The Role of the Serotonin Transporter as a Genetic Risk Factor in the Development of Drug Addiction

207 Pages·2016·1.69 MB·English

The Role of the Serotonin
Transporter as a Genetic Risk
Factor in the Development of
Drug Addiction
by
Bridget Williams Brox
Athesis
submittedtotheVictoriaUniversityofWellington
infulfilmentofthe
requirementsforthedegreeof
DoctorofPhilosophy
inPsychology.
VictoriaUniversityofWellington
2016

Abstract
Drugaddictionisaubiquitousphenomenonworldwidethatplacestreme-
ndousfinancialandpsychologicalburdenonsocieties,familiesandthein-
dividual. Interestingly, only a small percentage of individuals (∼20%), re-
gardlesstheirdrugofchoice,goontodevelopthecompulsivebehaviours
that define drug addiction. Clinical studies have shown that there is a
subset of the population with a genetically determined reduction in the
serotonin transporter that may increase vulnerability to developing a va-
rietyofpsychiatricdisorderslikedepression,anxietyanddrugaddiction.
To investigate the influence of reduced serotonin transporter function
in the laboratory we studied the effects of MDMA (‘ecstasy’) and heroin
in a genetically altered animal model: the serotonin transporter (SERT)
knockoutrat. Homozygous(HOM)animalslackSERTfunctioncompletely
while heterozygous (HET) have about 50% SERT function compared to
the wild type (WT). Groups of HOM, HET and WT animals completed
MDMA or heroin self-administration experiments. A robust genotype ef-
fectemergedforanimalsself-administeringMDMA;facilitationofMDMA
self-administration was inversely related to SERT function. HOM ani-
mals, without exception, reached acquisition criterion significantly faster
than theHET animals; HET animalsthen showed higheracquisition rates
compared to the WT animals. In contrast, there were no differences be-
tween the genotypes when animals self-administered heroin. To inves-
tigate the driving force behind facilitated MDMA self-administration in
animals with reduced SERT function locomotor activity and conditioned
taste aversion experiments were undertaken. In contrast to the drug self-
administrationexperiments,MDMAinducedhyperactivitywaspositively
related to SERT function. Thus, it was significantly reduced in HOM and
HET animals compared to the WT. Again, heroin treatment did not pro-
duce differences in locomotion between the genotypes. MDMA induced
conditionedtasteaversionrevealedonlyamaineffectofdosewithrobust
conditionedtasteaversionforbothdrugdoses,althoughatrendindicated
that HOM animals may have heightened sensitivity to MDMA. However,
heroin treatment failed to produce a conditioned taste aversion effect in
any of the groups regardless of dose. Beyond the aforementioned be-
havioural experiments striatal brain tissue from the animals that had pre-
viouslyself-administeredMDMAorheroinwasanalysedviaquantitative
reverse transcription polymerase chain reaction; five targets were evalu-
ated to quantify drug induced changes in brain derived neurotrophic fac-
tor gene expression (BDNF). Several BDNF isoforms (total BDNF, BDNF
III and BDNF IV) were significantly increased in animals that had self-
administeredMDMA;thiseffectwastrueacrossHOM,HETandWTsub-
jects. Comparatively, animals that had self-administered heroin did not
showadifferenceinBDNFexpressioncomparedtountreatedcontrolani-
mals.
This suite of experiments provides insight into the influence of a com-
promisedserotonergicsystemonthedevelopmentofdrugaddiction. That
is,whilereducedSERTfunctiondoesnotappeartoaugmenttheaddictive
properties of drugs like heroin there is reason to suspect that it does con-
feradditionalsusceptibilitytodevelopingaddictiontodrugslikeMDMA,
highlighting the hypothesis that different classes of addictive substances
actthroughdifferentneurobiologicalpathways.
Acknowledgements
Iwouldliketogratefullyacknowledgetheguidance,supportandencour-
agement of my primary doctoral supervisor, Professor Bart Ellenbroek.
This thanks extends also to my secondary supervisor, Professor David
Harper. From the start these two brilliant scientists have supported and
challenged me; they’ve helped me to extend my experience and expertise
asI’vegrownasaninvestigatorandprofessional.
I wouldlike tothank thefull CognitiveBehavioural Neurosciencelab-
oratory group particularly Professor Susan Schenk for advice regarding
drug self-administration. For technical advice and training in a variety
of laboratory techniques I would like to thank Dr. Katie Brennan and Dr.
JoyceColussi-Mas-theirhelpalongthewaywasinvaluable. Iwouldalso
like to thank Uta Waterhouse and Sophie Muller for their help with spe-
cific projects. Lastly, I would like to thank the animal technicians, Richard
Moore, Peter Vancompernolle and Michael Roberts for their support and
helpintheeverydaycareoftheanimalsinourlaboratory.
Additionally, I would to thank Dr. Darren Day for supporting the col-
laboration between our labs that allowed me to learn the molecular ana-
lytical technique, quantitative reverse transcription polymerase chain re-
action. This thanks also extends to the students working in his lab who
helped me with practical issues over many months of work; particularly,
VarunVenkatesh.
OnapersonalnoteIwouldliketoextendmybiggestthankstomyhus-
band,Tim,forhisunwaveringsupport,encouragementandhelpthrough-
iii
iv
out the entire PhD program and in every aspect of our lives. He encour-
