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 to dopamine transporter
mRNA.
25. Autoradiograph representing ['HJ WIN 35,428 binding in
the strianim foIIowing unilaterd intranigral uifusion of
oligonucleotide antisense to dopamine transporter mRNA.
26. Striatd vesicular monoamine transporter binding following
daterai mtranigrai fision of oligonucleotide antisense
to dopamine transporter mRNk
27. Effects of unilateral intranigral Uifusion of oiigonucleotide
antisense to dopamine transporter mRNA on Iocomotor
response to Ievodopa*
28. Effects of unilaterd intranigrd inhsion of oiigonucleotide
antisense to dopamine transporter mRNA on Iocomotor
response to amphetamine.
29. Stnatal dopamine transporter binding folIowing bilateral
intrastriatd infiision of the nernotoxin 6-hydroxydopamine
in anÏmais pretreated with daterai infirsion of oligonucleotide
antisense to dopamine transporter mRNk
30. Autoradiograph representmg [3~WIN 35,428 binding in the
stria- foIIowing bilaterd intmüïataI infiision of
6-hydt0xydopa.e in mimals pretreated with unilateral inksion of oligonucleotide antisense to dopamine transporter mRNA.
3 1. Striatd vesicdar monoamine transporter binding foiiowing
bilaterd infusion of the nemtoxin 6-hydroxydopamine in
animds pretreated with unilateral infusion of O iigonucleotide
antisense to dopamine transporter mRNk
3 2. Apomorphine-induced rotations in animds infused biIateraiIy
with 6-hydroxydopamine foUowing pretreatment with unilaterd
infusion with oligonucleotide antisense to dopamine transporter
mRNA.
33. Striatd dopamine content following bilateral MPP+ fision in
rats pretreated with unilateral infusion of O IigonucIeotide antisense
to dopamine transporter mRNA.
34. Apornorphine-induced rotations in animais infused bilaterally with
MPP' following pretreatment with unilaterai uifusion of
oligonucleotide antisense to dopamine transporter rnRNA. LIST OF TABLES
Table Description Page
1. Pharmacological profile of dopamine receptors. 13
2. Effects of oligonucleotide antisense to dopamine DIAreceptor
mRNA on SKF 38393-induced behaviours other than vacuous
chewing rnovements and grooming.
3. Effects of oligonucleotide antisense to dopamine DIAreceptor
mRNA following chronic fluphenazine on behaviours other than
vacuous chewing rnovements. ACKNOWLEDGEMENTS
Supported by the Medicd Research Foundation of Canada, the Parkinson's Foundation of
Canada, and the Ontario Mental Hedth Foundation. HeartfeIt thanks to Dr. A. Jon
StoessI for his caring support and @dance over the years and to Rick Kornelsen for his brain slicing prowess. Finally, extra speciai thanks to God who has been my staunchest supporter. Chapter 1
GENERAL REVIEW
1.1 Basal Ganglia
1.1.1 Introduction
The basa1 gangiia are a highly interconnected group of forebrain nuclei including
the striatum (caudate and putamen), giobus pallidus (internai and extemai segments),
substantia nigra (pars cornpacta and pars reticulata), and the subthaiamic nucleus (Parent,
1990). The basai ganglia are inthately involved in the control of movement and have traditionally been viewed as part of the 'extrapyramidal motor system', thought to control rnovement in pardel with, but independent of, the pyramidal or corticospinai motor system. However, we now know that these two systems do not hction independently but, rather, are extensively ktercomected (Smith et al., 1998). As welI, it is now clear that basal gmgiia fimction is not restncted to aspects of motor control, but aIso involves various cognitive and mneumonic functions related to the generation and execution of context-dependent behaviours (Graybiel, I 995; Schultz, 1997; Wurtz & Hikosaka, 1986).
1J.2 striirturn
The striatlmi, consishg of the caudate and putamen, Ïs the Iargest component of the basai gangIia and, because it is the main 'receiving area', it is also considered the moa auciai component. The stnatum receives input fiom various regions mcluding major afFerents nom cortex, thdamus, and substantia nigra pars compacta (SNC),as weU as less prominent ones fiom the @obus palLidus (GP), subthaiamic nucleus (STN), dorsal raphe, and peduncdoponthe nucleus (PPN), while sending projections only to the GP and SN.
Approximately 90% of miataI neurons are medium spiny GABAergic neumns, so named for the spines which densely cover their dendrites. These spiny neurons give nse to the major striata1 projection pathways. The remaining aspiny neurons in the striatum are generally thought to function as intemewons (Chang et ai., 1981; Kawaguchi et ai., 1990;
Wilson & Groves, 1%IO), and include cho Iinergic neurons, parvalbumin-containinp
GABAergic neurons, somatostatin/neuropeptideY-containing neurons, and calretinin- containing neurons (Kawaguchi et al., 1995).
The main sources of excitatory giutamatergic input to the striatum arise fiom the cerebrai cortex and the intraIminar thalamic nuclei. Excitatory cortical inputs make synaptic contact primariIy with the dendritic spines of medium sized spiny projection neurons (Kempt & PowelI, 1971)- while thalamostriata1 inputs terminate on the denciritic shds of these neurons pub6 et d., 1988; Kemp & Powell, 1971; Sadikot et al., 1992b;
Smith et ai., 1994). A small number of cortical afferents also contact striata1 intemeurons
(Bolam & Bemett, 1995; Kawaguchi et ai., 1995). Corticostriatd projechons form a topographicai organization which is maintained throughout the basa1 ganglia. in primates, the sensolimotor cortex projects mostiy to the putamen with a somatotopic representation, whiIe association cortices project prirnarily to the caudate nucleus. Limbic cortical areas terminate largeIy in the ventral stnatum including the nucleus accumbens and offactory tubercle (KiinzIe, 1975; Seiemon & Goban-Rakic, 1985; Haber et al.,
1990). Thus, inputs fkom different cortical areas may impart regional differences in striatai fimction. Howeva, much overIap exists, mahg distinct divisions difficdt. Thaiamostriatai projections have aIso recently been demonstrated to have a topographie organization, with input fkom the centromedian nudeus predonhantly innewating the sensorimotor putamen, while the parafascicular nucleus projects primarily to limbic- associative striatai regions (Groaiewegen & Berendse, 1994; Sadikot et al., 1992a;
Sadikot et ai., 1992b; Sidiié & Smith, 1996; Smith et al., 1994).
Cortical information is conveyed to the output nuclei of the basai ganglia via wo routes termed the direct and indirect pathways. The striatal medium spiny GABAergic oeurons comprising these two pathways are divided on the basis of their projection targets. Striatopailidal neurons, which give rise tu the indirect pathway, project to the extemal segment of the globus pallidus (GPe), while striatonigral neurons project to the nibstantia nigra, fonning the direct pathway (Kawaguchi et ai., 1990). nese neurons can dso be di~ting~shedon the basis of neuropeptide and dopamine receptor expression.
StriatopaiIidaI neurons contain the dopamine D2 receptors and express the neuropeptide enkephalin (Gerfen & Young, 1988; Gerfm et aI., 1990; Le Moine et al., 1990), whereas striatonigral neurons contain dopamine Dt receptoa and the neuropeptides substance P and dynorphin (Gerfen & Young, 1988; Gerfen et ai., 1990). Nthough reIativeIy distinct, the neurons in these two pathways give rise to extensive axon collaterals (Bevan et al.,
1996; Kawaguchi et ai., 1990) which may form the basis for their possible intercoanection (Smith & Bolam, 1990; Yung et ai., 1996).
Activation of the direct and indirect pathways produces functionally opposite effects in neurons of the target nuclei. Because GABA is an inhibitory neurotransmitter, activation of stxiatonigral newons in the direct pathway results in the inhibition of output nudei (GPiISNr) (Fig. 1). In contrast, activation of striatopdidai neurons results in enhanced GABAergic inhriition of tonicalIy active neurons in the GPe, thereby inhibiting
GABAergic projections to the STN. This inhibition of inhriitory neurons resuits in the disinhibition of STN giutamatergic aeurons, and the resultant excitation of inbi'bitory output nulcei. Such disinhiiition is a fundamentd feature of basal ganglia physiology
(Chevalier & Deniau, 1990; Wurtz & Hikosaka, 1986). It has been suggested that these two pathways rnay serve to transfomi the excitatory input from the cortex into baianced antagonistic inputs to the major output neurons of the basai ganglia (Gerfen, 1992). The nigrostriatai dopamine system may modulate this balance through its differential effects on striatonigral and sû-iatopaliidai activity, having excitatory actions through effects on the dopamine D t receptor and inhiiitory actions via the dopamine D2 receptor (Gerfen et al*, 1990).
Striatai architecture is divided into two different compartments termed patches
(stnosornes) and mani< (Gerfen, 1992; Gerfen & Wilson, 1996; Graybiel, 1990). The fint indication of thÎs patch-rnatnx organization came with the discovery of emkhed p- opiate receptoa in patches (Pert et al., 1976). These patches ah have weak acetyIcho1ùiesterase staining (Graybiel et al., 1978). By connast, the rnatnx is nch in somatostatin and caibindin irnmunoreactive neurons (Gerfen, 1985; Gerfen et ai., 1985).
Neuropeptides differentidly expressed in the direct and mdirect pathways do not show a distinct patch-ma& distribution (Gerfen & Young, 1988). However, the relative expression of neuropeptides by neurons in these two compartrnents varies regiondly
(Gerfen & Young, 1988) with reIativeIy higher IeveIs of dyaorphin expressed by neurons Figure 1 CEREBRAL COR-X
Feedbacic or output
projecrions as open umwo. The dopaminec neurones of the SN, exext a net excitatory effect on spiny ncufones gieg rise to the direct pathway by the activation of DLceceptors, whcrcls they excrt a net mhibitory effect on spiny neurones giving Ne to the indircct pathway by activation of 4 mepton.
Cortical mfomiation caa ais0 rcach the basal gangfia by way of de coricosubthaiamic projection. GPe = extemal segment of the @obus paiiidw; Gpi = mtdsegment of the globus pailidus; SN, = mbstantia nippars compaco; SN, = nibstantia nippars nticuiatr; SM = nibthahmic nuctau; 'ïhat = thafamw;
PPN = pedunculopontine aucleus; KBN = iatd habeauIar nuclnu; SC = -or coIlifuhu; and RF = ceticuiar focmatioa @dvdhm Smith a IL, L998) in patches than matrix in dorsal striatum while equd IeveIs of expression are seen in the ventral striatum. Conversely, substance P levels are relatively higher in the patches of ventrai than dorsal striatum.
Patch and matrix compartments are strictly segregated (Gerfen et al., 1985;
Kerkenharn et al., 1984; Bolam et al., 1988; Kawaguchi et al., 1989) and show some degree of differentiation O€ input and output connections. In primates, the striata1 matrix receives afKerents primariIy tiom motor and somatosensory cornces and ultirnately targets
GABAergic neurons of the SNr, while the patches derive their inputs largely from limbic- related areas and provide input to the dopaminergic neurons of the SNC(Gerfen, 1984;
Gerfen, 1985, limeriez-Castellanos & Graybiel, 1989). in the rat, however, this segregation is no t as clear, with most cortical areas innervahg both compartments. More prominent is the relation ofpatch-matrk compartments to the laminar organization of the cortex. Cortical inputs to the patch cornpartment originate Crom deep layer V and VI of the cortex, while inputs to the maeix originate nom superfkial layer V and supragranular layer (Gerfen, 1989). The huiction of this patch-rnatrix organization remains unclear, but it may provide an additional mechanism Tor regulathg the balance in activity of the two striata1 ouput pathways.
1.I 3 GIobus PallUus
The @obus pifidus is cornposed prharily of GABAergÎc nernoas divided into an mtemai segment (mi)and an extemal segment (GPc). The GP receives the rnajority of its inputs Eom the with enkephah-containing neurons temimathg in the GPe and substance P-containing neurons projecbing to the GPi, as part of the mdirect and direct pathways, respectively. The GP aiso receives minor projections from STN, SNC, dorsal raphe, and PPN. Striatal and STN fibers terminate in GPe in a band-like pattern (Parent,
1990; Smith & Parent, 1986), creating paraiiel charnels which remah segregated throughout the basai ganglia The GPe gives rise to a massive topogmphicaiiy organized projection which terminates throughout the entire extent of the STN. Minor projections also terminate in the striatum. SN, and reticuiar nucleus of the thalamus (Asmuma, 1989;
Asanuma, 1994; Bickford et ai., 1994; Hazrati & Parent, 1991)- The GPi sen& projections primarily to ventral tier thdamic nuclei, the centrornedian, the lateral habenula, and PPN (Smith & Parent, 1986). While information passing through the GP remains IargeIy in parailel segregated charnels, the intemal and extemal segments of this structure are reciprocdy linked (Parent, 1990).
