ofasignificanttimebyconditioninteraction,overall.Thispatternofresultsisvery

consistentwithourpreviousworkusingsimilarconditioningprotocols(Johnsonet

al.,2012).

Thedatafromthematernalcodingwereanalyzedusingarepeated

measuresANOVAwithpre-exposure(cocaineorsaline)asthebetweensubject

factorandpostnatalday(PND1,3,5,7,and9)asthewithinsubjectsfactor.Thedata

examiningtheoffspring’sacuteresponsetococainewereanalyzedusingarepeated

measuresANOVAwithmaternalhistory(cocaineorsaline)asabetween-subjects

factorsandcocainedose(0,10,and30mg/kg)asawithinsubjectsfactorinthefirst

10minoftesting.Inthiscase,carefulscrutinyofbehaviouraldataindicatedthat

between-groupdifferenceswererevealedonlyinthefirst10minofeachsession,

afterwhichactivitylevelsrapidlyreturnedtobaselinelevelsunder--aswasthe

caseinthetestforsensitizationinthedams--considerablevariabilitythat

interferedintheexpressionofasignificanttimebyconditioninteraction.

127

Thedatafromstress-inducedCORTwereanalyzedusingarepeated

measuresANOVAwithmaternalhistory(cocaineorsaline)asthebetweensubject

factorandtimeafterstressonset(0,20,and70mins)asthewithinsubjectsfactor.

Thedatafromgeneexpressionassayswereanalyzedusingstudent’st-testsforthe

factorofmaternalhistory(cocaineorsaline).

AllanalyseswereperformedusingIBMSPSSStatisticsversion21forMacOS.

Dataarepresentedasmeanvalues±SEMthroughout.Inallcases,statistical

significancewassetatp≤0.05.Incaseswheremaineffectsandinteractionswere

statisticallysignificant,posthoctestswithaBonferronicorrectionformultiple

comparisonswereusedtoclarifybetween-groupdifferences.

6.3.Results

6.3.1.Effectofrepeatedcocaineexposurepriortoconceptiononlocomotor

activityandmaternalbehaviourindams

6.3.1.1.Locomotoractivityduringcocainepre-exposure

Figure6.2showsthemean(±SEM)distancetraveled(cm)bydamsonDays1and

10ofthepre-exposurephase.Cocainerelativetosalinepre-exposeddams

exhibitedahigherleveloflocomotoractivityonbothDay1andDay10,andcocaine

pre-exposeddamsexhibitedahigherlevelofactivityonDay10relativetoDay1.

Overall,repeatedmeasuresANOVArevealedasignificantmaineffectofdrugpre-

exposure[F(1,16)=31.79,p<.0001],andasignificantinteractionofdrugpre-

exposurexday[F(1,16)=4.55,p<.05]thatcanbeattributedtoanincreasein

activitybetweenDay1toDay10incocainepre-exposeddams(p<.05).

128

6.3.1.2.Locomotoractivityduringtestforcocainesensitization

Approximately8weeksafterthelastpre-exposure,andwithinaweekofweaning

(i.e.,PND21),alldamsweregivenatestforlocomotorsensitizationinresponsetoa

cocainechallenge.Figure6.3showsthat,inthefirst30minoftesting(seedata

analysissectionforrationaleforlookingatfirst30min),damspre-exposedto

cocaineexhibitedasignificantlyhigherleveloflocomotoractivityinresponsetoan

acutecocainechallengerelativetodamspre-exposedtosaline(p<.05).Thus,the

effectsofrepeatedcocaineexposurepersistedoverasignificantperiodoftime,that

encompassedtheconception,birth,andweaningofagenerationofoffspring.

6.3.1.3.Maternalbehaviour

Figure6.4showsthemean(±SEM)percentageofmaternalbehaviouracrosseach

4-hobservationperiod,over5observationdays(seeSection2.2.1.3.fordetailson

thecalculationofthesevalues).Damlickingandgroomingofpupand/ornursing

pupinanarched-backposturewasmeasured,basedonpreviousevidencethat

thesebehaviourscanalterfunctionoftheHPAaxisandresponsetostressin

offspring(Liuetal.,1997).Althoughtherewasamaineffectofday[F(4,68)=3.47,p

<.05],therewerenoeffectsofmaternalhistory(Cocaine,Saline).

129

Fig. 6.2

Locomotoractivityduringcocainepre-exposureindams.Mean ± SEM distance

traveled (cm) by dams in 30 min, during Days 1 and 10 of the cocaine pre-exposure

phase. *Cocaine different than Saline, p<.001; ** Day 1 different than D10 cocaine,

p<.05.

0!

5000!

10000!

15000!

20000!

25000!

30000!

Day 1! Day 10!

Tota

l Dis

tanc

e Tr

avel

ed (c

m)!

Saline!Cocaine!

***"*"

130

Fig. 6.3

Locomotoractivityduringtestforcocainesensitizationindams.Mean ± SEM

distance traveled (cm) by dams in the first 30 min following a challenge injection of

cocaine (15mg/kg, ip). This test took place approximately 8 weeks after the last day of

the pre-exposure phase, and after the weaning of offspring. *Cocaine different than

Saline, p<0.05

0!

5000!

10000!

15000!

20000!

25000!

30000!

Dis

tanc

e Tr

avel

ed (c

m)!

Saline!Cocaine!

*"

131

Fig. 6.4

Maternalbehaviourindams.Mean percentage (%) ± SEM of the frequency of licking, grooming and/or arched-back nursing (ABN) on PND 1, 3, 5, 7, or 9.

0!10!20!30!40!50!60!70!80!

1! 3! 5! 7! 9!

% L

icki

ng/g

room

ing

AB

N!

Postnatal day!

Saline!Cocaine!

132

6.3.2.Effectofrepeatedcocaineexposurepriortopregnancyonbehavioural,

physiological,andgeneticresponsesinmaleoffspring

6.3.2.1.Acutelocomotorresponsetococaineinmaleoffspring

Figure6.5showsthemean(±SEM)distancetraveled(cm)bymaleoffspringduring

thefirst10minofeachlocomotortest(seedataanalysissectionforrationalefor

lookingatfirst10min).Thereweresignificanteffectsofcocainedose(0,10,30

mg/kg,i.p.)[F(2,40)=22.82,p<.0001]andmaternalhistory(Cocaine,Saline)

[F(1,20)=4.33,p=.05],andasignificantinteractionofmaternalhistorybydose

[F(2,40)=3.81,p<.05].Post-hoctestsrevealedasignificantdifferenceinlocomotor

activity,withthehighdoseofcocaine(30mg/kg,i.p.),betweenmaleoffspringof

cocaineandsalinepre-exposeddams(p<.01).

6.3.2.2.Immobilizationstress-inducedCORTresponsivityinmaleoffspring

Figure6.6showsthemean(±SEM)levelsofplasmaCORT(ng/ml)inthemale

offspringjustpriortotheonsetofimmobilizationstress(0min),justafterthe

terminationofthestress(20min),andagain50minlater(70min).TheANOVA

revealedonlyasignificantmaineffectoftime[F(2,42)=62.62,p<.0001].As

expected,therewasasignificantincreaseinplasmaCORTlevelsbetween0and20

min(p<.0001),andasignificantdecreasebetween20-and70-min(p<.0001).

Therewas,however,noeffectofmaternalhistory.

133

Fig. 6.5

Acutelocomotorresponsetococaineinmaleoffspring.Mean ± SEM distance traveled

(cm) by offspring in the first 10 min following an acute injection of cocaine (0, 10 and 30

mg/kg, i.p.). * Cocaine different than Saline, p<.05; ** 30 mg/kg different than Saline,

p<0.01.

0!

1000!

2000!

3000!

4000!

5000!

6000!

0! 10! 30!

Tota

l Dis

tanc

e Tr

avel

ed (c

m)!

Dose of Cocaine (mg/kg, i.p.)!

Saline!Cocaine!

**"

*"

134

Fig. 6.6

Immobilizationstress-inducedCORTresponsivityinmaleoffspring.Mean ± SEM

corticosterone (ng/mL) in offspring before (Time 0), 20 and 70 min after the onset of

immobilization stress. Black line shows the duration of stress. * Time 20 min different

from Times 0 and 70, p<.001.

0!100!200!300!400!500!600!700!800!900!

0! 20! 70!

Cor

ticos

tero

ne (n

g/m

L)!

(min after stress onset)!

Saline!

Cocaine!

***"

***"

135

6.3.2.3.Expressionofdopaminereceptorandstress-relatedgenesinmale

offspring

Table6.1showsthemean(±SEM)relativeabundanceoftranscriptforeachofthe

genesthatwereanalyzedinseveralrelevantbrainregions,includingmPFC,NAc,

VTA,AMG,HYPandHPC.Ofthegenesandregionsassessed,asignificanteffectof

maternalhistorywasfoundonlyfortheDRD1transcriptinthemPFC.Male

offspringofdamsthathadbeenpre-exposedtococaine,ascomparedtosaline,

beforepregnancyshowedahigherrelativeabundanceoftheDRD1transcriptin

mPFC(p<.01).

