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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.MaterialsandMethods............................................................................................................63
4.2.1.Animals.................................................................................................................................63
4.2.2.Proceduralmanipulationsofdams............................................................................63
4.2.3.Proceduralmanipulationsofoffspring....................................................................64
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.MaterialsandMethods............................................................................................................88
5.2.1.Animals.................................................................................................................................88
5.2.2.Proceduralmanipulationsofdams............................................................................88
5.2.3.Proceduralmanipulationsofoffspring....................................................................90
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.4.1.Experimentaltimelinefordamsandoffspring.
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.5.1.Experimentaltimelinefordamsandoffspring.
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.6.1.Experimentaltimelinefordamsandoffspring.
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
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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
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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
(correspondingtoSection5.2.3.2.Chapter5andSection6.2.3.2.Chapter6).
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
(correspondingtoSection3.2.3.2.Chapter3andSection5.2.3.5.Chapter5).
TheOpenfield(OF)consistedofanopaquesquarearena(40.3x40.3cm).Thetime
spentinthecenterandthedistancetraveledwererecordedusingeitherANY-maze
softwareorEthoVisionXToverthecourseofa10-15mintrial.Aftereachtest,the
25
mazewascleanedusinga70%ethanolsolutionandallowedtoairdrytoremoveor
homogenizeodorants.
2.3.3.3.Light-darktransitionbox
(correspondingtoSection3.2.3.4.Chapter3andSection5.2.3.6.Chapter5).
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
(correspondingtoSection3.2.3.5.Chapter3andSection6.2.3.3.Chapter6).
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
(correspondingtoSection3.2.3.6.Chapter3,Section4.2.3.6.Chapter4,Section5.2.3.7.
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
(correspondingtoSection3.2.3.6.Chapter3,Section4.2.3.6.Chapter4,Section5.2.3.7.
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
Thischapterisadaptedfrom:
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).
3.2.3.3.Elevatedplusmaze
Elevatedplus-mazewasrunasdescribedinChapter2(section2.3.3.1).
3.2.3.4.Light-darktransitionbox
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
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
0.2
0.4
0.6
0.8
1
1.2
1.4
Females Males
NF
kB (
rela
tive
ab
un
dan
ce)
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
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Females Males
Cen
ter/
Tota
l Tim
e R
atio
HFD CHD
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
0.2
0.3
0.4
0.5
0.6
0.7
Females Males
IL-I
Ra
(rel
ativ
e ab
un
dan
ce)
Hippocampus
#
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Females Males
MK
P-1
(re
lati
ve a
bu
nd
ance
)
Hippocampus
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Females Males Ik
Ba
(rel
ativ
e ab
un
dan
ce)
Amygdala
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Females Males C
ente
r/To
tal T
ime
Rat
io
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
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
0.02
0.04
0.06
0.08
0.10
0.12
Females Males
Cen
ter/
Tota
l Tim
e R
atio
HFD CHD
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.MaterialsandMethods
4.2.1.Animals
AdultmaleandfemaleLongEvansrats(7weeks)wereobtainedandprovidedad
libitumaccesstofoodandwater,asdescribedinChapter2(section2.1).
4.2.2.Proceduralmanipulationsofdams
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.Proceduralmanipulationsofoffspring
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
Experimentaltimelinefordamsandoffspring.(A)Damsreceivedeitherhighfatdiet
(HFD)orcontroldiet(CHD)forfourweekspriortopregnancy,duringpregnancy
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.
4.2.3.4.Testforcocainesensitization
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.MaterialsandMethods
5.2.1.Animals
AdultmaleandfemaleLongEvansrats(7week)usedwereobtainedandprovided
adlibitumaccesstofoodandwater,asdescribedinChapter2(section2.1).
5.2.2.Proceduralmanipulationsofdams
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).
5.2.2.4.Assessmentofmaternalbehaviour
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
observations/dam/day),usingacomputer-controlledbehaviouralcodingand
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.Proceduralmanipulationsofoffspring
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
Experimentaltimelinefordamsandoffspring.(A)Damsreceivedeitherhighfatdiet
(HFD)orcontroldiet(CHD)forfourweekspriortopregnancy,duringpregnancy
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).
5.2.3.6.Light-darktransitionbox
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
CHD CONCHD CVSHFD CONHFD CVS
#
@& &
* 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)
CHD CONCHD CVSHFD CONHFD CVS
0
200
400
600
800
1000
1200
0 20 80C
ortic
oste
rone
(ng/
mL)
( min after stress onset)
CHD CONCHD CVSHFD CONHFD CVS
** *
*
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
5.3.6.1.Elevatedplusmaze
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
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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.