1.1.4 Subthalamr'c Nucleus
The STN receives its main mput nom the GPe (Carpenter et al., 1981) but aIso receives a direct projection from cortex (Carpenter, 198 1). in tum, the STN exerts a powerfuI glutamatergic, excitatory influence on its target structures (Albin et ai.. 1989;
Kitai & Kit& 1987). STN projects primanly to GPi adSNr, the output nuclei of the basal ganglia (Smith & Parent, I986), but aiso sen& minor projections to striahm (Beckstead,
1983; Smith & Parent, 1986; Smith et ai., 1990), SNc (Kita & Kitai, 1987; Smith &
Grace, 1992; Smith et ai., 1WO), PPN (Hanmon et al., 1983; Kita & Kitai, 1987; Parent
& Smith, I987), and spinai cord (Takada et ai., 1987), as well as a dense feedback projection to GPe (Carpenter et ai., 1981). WhiIe, m rats, STN projections to GP and SN
&se hmthe same neurons via axon collaterais (Deniau et al., 1978; Parent & Smith, 1987; Van der Kooy & Hattori, 1987), these projections originate largely Eom separate cell groups (Parent et ai., L 995) in primates. Within the GP, SRJ terminais in GPe and
GPi also orighate from separate neurons, with STN projections to GPe &Wig tom more lateral areas than those projecting to GPi (Parent et al., 1989).
1.l.S Substantiu ivigra
The SN comprises GABAergic neurons of the pars reticulata and the dopaminergic neurons of the pars compacta The SNr is one of the main output nuclei of the basal ganglia, sending projections to ventrai anterior/ventroIateraI thaiarnic nuclei. the
PPN, superior colliculus, and reticular formation (Parent et ai., 1983). The SNc projects primarily to the striatum, but dopaminergic nigrosûiatal neurons extend denciritic arborizations into the underlying SN, where the release of dopamine regulates SN, activity (Abercrombie et al., 1998; Cheramy et ai., 198 1). SN, activity also regulates that of SN, (Tepper et al., 1995), although the source of such regulation remains unclear.
1.2 Basa1 Ganglia Disorders
1.2.1 Parkinson 's diseose
As mentioned, the basai ganglia are intimately involved m motor control. Thus, disniption in basd ganglia hction fonns the basis for several rnovement disorders.
Perhaps the best descnbed of these is Parkinson's disease (PD), a neumdegenerative disorder charactaized by tmor, rigÎdity, and akinesid bradykinka The gradud and progressive toss of dopaminergic neurons in the nigrostriatd pathway is the major patboiogical feature of PD. The Ioss of dopaminergic input to the resuits in increased activity of dopamine D2 receptor-bearing striatopaIlidal neurons, and decreased activity in dopamine Di receptor-bearing striatonigrai neurons (Albin et al., 1989;
Gerfen, 1992). Enhanced GABAergic inhibition of the GPe results in disinhibition of the
STN. Indeed, lesion of the STN ameliorate the cardinai signs of parkinsonism in monkeys rendered parkinsonian by the toxin I -methy14phenyI- lT2,3,6-tetrahydropyridine
(MPTP) (Aziz et al., 199 1; Bergman et ai., 1990; Wichman et al., 1994). Excessive activation of the STN resdts in increased excitation of inhibitory output pathways, and a resultant decrease in thaIamocorticaI activation. Decreased GABAergic inhibition O f output nuclei by striatonigral neurons aiso results in enhanced inhibition of thalamocortical projections (Albin et al., 1989; Ceballos-Baumann et al., 1994). which may underlie the abesidbradykinesia characteristic of this disorder. Lesioning of the
GP, (pdlidotomy) provides effective relief of most symptoms in patients with
Parkinson's disease (Baron et al., 1996; Cebailos-Bauman et al., 1994).
1.2.2 Huntington 's d-e
Huntington's disease is also associated wiîh basal gangLia dysfunction and is characterized by abnomai choreifonn movements. The early stages of disorder are rnarked by a selective Ioss of GABAergic stnatopallidai neurons (Reiner et al., L988).
The Ioss of inhiiitory input to the GPe disinbiiits mhibitory palIidaI neurons remking m excessive mhiiition of STN neurons, reduced activation of basai ganglia output nuclei, and reduced tonic inhibition of thaiamocorticd neurons (De Long, 1990). Reduced STN activity may be a key feature in the development of chorea. Lesions of the STN in both monkey and man, remit in hemibdlismus, an exaggerated form of chorea (Carpenter et ai., 1950; Hammond et al., 1979; Whittier & Mettler, 1949). By removing tonic inhibition of thaiamocorticai projections, these neurons may become increasingly responsive or spontaneously active, Ieadùig to involuntary movements. As the disease advances, degeneration progresses to include stnstnatonigraIneurons and symptomatology shi Rs towards rigidity and akinesia (Aibin et al., 1990). Thus, in these patients, basal ganglia output nuclei are disinhhited in a manner somewhat reminiscent of that seen in PD.
1.Z.3 Drug-induced dyskinesias
Drug-Uiduced dyskinesias aiso involve choreiform abnormal invo Iuntq movements resulbiig fiom alterations in basal ganglia hction. Dyskinesias include levodopa-induced dyskinesia (LID) which develops in PD patients following long-tm treatment with Ievodopa, or tardive dyskinesia (TD) which develops as a side effect of long-tem treatment with neuroleptics. Manifestations of dyskinesias in a variety of disorders rnay be mediated by simila.neural mechanisms, narnely, a shift in the balance between the direct and indirect pathways towards the direct pathway (Chesselet & Delfs.
1996; DeLong, 1990). AIthough the cause of this shia may Vary, the end resdt is similar.
According to our cment mode1 of basal ganglia fünction, this shift m balance wodd result in decreased basai gangIia output and a disinhr'bition of thaIamocoritcaI projections, which couid form the basis for a variety of hyperkinetic disorders. 1.3 Dopamine Receptors
1.XI Dopaniine Receptor CIassijkatiÔn
The traditional classincation of dopamine @A) receptors has been based ou their interactions with adenylyl cyclase. It was demonstrated in the early 1970's that DA was capable of stimulating the formation of cyclic adenosine monophosphate (CAMP) in neural tissue thmugh its activation of the enzyme adenylyI cyclase (Brown & Mackman,
1972; Kebabian et al., 1972). However, some antagonists known to modiS DA receptor- mediated actions (eg. neuroleptics) were found to have iittle or no effect on DA- stimulated adenylyl cyclase ac tivity (Iversen, 1975; Snyder et al., 1975). Also, compounds acting at DA receptors in the pituitary were shown to mirnic the effects of
DA but blocked the effects of DA on adenylyl cyclase (Kebabian et al., 1977; Pieri et al..
1978). Dopamine receptors were subsequently divided into those coupled to adenylyi cyclase and those which were not, or DIand Da respectively (Kebabian & Calne, 1979;
Spano et al., 1978). A short time later, Stoof and Kebabian (1981) showed that D2 recepton in the stria- were, in Cact, coupled to adenylyl cycIase but in a negative fashion. This abiw to inhibit adenylyl cyIcase activity was found to be characteristic of
D, recepton in some (DeCamiIli et al., 1979; Cote et ai., 1982; Weiss et al., 1985;
Cooper et ai., 1986) but not aiI (Memo et ai., 1986; Stoof & Verheijden, 1986; Stoof et al., 1987) cases. The Dt/D2 receptor classincation scheme was modified to inchde those
DA recepton which stlmiilate adenylyl cyclase activity (DI)and those which do not (Dz)
(Clark & White, 1987). Ushg molecdar cloning techniques, the dopamine receptor fdyhas since been expauded to mclude dopamine D,, D,, and D5receptors and more are still bekg isolated (Nimik & Van TOI, 1992). However, receptors are still generaIIy cIassÏfied as DI-iike (Dl, 03 and D&e (D2, D3,D4), not oniy on the basis of their actions on adenylyl cyclase, but also by vimie of their response to different agonists and antagonists (Table 1). Receptors within each cIass dso share common rnolecular characteristics and chromosornai Iocafization (Sibley & Monsma, 1992).
133 Dopamine DI Receptors
Cloning and characterization of the dopamine Dlreceptor (Dearry et ai., 1990;
Zhou et al., 1990; Monsma et al., I990) have revealed a receptor structure containing many features typical of the G-protein linked receptor family. It is characterized by seven transmembrane domains with four extraceiluiar and four intracellular regions (Niznik &
Van TOI, 1992). It can be distinguished nom the D-Iike receptors pharily by its srnaII thud intraceIIuIar Ioop and its long carboxy terminus (Sibley & Monsrna. 1992).
ExtraceIIdarIy directed consensus sites for N-linked glycosy Iation can be €0 und on the extracellular amino terminus and the second extracellular loop (Jarvie et al., 1994).
Consensus sites for phosphorylation by the regdatory kinases, protein kinase C and the
CAMP dependent protein kinase (PKA), are Iocated on the second and third intracel1uIar
Ioops (NiPiik & Van Tol, 1992). Dopamine DIreceptors are aIso known to contain an aspartate residue in the third transmembrane domain and two serine residues in the fifi transmernbrane domain, which are thought to be involved in ligand bmding (Strader et ai., 1989; Pollock et al., 1991). Nso characteristic of the dopamine DIreceptor €amiIy is Table 1 a cysteine residue located on the mtracelIular carboxyl-tail which may represent a putative paImitoyIation site (O'Dowd et al., 1989; Niznik & Van TOI, 1992).
Dopamine D, receptors are found in theù highest concentrations in the neuropii of the caudate-putamen, nucleus accumbens, and olfactory tubercle (Mengod et al., 199 1;
Mansour et al., 1992; Ciliax et al., 1994). immunocytochemical evidence indicates that, withùi the caudate-putamen, dopamine DIrecepton are concentrated in striatai 'patches" surmunded by regions of more moderate concentrations (Ciiiax et ai., 1994). These
"patches" may correspond to the striosomai cornpartment described above (Gerfen,
1984). Moderate amounts of dopamine D, receptor mRNA have also been found throughout the arnygdaia, particularly the intercaiated and basolateral nuclei, the granule ce11 Iayer of the hippocampus and various regions of cortex (Mengod et al., 199 1; Weiner et al., 1991). Low Ievels have been found in the grande ce11 layer of the cerebeIlum and the suprachiasmatic, paraventricular, and supraoptic nuclei of the hypothalamus, while very low levels have been detected in the thalamus (Mengod et al., 199 1; Weiner et al..
1991). No dopamine DIreceptor mRNA has been detected in striatal projection areas including the substantia nigra, globus palfidus, and endopeduncdar nucleus, despite evidence of immunoreactivity and binding in the neuropil of these regions (Dubois et al.,
1986; Weiner et al., 1991; Mansour et ai., 1992). Evidence Eom Iesion studies suggests bat dopamine DIreceptors are synthesized in the striatum and subsequently transported to these projection sites et al., 1987; Mansour et ai., 1992; Van der Kooy et al., t 986)-
SKF 3 8393 (2,3,4, 5-tetrahyh-7,8-dihydroxy-1-phenyu-H-3-benzazepine) has bem the most cornmon dopamine DI receptor-selective agonist used to shidy the functional role of this receptor. SKF 38393 was fint identified (Pendleton et al., 1978) and tested in the central nervous system (Setler et al., 1978) in 1978. It shows a high selectivity for the dopamine DIreceptor (Sibley et al., 1982; Hytell, 1984; Andersen et al., 1985) and readily crosses the btood-brain barrier (Hytell, 1984). h centrai nenrous system tissue, SKF 38393 has been shown to dose-dependently elevate CAMP but to a lesser extent than dopamine, indicating a partial agonist profile (Setler et ai., 1978). Other agonists selective for the D, receptor include SKF 82526, SKF 8 1297, SKF 82958, A
68930, A 77636, and A 86929. The dopamine DIreceptor aiso displays hi& affin@ for the antagonists SCH 23390, (+) butaclamol, and cis-fluopenthixol (Seeman & Van Tol.
1994).
