3.3 Effects of Oiigonucleotide Antisense to Dopamine Diareceptor Mrna on Sensitization of Apomorphine-Induced Rotations by Chronic

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3.3 Effects of Oiigonucleotide Antisense to Dopamine Diareceptor Mrna on Sensitization of Apomorphine-Induced Rotations by Chronic National tibmy Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie SeMces seMces biùliographiques 385 Welllngîon SB& 395, nie Weltington ûüawaON KIA ON4 ûttawaON KlAOiU4 Canada Cariada The author has granted a non- L'auteur a accordé melicence non exc1usi.e licence allowing the exclusive permettant à la National LÎbrary of Canada to Bibliothèque nationale du Canada de reproduce, loan, distrribute or sen reproduire, prêter, distribuer ou copies of this thesis in rnicroform, vendre des copies de cette thèse sous paper or electronic fornais. la forme de microfiche/fiIm, de reproduction sur papier ou sur format électronique. The anthor retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thése. thesis nor substafltid extracts f?om it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent Etre imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. The dopamine transporter @AT) hctions primady as a means for the termination of dopaminergic neurotransmission, but may aIso play a role in the pathogenesis of Parkkison's disease. Recent studies have implicated the DAT in the up take of two experimentai neurotoxins, 1-methyl4phenyI- 1,2,3,6-tetrahydrop yridine (MPTP) and 6-hydroxydopamine. Here, in Mvo administration of phosphorothioate adsense oügonucleotides targeting DAT mRNA in the left substantia nigra pars compacta resulted in reduced [3H]WIN 35,428 binding to DAT in the lefi striatum and signincant Ievodopa and amphetamine-induced conhalaterai rotations. Unilateral pretreatment with DAT antisense prior to bilaterd intrastriatal infiision of either neurotoxin resulted in asymrnetncd striatal DAT binding and dopamine content indicatuig significant preservation ipsilateral to antisense pretreatment. As well, significant apomorphine-induced ipsilaterai rotations were obsenred, suggesting neuroprotection of nigrostriatai neurons on the antisense-treated side. Thus, the DAT appears to play a critical role in determining susceptibility to these experimentd neurotoxins and rnay prove usefil as a marker for susceptibility to Parkinson's disease and as a target for therapeutic intervention. Regdation of dopamine neurotransmission by the DAT may aiso be an important factor in the development of dnig-induced dyskinesias. Dyskuiesias are abnomai involuntary movanents which deveIop as a side-effect of long-term treatment with either Ievodopa for Parkinson's (Ievodopa-induced dyskinesias) or antipsychotics (tardive dyskmesia) for schizophraiia The mechanhm tmderlyhg these dyskinesias remaÏns uncIear but may invoIve heightened activity in dopamine DIreceptor-bearing datonigral neurons. Here, intrastriataI infusion of antisense targeting dopamine DIAreceptor mRNA signifÏcantIy reduced striatai DIreceptor binding and attenuated behaviourd responses in rodent models of both levodopa-induced dyskinesia and tardive dyskinetia. Thus, the dopamine DIAreceptor rnay play a significant role in the expression of hg-induced dyskinesias. Recently, chronic pdsatiIe Ievodopa treatment in a rodent mode1 of levodopa- induced dyskinesias has been associated with increased expression of striatal dopamine D, receptors. These receptors are locaiized with DIreceptors on striatonigral neurons and their induction is dependent on dopamine DIreceptor activity. Here intmtriatal infusion of otigonucleotide antisense to dopamine D, receptor mRNA effectively reduced chronic Ievodopa-induced elevations in D, receptor expression and significantly reduced behaviourd responses in this modet. Thus, expression of levodopa-uiduced dyskinesias may aIso involve dopamine D3receptor activity, possibly through interaction with DI receptors. TABLE OF CONTENTS Page *. ABSTRACT fl TABLE OF CONTENTS iv LIST OF FIGTJRES X LIST OF TABLES xv 1.1 Basai Ganglia 1.1. I Introduction 1-1.2 Sm'atum 1. 1 -3 Globus Pallidus 1. f -4 Subthalamic Nucieus 1.1.5 Substantia Nigra 1.2 Basai Gangiia Disorders 1.2.1 Parkinson S Disese 12.2 Huntington 5 Diseare 12.3 Drug-lnduced Llyskinesras Page 1.3 Dopamine Receptors I t 1.3 .1 Dopamine Receptor CZassz~cation 11 1.3.2 Dopamine DI Receptors 12 1.3.3 Dopamine D3 Receptors 17 1.4 Dopamine Transporter 1.4.1 MolecuLar Structure 1.4.2 Lacaiization L .4.3 Functiun 1.5 Antisense Oligonucleotides i S. 1 Mechanism of Action 1 52Design 153 Therapeutic Potentiui 1.6 Rodent Models of Drug-Induced Dysbesias I -6.1 Levodopa-Induced Dysknesia 1-6.2 Tirdive Llyskinesia Page CHAPTER 2 EXPERIMENTAL DESIGN 37 2.1 Objectives 2.1.1 Rationale 2.1 -2Hypotheses 2.2 Gened Methods 2.2.1 Animafs 222surgq 2.2.3 Antisense OZigonucleotides 2.2.4 Chronic Drug Treatments 22.5 BehaviouraI Observations 2.2-6 A utoradiography 2.2.7 HPLC 2.2.8 Statistical AnaLys& CHAPTER 3 EXPERIMENTAL RESULTS 3.1 Effects of Ofigonucleotide Antisense to Dopamine D ,, Receptor mRNA on SKF 38393-Induced Behaviours 3 S.1 Introduction Page 3.1 2 Design 54 3 .1 -3 Results 54 3.1.4 Summary 63 3.2 Effects of Oligonucleotide Antisense to Dopamine DIAReceptor mRNA on Chronic Neuroleptic-Induced VCMs 3 2. 