6.4.Discussion

Thepresentstudyyielded4majorfindings.First,maternalexposuretococaine

priortopregnancyresultedinanenhancedsensitivitytothepsychomotor

activatingeffectsofcocaineintheadultmaleoffspring.Second,thisenhanced

sensitivitytococaineinthemaleoffspringwasassociatedwithaselective

upregulationoftheDRD1geneinthemPFC.Third,maternalexposuretococaine

priortopregnancyhadnoeffectonmaternalbehaviourduringlactation,andprior

toweaning;thusthepositivefindingscannotbeattributedtodifferentialrearingof

offspring.Finally,maternalexposuretococainepriortopregnancyhadnoeffecton

136

Table 6.1

Expressionofdopaminereceptorandstress-relatedgenesinmaleoffspring.Mean

±SEM relative abundance of transcripts for dopamine receptor D1 (DRD1) and dopamine

receptor D2 (DRD2) in medial prefrontal cortex (mPFC), nucleus accumbens (NAC) and

ventral tegmental area (VTA), and for glucocorticoid receptor (GR) and corticotropin

releasing factor (CRF) in hypothalamus (HYP), amygdala (AMG), and hippocampus

(HPC). Gene expression was assessed in male offspring of dams that had been pre-

exposed to Cocaine or Saline before pregnancy. *DRD1 in mPFC different in Cocaine

than in Saline condition, p<.01.

Mean relative abundance (± SEM)

Maternal Drug Treatment Gene Region Saline Cocaine DRD1 mPFC 0.87 ± 0.03 1.03 ± 0.03** NAC 0.92 ± 0.09 1.04 ± 0.75 VTA 1.64 ± 0.41 1.39 ± 0.34 DRD2 mPFC 1.07 ± 0.05 1.00 ± 0.05 NAC 1.07 ± 0.07 1.08 ± 0.10 VTA 1.24 ± 0.24 1.34 ± 0.12 GR AMY 1.38 ± 0.10 1.57 ± 0.18

HYP 0.95 ± 0.05 1.03 ± 0.06 HPC 0.88 ± 0.03 0.92 ± 0.04

CRF AMY 1.39 ± 0.18 1.49 ± 0.05 HYP 0.97 ± 0.10 0.89 ± 0.10 HPC 0.88 ± 0.09 1.07 ± 0.11

Saline < Cocaine, **p < .01

137

stress-inducedplasmaCORTlevelsinmaleoffspring,orontheexpressionofGRor

CRFgenesinkeylimbicregionsoftheseoffspring.Thus,undertheconditions

assessedinthepresentstudy,intergenerationaltransmissionoftheeffectsof

maternalcocaineexposureoccurredindependentofdirectexposureofoffspringto

thedruginutero;moreover,theeffectswereselectiveforaspecificalterationinthe

geneexpressionofcorticaldopamineneurons(i.e.,upregulationoftheDRD1in

mPFC),andaspecificbehaviourthatisknowntorelyontheintegrityofthe

mesocorticolimbicdopaminesystem(i.e.,acutecocaine-inducedpsychomotor

activation).

Oureffectofmaternalcocaineexposurepriortopregnancyontheacute

psychomotorresponsetococaineinmaleoffspringisconsistentwithseveralrecent

studiesdemonstratingsimilareffectsofmaternalexposuretocannabinoidsor

morphineondopamine-relatedbehavioursinasubsequentgeneration.For

example,femaleratsexposedtocannabinioidsduringadolescence,and

subsequentlymatedduringadulthood,producedmaleoffspringthatexpressed

enhancedsensitivitytomorphine-inducedconditionedplacepreference,relativeto

controls(Byrnesetal.,2012).Likewise,femaleratsexposedtomorphineduring

adolescence,andsubsequentlymatedduringadulthood,producedoffspringthat

expressedenhancedmorphine-inducedlocomotorsensitization,relativetocontrols

(Byrnes,2005).Ofrelevancetointerpretingthepresentfindings,bothdrug-

inducedconditioningofplacepreferencesanddrug-inducedlocomotorsensitization

arebehavioursthat,likeacutedrug-inducedpsychomotoractivation,aremediated

bythemesocorticolimbicsystem(SteketeeandKalivas,2011).

138

Toourknowledge,transgenerationaltransmissionofcocaineeffectson

behaviourhasonlybeenexaminedintheoffspringofsires(asopposedtodams)

thathadbeenexposedtococainepriortobreedingand,moreover,onlyinsiresthat

hadself-administeredcocaine(asopposedtobeengivennon-contingentinjections

ofcocaine).Morespecifically,maleratsthathadself-administeredcocainefor60

days,andweresubsequentlymatedtodrugnaïvedams,producedmaleoffspring

thatinfactacquiredcocaineself-administrationmoreslowlyandconsumedless

cocainerelativetocontrols(Vassoleretal.,2013).Whereasourfindingofenhanced

sensitivitytothepsychomotoractivatingeffectsofcocaineinmaleoffspringwould

seematoddswithwhatmightbeexpectedbasedontheseself-administration

results,ourfindingis,asdiscussed,consistentwiththepreviousresultsbasedon

maternalexposuretomorphineandcannabinoidspriortopregnancy.The

differencesinoutcomebetweenourstudyandthatofVassolerandcolleagues

(Vassoleretal.,2013)maybeattributedtoseveralfactors,includingwhether

transmissionofcocaineeffectsoccurredviadamsorsires,thebehaviouraloutcome

assessed(psychomotoractivationversusdrugself-administration),themethodof

parentalcocaineexposure(non-contingentinjectionsversusself-administration),

andthestringencyofcocaineexposure(7non-contingentinjectionsversus60days

ofself-administration).Indeed,itispossiblethatanyoneormoreofthesefactors

wouldproducedifferentialchangesinthecircuitsmediatingtheoutcomesobserved.

Correspondingtoanincreaseinsensitivitytothepsychomotoractivating

effectsofcocainethatweobservedinmaleoffspring,wefoundincreasedmRNA

expressionofDRD1inthemPFCofthesesameoffspring.Thisfindingisconsistent

139

withseveralrelatedfindingspointingtoanimportantroleforcorticalDRD1inlong-

lastingbehaviouraleffectsofcocaine.Mostnotably,activationofDRD1receptorsin

themPFChasbeenfoundtoplayacriticalroleinthereinstatementofcocaine

seekinginrats(e.g.,(Sanchezetal.,2003;SunandRebec,2005;SteketeeandKalivas,

2011;Lasseteretal.,2013;Sanchezetal.,2013)).Likewise,DRD1inmPFChasbeen

foundtoplayaroleintheexpressionofcocainesensitization,thoughinamanner

reflectinganinhibitoryratherthanfacilitoryrole(Sorgetal.,2001;Sorgetal.,2004).

Thus,althoughtheresultssuggestapossibleroleformPFCDRD1inthe

transgenerationaleffectsofmaternalcocaineexposure,theextenttowhichthis

effectisfunctionallyrelatedtotheincreasedsensitivitytothepsychomotor

activatingeffectsofcocaineisunknownatthistime.However,theDRD1and

correspondingbehaviouraleffectassociatedwithahistoryofmaternalcocaine

exposurepriortopregnancy,andthusintheabsenceofdirectexposureofoffspring

tococaineduringdevelopment,suggestapotentialroleforepigeneticmechanisms

inproducingchangesthataretransmissibletoasubsequentgeneration.

OfpossibleadditionalrelevancetoourfindingofupregulatedDRD1inmPFC

ofmaleoffspringistherecentfindingofanincreaseinBDNFmRNAexpressionin

themPFCofmaleoffspringofsiresthathadself-administeredcocaine;this

increasedBDNFexpressionwasattributedtoepigeneticreprogrammingofhistone

acetylationinthemalegermlineand,moreover,itcorrespondedtoanincreased

behaviouralsensitivitytococaine(Vassoleretal.,2013).Thisresultisnoteworthy,

giventhatdopaminereceptorsarethoughttoplayanessentialroleinregulating

BDNFexpressionincorticalregions,andthatDRD1signalinginthemPFCmaybe

140

especiallyimportantinthisregard(e.g.,(Zhangetal.,2002;Hasbietal.,2009;Xing

etal.,2012)).Inthecurrentstudy,ourrationaleforlookingatmRNAlevelswas

basedonthisandadditionalevidencethatbehaviouralsensitivitytococainecanbe

modifiedviaepigeneticmechanismsthataltermRNAlevels(e.g.,(Renthaletal.,

2009;Vassoleretal.,2013)).Thus,giventhatchangesintranscriptlevelsareclosely

associatedwiththeseepigeneticmechanisms,wefocusedongeneregulationatthe

mRNAlevel.Futurestudies,however,shouldalsoexaminefunctionalchangesin

proteinassociatedwithtranscriptionaldifferencesintheDRD1gene.

Inthepresentstudy,wemonitoredmaternalbehaviourpriortoweaning,in

ordertodeterminewhetheranyeffectsinthenextgenerationcouldbeattributedto

theeffectofdifferentialrearingbasedonmaternalhistory.Indeed,maternal

behaviour(e.g.,frequencyoflickingandgrooming)isknowntoalterstress

reactivityinpupsviaprogrammingofthehypothalamicpituitaryadrenal(HPA)axis

(Liuetal.,1997),andthesechangesare,inturn,relatedtocertainbehavioural

outcomes,suchasincreasedcocaine-inducedpsychomotoractivityofoffspringin

adulthood(Kippinetal.,2008).Thelackofchangeinmaternalbehaviour,eitherin

thefrequencyoflickingorgrooming,thatweobservedinthepresentstudyisinfact

consistentwiththeresultofapreviousexperiment,inwhichasimilarlackofeffect

ofpre-pregnancycocaineexposureonmaternalbehaviourwasobserved(Petruzzi

etal.,1997).Thus,undertheconditionsofthepresentstudy,wecanbereasonably

assuredthatthepositiveeffectsofmaternalhistorythatweobservedreflected

epigeneticchangesbroughtaboutbymaternalexposuretococainepriorto

pregnancy,andnotchangesinducedbydifferentialrearingofoffspring.