SKF 38393 has been incorporated in a number of studies attempting to uncover the behaviourai roIe of the dopamine DIreceptor. At first glance, dopamine D, recepton appeared to have no behavioural signilicance in and of themselves. Systemic SKF 38393 did not appear to induce any of the behaviourai effects typicaily associated with dopaminergic stimulation. It did not resdt in locomotor activation (Gower & Marriott,
1982; Braun & Chase, 1986; Jackson & Hashinune, 19861, stereotypy (Setier et al.,
1978; White et al., 1988; DeIfs & Kelly, 1990), reduction of exploratory behaviour
(CostalI et aI., 1981; Brown et ai., 1985), or yaTNning (Gower et al., 1984; Yarnada et al.,
1986) in intact animais; fded to elint rotation fouowing hemitransection or tmÏIateraI striatai quinohic acid lesions (Arnt, 1986; Barone et d., 1986% 1986b); and did not appear to play a major roIe in reward/motivation rdated behaviours nich as self- administration and conditioned pIace preference (Lippa et al., 1973; Woolverton et al.,
1984). Closer observation, however, has reveded a mild discontlliuous activation produced by SKF 38393 in weil-habituated rats (MoHoy & Waddington, 1983, 1984,
1985), but the most pronounced effect of systemic SKF 38393 treatment is enhanced flooming (MoEoy Br Waddington, 1983, 1984, 1985; Murray & Waddington, 1989;
Star & Starr, 1986) and vacuous chewing movements (VCMs) (Murray & Waddington,
1989; Rosengarten et ai., 1983), distinct nom the oral stereotypies induced by nonselective DA agonists (Cameron et ai., 1988). These behaviours remain wideiy accepted as a rodent behavioural mode1 of dopamine Dl-like receptor stimulation.
The highest density of Direceptors is Found in the basal ganglia, and it is not surprisin& therefore, that this receptor has been impticated in a number of disorders involving these structures. A variety of dopamine D, receptor selective agonists have been shown to be effective antiparkinsonian agents in both MPTP monkeys (Asin et ai..
1996; Bédard & Boucher, 1989; Bédard et ai., 1993; Domino, 1997; Gomez-Mancilla et al., 1993; Luquin et ai., 1990) and humans (Emre et ai., 1992; TemIett et al., 1989).
Dopamine DI receptor stimulation is also implicated in the pathophysiology of Lm
(Boyce et ai., 1986; Falardeau et al., 1988; Nomoto & Fukuda, 1993), as repeated treatment with dopamine DI receptor agonists elicits sensitization of apomorphine- induced rotations in hemipararksoni rats (Matsuda et al., 1992), and dyskinesias in
MPTP monkeys (Goulet et ai., 1996); two animai models of Ievodopa-induced dyskmesias. Also. chronic Ievodopa treatment in both modeIs has been associated with heightened activity in dopamine D, receptor-bearing striatonigral neurons (Crossman,
1990; DeLong, 1990). SmiiIarly, TD, which has traditionally been associated with supersensitivity of stnatal dopamine 4 receptors (Snyder, 1981), is now thought to hvoIve a shift in DI/DZreceptor bahce towards increased activation of dopamine D, receptocs (EUison et al., 1988; Lubin & Gerlach, 1988; Rosengarten et ai.. 1986) on striatonigrd neurons (Nbhet ai., 1989; Crossman, 1990; DeLong, 1990; Neisewander et ai., 1995)-
Dopamine DIreceptors aiso appear to be necessary for both the induction and expression of behaviod sensitization foffowing repeated exposure to psychostimulants
(Henry 8c White, 199 1; Mattingly et ai., 199 1; Mayfield et ai.. 1992; Vezina, 1996), a phenornenon which may have paraiiels with the pathophysiology of hg-induced dyskinesias. The dopamine DIreceptor may dso play a role in schizophrenia Data from animai studies has pointed to the dopamine DI receptor as a potentiai target for antipsychotic drugs (Chipkin et al., 1988; Gerlach et ai., 1995; Waddington. 1993). in agreement, Malemak et ai (1993) found elevations in the density of the low affiity state, and in the binding of the high affinity aate of Dl receptors in the caudate of schizophrenic patients. However, the clinical use of selective DIreceptor antagoni* has proved disappointhg (Barnes & Gerlach, 1995), as have attempts to associate the disorder with Dl receptor mutations (Carnpion et ai., 1994; Cichon et al., 1994). The dopamine DI receptor has dso been implicated in aspects OP rewardheinforcement
(Sutton & Beninger, 1999), and mernory (Castellano et ai., 1994; Sawaguchi et al.. 1994).
1.3 J Dopamine D, receptors
Dopamine D, receptors are classified as Drlike based largely on amino acid sequence and gene organization (SibIey et aI., 1993). in terms of genetic structure, one key featme distinguisbg dopamine D3receptors hmDI receptors is the presence of nitrons. The dopamine D, receptor gene con& five mtrons (Sokoloff et ai., 1990), a feature typical of dopamine D&ke receptors. These introns diow for the generation of spIÎce variants by alternative splicllig. Dopamine D3 receptor splice variants have been identified, but their functionaI sÏgnificance remains unclear as these variants failed to show any binding when transfected into ce11 iines (Guos et ai., 1991). It has been suggested that these variants may be expressed oniy in certain circumstances in order to control the density of hctioning D3receptor sites (Giros et ai., 1991).
Hydrop ho bicity andysis indicates that the most probable structure O f the dopamine D3 receptor is consistent with those of the seven transmernbme-spanning G- protein-coupled recepton. The D2 and D3 receptors exhibit 75% homology within transmembrane domains compared to ody 41% between dopamine DIand Dt recepton
(Monsma et al., 1990). The human (400 amino acids) and nt (446 amino acids) dopamine
Dj receptors share approxirnateiy 78% homology (Guos et al., 1990; Sokoloff et al..
1990). h contrast to dopamine DIreceptoa, D3 receptors have a long third intracellular
Ioop and a short carboxy terminus, features typicai of Dz-like receptor structure (Missale et ai., 1998). Studies of chimenc D2/D, receptors hplicate the third intracellular loop in conferring agonist binding properties (Robinson et ai., 1994), w hile transmembrane domains VI and W appear to play a role m the deiennination of antagonist amnity
(Norman & NayIor, 1994). The various residues desded above for the dopamine Di receptor are highly conserved across the various dopamine receptor types and can ais0 be found in dopamine D3 receptors. These indude two cysteine residues fo-g an extraceI1dar disuInde bond for rrceptor structure stability (Dohlman et al, 1990; Fraser,
19891, serine residues in transmembrane domain V which may be involved in the formation of H-bonds with the two hydroxyI groups of catechols (Malmberg et al., 1994; SokoIoff et al., I990), and an aspartate residue in transmembrafle domain III which may participate in binduig the amine group of the catecholamine side chah (Hibert et al.,
1993; Strader et al., 1988). A cysteine raidue on the carboxy terminus is also common to both receptors but its location mers. In InD,receptors, this cysteine residue is located near the of the carboxy terminus, whereas in the D, receptor, the carboxy terminus ends with this residue (Missale et al., 1998). The dopamine D, receptor has three consensus sites for wlinked glycosylation, two on the extrace1Iular amino terminus, and one on the third extracellular loop (Levant, 1997).
The distribution of dopamine D3receptors is best characterized in the rat bnin, although distri%ution patterns in humans appear to be generally simiIar. Dopamine D, receptor mRNA is most prominently expressed in granule cells of the islands of CalIeja
(Diaz et al., 1995). High densities are aiso seen in the nucIeus accumbens and olfactory tuberde (Bouthenet et al., 199 1; Diaz et ai., 1995; Mengod et al., 199 1; Sokoloff et al..
1990). Moderate amounts of D3 mRNA are also found in cerebrai cortex. ventral pallidum, substantia nigra pars compacts, arnygdala, nucleus of the horizontal limb of the diagonal band of Broca, the anteroventral, laterodoad, and ventral posterolateral nuclei of the thalamus, the paraventricuIar and ventromedial nuclei of the hypothalamus. superior collicus, inferior oIivary nucleus dentate gynis9 and olfactory bdb (Bouthenet et ai., 1991). Low IeveIs of D3 receptor MAare fond in caudate/putamen, substantia nigra pars reticuiata, ventral tegmentum, and cerebeuar cortex (Bouthenet et al., 1991;
Mengod et al., 1992). The distribution of dopamine D3receptors has not been mapped in detail due to the Iack of reIatively selective radioligands- However, evidence suggests that dopamke D, receptor binding cIosely paralIeIs the distniiotion of mRNA., with the highest denàty seen in the isIands of CaiIeja, olfactory bulb, and nucleus accumbens, and relatively Iittie binding seen in caudate/putamen (Booze & Wallace, 1995; Levant et al.,
1992; Lévesque et ai., 1992).
The signal transduction mechanisms of the dopamine D, receptor remain reIativeIy unclear. The lack of selective ligands for this receptor has limited studies largely to transfected ce11 lines which have yielded mixed results. Although the D, receptor is thought to share many of the structural characteristics of a G-protein-coupled receptor, it was initially thought that the Djreceptor rnight not be functionally coupled to
G-proteins, based on the lack of a guanine nucleotide shift in agonist binding (Sokoloff et al.. 1990). As well, contrary to typicd D2-like activity, dopamine D3 receptors fail to inhibit adenylyl cyclase activity in various ceIl lines (Freedman et ai., 1994; Lyon et al..
1987; Tang et al., 1994). However, other expression systems exhibit a variety of signahg events coupled to the D, receptor (Chioet al., 1994; Cox et al., 1995; PiIon et ai., 1994;
Potenza et ai., 1994; Seabrook et ai., 1994; Liu et aI., 1996). This variabiIity may depend upon the G-proteins or effector systems expressed by each ce11 line. More recently,
Griffon et ai. (1997) used a transfected neuroblastoma-glioma hybrid ce11 Iine (NG 108-
15) which expresses a variety of G-proteins and effectors (Pilon et al., 1994) and found a tight coupling of the D, receptor to G-proteins. UsÏng this ceiI he, they found that activation of the dopamine D3 receptor inhriits cyciic AMP formation and increases mitogenesis. The D,-mediated mitogenesis sÏgnaling pathway was reported to be hdependent fiom the inhibition of adenylyi cyclase activity, based on fLrndings that cyclic
AMP potentiates Dj receptor-mediated mitogenesis (Griffon et al., 1997; PiIon et al.,
1994). The mechanisms mvolved in this pathway remah tmdetermined, but rnay Involve a phosphatidyhositol 3-kinase and an atypical protein kinase C-dependent mechanim
(Cussac et al., 1999). Care must be taken when interpreting these data, as the agnding pathways seen rnay not necessady reflect those associated with the receptor in brain.
Udortunately, there is cmenfiy no conclusive evidence regarding dopamine D3recep tor signahg pathways in brain.
Although Little is known about the functiond role of the dopamine D, receptor, it may be involved in locomotor activity (Acdi et ai., 1996; DaIy & Waddington, 1993;
Svensson et al., 1994). reward (Caine & Koob, 1993; Kling-Petersen et al., 1994), and development (Swanenski et al., 1994). It has aiso been implicated in schizophrenia
(Kennedy et al., 1995; Griffon et al., 1996; Shaikh et ai, 1996), dnig abuse (Ebstein et al..
1997; Meador-WooM et ai., 1995; Staley & Mash, 1996). Parkinson's disease (Piggott et ai., 1999; Ryoo et al., 1998), and dyskinesias (Bordet et al., 1997: Steen et al., 1997).
The most consistentiy reported behaviourai effect of dopamine Dj receptor stimulation is modulation of locomotor activity. The D3receptor agonis 7-OHDPAT has been shown to reduce spontaneous locomotion at low doses and mcrease locomotion at high doses (AhIenius & Salmi, 1994; Daiy & Waddington, 1993; Depoortere et ai., 1996;
Khroyan et al., 1995; McElroy et al., 1993; Svensson et ai., 1994). Also, mice lacking the dopamine D3receptor have been shown to exhibit hyperactivity (AcciIi et al., 1996; Xu et al., 1997). Thus, it has been niggested that high affhîty postsynaptic dopamine D3 receptors may mediate inhicbition of locomotion by low doses of 7-OH DPAT, whiIe higher doses enhance locomotion through activation of D2receptors (Levant et al., 1996).
However, this biphasic dose-response is not signincantfy different nom that seen with similx doses of the D, receptor agoiiist quinpiroIe or the nonselective agonist apomorphine (Depoortere et al., 1996), and it has been suggested that the D, receptor may function as an autoreceptor (Lejeune & Millau, 1995; Rivet et al., 1994; Tepper et ai., 1997) SIarto presynaptic D2receptors. Low doses of 7-OHDPAT cm aiso induce yawnhg @amma et al., 1993; Van den Buuse, 1993), a behaviour typically associated with dopamine 4 autoreceptor fùnction (Stoessl et ai., 1997). Clearly, the functional role of the dopamine Dgreceptor wiIl not be clarified using the nonselective ligands currently available. Recentiy, work with in vivo dopamine D3 receptor antisense has provided support for a role for this receptor in locomotor inhibition (Menalled et ai., 1999). Further work with this technique rnay be necessary to uncover the frmctiond roIe of this receptor.
