Endogenous opioid peptides in drug addiction [Elektronische Ressource] / submitted by Britta Schürmann

Endogenous opioid peptides in drug addiction [Elektronische Ressource] / submitted by Britta Schürmann

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EndogenousopioidpeptidesindrugaddictionThesisSubmittedfora Doctoral Degree inNatural Sciences(Dr.rer.nat.)FacultyofMathematics andNaturalSciencesRheinischeFriedrich WilhelmsUniversity,BonnsubmittedbyBRITTA SCHÜRMANNBonnDecember2009Preparedwith the consentofthe Faculty ofMathematics andNatural SciencesRheinische Friedrich Wilhelms University,Bonn.This thesis is available online on HochschulschriftenserverofULB Bonnhttp://hss.ulb.uni-bonn.de/diss_online elektronisch publiziert.Publicationyear: 20101.Reviewer: Prof. Dr. rer. nat. Andreas Zimmer2.Reviewer: Prof. Dr. rer. nat. JörgHöfeldDate ofsubmission: 01/12/2009Date ofexamination: 14/03/2010Für H.-B. SchürmannAbbreviations5-HT 5-Hydroxytryptamine (serotonin)AAV Adeno-associatedvirusaca Anteriorcomissure,anteriorAcbC Nucleus acumbens,coreAcbShshellACTH AdrenocorticothropichormoneANOVA Analysis ofvarianceBlA BasolateralamygdalaBCIP 5-Bromo-4-chloro-3-indolylphosphateBCP 1-Bromo-3-chloro-propanecc Corpus callosumCDP-Star Chemiluminescentsubstrateforalkaline phosphataseCeA Central amygdalaCPu CaudateputamenCOGA Collaborativestudyonthegenetics ofalcoholismDAPI 4’,6-Diamidino-2-phenylindoleDEPC DiethylpyrocarbonateDIG DigoxigeninDMEM Dulbecco’s modifed Eagle’s mediumDMSO DimethylsulfoxideDNA DeoxyribonucleicacidDSM-IV Diagnosticandstatistical manualofmentaldisorders IVE.

