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The involvement of central L.type calcium channel subtypes Cavl ٠2 and Cavl ٠3 in alcohol dependence and comorbid mental disorders

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The involvement of central L.type

calcium channel subtypes Cavl

٠

2 and

Cavl

٠

3 in alcohol dependence and

comorbid mental disorders

Referees: Prof. Dr. Rainer S p ia g e l Prof. Dr· Christoph Schuster

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Inhalt

Abbreviations 11

Publierions..13 ״ . . . ״ . . ״ . . . ״ . . . ״ . . . . ״ . . . . . ٠٠

Introduction 15

1. Alcohol dependence...15 ...״״...״...״.״«..״....״״...״...״״...״...״ 1.1. Development o f Ле disorder...15 ...״...״...״...״ 1.2. Guidelines for ễ a g n o s i s . . . 18

1.3. Available pharmacological treatments 19

1.4. Animal models for alcohol dependence 20

1.5. Comorbidity wi٥ other mental disorders...23 ...״...״...״....״....״״..״....״״.״

1.5.1· Nicotine 23

15.2. ،؟á / z o p  r e w . a . 25

2. L־type calcium c h a ie ls ... 29 2.1. L-type calcium c h a n n e l s . . . 29

2.2. Subtypes Ca٧1.2 and Cavl.3 m n e u r o n s 30

2.3. Pharmacology o f L T C C s . 33

2.4. Animal models to investigate Cavl.2 and C avl.3... 35 2.5. Single nucleotide p o lş o ş h is m s as risk variants .···...37

2.6. LTCCs in mental disorders: støte o f knowledge 37

2.6.1 Alcohol depefidence.i.... . . .7ف

2.6.2. ٠co///ìể٠re/a/^rfốáw /or...38.... ״

2.63. ،؟á / z o á r e w / s l r . . . . . . . . 39

Π. Aims o f th e脱 ぬ…·……··…··……·…·…··……·……·…….… 41

III. Materials and 따 础 也…··………··“…·……·…·………"^ 43

1. Animal studies alconoi d e p e n d e n c e 43

1.1. Experimental i i i l s . ... 43

Wistar rats (Stody I and 11)………·……·…·………·…………···……… 43

CavL2flox X C a H I٠CreERT2 mice (Study III a n d 43

1.2 ن. Genotyping ofCavL2flox X € a M i- € r e E T 2^ ……··……·………·…·…………··…·…·…·…44 1.3. Locomotor activity ئ mice: Open ? ؛…·…·……·………·………·…··…·………·…·… 45 1.4. Induction o f alcohol dependence...45 .״...״.״.״״...״..״״...״....״

1.4.1. 요 다^ …·…··………·…"……·………··…………·…··…"^

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1.6. Chronic treatment with haloperidol or clozapine 46 1.7· In situ hybridization... 47

1.7.1. Preparation ofbrain sections,....,...47 ... . . . ״ 1.7.2. RNAprohegeneration.״ i , . , . ...

и з . ... 47

1.8. Western blot 紐 바 也·.…·………·………·………·…^ 49

1.9. blectrophysiology...…·………·……·……·…·…·………·………·49

1.9. L Preparation o f acutely dissociated

1.9.2. Eìectrophysiological r e c o r d i n g s 49 1.10. Operant alcohol self־a d _ is t o t i^ ^ experiments in rats (Study 1)……·……….………50

1.10.2. rra/m > ? g ، s r e ، y ، s ! / ơ / ? . 50

1.10.3. Stereotmic placement osỉntracerebrovenừicuỉar (Ì.C.V.) guide cannulas... 51

1.10.4. I.C.V. injections.,...ệ . . , . ...ế,...in. 51

1.10.5. Alcohol seìsadmỉnisừation祕사.·……·…·……·.…··………·………··…··…··…·……… 51

1.10.6. Extinction o f alcohol selføadministration ¿ á a v /o r... 52

1.10.7. C/Ueminducedreinstatement..,...,ềầ.,...ậệ.ềị...π... . í . . .1,1..ị1.4...ề....ệ..152

1.11· Operant alcohol self-admmistration experimente in mice (Study II)... 52

1.11.1. Saccharine f a d i n g p r o c e d u r e 52

1.12. Statistical a n a l y s i s 53

2. Animai studies — nicotine d e p e n d e n c e ว3

2.1· Experimental 53

2.2. Drugs...54 ...״.״...״....״....״...״..״.״.״...״

2.3. In situ hybridizatì0n·……·……··………·………·……·…54

2.4. Locomotor activity measurements 54

2.4.1. Nifedipine dose re،؟־/ ? o w ^ . 54

2.4.2. Locomotor s e n s i t i z a t i o n 54 2.5. Nicotine self·admınısfratìon………··………··………·…···…··…··…"^ 55 2.6. Statistical malysis ״...״...״...״...״H.55 ...״...״״..״...״