1.4 Dopamine Transporter
L .4.l Molecul.Sîructure
The dopamine transporter (DAT) is a member of a family of Na- and CI- dependent transporters (Amara & Kuhar, 1993) characterized by twelve hydrop ho bic traamembrane domains, five intracelluIar and six extracelluiar loops, induding a relatively long second extracellular Loop (Giros et ai., 1992). Both the amino and carboxy termini are Iocated inside the ceU (Guastella et al., 1990). There is a high degree of homology among the various members of this famiy, with DAT hakg the greatest homology &th the noradrenergic transporter (66%) (Giros & Caron, 1993). The greatest degree of sùniiarity can be sem in the nrst and second transmembrane domains (Giros &
Caron, 1993), suggestmg that these regions may be mvoLved m aspects of transporter fùnction common to al1 family members. The DAT is also reIatWeIy conserved across
çpecies, showkg 92% amino acid sequence homology between rat and human DAT
(Prishipa et a(,, 1994). The DAT has 2-4 N-Linked giycosylation sites found on the large second extracellular loop, and various potentid sites for phosphorylation by CAMP- dependent protein kinase PKA, PKC, and CaM kinase II Iocated within intracellular amino and carboxy termini and the second intraceilular Ioop (Cool et ai., 1991; Giros &
Caron, 1993). The DAT is also characterized by the presence of two Ieucine zippers
Iocated in the second and ninth ûansmembrane domains, which are traditionally thought to be involved protek-protein interactions (Landschulz et al., 1988) and may play a role in the structural or fimctional organization of DAT with the plasma membrane (Giros &
Caron, 1993).
1.4.2 Locdization
The dopamine transporter is located only in doparninergic neurons (Ciliax et al..
1995; Freed et ai.. L995; Lorang et ai., 1994; Revay et ai., 1996) where it is synthesized in the cell body and transported to dendrites, axons, and nerve terminds (Nirenberg et ai.,
1996). The DAT is absent nom regions of synaptic apposition or "active zones",
Uidicatuig that diasion rnun occur before uptake of dopamine OCCLUS (Garrîs et al.,
1994; Nirenberg et ai., 1996). The highest levels of DAT mRNA are expressed in midbrain dopaminergic neurons of the SNc with Iowa Ievels found in the nucleus parani*graIis and ventrai regmentai area (VTA) (Cerruti et al., 1993; Shimada et ai., 1992).
DAT protein expression demonstrates a striosomaI matrÎx distribution with highest Ieveis seen in the 'patch' compartments of the dorso1atera.I strÎatum (CiIiax et aI., 1995; Freed et al., 1995). Lower levels cmbe found in the nucleus accmbens, olfactory tubede, and
laterd habenda. Some DAT imrnunoreactivity is dso detectable in SNc and VTA (Freed
et al., 1995; Nirenberg et ai., 1996), consistent with proposed dendritic release of
dopamine (Robertson et al., 1991). Both in situ hybridization and immunohistochem*stry
studies indicate that the level of DAT expression varies among ciiffereut dopamine cell
groups (Nirenberg et al., 1997). This may reflect the type of dopaminergic signaling
occurrhg in a particular area, whether it is classicai synaptic, or a more paracrine or
voIume transmission type of signding (Garris & Wightman, 1994; Zoli et al., 1998). with
regions expressing a higher density of DAT, associated with les volume transmission.
1.4.3 Function
Dopamine reuptake by DAT is the primary means of terminating dopaminergic neurotransmission (Amara & Kuhar, 1993; Giros & Caron. 1993). DAT is a member of the NL-and Cr dependent neurotransmitter transporter family. Two ~a~ ions and one CI'
ion are cotransported with each dopamine molecde across the synaptosoma1 membrane
(Krueger, 1990; Gu et aI., 1994; McEIvain & Schenk, 1994). The cellular Na- gradient
needed to drive this type of transport is maintained by the Na'K' ATPase pmp
(Bogdanski & Brodie, 1969; Paton, 1973; White, 1976). Dmgs that block the biat/?Cr
ATPase or deplete extraceMar ~a',dramaticaIIy mipair dopamine uptake (Hom,1990).
Under conditions in which the concentration gradients are reversed, DAT cm mediate the caIcium-independent release of dopamine via reversa1 of the nomiai direction of transport
(Bannon et ai.. 1995; Ratieri et ai., 1979; Sulzer et ai., 1993). By regulating the leveI of synaptic dopamine, DAT cm have profomd effects on the intensity, duration, and quality
ofdopaminergic activity.
The DAT aiso serves as the phannacologicai target of various psychostimulants,
including cocaine and amphetamine. Amphetamine exerts numerous actions on dopamine
nerve termùiais (Seiden et al., 1993). Amphetamuie acts as a substrate for the DAT and is
actively taken hto the ceU with ~a+(Liang & RutIedge, 1982a,b; Seiden et al., 1993;
Sitte et ai., 1998; Zaczek et al., 199Ia,b), although it can also cross the plasma membrane by Iipophilic diffision (Jones et ai., 1998). inside the nerve terminal, amphetamine acts on synaptic vesicles to reduce normal pH gradients (Sulzer & Raypori, 1990; SuIzer et al., 1993), resulting in the depletion of vesicular stores and a subsequent elevation in cytoplasmic dopamine concentrations. Elevations in cytoplasmic dopamine would disrupt
the dopamine concentration gradient across the pIasma membrane, which could result in
the reversal of the DAT (Sulzer et al., 1993, 1995; Urwyler & von Wartburg, 1988). The
release of vesicular stores aiso involves release of Na' ions and the resulting elevation of
intracelIuIar ~a'could dismpt the ionic gradient and Mercontribute to transport
reversai. However, recent work with transgenic mice lacking the DAT, suggea that the
change in dopamine concentration gradient resuituig fkom depletion of vesicular stores
may not be SuffiCient to reverse transport (Jones et al., I998), in contrast to hdings of
eariier in vitro studies (EsHeman et al., 1993; Pi8 et al., 1995; Suber et al., 1995). Thus,
DAT reversa1 may require both the depletion of vesicular stores and a direct action at the
transporter. %y acting as a substrate, amphetamuie enters the cell W;,?h ~a+,hcreasing the
nimiber of inward-fa- transporter bindmg sites avdabk for carrier-mediated release
of dopéunine (Butcher et al., 1988; Liang & Rutiedge, 1982a; Sulzer et al., 1993). 1.5 Antisense Oligonucleotides
1.5.1 Mechanibn ofActrkn
The use of antisense oligonucIeotides experimentdly was triggered by the
observation that antisense RNA is used physiologicaiIy by some prokaryotic cells nich as
bacteria (Pilowsky et al., 1994). Chiasson et ai. (1992) were the fht to report the use of
in vivo antisense oligonucleotides to anmuate gene expression in the central nenrous
system. Since then, antisense oligonucleotides have becorne a usehl experimenral tool
for hvestigathg the hctional role of various gene products including recepton,
neuropeptides, and transporters.
Genes consist of a specific sequence of bases including adenine (A), thymine (T).
guanine (G), and cytosine (C). These bases can be covalently bound to each other to form
a strand, and two strands can hybridize into a double-stranded cornplex by lorming weak
bonds between specific base pairs. A can fom weak bonds with T, and G cm fom weak
bonds with C. DNA is doubIe-stranded with one strar~dcontainhg the genetic code while the other, antisense strand, is a complementary copy. When a gene is transcribed, an RNA copy of the sense strand is made by formiog a complement of the antisense strand. Once this RNA undergoes a series of enzymatic reactions to remove introns etc., it is
messenger RNA (&A). Antisense comprises a short sequence of deoxynucIeotides,
typicdly complementary to a portion of the target rnRNA. However, antisense sequences
can dso be designed to hybridize with DNA (van der Kr01 et a(., 1988) to interfére with
transcription, or with pre-&A to disnipt RNA spIicing (KuIka et al., 1989). The mechanism of action is primarily associated with translationd Hockade radtirtg nom hybridization to mRNA (Fig. 2). However, other secondary mechanisms may be involved including activation of RNase H, oiigonucleotide-induced cleavage of mRNA, and disnrption of secondary and tertiary structures (Bennett & Crooke, 1994;
Crooke, 1993; Hélène & Tolume, 1990). Hybrids of antisense o1igonucIeotides and mRNA are more rapidIy degraded by the enzyme RNase H (Cazenave & Hélène, 1991;
Crooke, 1993; Ramanathan et al., 1993). As weil, antisense rnay disrupt secondary and tertiary structures in mRNA which are UivoIved in RNA processing, transport, and stabilization, as well as translational regulation (Vicken et al., 199 1).
15.2 Design
Most antisense O tigonucleotides are designed to target the initiation codon (AUG) of the target mRNA (van der Krol et al., 198 8). However, some genes are more efficient1y inhibited by O ligonucleotides targeting regions farther downstream (Brysch et ai., t 994;
Jachimezak et al., 1993; Schingensiepen et ai., 1993). The rnechanisrn of action of these oIigonucIeotides is not cleariy understood but may be more attributable to mRNA cleavage than transIationaI arrest.
The strength of hybridization is determined by the length of the oiigonuc1eotide süands and the degree of complementarity (Crooke & Bennett, 1996). Thus, affïnity inmeases as the Iength of the oiigonucIeotide mcreases. However, the sequence must be short enough to dow uptake into the cd, hnitmg Iength to 20-25 bases. As weU, while a sequence must be Iong enough to avoid comcidentd hybridization, if an oligonucieotide is tao Iong, it wilI Iose às spe&city (Crooke & Bennett, 1996). Therefore, the im@ of Figure 2
Anrfjme mechenrh of odt'orr Aritisense ofigonuckotides caxt mtdicrt with the mdarion pmccss afkscqucnc:-âpciEc bindrrig to L6e tiuget -A by'a (mi) biock of rranslation dor activation of RStas~&(Deived hmBrysch & Schhgasicpcn, 1994) antisense oligonucleotides is an important factor in their design. A sequence of 15-17 bases provides an ideal length, hakg a high probability of binding to a single cellular mRNA (Cazenave & Hklène, 1991; Crooke, 1992) while readiiy behg taken up by cells.
Oligonucleotides are readily broken dom by endo and exonucleases, increasing nonspecific toxicity due to accumulation of metabolites, and limiting their effecbveness.
The half-life of unmodified oligonucleotides in senun is only 30 minutes (Wickstrom,
1986), although it tends to be longer ui cerebrospinai fluid (McCarthy et al., 1993; Ogawa et al., 1995). Resistaace to nucleases cm be enbanced through modification O€ the phosphate backbone. The moa widely used modification is phosphorothioate. which involves replacing one of the nonbridging oxygen atoms in the phosphate group with a sulfur (Akhtar et al., 1991; Campbell et al., 1990; Shaw et ai., 1991). Phosphorothioate oligonucleotides have a half-Iife of more than 24 houn (Robinson et al., 1997). They also exhibit enhanced anity (Crooke & Bennett, 1996) and absorption (Cossum et al.. 1994;
Robinson et ai., 1997). üdiortunately, phosphorothioate modification is aiso associated with increased nonspecific toxicity. However, owing to enhanced efficacy of these oligonucleotides, lower doses cm be used, rninimizîng toxic effects. As well, Iimiting modifkations to only a few bases, rather than the entire strand, minimizes toxicity while presenring etncacy (Hebb & Robertson, 1997).
Even the most well-designed oligonucIeotide may have nonspecific effects, such as nonspecinc bhding to proteins (Stein & Kneg, L994), UihibitÏon of various poIytnerases (Crooke et ai., 1995; Gao et al., 1992; Stein & Cheng, 1993), and inhibition ofRNA spIicing (Hodges & Crooke, 1995). When using antisense oligonucIeotides as an experimentai tool, it is important to inchde the proper controis. One possibility is the use of a sense strand which would have the same sequence as the homologous segment of target mRNA and, therefore, be unable to bind to it. However, the sequence may have a
Merent base composition resulting in different base concentrations when degraded.
Different bases might cause Merent metabolic effects (Crooke & Bennett* 1996). An alternative is to substitute a few of the bases of the antisense sequence to create a mismatch oligonucleotide. A single mismatch can decrease affinity by approximately 500 fold (Crooke, 1993; Fner et ai., 1992)- In this way, it would have most of the same bases but would not hybndize with the target mRNA. Another commonly used control is the use of a missense oligonucleotide, which contains the same bases as the antisense but in scrambled sequence (Wagner, 1994). In ail cases, sequences rnust be checked against gene data bases to ensure they wi11 not hybridize to unintended mWA.