141

Afinalobjectiveofourstudywastodeterminewhethermaternalexposure

tococainepriortopregnancywouldalter,inheroffspring,eitherthefunctionalityof

theHPAaxisortheexpressionofstress-relatedgenesinkeyregionsofthelimbic

system.Ourrationaleforexaminingthesemeasureswasbasedonconsiderable

evidencethatexposuretopsychostimulants,includingcocaine,altersHPAfunction

and/orstress-relatedneuralcircuits.Forexample,pharmacologicalmanipulations

ofcirculatingCORTlevelsarealteredbycocaineself-administrationinrats

(Mantschetal.,1998),andacutecocaineadministrationinducesactivationofthe

HPAaxisinrats(e.g.,(Sarnyaietal.,1992;Sarnyaietal.,1993)).Likewise,repeated

exposuretococaine,underconditionssimilartothoseusedinthepresentstudy,

inducesalong-termsensitizedlocomotorresponsetoanintracranialinjectionof

CRF,andthissensitizedresponsecorrespondstoincreasedneuronalactivityinthe

amygdala(Erbetal.,2003).Alsoofpossiblerelevanceforthepresentstudy,ithas

beenfound,bothinhumansandrodents,thatinuteroexposuretococainealtersthe

functionalityoftheHPAaxisinoffspring(Jacobsonetal.,1999;Campbelletal.,

2000;Chaplinetal.,2010;Salas-Ramirezetal.,2010).Althoughwefailedtoshow

anytransgenerationalalterationsinthestress-relatedmarkersthatweexamined,it

mustbeborninmindthatwedidnotcarryoutanexhaustivestudyofpossible

markersand,moreover,thatourregimenofmaternalcocaineexposurewas

relativelymild.Thus,thepresentfindingsdonotallowustoruleoutthepossibility

thatmaternalexposuretococainepriortopregnancycanproduce

transgenerationaleffectsonthedevelopmentandfunctionalityofstresssystemsin

theoffspring.Ontheotherhand,thepresentstudydoesallowustoconcludethata

142

relativelymildregimenofmaternalcocaineexposureissufficienttoproduce

transgenerationalalterationsinamarkerofdopaminefunction(i.e.,upregulationof

DRD1)andarelatedandrelevantbehaviour(i.e.,cocaine-inducedpsychomotor

activation).Thus,amoreextensivecharacterizationofpossibletransgenerational

effectsofcocaineexposure(thatmightincludemorestringentcocaineexposure

regimens)iswarrantedinfuturestudies.

143

Chapter 7: General Discussion

144

Chapter7:GeneralDiscussion

Thereare3majorfindingstoemergefromtheworkdescribedwithinthis

dissertation(seeAppendixTable7.1forasummaryofmythesisfindings).First,

maternalHFDresultedinincreasedanxietybehaviourandcorrespondingstress-

relatedgeneexpression.Second,maternalHFDandstressincreasedlocomotor

activity.Third,therewasanoveralllackofinteractionbetweenmaternalHFDand

cocaine-inducedbehaviourandgeneexpression.Inaddition,itwasfoundthat

maternalHFDconsistentlyleadtoincreasedbodyweightindamsandpre-weaning

offspring,andthatmaternalmanipulationsdidnotaltertheoverallqualityof

maternalcare(saveforamodestsignificanteffectofmaternalHFDonmaternalcare

inChapter5).Below,Iwilldiscusseachoftheseoutcomesinturn.

7.1.MaternalHFDresultedinincreasedanxietybehaviourandcorresponding

geneexpressionchanges

TheexperimentsinChapter3revealedchangesinglucocorticoidsignalingin

responsetoperinatalHFDexposureinlimbicareasknowntoregulateHPAfunction

andanxiety-likebehaviour.TheresultsshowedthatmaternalHFDexposureis

associatedwithalteredexpressionofcorticosteroidreceptorsandinflammatory

genesinthehippocampusandamygdalaofadultoffspring.Thesechangesinneural

geneexpressionmaycontributetotheobservedincreasedCORTresponsetostress

andanxietybehaviour.Importantly,theresultsindicatethatexposuretoHFDonly

duringtheperiodpriortoweaningissufficienttoinducethesechanges.

145

InChapter4,offspringweregivenrepeatedexposuretoeithercocaineor

salineinadulthood,andwerethentestedontheEPM.Inthesestudies,thedrug-

naïveconditionwassimilartotheconditionsinchapter3,providingameansto

comparethebehaviouralresponseintheEPMtopreviousresultsobtainedin

Chapter3andexaminethereplicabilityofthefindings.Consistentwiththefindings

inChapter3,saline-treatedHFDfemalesshowedincreasedanxietybyenteringthe

openarmslesscomparedtosaline-treatedCHDfemaleoffspring.

MaleandfemaleoffspringshowedasimilareffectofmaternalHFDonthe

CORTresponsetostressinadulthood.IntermsofbasallevelsofCORTamong

offspringexposedtomaternalHFD,malesshowedsignificantlylowerlevelsofCORT

andfemalesshowedatrendtowardslowerlevelsofCORT.Overall,offspring

exposedtomaternalHFDshowedaheightenedCORTresponsetorestraint

comparedtocontrols,aneffectthatwasparticularlypronouncedamongfemale

offspring.ThesedatasuggestimpairednegativefeedbackinhibitionamongHFD

offspringandconcurwithsomebutnotallpreviousstudiesontheeffectsof

maternalHFDexposureontheCORTresponsetostresschallenge(Walkeretal.,

2008;Shalevetal.,2010;Auvinenetal.,2011).Interestingly,thedataarealso

consistentwiththeincreasedCORTresponsetorestraintstressobservedinfemale

offspringfromdamsexposedtopsychosocialstressduringgestation(McCormicket

al.,1995;Weinstock,2007),aswellasdatafromhumanfemales(Kudielkaand

Kirschbaum,2005).ChangesinbasalCORTandstress-activatedCORTare

commonlyassociatedwithanxietybehaviour(Lupienetal.,2009b),providing

146

evidencethatmaternalHFDandmaternalstressmayaffectbehaviourviasimilar

neuralmechanisms.

Limbicbrainareas,includingthehippocampusandamygdala,mediatethe

endocrineresponsetopsychosocialstressviatheactivitiesofcorticosteroid

receptors(Groenewegetal.,2011).BasallevelsofCORTandthethresholdof

activationofthestressresponseareprimarilysetbylevelsofMR.Incontrast,the

loweraffinityGRisinvolvedintheimmediateadaptiveCORTresponseto

psychosocialstress(Joelsetal.,2008).Assuch,GRactivityintheAMYisassociated

withincreasedCORTactivation,andGRactivityintheHPCisassociatedwith

inhibitionofthereleaseofCORTinresponsetoastressor.Theselimbicareasdirect

responsestostressviaprojectionstonucleiintheHYPsuchastheparaventricular

nucleus.Inturn,theparaventricularnucleusdirectsthereleaseof

adrenocorticotropinreleasinghormone,whichthenleadstothereleaseofACTH

fromthepituitaryandintocirculation,regulatingCORTreleasefromtheadrenal

cortex.

Similarly,myfindingssupportpreviousobservationsthatlevelsofMRand

GRinlimbicareasareassociatedwiththeresponsetostress,extendingthese

observationstotheeffectsofmaternalHFD.MaternalHFDoffspringshowed

increasedlevelsofMRandGRintheAMYinassociationwithdecreasedbasallevels

ofCORTandanincreasedCORTresponsetostress.Thesedataareconsistentwith

otherstudiesshowingthatincreasedGRintheAMYincreasestheCORTresponseto

stress(Joelsetal.,2008).Thedataalsoconcurwithapreviousstudyshowingthat

147

diet-inducedobesityisassociatedwithincreasedMRintheAMY(Johrenetal.,

2007).

MaternalHFDalsoledtosexdifferencesingeneexpressioninthisstudy,

withfemaleoffspringshowingthemostchangesingeneexpression.Fewother

studieshaveexaminedtheeffectsofmaternalHFDonglucocorticoidand

downstreamimmunesignalingmechanisms.However,otherstudiesexaminingsex

differencesinplacentaltissueindicatethatfemaleoffspringshowagreaternumber

oftranscriptionalalterationswithexposuretomaternalHFDthanmales,includinga

greaternumberofgenesinvolvedinimmuneresponses(Maoetal.,2010;Gaboryet

al.,2012).ThefindingsinChapter3andtheresultsfromChapter4alsoindicated

thattheeffectsofmaternalHFDleadtoincreasedanxietyinfemaleoffspring.Other

studieshaverevealedthatfemalesareparticularlysensitivetoalterationsin

immunegenesthatareknowntoaffectanxietybehaviour(Bouret,2009;Schwarz

andBilbo,2012).Togetherwithourdata,thesefindingssuggestasexually

dimorphicresponsetomaternalHFDduringdevelopmentthatmayariseasa

functionofchangesinglucocorticoidanddownstreamimmunegeneexpression,

particularlyinfemales.Themannerbywhichsexsteroidsmayimpactthesesex

differencesremainstobedetermined.Notably,thesechangesappeartobelong-

lastingandariseasafunctionofdietexposure,suggestingthepotentialinvolvement

ofepigeneticmechanisms.