agedmetopushmyself,tonotbesoterriblyhardonmyselfandtopursue
ambitiousgoalsbothinsideandoutsidethelaboratory.
I must extend many thanks to both of our families for their continued
supportthroughoutthisprocess. Thepackagesfilledwithgiftsandtreats,
thephonecallsandSkypechatshavemademefeelthattheyarenotsofar
away.
Lastly, I would like to thank the Vic Books cafe and Red Bull energy
drinksforcaffeinesupportthatkeptmegoingthroughoutmanylongterm
experiments.
Contents
1 GeneralIntroduction 1
1.1 DrugAddiction . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 CostsAssociatedwithDrugAddiction . . . . . . . . 2
1.1.2 Terminology . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1.3 BehaviouralChangesthatDefineDrugAddiction . . 3
1.1.4 NeurochemicalEffectsthatCoincidewithBehavioural
Changes . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.1.5 TransitiontoDrugAddiction . . . . . . . . . . . . . . 7
1.1.6 TheoriesofDrugAddiction . . . . . . . . . . . . . . . 9
1.2 IndividualDifferences . . . . . . . . . . . . . . . . . . . . . . 10
1.2.1 TheSerotoninTransporter(SERT) . . . . . . . . . . . 11
1.2.2 The Serotonin Transporter Gene (SLC64A) and Pro-
moterRegion . . . . . . . . . . . . . . . . . . . . . . . 11
1.2.3 HumanStudiesoftheSERT . . . . . . . . . . . . . . . 12
1.3 ModellingIndividualDifferencesintheLaboratory . . . . . 18
1.3.1 SerotoninTransporterKnockoutPreclinicalModel . . 18
1.3.2 PointMutationintheSLC6A4Gene . . . . . . . . . . 19
1.4 DrugsofAbuse . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.4.1 3,4-methylenedioxymethamphetamine,MDMA,“Ec-
stasy” . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
1.4.2 (5α,6α)-7,8-Didehydro-4,5epoxy-17-methylmorphinan-
3,6-dioldiacetate(ester),Heroin,diamorphine . . . . 26
v
vi CONTENTS
1.5 MolecularAnalysis . . . . . . . . . . . . . . . . . . . . . . . . 30
1.5.1 BrainDerivedNeurotrophicFactor(BDNF) . . . . . . 31
1.5.2 BDNFandNeuropsychiatricIllness . . . . . . . . . . 31
1.6 BriefOutlineofBehaviouralandMolecularExperiments . . 32
2 NoveltySuppressedFeedingand
DrugSelf-Administration 35
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.1.1 Predictions . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.2.1 NoveltySuppressedFeeding . . . . . . . . . . . . . . 38
2.2.2 DrugSelf-Administration . . . . . . . . . . . . . . . . 39
2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.3.1 MDMA . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.3.2 Heroin . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3 LocomotorActivityandConditioned
TasteAversion 65
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.1.1 Predictions . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.2.1 LocomotorActivity . . . . . . . . . . . . . . . . . . . . 68
3.2.2 ConditionedTasteAversion . . . . . . . . . . . . . . . 69
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.3.1 LocomotorActivity . . . . . . . . . . . . . . . . . . . . 71
3.3.2 ConditionedTasteAversion . . . . . . . . . . . . . . . 74
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4 QuantitativeReverseTranscriptionPolymeraseChainReaction 83
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.1.1 Predictions . . . . . . . . . . . . . . . . . . . . . . . . . 87
CONTENTS vii
4.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.2.1 QuantitativeReverseTranscriptionPolymeraseChain
Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.3.1 AverageHousekeepingGenes . . . . . . . . . . . . . 96
4.3.2 TotalBDNF . . . . . . . . . . . . . . . . . . . . . . . . 97
4.3.3 BDNFtranscriptIII . . . . . . . . . . . . . . . . . . . . 99
4.3.4 BDNFtranscriptIV . . . . . . . . . . . . . . . . . . . . 101
4.3.5 BDNFtranscriptVI . . . . . . . . . . . . . . . . . . . . 103
4.3.6 BDNFtranscriptIXa . . . . . . . . . . . . . . . . . . . 106
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5 GeneralDiscussion 113
5.1 Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
5.2 BriefSummary . . . . . . . . . . . . . . . . . . . . . . . . . . 113
5.3 ExperimentalSignificance . . . . . . . . . . . . . . . . . . . . 114
5.4 Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
5.4.1 NoveltySuppressedFeeding . . . . . . . . . . . . . . 121
5.4.2 DrugSelf-Administration . . . . . . . . . . . . . . . . 122
5.4.3 LocomotorActivity . . . . . . . . . . . . . . . . . . . . 124
5.4.4 ConditionedTasteAversion . . . . . . . . . . . . . . . 125
5.4.5 QuantitativeReverseTranscriptionPolymeraseChain
Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . 126
5.4.6 TranslatingAnimalOutcomestoHumans . . . . . . . 127
5.5 FutureDirections . . . . . . . . . . . . . . . . . . . . . . . . . 128
5.5.1 MicrodialysisandInVivoVoltammetry . . . . . . . . 128
5.5.2 AmbiguousCueInterpretation . . . . . . . . . . . . . 129
5.5.3 EmulsionPCR . . . . . . . . . . . . . . . . . . . . . . . 130
5.6 FinalConclusion . . . . . . . . . . . . . . . . . . . . . . . . . 130
A SupplementalqRT-PCRAnalysis 133
A.1 Section1: HousekeepingGenes . . . . . . . . . . . . . . . . . 133
viii CONTENTS
A.2 Section2: TotalBDNF . . . . . . . . . . . . . . . . . . . . . . 138
A.3 Section3: BDNFTranscriptIII . . . . . . . . . . . . . . . . . . 142
A.4 Section4: BDNFTranscriptIV . . . . . . . . . . . . . . . . . . 147
A.5 Section5: BDNFTranscriptVI . . . . . . . . . . . . . . . . . . 151
A.6 Section6: BDNFTranscriptIXa . . . . . . . . . . . . . . . . . 156