The duration of adsense treatment required to sigm*ficantly reduce protein expression varies greatly and depends pruriarily on the basal expression level of the protein and its turnover rate. Those with a low basai expression and rapid turnover rate, as seen with some immediate early genes, require ody a single injection to reduce their expression (Heilig et ai., 1993; Suniki et al., 1994). By contrast, receptors generally require three or more days of antisense treatment to see a significant effect (Wahlestedt et al., 1993; Zhang & Creese, 1993). This may be achieved with twice daily administrations or with co~tinuousinfirsion by osmotic minipump, which may reduce the incidence of toxicity (whitesell et ai., 1993). 1.53 Thmapeutic Putentid
Although antisense technology is still being perfected it is thought to have some
therapeutic potentid. The use of antisense oIigonucIeotides is currently being explored as
a possibie treatment for various Wal infections incIuding HIV-I (Agrawal et al., 1988;
Anazodo et al., 1995). measles (Koschel et al., 199S), hapatitis B (Korba & Gerin. 19951,
and herpes (Kean et al., 1995). Preciinicai studies have aiso exarnined the effects of
antisense to tumor ce11 proteins on cancer ceIl proliferation (Hélène, 1994). However.
oligonucleotides do not cross the blood-brain-bmier very efficiently (Agrawd et al..
L99 1), and will require improvements ui their delivery system before they cm be useful
as therapeutic agents for central nervous system disorders.
1.6 Rodent Models of Dmg-Induced Dyskinesias
1.6.1 Levudopa-Induced Dyskinesios
Lise of the neurotolrin 6-hydroxydopamine (6-OHDA) to create chemicai lesions of
forebrain dopamine projections in rodents provides a useful mode1 of parkinsonism. WhiIe bilaterd lesions are associateci with extreme aphagia and adiptia, oflen resulting in death,
uniIaterai Iesions Leave the animal m an otherwise normal state. The unilaterai mode1 aiso dows withm-subject cornparisons of pathophysioIogicd mechanisms and provides a more qua11titative means of rneaSunng both dydbncîion and responsiveness to antiparkinsonian agents. Thus, rotationai response to dopamine agonists such as apomorphine in this model is predictive of antiparkinsonian efficacy in humans (Ungerstedt, 1971).
As descn'bed earlier, levodopa remains the most widely used treatment for
Parkinson's disease (Cotzias et ai., 1969; Homykiewicz, 1998; Yahr et ai., 1969). However, foflowing sevaal years of treatment, abnormal involuntary movements termed levodopa- induced dyskinesias (LID)are observed (Barbeau, 1974). Such dyskinesias are aiso seen in
MPTP-treated rnonkeys followïng chronic pdsatile Ievodopa treatment (Bédard et ai.,
1986; Burns et al., 1983; CIarke et ai., 1987; Henry et ai., 1997; Pearce et ai., 1995). While the appearance of dyskinetic behavioun in MPTP-treated monkeys fo tlowing chronic levodopa rnay provide a good mode1 for the human condition, experimentai progress can be hindered by ethicai and logisticai limitations inherent to primate work, in generai. By contrast, rodent modeIs provide an inexpensive meaw of testing several compounds de novo in short periods of time. Also, chroaic studies in rodents may be carried out in a much shorter tirne fiame than is possible Ïn primates. The use of rodents dso readily permits the investigation of molecdar, biochemical, and receptor changes fo Ilowing each treatment-
Udorhniately, the repertoire of rodent behavioun is rather limited while the choreiform movements characteristic of hman LID are quite cornplex. Thus, rats do not exhibit the same dyskinetic behaviours seen in patients or MPTP-treated monkeys. These anhals do, however, demonstrate alterations in behaviourai responses, which may be andogous to LI. m humans. ArfmMistration of apomorphine to hemiparkinsonian rats redts in contraversive rotations, as desm'bed above. Foffowing chronic pdsahle administration of Ievodopa, this rotationai response to apomorphine becomes sensitized
(Bevan, 1983). It is this sensitization, rather than the behaviour per se, which is thought to be analogous to the emergence of dyskinesias in hirmans. Understanding the mechanims underlyhg this sdtization may provide valuable insight into himian LID.
Though not an ideal model, it appears to share many characteristics with the human disorder (Henry et al., 1998). As with human LID, the development of sensitized apomorphine-induced rotations in rodents requires extensive dopamine depletion in the striatum (Markham & Diamon4 L981; Papa et al., 1994; Thomas et ai., 1994) and a pdsatile mode of hgadministration (Engber et al., 1992). As well, its rate of onset is closely related to both the dose and duration of treatment, as is seen clinicaliy (Nutt, 1990).
Chronic levodopa-induced sensitization of apomorphine-induced rotation also has a very similar phatmacological profile to that of LID in primates. Those dopamhergic treatments which typically eIicit dyskinesias in primates, also trigger enhanced behavioural responses in this rodent mode1 (Henry et al., 1998), while br~rno~ptineand Lisinide are associated with fewer dyskinesias in both primates and rats (Bédard et al., 1986; Rime, 1989).
Behaviours in rodent and primate models dso respond timiIarly to nondopaminergic compormds. a, adrenergic receptor antagonists, 5-HT uptake inhibitors, and P-adrenergïc receptor antagonists aii reduce rotations in a rodent model of UD (Henry et ai., 1998). This is nmilar to reports fkom studies of both MPTP-treated monkeys (Ashby et ai., 1996;
Herrero et ai., 1995) and Parkinson's patients (Carpentier et aI., 1996; Durif et ai., 1995;
Rascol et al., 1997). Finaiiy, both primate and rodent models exhibit qualitahvely very
SimiIar effects on strïatd opioid peptide expression (Engber et ai., 1992; Herrero et aI.,
1995; JoIkkonen et aL, 1995; Zeng et ai., I995), with nomakation of Iesion-induced reductions m subûtance P expression, no signincant changes in enkephaIin expression, and a substa~ltialeIevation m dynorphui expression. These neuropeptides play a signincant role in the regdation of stnatal function and may be an important fator in the expression of Lm.
Thus, while the behaviod responses in this rodent mode1 of LID hi1 to mVnick those seen in primates, this model rnay be usefiil in investigating the mechanisrns underlying the development of dyskinesias induced by chronic levodopa therapy.
1.6.2 Tardnte Dyskinesia
AIthough an ideal rodent mode1 of tardive dyskinesia has not yet been established, current paradigms rnay provide usehl insights. Long-term administration of neuroleptics to rodents results in the anergence of vacuous chewing movements (VCMs)(Clow et al..
1979; Iversen et al., 1980; Waddington et ai., 1983). These VCMs are nondirected mouth movements including chewing, tongue protrusion, and teeth grinding and are cornmonly used as a model of TD, WhiIe this model remains somewhat controversial, these VCMs share many characteristics typicd of TD including similarities in phenotypic expression, epidemioIogy, time course of development, and response to dopaminergic dmgs (Glenthoj,
1995). TD is characterized by repetitive, UivoIuntary movements, typicdly of the orofacid region, aithough may ako involve di& hbs, As with human TD, chronic neumleptic- induced VCMs are evident in only a proportion of animds (Waddington et al., 1985) and are seen more often ui older than yomger animds (Waddington et ai., 1985. 1986). These
VCMs deveIop oniy afler Long-term treatment and persist long derdiscontinuation (Rlison et ai., 1987; Glenthoj et al., 1990; Levin et al, 1987; See et al., 1988; Waddington et al.,
1983, 1985,I986). Abnormai mouth movements have been used as a rodent mode1 for a number of human movement disorders, a fxtor which underiies much of the controversy sumiunding its use as a model of TD. Vacuous jaw movements have been proposed by Salamone as a model of parkinsonian tremor. These mouth movements cini be elicited by administration of cholinomimetic dmgs (I3aski.n et al., 1994), lesioning the ventrolateral Stnaturn with 6- hydroxydopamine (Jicha & Sdamone, 199 I ), acute administration of dopamine antagonists
(Rupniak et al., 1985), and acute dopamine depletion by reserpine (Baskin & Salarnone,
1993). These oral rnovements, however, mer fiom VCMs in that they tend to occur in a cange of 3-7 Hz while VCMs occur in a range of 1-2 Hz, similar to that seen in TD (Ellison
& See, 1989; See et al., 1988). Mso, vacuous jaw rnovements respond to antiparkinsonian dmgs while VCMs do not (Salamone et ai., 1986; Stoessl et ai., 1989; Waddington 1990).
Abnormal oral movements have also been observed by sorne researchers to occur early in the course of neuroleptic treatrnent (Rupniak et ai., 1985; GIassman & Glassman,
1980). As a result, these mouth movements have been proposed to be a mode1 of acute dystonia which invoIves intermittent or nistained muscle spasrns often seen in patients in the initial stages of neuroleptic treatment (Rupniak et al., 1986). These mouth movements differ fbm VCMs in their time course and appearance (abnomai versus normal) and, Iike the vacuous jaw movements discussed above, these mouth rnovements diffa fiom VCMs in their kquency range (Gienthoj, 1995). Mthough controversy stil1 exists, they also appear to differ in theu response to choIinergic manipulations (Glenthoj. 1995; StoessI et ai., 1989;
Waddington et ai., 1986) and acute neurokptic challenge (Gunne et al., L986; Stoessl et ai.,
1989). Thus, these VCMs cmbe disiinguished hmother abnomal mouth movements by their the course, kquency range, and response to chohergic and dopaminergÏc manipulations (Glenthoj, 1995; Stoessi et al, 1989). h this context, chnic neuroteptic- induced VCMs may provide a couvenient and effective means of investigathg the pathophysioIogicai mechanisms uivoIved in the development of TD. Chapter 2
EXPERtMENTAC DESIGN
2.1 Objectives
2J.1 Ran'ontrle
Parkinson's disease resdts hmthe graduai and near total to total Ioss of striata1 dopamine- While dopamine replacement in the fom of Ievodopa remains the primary mode of treatment, long-term exposure is complicated by the emergence of fluctuations in motor bction and a variety of involuntary movements, termed levodopa-induced dyskinesias
(LD)(Manden, 1982). Another form of dmg-induced dyskinesia is tardive dyskinesia
(TD),which develops as a complication of long-term neuroteptic exposure in approximately
20-30% of patients so treated (Kaae & Smith, 1982). TD and Lm are characterized by repetitive involuntary movements, primarily of the orofacid region and distai limbs. respectively.
The mechanranrsmslmderlying these dyskinesias rernain poorly understood but may involve an imbaiance in striahi output pathways. While studies of dopamine D, receptor changes in rnodels of LID have yielded mixed fesults, there is evidence to suggest heightened activity in dopamine D, receptor-bearing striatonigrai neurons. Chronic pulsatile administration of levodopa has been found to elevate depresseci IeveIs of dynorphin and nomalise substance P 1eveIs m striatonigrai neurons (Engba et ai., 1991; Herrero et al.,
1995). Similar eievations in nempeptlde expression are seen foIIowing chronic pulsatiIe administration of dopamine DIreceptor seIective agonists (Engba et al., 1992) and are positively correlateci with rotationai mponses (Steiner & Gerfien, 1996). Such changes are not seen following a more continuous mode of administration (Chase, 1998), which has been associated with reduced behaviourai sensitisation in experimental models (Juncos et ai., 1989) and fewer dyskinesias in dinical -dies (Chase et ai., 1994; Mouradian et ai.,
1990). Exposw of6-OHDA lesioned rats to Ievodopa potentiates subsequent dopamine Dl receptor-mediated cyclic AMP production in striatd tissue (Pinna et al., 1997), thus resulting in increased effectiveness of dopamine D,receptor signal transduction, which may play a role in the development of UD. It has been suggested that immediate early genes may play a dein the signal transduction events responsible for subsequent alterations in neuropeptide gene expression (Naratljo et al., 1991; Sonnenberg et ai., 1989). Chronic levodopa administration has been associated with increased expression of the immediate early gene *SB, whose induction is dependent upon dopamine DI receptor activity
(Doucet et d., 1996).
Chronic neuroleptic-induced VCMs, kqyently regarded as a rodent mode1 of TD, ako appear to involve relatively heightened activity at dopamine DIreceptoa (Ellison et al.,
1988). niese mouth movements are readiIy attenuated by dopamine DIreceptor antagonists
(Stoessl et al., 1989) and enhanced by dopamine DIreceptor agonists (LubIin et al., 1992;
Rosengarten et ai., 19833. AIso, a SimiIar syndrome can be elicited in hg-naive animais by acute administration of a dopamine DIreceptor agonist (Rosengarten et al, 1983b).
Dopamine DIand D2receptors have opposing effects on adenylyI cyclase activity, such that the dopamine D2 receptor is thought to exert a tonic inhibition of dopamine DIreceptor- mediated CAMP efflux possibIy via collaterd projections Thus, disinhicbition of this respo~eby chmkdopamine D2receptor blockade, coupled with stimulation of dopamine
Dl receptors by endogenous dopamine, may resuit in enhanced DI-reIated activitr
(Tnigmm et al., 1994). hteresthq&, clozapine, a neuroleptic drug associateci with reduced incidence of TD, displays antagonistic action at dopamine Dlreceptors (Ashby et ai., 1996) in addition to blockade of 4 receptors. Vacuous chewing movements have been Linked to dopamine DI receptoa in the ventroiaterai striahan (Neiswander et al., L995), an area thought to project prirnarily to targets in the substantia nigra pars reticulata Thus, behaviours in experimental models of TD may be mediated by dopamine D, receptor- bearing striatonigmi neurons.