EpigeneticmodificationstochromatinstructureandDNAmethylationcan

altergeneexpressionandcellularphenotypeinalong-termmannerintheabsence

ofvariationsinDNAsequence.Others',andourpreliminary,dataindicatethatthe

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epigeneticmachineryisalteredinadultoffspringbrainsasafunctionofmaternal

HFDconsumption.Epigeneticmechanismsappeartosomeextentresponsiveto

environmentalfactors.Futurestudieswithepigeneticprofilesindicativeof

transcriptionalenhancementorrepressionwillhelpidentifymechanismsofchronic

HPAdysregulationacrossavarietyofenvironmentalconditions.Iwilldiscuss

epigeneticmechanismsinthesectiononfuturedirectionsbelow(seeSection7.6.2).

ThephenotypesofoffspringexposedtomaternalHFDincludealtered

glucosemetabolism.Althoughglucosemetabolismisimportantinthebodyaswell

asinthebrain,wheretheactivityofneuronsisaffectedbyglucoseavailability,the

effectsofglucosemetabolismhavenotbeenshowntoinfluencebehavioural

outcomeunderconditionsofnormalbodyweight.Sinceinmyworktherewasno

differenceinbodyweightbetweenmaternalHFDandCHDoffspringinadulthood,

whenbehaviouralandphysiologicalmeasuresweretaken,itmaythereforebe

concludedthattheobservedphenotypewasassociatedwithperinatalHFD

exposureratherthanalteredglucosemetabolisminthebrain.

7.2.MaternalHFDandstressincreaselocomotoractivity

TheresultsofmythesishaveshownthatmaternalHFD,whilenotleadingto

enhancedsensitivitytothelocomotor-activatingeffectsofcocaine,doesleadtoan

overallincreasedleveloflocomotoractivity.Inthissection,Iwilldiscussevidence

concerningpotentialmechanismsthatareknowntoalterlocomotoractivityinthe

contextofmaternalHFD.Iwillalsoexamineevidencefromstudiesofmaternal

stresswhichhavebeenshowntoincreaselocomotoractivityinoffspring.

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TheresultsofmythesisindicatingthatmaternalHFDleadstoanoverallincreasein

thelocomotoractivityofoffspringareinkeepingwithpriorresultsoftheeffectsof

prenatalstressonlocomotoractivity.Inparticular,prenatallystressedanimals

showenhancedCORTresponsestostressaswellasincreasedlocomotorandCORT

responsestonovelty(Piazzaetal.,1991).Itispossiblethatthechangeinlocomotor

activitymayresultfromtheeffectsofglucocorticoidsondopamineoutput,as

injectionsofCORTatlevelsthatmimichighstressalsoleadtoincreaseddopamine

levels(Piazzaetal.,1996).Indeed,higherbasaldopamineactivityinthestriatum

wasassociatedwithincreasedlocomotoractivityinprenatallystressedanimalsina

numberofstudies(Diazetal.,1995;Muneokaetal.,1997;Silvagnietal.,2008).

FewstudieshaveaddressedtheeffectsofmaternalHFDonlocomotor

activityintheoffspring.Thefindingsacrossstudiesareinconsistentpossibly

becausetheeffectofmaternaldietonphysicalactivityleveldependsonthetypeof

fatinthediet(Sullivanetal.,2011).Intwostudies,ratoffspringfromdamsfedwith

aHFDorajunkdietshowednodifferenceinlocomotoractivitycomparedto

offspringfromdamsfedwithacontroldiet,althoughtheinformationofthetypeof

fatwasnotprovided(Bayoletal.,2007;Naefetal.,2008).Ontheotherhand,rat

offspringfromdamsfedadietrichinpolyunsaturatedfat(i.e.,sunfloweroil)

displayedincreasedlocomotoractivitywhencomparedtooffspringfromdamsfeda

saturatedfat(i.e.,coconutoil)orstandardlaboratorydiet(Brennemanand

Rutledge,1982).Locomotoractivityinratsofvariousageswasmeasuredover60

minandthedifferenceinthelocomotoractivitywasevidentatPND20and

increasedastheratsreachedadulthood,bothinmalesandfemales.Morerecently,it

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wasshownthatmiceoffspringfromdamsfedadietrichinpolyunsaturatedfat(i.e.,

cornoil)showedincreasedlocomotoractivityinadulthood,bothinmalesand

females,(Raygadaetal.,1998).Thusitmaybethatthefattyacidcompositionofthe

maternaldietiscrucialinprogrammingactivitylevels(Sullivanetal.,2011).It

shouldbenotedthattheHFDemployedinmystudiescontained60%fatoverall.Of

this,36.7%wassaturatedfatand15.7%waspolyunsaturatedfat.Thecontroldietin

mystudiescontained13.42%fatoverall.Ofthis,11%wassaturatedfatand6.7%

waspolyunsaturatedfat.Therefore,itcouldbethatthehigheroveralllevelsof

polyunsaturatedfatcontentintheHFDcomparedtocontroldietcontributedtothe

observeddifferenceinlocomotoractivity.

Regardlessofthespecificanimalmodelofmaternalovernutritionused

(variousformulationsofHFD,‘junkfood’dietorreducedlittersize),thesetypesof

dietsappeartoleadtoacommonoffspringphenotypeofhyperphagia,

hyperleptimia,insulinresistance,increasedadiposity,andhypertension.Insulin

resistanceandhypertensionbotharisebeforethedevelopmentofincreased

adiposity,suggestingindependentprogrammingofthesesystemsoccursin

responsetomaternalovernutrition(AlfaradhiandOzanne,2011).

Skeletalmuscletissue,whichstoresglucose,ishighlyresponsivetochanges

ininsulin,andabnormalmuscledevelopmentinterfereswithgeneralphysical

activity,includinglocomotoractivity.Infact,abnormalskeletalmuscledevelopment

hasbeenreportedinanumberofmaternalovernutritionmodels.Forexample,

offspringofdamsexposedtoHFDshowincreasedadiposityandreducedskeletal

musclemass(Samuelssonetal.,2008).Inaddition,amaternal‘junkfood’diet

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impairedskeletalmuscledevelopmentinoffspring,andpromotedadiposetissue

development(Bayoletal.,2007).Thus,oneconsequenceofmaternalHFDexposure

isareducedmuscletofatratio.Ithasbeenproposedthatsomeofthese

programmingeventscouldreducegenerallocomotoractivityinoffspring.SinceI

observedanincreaseinlocomotoractivity,metaboliceffectsonmuscleasa

consequenceofexposuretomaternalHFDarenotlikelytoexplainthefindingsin

mystudies.

Theincreasedsecretionofleptinasaresultofadiposityandexposure

throughmothersmayalsobeakeyeventintheresultingleptinresistancewithin

hypothalamicneuronsleadingtoalteredlocomotoractivity.Leptin,anadipocyte-

derivedhormonethatactsonhypothalamicneuronslocatedinthearcuatenucleus

ofthehypothalamus,haswellcharacterizedeffectsonlocomotoractivity.For

example,ithasbeenfoundthatmicelackingleptinhaveimpairedinsulin/glucose

homeostasisandarehypoactive;however,theirlocomotoractivitycanbe

normalizedbyleptintreatment(Pelleymounteretal.,1995).Inaddition,leptin

signalingonlyinARHneuronsissufficienttopreventhypoactivity,asre-expressing

leptinreceptorsinneuronsintheARHofmicerestored24-hrlocomotoractivity

(Copparietal.,2005).However,knockingoutleptinreceptorsintheVTAhadthe

oppositeeffect.Morespecifically,long-termgeneticknockdownofleptinreceptors

intheVTAledtoanincreaseindark-cyclelocomotoractivity(Hommeletal.,2006).

However,sinceleptinlevelswerenotmeasuredinthisstudyitisunknownwhether

therewasacompensatoryincreaseinleptinproductionassociatedwiththe

increaseinlocomotoractivity.Basedonthesestudies,theincreasedlocomotor

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activityinoffspringobservedwithmaternalHFDexposureinmystudiescould

occurasaresultofincreasedleptinlevels.

Thestudiesreviewedaboveprovideevidencethattheeffectofleptinon

locomotoractivityismediatedbyneuralcircuitry,specificallytheARHandtheVTA.

Basedonthesestudies,itwouldbepossibletodesignanexperimenttoaddressthe

influenceofincreasedleptinasaresultofmaternalHFDexposureonbrain

mechanismsinvolvedintheregulationoflocomotoractivity.Todoso,locomotor

activityshouldbemeasuredinHFDoffspringinthecontextoflocalleptinreceptor

knockdownbyRNAiinARHorVTA,whichshouldleadtoareducedlocomotor

activitytolevelsobservedincontroloffspring.Theseexperimentswouldbeneeded

toidentifywhetherthesamemechanismsdescribedabovemediatethe

hyperactivitywithmaternalHFDexposureobservedinmystudies.

ThemetaboliceffectsofmaternalHFDexposureonskeletalmusclewere

reportedtohaveeithernoeffectoranegativeeffectonlocomotoractivityinthe

offspring.Therefore,thesemetaboliceffectsareunlikelytoexplaintheincreasein

locomotoractivityobservedinmystudies.NointeractionbetweenmaternalHFD

andcocaine,whichisknowntoincreasedopamineactivation,wasobservedinmy

studies.