3. Human 56

3 .1 Postmortem tissue s a ş l e s . . . ... 56

3.1.1. Л/сойо//с/7а//ел/..56...؛׳.״

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3.1,3. 57 3.2. Genotyping for the CACNAIC single nucleotide po_O T pl1ism rsl006737···.·.··· 57 3.3· (Cantitative real-time PCR (qRT-PCR)57 ... ٠٠״ 3.4. Støtistical analysis ....״...״״...״...״...״..״....n58 ..״...״.״.״...״

IV. Results 59

1. Study I: Differential roles forL־type calcium channel subtiles in alcohol dependence... 59 1.1· Increased Cacnalc mRNA in the hippocampus and amygdala ofalcohoi dependent rats... 59 1.2. Cavl٠2 protein increase m the CAI matches observations on mRNA lev e l... 61

1.3. Cacnalc expression levels show dynamic regulation from acute intoxication to prolonged

1.4. it ! rats display increased Cavl٠2 currents in the hippocampus···..·.····.···· 63 1.5. Alcohol sels.adimmsữation is not susceptible to LTCC ẩ a g o n ism ... 64 1.6. Cue-induced reinstatement o f alcoholseeking is blocked by verapamil in alcohol dependent rats 64

1.7. Summary o f Study I 66

2. Study II: Characterization ofL-type calcium channel subtype expression in animal models o f alcoholism ...丨.٠..丨.٠...._..٠.丨...٠....٠...״...丨丨.٠״ 丨丨.丨٠...__٠٠..٠..丨٠...丨.٠.丨._丨..6 ״/

2.1· Differential Cacnalc and C a c n a l d m l A in the amygdala and hippocampus o f alcohol preferring AA and non-preferring ANA 대 호 노…………··……·………··……·………·… 67 2.2. bduction o f the alcohol deprivation effe^ (ADE) does not influence central LTCC

expression in the amygdala and hippocampus 68

2.3. 4 weeks o f alcohol vapor inhalation is sufficient to increase Cacnalc i i N A levels ..···.···· 69 2.4. Voluntary alcohol consumption decreases Cacnalc l i N A expression and differentially

regulates i acnald i N A levels 71

2.5. Stress increases C ữ c n a ỉ d v ẫ Ấ expression in the hippocampus····.·.··.···.···.·.···.··· 73 2.6. Summary o f Study II... 74 3. Study III: L-type calcium channel subtype Cavi .2 mediates dependence-induced increase in alcohol self-admimsfration..." 3.1. Locomotor activity does not differ between mutants and wildtypes.···.···.···...■···.11 ·,·..···.·״ 3.2. Lack o f Cavl.2 blocks alcohol dependence.mauced increase in sels-admmisừation... 78

3.3. Demonsfration o f the Cavl.2 t o o c k o u t . 79

3.4. Summary o f Study……·.·……·………··……….………··……··…… 81

¡4. Study IV: Functions ofL.type calcium charnel subtypes Cavl٠2 and Cavl٠3 in nicotine-related

behavior... .

4.1. Cacnalc and Cacnald Ể N A levels change during acute and chronic nicotine exDosure and

abstinence...82. . . . ״...״

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4.4. Nifedipme prevents increased mcotine sensitization after abstinence 85 4.5. Decreased Cavi •2 attenuates nicotine self-administration 86

4.6. Summary o f S t u d y 88

5. Study V: Analysis o f L-type calcium chaimel subtype expression in human postmortem

samples o f patients with mental disorders...89 ...״...״...״..״..״...״ 5.1· No changes in CACNAIC mRNA expression in the striatum o f human alcoholics."··”.·..··· 89 5.2· Decreased CACNAIC and CACNAID n i A in forebrain regions o f schizophrenia patients

90

5.3. No effect ofhaloperidol and clozapine on Cacnalc and CacnaJdmRNA expression.···.·.·. 92 5.4. m i A expression o f LTCC subtypes is not altered in the dorsolateral prefrontal cortex o f suicide completers with major depression...93 ...״...״״...״..״...״.״...״

5.5. S u m a r y o f Study V . . . ....93

V. Discussion…·…··………·……··…·…

1. Study I: Differential roles for L-type calcium c h a i e l subtypes in alcohol dependence..·..·.··· 95 2. Study II: Characterization 0f L | e calcium channel subtype expression in animal models o f

alcoholism و و

3. Study Ш: L־type calcium channel subtype Cavl.2 mediates dependence-induced increase in

alcohol sels-adminisừation..104. . . . ״....״

4. Study IV: Functions o f L־type calcium channel subtypes Cavl.2 and Cavl.3 in nicotine-related behavior...107 5. Study V: Analysis ofL-type calcium channel subtype expression in human postmortem

samples o f patients with mental d i s o r d e r s . . . 110

6. General discussion LTCC involvement in mental disorders...114 ...״..״״...״...״...״ VI. Summary and Outlook... 117

VII. A c k n o w l e d g e m e n t s 121

V in. References....123 ...״.״...״״...״...״...״...״..״...״...״...״...״..״....״

IX. A p p e n d i x . . . . 151

References

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