Dopamine Dg receptors have aiso recently been implicated in hg-induceci dyskinesias. These recepton are localized on dopamine DI receptor-bearing neurons primariIy in the sheII of the nucleus accumbens and islands of CaIIeja with relatively Iow expression in the caudatdputamen under normal conditions (Ariano & Sibley, 1994;
Schwartz et ai., 1998). However, chronic pulsatiIe administration of levodopa to hemiparhonian rats redts in the induction of dopamine D, receptor mRNA expression and D3 receptor binduig which is localued to dopamine D, receptor-bearing striatonipI neurons (Bordet et al., 1997). This induction appears to be dependent on dopamine Dl receptor stimulation and, thus, rnay result nom increased activity of striatonigrd neurons occunulg in response to repeated levodopa treatment. These changes in dopamine D3 receptor expression cIosely paraHe[ the sensitization of apomoiphine-induced rotation in these anmials which is readily attmuated by a dopamine Dj receptor antagonist and enhanced by a D3agonist (Bordet et ai, 1997; Schwartz et ai., 1998). Dopamine D3receptor mduction aIso cIosely paraIIeh changes in neuropeptide mRNA expression wEch may be regdated by the D, receptor (Tremblay et aI., 1998). Thus, the dopamine D, receptor may play a miticai role in LJD.
The dopamine transporter rnay play a roIe in the development of hg-uiduced dyskinesias. The DAT, through dopamine reuptake, serves a vital fùnction in regulating dopaminergic neurotransmission. The DAT maintains low steady-state concentrations of extracellular dopamine and Iimits the efnux of dopamine hm the synaptic cleft to extrasynaptic compartrnents (Parsons & Justice, 1992). Thus, a particular dopamine synapse acts on cells within a certain perheter, which is specified, in large part, by dopamine uptake. In this way, the DAT imparts both temporal and spatial regulation of doparninergic nemtransmission. The majority of striatai dopamine Direceptors are located outside the synaptic zone (Caillé et ai., 1996; Levey et al., 1993) and are acted on by dopamine difhed away Eom the synapse, a process refmd to as volume transmission (Zoti et ai., 1998). By controlhg dopamine overflow, the DAT can regdate activation of these extrasynaptic recepton. Blockade of DAT by nomifensine enhances the amplitude and duration of stimulus-evoked dopamine overflow and increases stimulation of extrasynaptic dopamine
D, receptors (Gonon, 1997). Chronic alterations in DAT Function can aiso cause long-term changes m gene expression, which codd aIter striatai activity. Repeated treatment with the DAT blocker GBR-12909 increases striahi dynorphlli expression
(Sivam, 1996). Similariy, DAT bockout mice exhibit a 100 fold increase in extraceilular dopamine concentration (Giros et d., 1996), accompanied by neuropeptide aiterations reminiscent of those seen m models of LID, incIudmg efevations in striatai dynorphin, thought to be regdated by dopamine D, receptor actMty (Stemer & Gerfh,1998). In PD, the main risk factor for the deveIopment of LID is disease seventy (Horstid et d., 1990; Markham & Di-amond, 1981). As weiI, the magnitude of dopa-induced dyskinesias in parkinsonian monkeys is related to the extent of nigrostriatai damage
(Schneider, 1989). This has 1ed some to suggest that the appearance of LID may be related to the continual Ioss of buffaing nonndy afEorded by dopamiuergic temhds, Uicluding an increasing inability of degrnerathg neurons to reuptake dopamine (Homykiewicz, 1979;
Mouradian & Chase, 1988; Shaw et al., 1980). The development of tardive dyskinesia rnay dso involve alterations in DAT hction. Neuroleptic dmgs have been show to inhibit
DAT-rnediated dopamine uptake (Lee et al., 1997; Rothblat & Schneider, 1997) thereby hcreasing dopamine overtlow in the synaptic cleft. uiterestingiy, the atypical neuroleptic clozapine, a hgassociated with significantly fewer dyskinesias, is a much less potent uihibitor of dopamine uptake. Thus, by virtue of the signincant role of the DAT in regulating both temporal and spatial aspects of dopaminergic neurofransmission, aiterations in its bction could contribute to the development of hg-Ïnduced dyshesias.
The DAT may dso contniute to the pathogenesis of PD. There is some correlation between the denmty and distribution of DAT expression and the pattern of ce11 loss in PD (Miller et ai., 1997; Sanghera et al., 1997; Clhl, 1990; Uh( & Kitayama L993;
IJhi et al., 1994). As welt, the DAT may be responsible for the uptake of neurotoxins used to produce experimentai parkins~nism~The neurotoxic effects of MPTP can be blocked by dopamine qtake blockers including nomifensine, cocaine, and GBR compounds
(Giros & Caron, 1993). In vitro, ceIIs nomaDy resistant to MPP' can be rmdered
vuherable when transfected wah cloned human and rat dopamine tramporten (Kitayama
et A,1998; Pifl et aI., 1993), whiIe ~Iigonucleotideaatisense to the DAT has been shown to decrease the toxic effects of 6-hydroxydopa~nine(Simantov et al., 1996). In vivo, genetic manipulations have also reveaied a between DAT and the toxic effects of
MPTP. The overexpression of DAT in mice makes these animais more vulnerable to the toxic effects of MPTP (UN, 1998) while, conversely, DAT bockout mice appear to be resistant to the toxin (Gainetdinov et al., 1997). Thus, individuai variations in DAT expression or its anüiity for neurotoxins, codd determine susceptibiiity to PD.
2.1.2 Hypotheses
1. intrastriatal antisense 'knockdown' of dopamine DIA receptors will attenuate both
neuroleptic-induced VCMs in a rodent mode1 of TD, as we11 as sensitization of
apomorphine-induced rotations in a rodent model of Lm.
2. Sensitization of apomorphine-induced rotations in a rodent model of LEI will also be
attenuated by intrastriatai antisense 'knockdown' of dopamine D, receptors.
3. Reductions in striatai DAT expression by intranigral antisense will attenuate the
neurotoxic effects of both MPP' and 6-hydroxydopamine. 2.2 Generai Methods
2.2.1 Animais
AU studies used male Sprague-Dawley rats (Charles River, Montreal), weighing approximately 250 g at the start of the experiment. Animais were housed in a temperature controlled environment with a 12 h light/dark cycle (lights on at 0700) and ad libitum access to standard rat chow and water.
2.2.2 Surgety
For ai1 experiments involving dopamine transporter antisense, animals were anaesthetized using ketamine (MTC Pharmaceuticds; Ont., Canada: 40 mgkg, km.) and xylazine (Bayer; Ont., Canada: 10 mgkg, Lm.). However, Eiollowing these studies, it was reported that ketamine dose-dependently bIocks uptake by DAT (Nishimura et ai., 1998).
This raised concerns regarding the eflicacy of 6-hydroxydopamine lesioning under this anaesthetic, particuiarly in iight of recent suggestions that 6-hydroxydopamine-induced neurotoxicity may be mediated by the DAT (see chapter 3.6). A brief cornparison of
Iesion efficacy under ketamine versus halothane gas anaesthesia suggest that ketarnine may, indeed, be interferhg with the neurotoxic effects of 6-hydroxydopamine, as a sipnincantly greater reduction in striatal WIN 35-428(NEN; MA, USA) binding was seen m those Iesioned under gas anaesthesia (Fig. 3). Thus, dl other surgeries were performed using halothane ( 1-2%). Figure 3
Striatal Dopamine Transporter Binding Following Unilateral Infusion of GOHDA Under Ketamine versus Halothane Anaesthesia
halothane
Denritotnaric nteus~r~mentof aururadiograpks rrprtsentlng HI- WIN 35,428 binding in the striatum. Two weeks fotlowing unilateml infusion of 6-hydroxydopaminc inro the lcft MFB, ipsilaterd striatat dopamine transporter binding was significantIy reduced in dl animais, This rcduction was significantly greater in those animals that had been anaesthetized usîng halothane than those anacsthetizcd using ketamine. Each bar rcpresents the mcan (r S.E.M-) (n-8) opticaI density (nCllmg). ** sig. difE from right hcmisphcrc, p <- 0.00 1 ; p c 0-0 t ## sig, diff- from ketamine, p < 0.00 1 Unilateral nigrostriatai lesions were created using the dopaminergic nemtoxui 6-
hydroxydopamine HCI (6-OHDA)(8 pg/4@ saline with 0.05% ascorbic acid; Sigma,
MO). Anirnals were pretreated with desipramine (20 mgkg, s.c.; Sigma, MO) in order to
protect noradrenergic neurons. AnimaIs were then anaesthetized and placed in a KopP
stereotaxic he.6-OHDA was iofised unilateraiiy into the left medial forebrain bundle
(AP -4.30, ML +1.70, DV -8.40) at a rate of 1 pUmin for 4 minutes using a Harvard
pump. The cannula was left in place for another 2 minutes to allow diffision away from
the tip. Coordinates were derived from the atlas of Paxinos and Watson (1986). For DAT
antisense experiments, either 6-hydroxydopamine or 1-rnethyI4p henylp yridiniun iodide
(MPP') (8 pg/3pI; Sigma, Ont.) were infused via an infusion cannufa inserted through
previously implanted guide cannulae. The infiision cannula was approximately 3.7 mm
longer than the guide cannulae, designed for a DV iarget of -7.70. These animais were
not anaesthetized. AnirnaIs were allowed to recover for 2 weeks following Iesioning.
For implantation of cannulae, animais were anaesthetized and placed in a Kopt'
stereotaxic £&ne. Stainless steel indwelling ca~ulaewere placed either in the striatum
or nibstantia nigra using coordinates derived from Paxinos and Watson (1986) (for exact
coordinates see chapter 3). The canntdae were fked to the skul1 using dental aqlic and jeweIer's smws. When used for antisense infirsion, each cannda was attached, by 50 PE
polyethylene tubin& to an osmotic mmipump (ha,California) which was placed under
the skin at the base of the neck. 22.3 AnhseOligonucleotides
Oligonucleotide sequences rangkg fkom 17 to 18 mer, were custom synthesized
(Oligonucleotide Synthesis Laboratory, UBC). The antisense oligonucIeotides were complementary to mRNA encoding the initiation site of the rat dopamine DIAreceptor
(5'-AGG-AGC-CAT-CTT-CCA-GA-3') (7 nmoUday, 3 days), dopamine D, receptor (5'-
GCT-C AG-AGG-TGC-CAT-GGC-3') (7 moUday, 5 days), or dopamine transporter (5' -
AGA-TTC-AGT-GGA-TCC-AT-3')(1 nmoi/day, 7 days). Control missense otigonucIeotides, consisting of the same bases in scrambled sequence (Wagner, 1994;
SEE DI PAPER), were aiso synthesized for the DIAreceptor (5'-CGC-GGT-AAT-CCA-
GAT-CA-3'), D3 receptor (5'-AGC-CAG-AGT-GTC-GCG-CATT3'),and dopamine transporter (5'-AGC-ATT-GAA-CAA-GCC-AT-3').None of the oligonucleotides described here display significant homology with any sequences round in current databases (Genbank). OIigonuc teotides were dissolved in sterile saline and adrninistered continuously via osmotic minipumps (Alza, California) ai a rate of I pVhr. Each pump was fiIIed with either antisense, missense, or steriIe saline.
2.2.4 Chronic Dmg Treahnents
For chronic levodopa studies, treatment began approlamately 2 weeks Colollowing unilaterai Iesioning with 6-OHDA. Animais were mjected with either the peripherai decarboxylase mhibitor benserazide (IO mgkg, ip.; Sigma, MO) (to enhance cenb?llly avdable concentrations) aIone, or a combination of benserazide and L-34- dihydroxyphenyIaIanine methyl ester (Ievodopa) (50 mgkg, Lp.; Sigma, MO) hKice daiIy for 3 weeks, For chronic neuroleptic studies, animds were treated with either fluphenazine decanoate (25 mglkg, Lm.; Squibb, Quebec) or its vehicle sesame oil (1 mikg, Lm.;
Sigma, MO) every 3 weeks for a minimum of 18 weeks.