7.3.MaternalHFDhadnoeffectoncocaine-inducedlocomotoractivity

Therearetwomajorstress-relatedhormonesimplicatedinresearchofcocaine

intake,CRFandcorticosterone.CRFcanactdirectlyatextra-hypothalamicsitesin

thebrain,aswellasinthepituitarytomediatemanyofthebehaviouraland

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physiologicalsymptomsofthestressresponseviaitseffectsontheHPAaxis(Smith

andVale,2006).Corticosteroneisinvolvedinavarietyofbehaviouraland

neurochemicaleffectsofexposuretostress(BaleandVale,2004).Therearereports

thatexposuretocorticosteronefacilitatestheinitiationofself-administrationoflow

dosesofpsychostimulants,whereasadrenalectomy(ADX)andchronicexposureto

thecorticosteronesynthesisinhibitorsdecreasecocaineself-administration(Piazza

etal.,1994;GoedersandGuerin,1996;Mantschetal.,1998).

Intermsofthelong-termeffectsofcocaine,theHPAaxisplaysapermissive

role.Inindividualswithahistoryofcocaineself-administration,relapsetococaine

taking,evenafterprolongedperiodsofabstinence,ishighlyprobableespeciallyin

thecontextofexposuretostress.Usingananimalmodelofrelapse,abriefexposure

toastressorhasbeenshowntoserveasapowerfulstimulusforreinstatementof

cocaineseekingafterextendeddrug-freeperiods(Erbetal.,1996).Furthermore,in

animalstrainedtoself-administercocaine,stress-inducedrelapsecanbeattenuated

bysystemicinjectionsofCRFreceptorantagonists(Shahametal.,1998).Thusin

cocaine-trainedanimals,CRFappearstoplayasignificantroleinstress-induced

relapse.Indeed,CRFreceptorantagonistssuppressedstress-inducedrelapseto

cocaineseekinginintactanimalsandinadrenalectomizedanimalsgiven

corticosteronereplacement(Erbetal.,1996).ThesedatasuggestthatCRFcanact

directlyinthebrain,independentofitseffectsontheendocrineHPAaxis,to

mediatetheeffectsofstressonrelapse.Thus,theHPAmayplayapermissiverolein

thebehaviouraleffectsoflong-termexposuretococaineinresponsetostress.

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GiventhattheHPAplaysapermissiveroleinthebehaviouraleffectsoflong-

termexposuretococaineinresponsetostress,itispossibletospeculatethat

maternalexposuretoHFDmayinfluencethecocaine-inducedbehaviourinoffspring

inamannerthatisindependentoftheHPAaxis.Indeed,Ihaveshownthatmaternal

HFDincreasedtheHPAactivityinfemaleoffspring,butmaternaldietdidnot

interactwithoffspringexposuretolong-termcocaine.Ontheotherhand,giventhe

permissiverolethattheHPAaxishasbeenshowntoplayinthelong-termeffectsof

cocaine,itispossiblethatImayhaveobservedaneffectofmaternalHFDon

cocaine-inducedlocomotoractivityunderdifferentparametricalconditionsthanthe

onesIemployed.Indeed,GrissomandRyesfoundthatmalemiceoffspringfrom

maternalHFDshowedincreasedlocomotorresponsetococaine(20mg/kg.,i.p.)

(2013).Inmythesis,asimilardosageofcocaine(30mg/kg.,i.p.)wasappliedtorats;

however,assomestudieshaveindicated,cocainedosageinmiceof20mg/kgmay

bemorepotentthanthedosageinratsof30mg/kgofcocaineusedinmythesis.

Althoughithasnotbeenexploredsystematically,itcanbespeculatedthatmicemay

bemoresensitivetotheeffectsofpsychostimulantsthanrats.Ifso,inconsistencies

infindingscouldbeattributedtospeciesdifferencesinthecocainemetabolism.

Futureexperimentsexaminingdose-relateddifferencesintheresponsetococaine

inratswouldbehelpfulindeterminingwhetherthisisthecase.Investigations

involvingamorestringentdrugschedulearewarrantedforfuturestudies(changing

thedrugschedulewillbediscussedinmoredetailinsection7.6.Limitationsand

futuredirectionsbelow).

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InsupportoftheinterpretationthattheHPAplaysapermissiveroleinthe

long-termeffectofcocaine,Ihaveshownthatmaternalexposuretococainepriorto

pregnancyresultedinanenhancedsensitivitytothepsychomotoractivatingeffects

ofcocaineinadultmaleoffspring,independentofchangesintheresponsivenessof

theHPAaxis.Importantly,undertheconditionsassessedinthepresentstudy,

intergenerationaltransmissionoftheeffectsofmaternalcocaineexposureoccurred

independentofdirectexposureofoffspringtothedruginutero.Indeed,maternal

exposuretococainepriortopregnancyhadnoeffectonstress-inducedplasma

CORTlevelsinmaleoffspring,orontheexpressionofGRorCRFgenesinkeylimbic

regionsoftheseoffspring.Instead,theeffectswereselectiveforaspecificalteration

inthegeneexpressionofcorticaldopamineneurons(i.e.,upregulationoftheDRD1

inmPFC),andaspecificbehaviourthatisknowntorelyontheintegrityofthe

mesocorticolimbicdopaminesystem(i.e.,acutecocaine-inducedpsychomotor

activation).

7.4.MaternalHFDincreasedbodyweightsindamsandpre-weaningoffspring

Maternalbodyweightswerecomparableacrossstudies,whereHFDdamswere

heavierthanCHDdamspriortomatingandthroughoutgestationandlactation.This

observationisconsistentwithpreviousliteratureonmaternalovernutritionmodels

thatleadtotheprogrammingofmetabolicdysregulationssuchasincreased

adiposityandinsulinresistanceinoffspring(AlfaradhiandOzanne,2011).Itis

interestingtonotethatchangesinmaternalbodyweightsarenotnecessaryto

inducethesemetaboliceffectsinoffspringasexposuretomaternalHFDatdifferent

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pointsintime(i.e.HFDexposureuntilweaningorrestrictedtopregnancyand

lactation)bothleadstosimilarmetabolicphenotypesinoffspring,showingthat

thesemetaboliceffectsareindependentofmaternalbodyweights(Howieetal.,

2009).

HeaviermaternalbodyweightsinHFDdamsweretransmittedtoHFD

offspring,whowereheavierthanCHD-exposedoffspringpriortoweaning(except

inChapter5wherenodifferenceinpre-weaningbodyweightswasfound).This

resultisconsistentwiththeliteratureshowingthatincreasedpre-weaningbody

weightsaregenerallyobserved(Vuceticetal.,2010;Purcelletal.,2011;Sunetal.,

2012).ThetimingoftheexposuretomaternalHFDplaysakeyroledetermining

pre-weaningbodyweightsinoffspring.Ithasbeenshownbycross-fosteringlitters

toCHDorHFDdams24hrsafterbirththatmaternalHFDduringlactationhasa

greaterinfluencedeterminingoffspringmetabolicphenotypessuchaspre-weaning

bodyweights,adiposity,leptinlevelsandglucosetolerance(Sunetal.,2012).It

shouldbenotedthatbecausetherewerenoobserveddifferencesinoverallbody

weightbetweenHFD-andCHD-exposedoffspringinadulthoodwhenbehavioural

andphysiologicalmeasuresweretaken,theobservedadultphenotypewas

associatedwithperinatalHFDexposureratherthanphenotypiceffectsrelatedto

currentbodyweight.

7.5.Maternalmanipulationsdidnotalterthequalityofmaternalcare

MaternalmanipulationsofHFD,stressorcocainedidnotsignificantlyalterthe

qualityofmaternalcare,withtheexceptionofamodesteffectofHFDonmaternal

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careobservedinChapter5.Itwasimportanttoexaminethequalityofmaternalcare

becausemanystudieshaveshownthatmaternalcarecanmodifystressreactivityin

pups(Liuetal.,1997).Thesechangesare,inturn,relatedtoalteredbehavioural

phenotypes,suchascocaine-inducedpsychomotoractivityofoffspringinadulthood

andstress-relatedbehaviour,asmeasuredinmythesis.Poormaternalcare(i.e.

lowerlevelsoflickingandgrooming)isassociatedwithdetrimentaleffectsin

offspringsuchasincreasedreactivitytopsychostimulants(Kippinetal.,2008),

increasedCORTresponsetostress,andincreasedanxiety-likebehaviourin

offspring(Liuetal.,1997;Caldjietal.,2000).Thereforeitwasimportanttoexclude

thepossibilityofeffectsassociatedwithdifferentialmaternalcareofoffspringinmy

studies.

IexaminedtheeffectsofmaternalHFDonthequalityofmaternalcarethe

offspringreceived.InChapter4,therewasnodifferenceinmaternalcarereceived

betweenHFDandCHDoffspring.InChapter5,therewasamodesteffectof

increasedmaternalcareinHFDoffspring.Theseresultsareconsistentwith

previousliterature.OnestudyfoundthatdamsconsumingHFDfrommating

throughlactationdisplayedincreasedmaternalnursing(i.e.arched-backand

passivenursingpostures),butnochangeinlickingandgroomingbehaviourduring

thefirstweekpostpartumcomparedtoCHD-fedcontrols(Purcelletal.,2011).

AnotherstudyfoundthatdamsconsumingHFDfrommatingthroughlactation

exhibiteddecreasedmaternallickingandgrooming,butexhibitednochangein

nursingbehaviourbetweenpostnataldays3and8(Connoretal.,2012).These

disparateresultsindicatethatmoreresearchisneededtoelucidatetheroleof

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maternalbehaviourinoffspringphenotypeinthecontextofmaternalHFD.

However,consideringthatittherewasnodifferenceoramodestofeffectofHFDof

increasedmaternalcareobservedinmystudies,itisunlikelythatmaternalcarewas

theprimaryfactortoinducechangesobservedinoffspringsuchasincreased

anxiety,stress-inducedCORTandgeneexpressionchanges.