1 Introduction 3.2.2 Design 3 2.3 Redts 32.4 Summav 3.3 Effects of OLigonucleotide Antisense to Dopamine DIAReceptor mRNA on Sensitization of Apomorphine-Induced Rotations by Chroaic Levodopa in Hemiparkinsonian Rats 3.3.1 Introduction 3.3 -2 Design 3-3 -3 ResrrIts 3.3.4 Smmm 3.4 Effects of OligonucIeotide Antisense to Dopamine DjReceptor mRNA on Sensïtization of Apomorphine-hduced Rotations by Chronic Levodopa m HemiparkÏnsonÏan Rats Page 3 .4.l Introduction 84 3.4.2 Design 85 3*4,3 Results 86 3 -4.4 Sumrnary 89 3.5 Effects of Oligonucleotide Antisense to Dopamine Transporter mRNA on Locomotor Responses to Levodopa and Amphetamine 3 S. I htmduction 3.5.2 Design 3 53Resuits 3 S.4 Summav 3.6 Effects of Oügonucleotide Antiseose to Dopamine Transporter mRNA on the Neurotoxicity of MPP' and 6-OHDA 3,6.1 Introduction 3 -6.2 Design 3.6.3 Results 3.6.4 Summmy CHAPTE.4 GENERAL DISCUSSION Page 4.1 Discussion 112 4.2 ConcIusions l29 LIST OF FIGURES Fig. Descriptioa Page Basai gangIia circuitry in primates. 5 Antisense mechanism of action. 28 Stnatai dopamine transporter binding following unilateral infiision of 6-OHDA under ketamine versus halothane anaesthesia. Effects of in-atal infusion ofoligonuc teotide antisense to dopamine DIAreceptor mRNA on SKF 38393-hduced mouth movements. 56 Effects of Uitmstriatal infusion of oligonucleotide antisense to dopamine DIAreceptor mRNA on SKF 38393-hduced groomuig. 57 Dopamine DI receptor binding in the striatum folIowing intmtriatal Uifusion of oIigonuc1eotide antisense to dopamine D receptor &A. Autoradiograph representing [%j SCH 23390 binding in the striatinn foiIowing bilateral Uitrastriatai infusion of oligonucleotide antisense to dopamine DIAreceptor mRNA. Dopamine D2receptor bbding in the striahm following intrastriatai uifusion of oligonucIeotide antiseme to dopa-mhe: DIAreceptor mRNk Antoradiograph representing [%Ilracfopride bindhg in the sûiahun Page following intrastriatal infusion of oligonucIeotide antisense to dopamine DIAreceptor rnRNA. 62 10. Chronic fiuphenazine-induced mouth movements following intrastriatd infusion of oiigonucleotide antisense to dopamine DIAreceptor mRNA. 1 1. Dopamine D , receptor binding foUowing htrastriatal infusion of oiigonucleotide antisense to dopamine DIAreceptor mRNA in rats treated chronically with fluphenazine. 12. Dopamine D2receptor binding following intrastriatd infiision of oiigonucleotide antisense to dopamine DIAreceptor mRNA in rats treated chronicaIIy with fluphenazine. 13. Dopamine D3receptor binding followhg intrastn*atai&sion of oiigonucIeotide antisense to dopamine DIAreceptor mRNA in rats treated chronicdIy with Buphenazine. 14. Effects of intrastriatd Uifiision of oiigonucleotide antisense to dopamine DIAreceptor mRNA on apomorphine-induced rotations in a rodent mode1 of LID. 15. StnataI dopamine Direceptor buiding followhg unilatmai intrastriatal infuson of oligonucleotide antisense to dopamine DIAreceptor mRNA in a rodent mode1 of LID, 16. Autoradiograph representhg [%Q Sa23390 binding in the siriahmi foiiowing unilaterd htrastn'*atai mfiision oligonucIeotide Page antisense to dopamine DIAreceptor mRNA in hemiparkuisonian rats. 78 17. Striatal dopamine D2receptor binding foiIowing daterai intraStnatal Uifusion of oligonucleotide antisense to dopamine DI, receptor mRNA in a rodent model of Lm. 18- Autoradiograph representing ['Hl radopride binding in the striatum following unilateral intrastriatal infusion of 01igonucIeotide antisense to dopamine DIAreceptor -A. 19. Striatai dopamine D, receptor binding following intrastriatai infusion of oligonucleotide antisense to dopamine D ,, receptor mRNA in a rodent model of LID, 20. E ffects of inWataI uifusion of o ligonucleotide antisense to dopamine D, receptor mRNA on apomorphine-induced rotations in a rodent model of LID. 2 I . Shiatai dopamine D3 receptor binding foilowing intrastriatal uifusion of oligonucIeotide antisense to dopamine D3 receptor mRNA in a rodent model of Lm. 22. Striatai dopamine DIreceptor binding folIowing unilateral intrastriatai mtllsion of oligonucleotide antisense to dopamine D3receptor mRNA in a rodent model of LID. 23. Striatai dopamine 4 receptor binding folIowing intrastriatai Wonof oligonucleotide antiseme to dopamine D3receptor mRNA in a rodent mode1 of LID. 24. Striatal dopamine transporter binding folloaring unilaterai infusion of oligonucleotide antisense
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