2.2.5 BehaviouruL Obsewatiims
For direct observations, animais were habituated to plexiglas boxes (50 x 50 x
30 cm) for at least two hours prior to testing. Animals were then either tested for spontaneous behaviours (as for chronic fIup henazine-treated animais) or were injected with the dopamine Dl receptor agonist SKF 38393 (5 mgkg, s.c.; Research
Biochemicais, MA) and tested 30 minutes later. During testing, anmials were observed continuously for three minutes out of every six minute block, altemating between two animais, for a total of IO blocks (60 min) each. The Eequency and duration of varÏous behaviourai responses were recorded using a microcornputer keyboard and custom- designed software (BEBOP: Dr. Martin-Iverson, University of Western Australia). For each response of interest, a coded key was pressed upon onset and offset of each behaviour.
The following behaviourd responses were recorded: VCMs, grooming, locomotion, sniffing, and rea~g.VCMs were dehed as al1 nondirected mouth movements incIuding chewing and tongue prohusions but excluding jaw tremor and directed movements such as kickin& eating, groommg, and yawning. Saw tremor was excluded, as it is thought to be distinct fiom VCMs and not correIated with neuroleptic treatment (Glenthoj et al., 1990). Grooming was defhed to include scratcbg, forepaw licking, body fur groomin& and face washing. PeniIe grooming was excluded as it is thought to reflect selective activation of hi& afnnity dopamine D2 receptors (Stoessl et ai., 1987). Sniffing was marked by head bobbing and whisker movements. Rearing was defined as the time spent with both forepaws off the ground and the head elevated.
Locomotion was defined as the thespent in forward movement invoIving ail four limbs.
Animals were tested for rotation ushg a muitichannel hamess rotometer linked to a microcornputer (RotaCount, Omnitech). Animais were habituated to the test chamber for 15 minutes priot to testing and then injected with either methylamphetamine hydrochloride (2 mgkg, se.; BDH Inc., Ont.), benserazide hydrochloride ( 15 mgkg, i .p .;
Sigma, MO)/L-3,kiihydroxyphenyIdanine methy 1 ester (levodopa) (50 mgkg, i.p .;
Sigma, MO), or R(-)-apomorphine hydrochlonde (0.3 mgkg, SC.; Research
Biochemicals, MA). Levodopa and amphetamine were administered 30 minutes prior to testing, while apomorphine was given immediately before testing. Animals were tested for 1 hour, during which, the direction and number of each 360' turn was recorded and grouped into five eutebins.
2.2 -6Auto radiography
Six to tweIve hom followhg behaviourai testuig, animais were decapitated and the brains were removed and rapidy fiozen using dry ice and isopentane and stored at -
80°C lmtil being sectioned ( IOp)using a cryostat.
For DAT binduig, sections were preincubated in 50 mM Tris-HCI baer (pH 7.4) containhg 120 mM NaCI, and 5 mM KCI for 15-30 minutes at 4°C. SIides were then incubateci in humidified boxes and sections overfaid with 50 mM Tris-HCI bui3er (pH
7.4) containing 300 mM NaCI, 5 mM KCI, and IO nM [m35-428 (SA = 84.5 Cilmol; Dupont NEN, MA) for 40 minutes at 4 OC. Non-specific binding was defhed with 10 pM nomifensine (Research Biochemicds, MA). FoUowing incubation, slides were washed in the assay bfler twice for 60 seconds at 4 OC and dried under a cool air stream.
For vesicular monoamine transporter (VMAT2) binding, sections were preincubated in a sucrose b&er consisting of 300 mM sucrose, 50 mM Tris-HCI, and 1 mM EDTA (pH 8.0) for 5 minutes at 25OC. Slides were then incubated in humidified boxes and sections overlaid with sucrose buffer containing 1 nM
[3~methoxytetrabenazine(MTBZ) (SA = 80 Cilmmol; provided by Dr. M. filboum,
University of Michigan, Ann Arbor, MI) at 25°C for 3 hom. Nonspecific binding was defined in the presence of 10 pM uniabeled tetrabenazine (Roche). Following incubation, dides were washed in sucrose buf3er 3 times for 3 minutes at 2S°C, bnefly dipped in distilled water at 4OC, and dried under a cool air stream.
For dopamine Dl receptor binding, sections were preincubated in 50 mM Tris-
HCI bser (pH 7.4) contaîning 120 mM NaCl, 5 mM KCI, 2 mM CaCL, and 1 mM
MgCl, for 15 minutes at room temperature. SIides were then placed in humidified boxes and sections ovedaid with the same buffer containing 2 nM 3~-SCH23390 (SA = 81.4
Cifmmol; Dupont NEN, MA) and 30 nM ritanserin (Research Biochemicds International.
MA) (to occhde 5HTaK receptors) for 45 minutes at room temperature. Non-specific binding was defhed with 10 pM (+)-butacIamol (Research Biochemicak, MA).
FoUowing incubation, slides were washed in the same bu&r twice for 3 minutes at 4OC and dried under a cool air stream- For dopamine D, receptor bindÏng, sections were preincubated in 50 mM Tris-
HCI buffer, similar to that used for D, binding, for 15 minutes. Slides were placed in humidined boxes and ovedaid with the same baer containhg 2 nM [3~]-raclopride(SA
= 79.3 Ci/mmol; Dupont NEN, MA) for 60 minutes at room temperature. Non-specific binding was dehed using 1 pM (+)-butaclamol (Research Biochemicds, MA). Sections were then washed in the same baer 4 times for 60 seconds at 4 OC and dned under a cool air stream.
For dopamine 4 receptor bhding, sections were preincubated three times for 5 minutes each at 21°C in 50 mM HEPES buffer (pH7.5) containing i mM EDTA and
0.1% bovine senim aibumin (Sigma, MO). Slides were then placed in humidified boxes and overiaid with the same bufTer containhg 0.5 nM R(+)-?-~~~~OX~-~H~DPAT(SA =
154 Ci/mmol; Amersham, Ont.) for 90 minutes at 21 OC. Nonspeci fic binding was defined in the presence of 10 pM (+)-butaclamot (Research Biochemicds, MA). Following incubation, siides were then rinsed 3 times for 30 s in KEPES buffer containhg IO0 rnM
NaCl at 4*C, bnefly dipped in distiIled water at 4OC, and dried under a cool air stream.
Ai1 sections were apposed to tritium-sensitive ~yperfilm3~(Amenham.
Toronto, Canada) dong with tritirmi standards for 4-6 weeks and developed with D 19 deveioper (Kodak, NY). Opticai densities were detemiuied using a cornputer-assisted image analysis system. Measurements were taken nom four distinct quadrants of the Iett and nght striatum of 3-6 individual sections per mimai and the numbers were averaged to obtain a single measurement for each quadrant. Where no signincant regionai differences were formd, numbers were averaged to obtah a sbgIe meamernent for each animai. 2.2.7 RPLC
Striatai tissue was dissected, weighed, and fiozen at -80°C until the time of assay.
The tissue concentration of dopamine was determined by reverse phase ion-pair high pressure Iiquid chromatography with eIectrochemicd detection. Frozen tissue samples were sonicated in 10 vols. of 0.1 M H2C104containhg 0.1% sodium metabisuifïte, 0.02% disodium EDTA and 50 ng/d of 3,4-dihydroxybellzylamine as an intemal standard.
Mer cenhifugation, the supematants were nItered and 30 pl aliquots were injected onto the isocratic chromatograph by a renigerated WSP 712 autoinjector. The column was a 5 pm spherical octyldecylsilane 32 cm anaiyticai cartndge equipped with a 1.5 cm New-
Guard cddge. The identity of the compounds of interest was checked by the method O € standard addition. HPLC andysis was pdonned by the lab of Dr. E. McGeer.
2.2.8 Stati;stical Anatysis
Data were anaiyzed usuig a multivariate anaiysis of variance and .where ngnificant F-vaiues were fotmd, planned pairwise cornparisons were made ushg a
Newman-Keds test, Chapter 3
EXPERIMENTAL RESULTS
3.1. Effects of Oligonucleotide Antisense to Dopamine DIAReceptor
mRNA on SKI? 38393-hduced Behaviours
3.1.1 htroduchton
SKF 38393-induced VCMs and gmoming have corne to be regarded as a behavioural mode1 for dopamine D, receptor stimulation (Murray & Waddington, 1989).
However, not ail DIagonists elicit these responses. ui fact, stiucturally distinct dopamine
DIreceptor agonists elicit different behavioural pheaotypes (Downes & Waddington, 1993;
Deveney & Waddington, 1997; Clifford et al., 1998). There is growing evidence for the existence of dopamuie DI-iike receptors that are coupIed to alternative transduction mechanismsother than adenytyl cyclase (Amt et ai., 1988; Giambaivo & Wagner, 1994;
Johansen et ai., 1991; Laitmen, 1993; Mahan et ai., 1990; Murray & Waddington, 1989;
Schoors et ai., I99 1; Undie & Friedman, 1990; Undie et ai., 1994; Waddington et ai.,
1995). Thus, the abiIity of these DI-Like agonists to mduce VCMs and grooming may be unrelated to their abiIity to stimdate adenyIy1 cyclase (Devaiey & Waddington, 1995;
Murray & Waddington, 1989; Waddington et ai, 1995). While the selective DIreceptor agonist SKF 83959 does selectiveIy bmd to the Direceptor and cm hduce VCMs and groom& it fds to stimuiaîe adenyIyl cycIase and mhibits stundation of adenyIyI cycIase by dopamine (Amt et al., 1992). As weU, dopamine DI-fie agonists are lmable to stimulate adenylyl cycIase in dopamine DIAreceptor deficient mice, yet these anÏmaIs demonstrate elevations in behaviours etaditiomIly Lùiked to the D, receptor (Clinid et ai, 1998; DeE
& Kelly, 1990), and continue to exhibit dopamine DI-like agonist-induced behaviours
(Clifford et al., 1999). Thus, it has been suggested that there may be different subtypes of the dopamine Dlreceptor that have not yet been identined which may tecognize different chemical classes of dopamine DI-iike compolmds and rnay be coupled to transduction mechanisrns other than adenylyl cyclase (Adachi et ai., 1999; Daiy & Waddington, 1993;
Deveney & Waddington, 1997; Munay & Waddington, 1989).
The use of oligonucleotide antisense may provide a more selective meaw of studying the role of dopamine DI, receptors m VCMs and grooming. While gene knockouts also afXord a high degree of specificity, behaviourai reports have been rnixed
(Cromwell et ai., 1998; Deüs & Kelly, 1990; Smith et ai.. 1997) and concems have been raised regatding possible compensatory changes occmruig during development in these animais (Deffs & Kelly, 1990). ï'his approach dso lacks the anatornicai specificity. which cari be afforded by direct infitsion of oligonucleotides mto the site of mterest. Behavioural changes induced by a globai reduction or absence of a receptor are often difficuit to interpret.
Thus, m the study descnbed here, 1 use in vivo oiigonucIeotide antisense to dopamine DI, receptor mRNA m an attempt to cIariQ the mie of the DiA receptor in SKF
38393-induced VCMs and gmoming. 3.1 2 Design
In animais (N=12) anaesthetized with halothane, bilaterai cannulae were implanted (AP +0.50, ML E2.50, DV -620) into the ventrolaterd stnatum, an area thought to mediate VCMs and grooming (Fletcher & Starr, 1987; Neisewander et al.,
1995). Attached to the cannuiae, by polyethylene tubing, were osmotic minipurnps
(mode1 L003D; ka,CA) which delivered either dopamine DlA receptor antisense (5'-
AGG-AGC-CAT-CTT-CCA-GA-3'), missense (5'-CGC-GGT-MT-CCA-GAT-CA-37, or saline at a rate of 1p.M~or 7nmoVday for 3 days. Following antisense infusion, animais were habituated to the test chamber for at Ieast two hours, injected with saline and then tested for VCMs and grooming 30 minutes Iater. Animais were then injected with SKF 38393 (5 rng/kg, SC.) and tested again 30 minutes later. Approximately six hours after testing, animais were decapitated and the brains were removed and rapidly fiozen. Once shed (10 p),sections were tested for dopamine DI and D2 receptor binding. AU data were anaiyzed using a two-way anaiysis of variance (ANTISENSE X
SKF) (ANTISENSE X REGION) with repeated measures on one tàctor (SU or
REGION). Where significant F values were foui, pIanned pairwise cornparisons were made using a Newman-Keuis test
3.1.3 Resulis
Vacuous chewing movementr: SKF 38393 (5 mgkg) ùiduced VCMs which were signincantiy attenuated by dopamine DIA receptor antisense (n=4) but remauied rmaffiected by either missense (n=4) or saline (n=4) treatment (Fi.iz= 71 S9, p < 0.000 1, SKF main effect; Fu = 7.73, p = 0.011 1, ANTISENSE main effect; F,-. ,, = 9.55, p =
0.006, SKF X ANTISENSE interaction effect) (Fig. 4).