Gene Region Gene RegionControl chow High fat chow Control chow High fat chow
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
Thischapterisadaptedfrom:
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.MaterialsandMethods
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.Proceduralmanipulationsofdams
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).
6.2.2.2.Testforcocainesensitization
Approximately8weeksaftertheterminationofthecocaineexposurephase,and
withinoneweekofweaning(whichoccurredonPND21),alldamsweretestedfor
theirlocomotorresponsetoachallengeinjectionof15mg/kgofcocaine(i.p.).For
thistest,ratswereplacedinthelocomotorchambersfor30min,injectedwith
cocaine,andplacedbackinthelocomotorchambersforanadditional60minperiod.
6.2.2.3.Assessmentofmaternalbehaviour
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
observations/dam/day),usingacomputer-controlledbehaviouralcodingand
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
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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
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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
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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
164
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
165
andstress-relatedbehaviour,whereasmaternalexposuretococaineinfluencesthe
rewardsysteminoffspring.Furtherexplorationofthenatureoftheseinteractions
mayhaveimportantimplicationsforunderstandingthemechanismsunderlyingthe
recentepidemicriseinobesity.
166
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.
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 ± 0.10HYP 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*
Maternal diet Maternal diet
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.
Gene Region Gene RegionControl chow High fat chow Control chow High fat chow
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 diet Maternal diet
Maternal stress Maternal stress
168
AppendixTable7.1
Mat
erna
l HFD
Offs
prin
g (b
oth
sexe
s)B
ody
Wei
ght
Mat
erna
l Car
eP
re-w
eani
ng B
WA
dult
BW
Acu
te C
OC
EP
M a
nxie
tyO
Fba
sal C
OR
Tpe
ak C
OR
T70
min
CO
RT
HP
CA
MY
PFC
NA
CA
dult
BW
Acu
te C
OC
EP
M a
nxie
tyO
Fba
sal C
OR
Tpe
ak C
OR
T70
min
CO
RT
HP
CA
MY
PFC
NA
CC
hapt
er3
∧N
.T.
∧=
N.T
.=
∧∨
==
=M
R=G
R∧T
H=C
RF
N.T
.N
.T.
=N
.T.
∧=
==
∧∧M
R∧G
R∧T
H∧C
RF
N.T
.N
.T.
Cha
pter
4∧
=∧
==
=N
.T.
N.T
.N
.T.
N.T
.∧C
RF
=∧
∧ w
ithS
AL,
∨ w
ithC
OC
N.T
.N
.T.
N.T
.N
.T.
=C
RF
Cha
pter
5∧
∧ =
=∧,
∧ w
ithC
VS
∨
N.T
.=
==
∧CR
F∨D
RD
1∨D
RD
2=
∧ w
ithC
VS
∨=
==
= =
CR
F =
DR
D1=
DR
D2
Mat
erna
l Coc
aine
Cha
pter
6N
.T.
=N
.T.
=+
N.T
.N
.T.
==
= =
CR
F =
GR
=CR
F +
DR
D1
N.T
.N
.T.
N.T
.N
.T.
N.T
.N
.T.
N.T
.N
.T.
N.T
.N
.T.
N.T
.
Lege
nds
∧: in
crea
sed
in M
ater
nal H
FD v
s. C
HD
∨: d
ecre
ased
in M
ater
nal H
FD v
s. C
HD
with
CV
S: H
FD e
ffect
dep
ends
on
CV
S c
ondi
tion
with
SA
L: H
FD e
ffect
in s
alin
e co
nditi
onw
ithC
OC
: Coc
aine
effe
ct in
HFD
con
ditio
n +
: inc
reas
ed in
Mat
erna
l coc
aine
vs.
sal
ine
N.T
.: no
t tes
ted
in th
is c
hapt
er
Dam
Beh
avio
urC
OR
T re
spon
se
Offs
prin
g
CO
RT
resp
onse
Beh
avio
urM
ale
Fem
ale
Gen
e E
xpre
ssio
nG
ene
Exp
ress
ion
Mat
erna
l HFD
Offs
prin
g (b
oth
sexe
s)B
ody
Wei
ght
Mat
erna
l Car
eP
re-w
eani
ng B
WA
dult
BW
Acu
te C
OC
EP
M a
nxie
tyO
Fba
sal C
OR
Tpe
ak C
OR
T70
min
CO
RT
HP
CA
MY
PFC
NA
CA
dult
BW
Acu
te C
OC
EP
M a
nxie
tyO
Fba
sal C
OR
Tpe
ak C
OR
T70
min
CO
RT
HP
CA
MY
PFC
NA
CC
hapt
er3
∧N
.T.