Ialsoexaminedwhetherpregestationalcocaineinfluencesmaternalcare.I

foundnochangeinthefrequencyoflickingandgroomingincocaine-exposeddams,

supportingtheresultsofapreviousstudyusingasimilarmanipulationduring

gestation(Petruzzietal.,1997).Asaresult,itisunlikelythattheeffectsof

pregestationalcocaineonbehaviouranddopamine-relatedgeneexpression

occurredasaresultofdifferencesinmaternalcare.Atleasttwopossibilitiesother

thandifferencesinmaternalcaremayexplainthetransmissionofchangestothe

dopaminesysteminoffspringwithpregestationalexposuretococaine.First,itis

possiblethatexposuretococaineorcocainemetabolitesindamsmayhavehad

directeffectsongametespriortofertilization,oraffectedtheselectionofparticular

gametesthatwereultimatelyfertilized.Second,itispossiblethatexposureto

cocainepriortopregnancyaffectedthematernalphysiologyduringgestation.Inthis

case,maternalphysiologymayhavebeenaffectedduringcriticalperiodsof

gestationcorrespondingtothedevelopmentofthedopaminesysteminoffspring.In

futureexperiments,astudydesignemployinginvitrofertilizationcouldbehelpful

indissociatingthesepossibleeffectsofcocaineexposureonoffspringphenotype.In

thenextsection,wewillconsideradditionalexperimentsthatcouldaidinthe

interpretationofmyfindings.

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7.6.Limitationsandfuturedirections

Inthissection,Iwilldiscusslimitationsintheinterpretationofmyfindingsasa

resultofthespecificapproachesIemployed.Inlightoftheselimitations,Iwill

discusshowmyfindingscouldinformfuturestudiesofbehaviouralandneural

mechanismsassociatedwithmaternalHFDexposure.

7.6.1.Anxietybehaviour:Drugexposureconditionsandbehavioural

assessmentsofanxiety

Repeatedexposurestococaineareknowntoinduceanxietyinrodents,particularly

inthefirst24-48hoursaftertheirtermination.InChapter4,theoffspringwere

testedforanxietybehaviourtwodaysafterthelastexposuretodrugtreatment.The

drugregimenusedinourstudy(i.e.,6exposurestococaineat30mg/kg,i.p.with2

daysofabstinence)issimilartootherstudiesthatfoundanxiogeniceffectsof

cocainewithdrawal.However,ourstudyhadatestforconditionedlocomotion24h

priortotheEPMtaskusedtomeasureanxietybehaviour.Conditionedlocomotion

referstoaclassicalconditioningofdrugeffectstocontextualstimuli,suchasthe

environmentwhereadrugsuchascocainewaspreviouslyadministered,in

responsetoasalineinjection(i.e.,intheabsenceofcocaine).Inthefuturestudies,

animalsshouldremainundisturbedbetweenthecocaineexposureandEPMtestin

ordertomaintainthememoryforthepairingbetweencocaineandenvironmental

cuesandthereforeelicitastrongerwithdrawal-inducedanxietyeffect.Alternatively,

itisknownthatcuesassociatedwithdrugadministrationenhanceanxietyduring

160

thewithdrawalperiod(Erbetal.,2006).Futurestudiesusingpre-exposuretocues

associatedwiththecontextinwhichcocainewasadministeredcouldbeusedto

enhanceanxietyintheCHDandHFDgroupsexposedtococaine.

ThelackofanxietyinHFDoffspringcomparedtoCHDcontrolsobservedin

Chapter5maybeduetothefactthattheanimalstestedwereexposedtococaine

48-72hpriortotheanxietytests.Thiswasbecausethedesignoftheacutecocaine

paradigmrequiredallindividualstobeexposedtococaineinjections(i.e.,salineand

cocainegroupswerenotindependent,unlikeinthechroniccocaineparadigmused

inChapter4).Acutecocaineexposureisnotgenerallyassociatedwithincreased

anxiety,butsomestudieshavereportedanxiolyticresponseswithacutecocaine

exposure[e.g.see:(VanSwearingenetal.,2013)].Infuturestudies,aseparatesetof

drug-naïveanimalscouldbeusedtoexcludethepossibleinteractionbetween

cocaineexposureandmaternaldietthatweobservedinChapter4.

Itmayalsobepossibleinfuturestudiestoincreasethesensitivityofour

anxietytestingbyrefiningthetasksprocedurallyandanalytically.Forexample,in

Chapter3weevaluatedriskassessment(i.e.headentries),whichhasbeen

proposedtobemoresensitivetotheeffectsofanxiogenicdrugsthanstandard

measuresoftimespentintheopenarms(CarobrezandBertoglio,2005).Dueto

technicallimitationsintheequipmentusedinChapters4and5,trackingrisk

assessmentwasnotpossible.Inaddition,previousliteratureindicatesthatover-

handlingcanreducetheanxiogenicimpactoftheEPM(Hogg,1996).Thus,forthe

experimentsdescribedinChapters4and5,theuseofasubgroupofnaïveanimals

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fortestinganxietybehaviourcouldbeinformativeincomparingtheiranxiety

behaviourtothatofanimalsrepeatedlyhandledforthepurposeofdruginjections.

7.6.2.Cocaine-inducedlocomotoractivity:self-administrationanddopamine

function

MaternalHFDincreasedlocomotoractivitywhentheanimalswereexposedtoa

newenvironmentforthefirsttime(habituationsessioninChapter5).Itispossible

thattheincreaseinspontaneouslocomotoractivitywascarriedovertothetestfor

cocaine-inducedlocomotoractivity.Otherstudiesofgroupsthatdifferinbaseline

locomotoractivity[forexample:(Kippinetal.,2008)]useself-administration

paradigmstoassesscocainesensitivity,astheoutputmeasureisindependentof

locomotoractivity.Theself-administrationparadigmisaformofoperant

conditioningthatinvolvesabehavioursuchasleverpressingthatisreinforcedand

maintainedbydrugdelivery.Themostcommonlyusedoutputmeasureistoreplace

salineforthedrugsolutionanddeterminewhetherthebehaviourundergoes

extinction,thereforemeasuringdrug-reinforcedbehaviour.Thus,aself-

administrationparadigmcouldbeusedinfuturestudiestodissociatemotivationto

seekcocainefromspontaneouslocomotoractivity.

Cocaineinducesdopaminereleaseinthemesolimbicpathway,whichhas

beenproposedtocontroldrug-relatedbehavioursuchascocaine-induced

locomotoractivityandself-administration.Thesebehavioursarethoughttoreflect

dopaminelevelsinthemesolimbicpathway.In-vivomicrodialysistogetherwith

cocaine-inducedlocomotoractivityandself-administrationmayprovidefurther

162

insightsontheresponsivityofthemesolimbicdopaminepathway.With

microdialysis,itwouldalsobepossibletomeasurethelocalrateofchangeof

dopamineinresponsetoinfusionsofvariousdosesofcocaineintocirculation.

Thesestudieswouldenablethequantificationoftheneurophysiologicalresponseto

cocaineintheHFDandCHDgroups.

7.6.3.Epigeneticmechanisms

Inmythesis,expressionanalyseswerecarriedoutonanumberofcandidategenes

inassociationwithbehaviouraloutcomesinoffspring.Theaffectedbrainregions

andthedirectionalityoftheeffectswerefoundtobecontext-andsex-specific.These

findingsareconsistentwiththeliterature.Forexample,thereissupportinthe

literatureforthesuggestionthatmaternalHFDleadstoalterationsinthe

expressionofdopaminergicgenesoverall,howeverwhatdopaminergicgenesare

altered,andinwhatbrainregionsinthemesolimbicpathway,havenotbeen

consistentacrossstudies(Naefetal.,2008;Vuceticetal.,2010;Naefetal.,2011;

OngandMuhlhausler,2011).Thisisperhapsnotunexpectedgiventhedifferences

inexposureregimens,species/strainofanimal,andenvironmentalconditionsunder

whichtheexperimentswerecarriedout,asthesefactorsmaybeexpectedtoaffect

theexpressionofmultiplegenes.

Ourrationaleforlookingatlevelsofgeneexpressionwasbasedonevidence

suggestingaroleforepigeneticmechanismsinmodifyinggeneexpressionand

producingchangesinbehaviourthatcanbetransmittedfromparenttooffspring

(McGowanetal.,2011;Vassoleretal.,2013).Inchapter3wefoundchangesingene

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expressionassociatedwithanxietyinoffspring.Inchapter6,wefoundchangesin

DRD1expressionassociatedwithpregestationalcocaineexposure.Giventhatlevels

ofgeneexpressionareknowntodirectlong-termchangesingeneexpression,future

studiesstemmingfromtheworkdescribedinmythesisshouldincludeananalysis

ofepigeneticmodifications.

Epigeneticmechanisms,whichmodifygenefunctionintheabsenceofa

changeingenesequence,havebeenproposedtoprogramgeneexpressionasa

functionofearlylifeexperience(Sasakietal.,2013).Long-termchangesingene

regulationcanoccurviaepigeneticmodificationsofDNAandchromatinstructure.

Incontrasttochromatinmodifications,whichmaybetransientandaretightly

coupledtogeneexpression,DNAmethylationisarelativelystablemodificationthat,

inregulatoryelements,typicallyleadstopersistentrepressionofgeneexpression

(CedarandBergman,2009).Forexample,levelsofmaternalbehaviourreceived

withinthefirstweekoflifeareassociatedwithoffspringHPAfunction,andlevelsof

DNAmethylationinstress-relatedgenes(McGowanetal.,2011;Panetal.,2014).