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Publié le 01 janvier 2010
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Endogenousopioidpeptides
indrugaddiction
Thesis
Submittedfora Doctoral Degree inNatural Sciences
(Dr.rer.nat.)
FacultyofMathematics andNaturalSciences
RheinischeFriedrich WilhelmsUniversity,Bonn
submittedby
BRITTA SCHÜRMANN
Bonn
December2009Preparedwith the consentofthe Faculty ofMathematics andNatural Sciences
Rheinische Friedrich Wilhelms University,Bonn.
This thesis is available online on HochschulschriftenserverofULB Bonn
http://hss.ulb.uni-bonn.de/diss_online elektronisch publiziert.
Publicationyear: 2010
1.Reviewer: Prof. Dr. rer. nat. Andreas Zimmer
2.Reviewer: Prof. Dr. rer. nat. JörgHöfeld
Date ofsubmission: 01/12/2009
Date ofexamination: 14/03/2010Für H.-B. SchürmannAbbreviations
5-HT 5-Hydroxytryptamine (serotonin)
AAV Adeno-associatedvirus
aca Anteriorcomissure,anterior
AcbC Nucleus acumbens,core
AcbShshell
ACTH Adrenocorticothropichormone
ANOVA Analysis ofvariance
BlA Basolateralamygdala
BCIP 5-Bromo-4-chloro-3-indolylphosphate
BCP 1-Bromo-3-chloro-propane
cc Corpus callosum
CDP-Star Chemiluminescentsubstrateforalkaline phosphatase
CeA Central amygdala
CPu Caudateputamen
COGA Collaborativestudyonthegenetics ofalcoholism
DAPI 4’,6-Diamidino-2-phenylindole
DEPC Diethylpyrocarbonate
DIG Digoxigenin
DMEM Dulbecco’s modifed Eagle’s medium
DMSO Dimethylsulfoxide
DNA Deoxyribonucleicacid
DSM-IV Diagnosticandstatistical manualofmentaldisorders IV
E.coli Escherichia coli
EDTA Ethylenediaminetetraaceticacid
EGTA Ethyleneglycolacid
EIIa Adenoviralpromoter
EScell Embryonicstemcell
FCS Fetal calfserum
FIAU Fialuridine
FITC Fluorescein isothiocyanate
g Gravity
G418 Geneticin
GABA γ-Aminobutyrcaccid
γ-MSH γ-Melanocyte stimulatinghormone
GFP Greenfluorescentprotein
+GIRK G-proteinactivatedinwardlyrectifying K channelGPCR G-protein coupledreceptor
HBSS Hank’s balancedsalt solution
HET Haus fürexperimentelleTheraphie
HPA Hypothalamic-pituitary-adrenal
HRP Horseradish peroxidase
HWE Hardy–Weinbergequilibrium
i.v. Intravenous
ICD-10 Internationalstatisticalclassificationofdiseases and related
healthproblems
IgG ImmunoglobulinG
kb Kilobase
lacZ Gene encoding ß-galactosidase
LD Linkage disequilibrium
LIF Leukemiainhibitoryfactor
LV Lateral ventricle
MALDI-TOF Matrix-assistedlaserdesorption/ionization timeofflight
MEF Mouse embryonicfeeder
NAc Nucleus accumbens
nAChR Nicotinic acetylcholinereceptor
neo Gene providinga neomycinresistance
NGFN National genomeresearch network
NMDA N-methyl-D-aspartic acid
nor-BNI Nor-binaltrophimine
ns Not significant
NTP Nucleoside triphosphate
PBS Phosphatebufferedsaline
PCR Polymerasechainreaction
PDYN Preprodynorphin
PENK Preproenkephalin
PFA Paraform aldehyde
Pgk-1 Phosphoglyceratekinase 1promoter
POMC Proopiomelanocortin
QTL Quantitativetraitlocus
RIA Radioimmunoassay
RNA Ribonucleicacid
rpm Rotations perminute
SCID Structured clinicalinterviewforDSM disorders
SDS Sodium dodecylsulfateSEM Standarderrorofmean
SNP Single nucleotidepolymorphism
SSC Saline sodiumcitrate
ß-LPH ß-lipotripichormone
SYBR Cyaninedyetostain DNA
TE Tris EDTA
Tris Tris(hydroxymethyl)aminomethane
UV Ultraviolet
vg Virusgenome
VTA Ventraltegmentalarea
WHO Worldhealthorganization
wt Wildtype
X-gal Bromo-chloro-indolyl-galactopyranosideContents
1 Introduction 1
1.1 The opioidsystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 The opioid system andnicotineaddiction . . . . . . . . . . . . . . . . . . . . 4
1.3 The opioid system andalcohol . . . . . . . . . . . . . . . . . . . . . 6
1.4 Aim ofthis work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2 Material 11
2.1 Enzymes andantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2 Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.3 Bacteria, cells and plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.4 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.4.1 DNA isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.4.2 Agarosegelelectrophoresis . . . . . . . . . . . . . . . . . . . . . . . 13
2.4.3 Cellcultureexperiments . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.4.4 Southernblot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.4.5 In situhybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.4.6 LacZ staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.4.7 Immunofluorescencestaining . . . . . . . . . . . . . . . . . . . . . . 18
2.4.8 Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3 Methods 21
3.1 Cellculture methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.1 Cultivation ofcells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.2 Generationofembryonicmousefibroblasts . . . . . . . . . . . . . . . 22
3.1.3 Storage and re-cultivation ofcells . . . . . . . . . . . . . . . . . . . . 22
3.1.4 ElectroporationandisolationofrecombinantEScellclones . . . . . . 22
3.2 Molecularbiologymethods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2.1 DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2.2 Determinationofnucleic acidconcentration . . . . . . . . . . . . . . . 23
3.2.3 DigestionofDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
I3.2.4 PurificationofDNAfragments . . . . . . . . . . . . . . . . . . . . . . 23
3.2.5 Reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2.6 Polymerase-chainreaction . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2.7 In vitrotranscription . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2.8 Southernblot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2.9 In situhybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.10 RNA isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.3 SNP genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3.1 Humansubjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3.2 SNP selectionandgenotyping . . . . . . . . . . . . . . . . . . . . . . 29
3.4 Proteinbiochemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.4.1 Radioimmunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.4.2 LacZ staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.4.3 Immunofluorescence staining . . . . . . . . . . . . . . . . . . . . . . 30
3.5 Animalexperiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.5.1 Tissue preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.5.2 Preparationoftissue sections . . . . . . . . . . . . . . . . . . . . . . . 31
3.5.3 Generationofchimericmice . . . . . . . . . . . . . . . . . . . . . . . 31
3.5.4 Deletionoftheneomycingene . . . . . . . . . . . . . . . . . . . . . . 31
3.5.5 Anxiety measurement . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.5.6 Stereotacticinjection . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.5.7 Nicotine self-administration . . . . . . . . . . . . . . . . . . . . . . . 33
3.6 Statisticalmethods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.6.1 Generalstatistic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.6.2 Casecontrol study . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
4 Results 37
4.1 Generationoffloxedpreproenkephalinmice . . . . . . . . . . . . . . . . . . . 37
4.1.1 Targetingstrategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.1.2 Generationofconditional knockout mice . . . . . . . . . . . . . . . . 39
4.1.3 Quantitativeexpression analysis . . . . . . . . . . . . . . . . . . . . . 42
4.1.4 Qualitative e analysis . . . . . . . . . . . . . . . . . . . . . . 45
4.1.5 Behavioral phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . 47
4.1.6 Viralinjection intothe nucleus accumbens . . . . . . . . . . . . . . . 47
4.2 SNP genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.2.1 Exploratoryanalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.2.2 Detailedgenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.2.3 Haplotype analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
II