∧=
N.T
.=
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==
=M
R=G
R∧T
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.T.
=N
.T.
∧=
==
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R∧G
R∧T
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RF
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.T.
Cha
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=N
.T.
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.N
.T.
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RF
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AL,
∨ w
ithC
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N.T
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.T.
N.T
.N
.T.
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RF
Cha
pter
5∧
∧ =
=∧,
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ithC
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∨
N.T
.=
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∧CR
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RD
2=
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ithC
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∨=
==
= =
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F =
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D2
Mat
erna
l Coc
aine
Cha
pter
6N
.T.
=N
.T.
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N.T
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==
= =
CR
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=CR
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D1
N.T
.N
.T.
N.T
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.T.
N.T
.N
.T.
N.T
.N
.T.
N.T
.N
.T.
N.T
.
Lege
nds
∧: in
crea
sed
in M
ater
nal H
FD v
s. C
HD
∨: d
ecre
ased
in M
ater
nal H
FD v
s. C
HD
with
CV
S: H
FD e
ffect
dep
ends
on
CV
S c
ondi
tion
with
SA
L: H
FD e
ffect
in s
alin
e co
nditi
onw
ithC
OC
: Coc
aine
effe
ct in
HFD
con
ditio
n +
: inc
reas
ed in
Mat
erna
l coc
aine
vs.
sal
ine
N.T
.: no
t tes
ted
in th
is c
hapt
er
Dam
Beh
avio
urC
OR
T re
spon
se
Offs
prin
g
CO
RT
resp
onse
Beh
avio
urM
ale
Fem
ale
Gen
e E
xpre
ssio
nG
ene
Exp
ress
ion
Mat
erna
l HFD
Offs
prin
g (b
oth
sexe
s)B
ody
Wei
ght
Mat
erna
l Car
eP
re-w
eani
ng B
WA
dult
BW
Acu
te C
OC
EP
M a
nxie
tyO
Fba
sal C
OR
Tpe
ak C
OR
T70
min
CO
RT
HP
CA
MY
PFC
NA
CA
dult
BW
Acu
te C
OC
EP
M a
nxie
tyO
Fba
sal C
OR
Tpe
ak C
OR
T70
min
CO
RT
HP
CA
MY
PFC
NA
CC
hapt
er3
∧N
.T.
∧=
N.T
.=
∧∨
==
=M
R=G
R∧T
H=C
RF
N.T
.N
.T.
=N
.T.
∧=
==
∧∧M
R∧G
R∧T
H∧C
RF
N.T
.N
.T.
Cha
pter
4∧
=∧
==
=N
.T.
N.T
.N
.T.
N.T
.∧C
RF
=∧
∧ w
ithS
AL,
∨ w
ithC
OC
N.T
.N
.T.
N.T
.N
.T.
=C
RF
Cha
pter
5∧
∧ =
=∧,
∧ w
ithC
VS
∨
N.T
.=
==
∧CR
F∨D
RD
1∨D
RD
2=
∧ w
ithC
VS
∨=
==
= =
CR
F =
DR
D1=
DR
D2
Mat
erna
l Coc
aine
Cha
pter
6N
.T.
=N
.T.
=+
N.T
.N
.T.
==
= =
CR
F =
GR
=CR
F +
DR
D1
N.T
.N
.T.
N.T
.N
.T.
N.T
.N
.T.
N.T
.N
.T.
N.T
.N
.T.
N.T
.
Lege
nds
∧: in
crea
sed
in M
ater
nal H
FD v
s. C
HD
∨: d
ecre
ased
in M
ater
nal H
FD v
s. C
HD
with
CV
S: H
FD e
ffect
dep
ends
on
CV
S c
ondi
tion
with
SA
L: H
FD e
ffect
in s
alin
e co
nditi
onw
ithC
OC
: Coc
aine
effe
ct in
HFD
con
ditio
n +
: inc
reas
ed in
Mat
erna
l coc
aine
vs.
sal
ine
N.T
.: no
t tes
ted
in th
is c
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offspring.∧:increasedinMaternalHFDvs.CHD,∨:decreasedinMaternalHFDvs.CHD,withCVS:HFDeffect
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+:increasedinMaternalcocainevs.saline,N.T.:nottestedinthischapter.
169
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