Recently,anumberofstudiesofcandidategeneshaveindicatedthatmaternal

overnutritionalterslevelsofDNAmethylationingenepromotersinoffspring

(Vuceticetal.,2010;Dudleyetal.,2011;Jiangetal.,2011;Palouetal.,2011;Attiget

al.,2013;YoungsonandMorris,2013).

Withthesupportoftechnologicaladvancesinhigh-throughputDNA

sequencing,itisnowpossibletoextendthisworkfromaconsiderationofcandidate

genestocandidatepathways.Manyofthewaysinwhichenvironmentalexposures

alterepigeneticmechanismsinoffspringremainunknown.Improvedmethodsfor

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genome-widedetectionofepigeneticalterations,however,havegreatlyadvanced

complexdiseaseresearchbyprovidingthemeanstoidentifymechanismsleadingto

stablechangesincellularfunction(LegendreandHarris,2006).Howthesechanges

mightdistributeacrossdopaminergicandHPA-relatedgenenetworksisonerelated

topicofactiveresearch.

Infutureresearch,itwillbeofinteresttoidentifyhowepigeneticmarks

indicatingenhancedorrepressedtranscriptionalpotentialmayrelateto

dysfunctioninrewardandstresspathwaysacrossavarietyofconditions.Because

epigeneticmarksarepotentiallyreversible,identifyingthemannerinwhichthey

arealteredinthedopaminergicandHPA-relatedpathwaysofoffspringwhose

motherswhowerefedahighfatdiet,willofferinsightintomechanismsleadingto

stablediseasestates;thisworkmayalsoinformnovelroutestopharmacological

intervention.Altogether,suchstudieswillilluminateourunderstandingofrisk

factorsfordisorderscharacterizedbydysregulatedemotionalprocessing.

7.7.Conclusions

Identifyingthemechanismsthroughwhichmaternalovernutritionresultsinaltered

rewardandstresspathwayslaterinlifewillenabletheunderstandingofriskfactors

fordisorderscharacterizedbydysregulatedhedonicandnegativeemotionalstates.

Theexperimentscomprisingthisdissertationareamongthefirsttoinvestigatethe

effectsofmaternalHFDinmediatingcocaine-andanxiety-relatedbehaviour.My

findingssuggestdistincteffectsofmaternalHFDandmaternalexposuretococaine

onlocomotoractivityandanxietybehaviour.MaternalHFDaffectsthestresssystem

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andstress-relatedbehaviour,whereasmaternalexposuretococaineinfluencesthe

rewardsysteminoffspring.Furtherexplorationofthenatureoftheseinteractions

mayhaveimportantimplicationsforunderstandingthemechanismsunderlyingthe

recentepidemicriseinobesity.

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AppendixTable4.1

Cocaine-primedgeneexpressionanalysesinoffspring.Twenty-fourhoursaftera

challengeinjectionofcocaine(10mg/kg,i.p.),thebrainswerecollectedforanalyses

ofcocaine-activatedgeneexpressionlevels.Mean± SEM relative abundance of

transcripts for dopamine receptor D1 (DRD1) and dopamine receptor D2 (DRD2) in

medial prefrontal cortex (mPFC), nucleus accumbens (NAC) and ventral tegmental area

(VTA) and for glucocorticoid receptor (GR) and corticotropin releasing factor (CRF) in

hypothalamus (HYP), amygdala (AMY), and hippocampus (HPC). Gene expression was

assessed in male (left) and female offspring (right) that had been pre-exposed to maternal

high fat diet and to repeated injections of cocaine or saline in adulthood. *P < .05 main

effect of diet, #P < .05 main effect of drug treatment, &P < .05 diet by drug interaction.


Saline Cocaine Saline Cocaine Saline Cocaine Saline CocaineDRD1 mPFC 0.95 ± 0.15 0.88 ± 0.11 0.83 ± 0.08 0.85 ± 0.08 DRD1 mPFC 1.46 ± 0.14 1.48 ± 0.13 1.52 ± 0.17 1.51 ± 0.13

NAC 1.77 ± 0.22 1.22 ± 0.17 1.31 ± 0.15 1.25 ± 0.20 NAC 1.16 ± 0.01 1.04 ± 0.04 1.17 ± 0.09 1.07 ± 0.04VTA 1.23 ± 0.14 1.23 ± 0.06 1.24 ± 0.06 1.26 ± 0.12 VTA 0.89 ± 0.05 0.93 ± 0.05 0.97 ± 0.05 1.04 ± 0.10HYP 1.08 ± 0.17 1.18 ± 0.13 1.29 ± 0.18 1.03 ± 0.07 HYP 0.95 ± 0.07 0.94 ± 0.03 0.91 ± 0.08 0.86 ± 0.03

DRD2 mPFC 1.07 ± 0.09 1.09 ± 0.08 1.05 ± 0.06 1.06 ± 0.03 DRD2 mPFC 1.74 ± 0.11 1.70 ± 0.11 1.95 ± 0.06 1.82 ± 0.07NAC 1.64 ± 0.19 1.75 ± 0.24 1.72 ± 0.40 1.11 ± 0.14 NAC 0.84 ± 0.05 0.82 ± 0.02 0.89 ± 0.04 0.74 ± 0.04VTA 1.23 ± 0.16 1.16 ± 0.08 1.19 ± 0.10 1.22 ± 0.08 VTA 0.96 ± 0.11 0.95 ± 0.07 1.07 ± 0.08 0.96 ± 0.11HYP 0.98 ± 0.05 1.64 ± 0.24 1.58 ± 0.24 1.40 ± 0.19*# HYP 1.88 ± 0.14 1.81 ± 0.07 1.74 ± 0.07 1.86 ± 0.07

GR AMY 1.26 ± 0.04 1.26 ± 0.01 1.22 ± 0.03 1.15 ± 0.06 GR AMY 1.12 ± 0.03 1.11 ± 0.04 1.11 ± 0.05 0.97 ± 0.05HYP 1.03 ± 0.11 0.82 ± 0.06 0.94 ± 0.06 0.85 ± 0.05 HYP 0.76 ± 0.06 0.71 ± 0.04 0.71 ± 0.06 0.69 ± 0.03HPC 1.19 ± 0.10 1.31 ± 0.08 1.33 ± 0.06 1.40 ± 0.08 HPC 1.28 ± 0.05 1.10 ± 0.03 1.10 ± 0.04 1.04 ± 0.02#mPFC 0.75 ± 0.02 0.75 ± 0.04 0.78 ± 0.02 0.71 ± 0.04 mPFC 1.88 ± 0.13 1.67 ± 0.06 1.94 ± 0.07 1.58 ± 0.06#NAC 0.95 ± 0.07 0.90 ± 0.05 0.85 ± 0.04 0.88 ± 0.04 NAC 0.82 ± 0.05 0.73 ± 0.03 0.86 ± 0.04 0.72 ± 0.02#

CRF AMY 1.49 ± 0.14 1.43 ± 0.14 1.41 ± 0.13 1.29 ± 0.09 CRF AMY 1.33 ± 0.08 1.37 ± 0.08 1.34 ± 0.19 1.11 ± 0.05HYP 1.26 ± 0.11 1.09 ± 0.21 1.32 ± 0.22 1.30 ± 0.11 HYP 1.02 ± 0.20 0.93 ± 0.13 1.22 ± 0.18 1.00 ± 0.13HPC 1.30 ± 0.07 1.40 ± 0.15 1.65 ± 0.19 1.85 ± 0.09* HPC 1.12 ± 0.07 1.33 ± 0.08 1.31 ± 0.05 1.15 ± 0.06&

TH mPFC 0.81 ± 0.06 1.13 ± 0.14 0.98 ± 0.08 1.00 ± 0.05 TH mPFC 1.50 ± 0.06 2.05 ± 0.16# 1.56 ± 0.11 2.04 ± 0.22#NAC 1.25 ± 0.12 1.66 ± 0.26 1.35 ± 0.19 1.21 ± 0.13 NAC 1.35 ± 0.18 1.13 ± 0.12 1.04 ± 0.07 1.20 ± 0.12VTA 1.23 ± 0.16 1.12 ± 0.11 1.11 ± 0.11 1.25 ± 0.06 VTA 0.91 ± 0.18 0.74 ± 0.07 0.84 ± 0.07 0.71 ± 0.10HYP 0.97 ± 0.05 1.60 ± 0.17 1.40 ± 0.10 1.69 ± 0.14*# HYP 1.36 ± 0.14 1.60 ± 0.10 1.82 ± 0.10 1.67 ± 0.13HPC 1.09 ± 0.15 1.87 ± 0.73 1.29 ± 0.33 14.15 ± 12.38 HPC 3.19 ± 1.26 2.89 ± 0.89 1.01 ± 0.15 3.46 ± 1.08AMY 1.54 ± 0.20 1.68 ± 0.21 0.97 ± 0.08 1.92 ± 0.37# AMY 1.04 ± 0.19 1.42 ± 0.39 1.17 ± 0.16 0.93 ± 0.08

DARPP mPFC 1.07 ± 0.06 1.10 ± 0.08 0.92 ± 0.08 0.94 ± 0.05 DARPP mPFC 1.08 ± 0.11 1.13 ± 0.08 1.18 ± 0.08 1.15 ± 0.04NAC 1.10 ± 0.13 1.12 ± 0.05 1.12 ± 0.06 1.11 ± 0.04 NAC 0.88 ± 0.05 1.06 ± 0.05 0.99 ± 0.05 0.92 ± 0.09VTA 1.18 ± 0.10 0.99 ± 0.10 0.98 ± 0.05 0.90 ± 0.12 VTA 1.15 ± 0.02 1.14 ± 0.14 0.94 ± 0.06 0.99 ± 0.05*HYP 1.18 ± 0.15 1.36 ± 0.08 1.36 ± 0.22 1.61 ± 0.14 HYP 1.93 ± 0.26 2.10 ± 0.11 1.94 ± 0.08 1.88 ± 0.13HPC 0.81 ± 0.15 0.60 ± 0.07 0.56 ± 0.03 0.51 ± 0.04 HPC 1.38 ± 0.12 1.36 ± 0.14 1.06 ± 0.06 1.15 ± 0.17AMY 1.57 ± 0.20 1.38 ± 0.15 1.37 ± 0.14 1.47 ± 0.13 AMY 1.43 ± 0.18 1.23 ± 0.13 1.12 ± 0.14 0.95 ± 0.06*


Offspring drug treatment Offspring drug treatment

167

AppendixTable5.1

Geneexpressionanalysisinoffspringpreviouslyexposedtoacutecocaine.Mean±

SEMrelativeabundanceoftranscriptsfordopaminereceptorD1(DRD1)and

dopaminereceptorD2(DRD2)inthemedialprefrontalcortex(mPFC),nucleus

accumbens(NAC)andventraltegmentalarea(VTA),andforglucocorticoidreceptor

(GR)andcorticotropinreleasingfactor(CRF)inthehypothalamus(HYP),amygdala

(AMY),andhippocampus(HPC).Geneexpressionwasassessedinmale(left)and

femaleoffspring(right)thathadbeenpre-exposedtomaternalhighfatdietandto

acuteinjectionsofcocaineinadulthood.*P<.05maineffectofdiet,%P<.05main

effectofmaternalstressand^P<.05interactionofmaternaldietandstress.


Control Stress Control Stress Control Stress Control StressDRD1 mPFC 1.05 ± 0.09 1.14 ± 0.08 1.04 ± 0.13 1.00 ± 0.08 DRD1 mPFC 1.03 ± 0.07 1.20 ± 0.09 1.29 ± 0.13 1.15 ± 0.08

NAC 1.27 ± 0.09 1.18 ± 0.10 1.03 ± 0.06 0.80 ± 0.12* NAC 0.75 ± 0.13 0.80 ± 0.05 0.72 ± 0.17 0.85 ± 0.07VTA 1.51 ± 0.57 2.57 ± 0.86 0.74 ± 0.05 1.56 ± 0.80 VTA 1.01 ± 0.03 0.99 ± 0.07 1.02 ± 0.08 1.10 ± 0.06HYP 1.02 ± 0.08 0.96 ± 0.07 0.94 ± 0.04 1.07 ± 0.17 HYP 1.05 ± 0.07 1.22 ± 0.08 1.02 ± 0.03 1.16 ± 0.05

DRD2 mPFC 1.29 ± 0.16 1.38 ± 0.13 1.06 ± 0.06 1.23 ± 0.07 DRD2 mPFC 0.90 ± 0.08 0.93 ± 0.06 0.77 ± 0.08 0.93 ± 0.07NAC 0.96 ± 0.11 0.90 ± 0.07 0.80 ± 0.06 0.64 ± 0.10* NAC 0.60 ± 0.10 0.75 ± 0.08 0.58 ± 0.16 0.78 ± 0.11VTA 0.89 ± 0.06 0.87 ± 0.06 0.85 ± 0.03 0.96 ± 0.07 VTA 0.75 ± 0.07 0.85 ± 0.05 0.90 ± 0.08 0.81 ± 0.06HYP 0.9 ± 0.07 0.89 ± 0.05 0.94 ± 0.06 0.90 ± 0.05 HYP 1.23 ± 007 1.47 ± 0.12 1.35 ± 0.13 1.49 ± 0.13

GR AMY 1.09 ± 0.07 0.93 ± 0.06 1.06 ± 0.09 1.03 ± 0.07 GR AMY 1.20 ± 0.05 1.23 ± 0.04 1.43 ± 0.09 1.28 ± 0.08HYP 1.05 ± 0.02 0.91 ± 0.04 1.06 ± 0.04 1.04 ± 0.03 HYP 1.15 ± 0.07 1.09 ± 0.04 1.73 ± 0.66 1.08 ± 0.08HPC 2.0 ± 1.04 0.81 ± 0.09 4.03 ± 1.61 2.05 ± 1.29 HPC 1.20 ± 0.23 1.39 ± 0.37 8.29 ± 5.11 1.78 ± 0.72mPFC 1.09 ± 0.04 1.20 ± 0.08 1.12 ± 0.04 1.19 ± 0.05 mPFC 1.13 ± 0.05 1.04 ± 0.03 1.39 ± 0.03 1.25 ± 0.05*%NAC 1.32 ± 0.10 1.19 ± 0.05 1.11 ± 0.11 1.04 ± 0.04* NAC 1.05 ± 0.05 1.12 ± 0.06 1.01 ± 0.03 1.02 ± 0.05

CRF AMY 1.13 ± 0.10 0.78 ± 0.05 1.12 ± 0.10 1.08 ± 0.07% CRF AMY 1.36 ± 0.09 1.28 ± 0.07 1.24 ± 0.14 1.17 ± 0.06HYP 1.42 ± 0.25 1.02 ± 0.32 1.64 ± 0.31 0.99 ± 0.21 HYP 1.31 ± 0.24 1.44 ± 0.23 1.46 ± 0.30 1.82 ± 0.25HPC 1.14 ± 0.03 1.09 ± 0.02 1.24 ± 0.11 1.28 ± 0.05* HPC 1.17 ± 0.12 1.27 ±0.06 1.19 ± 0.10 1.28 ± 0.05

TH mPFC 1.95 ± 0.68 1.94 ± 0.74 1.64 ± 0.34 1.22 ± 0.07 TH mPFC 1.35 ± 0.14 1.36 ± 0.10 1.60 ± 0.29 1.27 ± 0.07NAC 1.36 ± 0.20 1.02 ± 0.07 1.27 ± 0.10 1.47 ± 0.30 NAC 1.20 ± 0.08 1.21 ± 0.14 1.31 ± 0.13 1.18 ± 0.04VTA 1.00 ± 0.08 0.97 ± 0.07 0.91 ± 0.06 1.04 ± 0.07 VTA 0.71 ± 0.11 0.83 ± 0.6 0.92 ± 0.06 0.83 ± 0.09HYP 1.08 ± 0.08 0.95 ± 0.10 1.07 ± 0.06 1.01 ± 0.10 HYP 1.16 ± 0.13 1.44 ± 0.10 1.24 ± 0.22 1.34 ± 0.08HPC 1.21 ± 0.40 2.31 ± 0.82 0.72 ± 0.04 1.43 ± 0.73 HPC 0.87 ± 0.08 0.99 ± 0.09 2.54 ± 1.25 1.12 ± 0.41AMY 1.04 ± 0.27 0.54 ± 0.07 1.54 ± 0.44 1.31 ± 0.32* AMY 0.96 ± 0.15 0.91 ± 0.27 0.91 ± 0.08 1.00 ± 0.19

DARPP mPFC 0.98 ± 0.13 1.11 ± 0.11 1.07 ± 0.10 1.08 ± 0.19 DARPP mPFC 0.97 ± 0.19 1.10 ± 0.13 1.06 ± 0.21 1.19 ± 0.24NAC 0.94 ± 0.09 0.89 ± 0.06 1.04 ± 0.12 0.69 ± 0.12 NAC 0.66 ± 0.11 0.68 ± 0.11 0.78 ± 0.22 0.85 ± 0.14VTA 0.84 ± 0.05 1.01 ± 0.04 1.02 ± 0.07 0.85 ± 0.04^ VTA 0.95 ± 0.08 1.04 ± 0.05 0.99 ± 0.08 1.21 ± 0.08HYP 0.90 ± 0.10 0.88 ± 0.15 0.89 ± 0.15 0.96 ± 0.12 HYP 1.05 ± 0.07 1.39 ± 0.11 1.17 ± 0.17 1.30 ± 0.14HPC 1.06 ± 0.13 1.07 ± 0.18 1.03 ± 0.16 1.42 ± 0.43 HPC 1.30 ± 0.22 1.37 ± 0.14 1.25 ± 0.20 1.27 ± 0.17AMY 0.83 ± 0.07 0.88 ± 0.09 1.03 ± 0.11 1.01 ± 0.12 AMY 1.10 ± 0.19 0.93 ± 0.16 1.16 ± 0.09 1.01 ± 0.10


Maternal stress Maternal stress

168

AppendixTable7.1

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A B Summaryofthesisfindingswithhighlightsindicatingsimilaritiesacrossstudiesform

ale(A)andfemale(B)

offspring.∧:increasedinMaternalHFDvs.CHD,∨:decreasedinMaternalHFDvs.CHD,withCVS:HFDeffect

dependsonCVScondition,withSAL:HFDeffectinsalinecondition,withCOC:CocaineeffectinHFDcondition,

+:increasedinMaternalcocainevs.saline,N.T.:nottestedinthischapter.

169

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Influence of Maternal High Fat Diet, Stress and Cocaine on Neural … · 2017-11-07 · 2.3.5. Gene expression analyses ... Chapter 5: Effects of maternal high fat diet and stress