Gmoming: SKF 38393 (5 mglkg) produced a significant hcrease in grooming which was atîenuated by dopamine DIAreceptor antisense but rernahed dected by either missense or saline treatment = 71.61, p < 0.0001, SKF main effect: FI, =
6.1 1, p = 0.02 1, MSENSEmain effect) (Fig. 5).
Other behiviours: SKF 38393 (5 mgkg) induced a significant sniffing response
= 27.08, p = 0.0006, SKF main effect) wbich was not significantly affected by either antisense, missense, or saiine treatment (F,, = 0.49, p = 0.6303, ANTISENSE main effect) (Table 2). Neither rearing (F,*,z= 1.65, p = 0.23 14, SKF main effect; F2*9=
0.83, p = 0.4686, ANTISENSE main effect), aor locomotion = 1.3 1, p = 0.282 1,
SKF main effect; Fu = 0.3 1, p = 0.7439, ANTISENSE main effect) were afiected by any of the treatments.
Dopamine D, receptor binding: Dopamine D, receptor binding, as assessed using ['HJscH 23390, was significantly reduced in the ventrolateral and donolaterai striatum of those animals treated with dopamine DI, receptor antisense, but no significant difference in binding was seen foilowing either missense or saline treatments (F2,=
7020, p < 0.000 1, ANTISENSE main effect; FIJ6= 37.26, p < 0.0001, REGION main effect; FSJ6= 36.39, p c 0.000 1, ANTISENSE X REGION interaction effect) (Fig. 63.
Dopamine D2 receptor binding: Dopamine 4 receptor bbding, as assessed using
[3H]rac1oPride, was not affected by either antisense, missense, or saline treatment (F, =
0.87, p = 0.45, ANTISENSE main effect) (Fig. 8,9). Some regionai variation in Dt receptor binding was detected (F3,36= I0.86,p c 0.0001, REGION main effect). but Figure 4
Efbcts of Intrasttiatal Infusion of Oligonucleotide Antisense to Dopamine DIA Receptor mRNA on SKF 38393-lnduced Mouth Movements
RSisaline SKF38393
w saline missense an tisense
SKF 38393-htduced vacuous chmitcg nrovements- SKF 38393 (5 mglkg) induced a tignificant vacuous chewing response which was tignificantly attenuated by mtnstriata1 infiision of 01igonucIeotidt antisense to dopamine DIAreceptor mRNA (7 nmoVday, 3 days) but remained unaffected by either saline or missense treatment. Each bar represents the mean (+ S.E,M,) (n=4) duration (s) scored over 10 bIocks of 3 min. ** sig. diff. fkom acute saline injection, p < 0.00 t ## sig. diff. fiom mtrastriatal saIine intùsion, p c 0.00 1 Figure 5
Effects of lntrastriatal Inhision of Oligonucleotide Antisense to Dopamine DIA Receptor mRNA on SKF 383934nduced Grooming
RSl saline DSKF 38393
saline antisense
SKF 38393-induced groonirirg. SKF 38393 (5 rnglkg) induced a significant groorning response which was significantiy attenuated by mtrastnatat infusion of oligonucIeotide antisense to dopamme DtAreceptor mRNA (7 nmovday, 3 days) but remained unaffected by either salirie or missense treatment. Each bar represents the mean (k S-E-M-) (n=4) duration (s) scored over 10 b1ocks of 3 mm, * * sig, diff. Eom acute saline injection, p < 0.00 1; * p c 0.0 1 # sig. diff. 60m mtfastnfaStnatatsaime infision, p c 0.0 1 Table 2
Saline Milrsense Anthense Locomotion 9-75 593 8.48 f1.89 17.85 k7.82 Reariag 1.20 *O-72 1.3 fi055 632 fi.59 Snifflng 89.53 k48.33 84.27 S0.8 1 93.86 a4.3L
38393 Salrne Mksense Antisense Locomotion 21-01 fi.93 1623 s.05 17-85 r12.67 Rearina 42-16 fi4.53 31-94 f10.f6 41.34 k13.42
Effects of olr'gonucieotide antikense to dopamine DIAreceptor ntRNA on SgF 38393-induced behaviouts other than vacuous chewing movewtents and grooming. SKF 38393 (5 mgkg) did not significantly affect locomotion or rearing but did signifcantly elevate sniffmg. However, noue of these behaviours was signficmtly affécted by saiine, missense, or antisense to dopamine DIAreceptor mRNA (7 nrnoUday, 3
&YS). Each number represents the mean (fS.E.M.) (rH) duration (s) scored over 10 blocks of 3 min.
* sig. diff. ftom acute saline injection, p c 0.0 t Figure 6
Dopamine D, Receptor Binding in the Striatum Following lntrastriatal lnhrsion of an Oligonucleoüde Antisense to Dopamine DfAReceptor mRNA
0saline misense =antisense
D ensitansetrfe nt easurent ent of autondiagraphs representiitg ~H]-SCIZ 233 90 binding in the striatuni. Dopamine DI receptor binding was reduced in the dorsotateral and ventroQtera1 regions of the striatum fo llowing intrastriatal infusion of O ligonucleotide antisense to dopamine Dt~receptor mRNA (7 nmoüday, 3 days) but was unaffected by either saline or missense treatment. Each bar rcpresems the mean (k S.E.M.) (n=3) opticaI density (nCi/mg). (DM = dorsornedial, DL = dorsolateral, VL = ventrotateral, VM = ventromediai) ** sig diff from intrastriatal saline infirsion, p < 0.00 t Figure 7
Autoradiograph representing &kH2!€ 23390 bhding. Autotodioiogph of [~H]SCH23390 bindmg in a section taken through the striatum of an animal treated with bilaterai uitrasûiatal oligonucleotide antisense to dopamine D ,, receptor mRNA (7 nrnoilday, 3 days). Figure 8
Dopamine D2 Receptor Binding in the Striatum Following lntrastriatal lnfusion of an Oligonucleotide Antisense to Dopamine DIA Receptor mRNA
0saline 5 misense antisense
D ensirom etrie nr easurement of aotoradiographs represenrlng f~j-R adopride binding in the striarum. Dopamine Dz reccptor binding was not sîgnificantly affected by eithcr saline, missense. or antisense to dopamine DIA receptor mRNA (7 nmoyday, 3days). Each bar rcprescnts the mean (î S.E.M.) (n=4) opticd density (nCi/mg). (DM = dorsarnedial, DL = dorsolatcral, VL = ventrolateral, VM = ventromedid) Figure 9
A utoradiogrizph representittg ~~-racloPridebinding. Autoradiograpti of ['HI- radopride bindhg in a section taken through the striatum of an ahai treated with biIateral intrastnatal ~Iigonucleotideantisense to dopamine DIA receptor mEWA (7 nrnoVday, 3 days). conservative post hoc tests reveded only scattered differaices. A one-way analysis of variance (REGION) reveaied significantly higher dopamine D, receptor binding, overall, in the laterd regions of the stria- (F336= 11.74, p c 0.0001).
3.1.4 Summary
The data presented here, confi the role of the dopanine DIA receptor in mediating SW 38393-induced VCMs and grooming. intrastriatal infusion of oligonucleotide antisense to dopamine DIAreceptor mRNA was able to reduce dopamine
D, receptor buidhg without aflecting dopamine D2 receptor binding, and this selective reduction was accompanied by a significant attenuation of D, agonist-induced VCMs and grooming. While the possible hvo~vementof other putative dopamine DI receptor subtypes in the expression of these behaviours cannot be completeiy mled out. the
importance of the DI, receptor remains cIem
These hdings rnay have important implications for the study of tardive dyskinesia The VCMs syndrome induced acutely by dopamine DIreceptor agonists, is similar to that seen following chronic neuroleptic treatrnent, and is O ften used as a rodent
mode1 of TD. Thus, understanding the mechanism of dopamine D, agonist-induced
VCMs rnay help hrther our understanding of TD. 3.2.Effects of Oiigonucleotide Antisense to Dopamine DIA Receptor
mRNA on Chronic Neuroleptic-Induced VCMs
3.2.1 Introduction
Tardive dyskinesia is characterized by abnormal involuntary movements which develop as a side-effect of long-tem treatment with neuroleptics (Jeste & Wyatt, 1979;
Klawans & Rubovits, 1972). While TD has been traditionally associated with biochemical and behavioural supeaensitivity of dopamine D2receptors (Burt et al., 1977;
Kane & Smith, 1982), poor temporal and spatial correlation between the development of supmeusitivity and TD (Fibiger & LIoyd, 1984) suggests that other factors may be involved. It has been suggested that these dyskinesias may involve a shifi in the balance between the direct and indirect striatal output pathways towards heightened dopamine DI receptor activation (Ellison et ai., 1988; Lublin & Gerlach, 1988; Rosengarten et al..
1983).
When rats are treated chronically with neuroleptics, they deveIop a syndrome of vacuous chewing movements, which shares many characteristics typical of TD (Glenthoj,
1995). These VCMs are thought to involve relatively heightened activity at dopamine DI receptors, and can be readiIy triggered by acute injection of a selective dopamine D, agonist (Rosengarten et ai., 1983). As mentioned, not aU D, agonists eticit this respoose,
Ieading some to suggest the involvement of other (not DIAor DIB),as yet unidentified. subtypes of the dopamine Dtreceptor (Deveney & Wadduigtoa, 1997).
However, based on the data just presented, it appears that VCMs mduced by the dopamine D, agonist SKF 38393 are, indeed, mediated by the dopamine DIAreceptor- In the study descnbed here, 1 use in vivo oligonucleotide antisense to dopamine DIAreceptor mRNA to examine the role of the dopamine DIAreceptor in chronic neuroleptic-induced
Vas, a rodent mode1 of TD.
3.2.2 Design
Animais (N-30) were hjected with either fluphenazine decanoate (25 mgkg, im.) or its vehicle sesame oil every the weeks for a totaI of 18 weeks. Animais were then anaestheîized using haIothane and bilaterai cannulae were implanted stereotaxically into the ventrolateral striahm (AP i0.50, ML f2.50, DV -6.201, an area thought to mediate VCMs (Fletcher & Starr, 1987). Attached to the cannulae, by polyethyiene tubing, were osmotic minipumps (mode1 1003D; ha, CA) which delivered either dopamine DIAreceptor antisense (5'-AGG-AGC-CAT-CTT-CCA-GA-3'),missense (5'-
CGC-GGT-AAT-CCA-GAT-CA-3'),or saline at a rate of 1pIh.r or 7nmoVday for 3 days. FoIIowing antisense infusion, animais were habituated to the test chamber for at least two houn and then observed for one hour. Animais were decapitated and the brains removed and flash hzen. Once the brains were sliced, sections were tested for dopamine
DIand D2 receptor bùiding. Behavioural data were anaiyzed using a two-way analyàs of variance (FLUPHENAZINE X ANTISENSE), while binding data were andyzed ushg a the-way anaiysis of variance (FLWHENAZINE X ANTISENSE X REGION) with repeated measures on one factor (REGION). Where significant F vdues were foud, planned pauwise cornparisons were made using a Newman-KeuIs test 3.23 Resufts
Vacuous chewing movements: Chronic treatment with fluphenazine (25 m@g/3 weeks, im.; 18 weeks) resulted m enhanced spontaneous VCMs (Fi, = 7593, p <
0.0001. IKUPHENAZINE main eEect) (Fig. IO), which were signüïcantiy attenuated lollowing intrastriatd infusion of dopamine DIAreceptor antisense (F2j4= 12.51, p =
0.0002, ANTEENSE main effect; Fu = 20.13, p < 0.0001, EXUPHENAZNE X
ANTISENSE interaction effect), but remallied unaffected by either saline or missense treatment .
Other behavioun: Grooming (F,, = 0.13, p = 0.7178, FLWHENAZINE main effect; F2j4= 0.21, p = 0.8146, ANTISENSE main effect), snifing (F2, = 0.03, p =
0.8754, EXUPHENAZINE main effect; FZs4= 0.17. p = 0.8451, ANnSENSE main effect), rearing (Fz, = 0.32, p = 0.5778, FLUPHENAZINE main effect; FI,, = 0.27, p =
0.7689, ANTISENSE main effect), and locomotion (F:, = 0.58, p = 0.4541,
FLUPHENAZINE main effect; FU, = 0.60, p = 0.5575, ANTTSENSE main effect) were not aEected by any of the treatments (Table 3).
Dopamine D, receptor binding: Dopamine DI receptor binding, as assessed using ['H]SCH 23390, was significantiy attenuated in the ventrolateral, dorsolateral, and ventromedial stn'atum of those animais treated with inhatai infusion of dopamine D,, receptor antisense (F2j4= 97.20, p c 0.0001, ANnSENSE main effect; FI$()= 11 - 19, p <
0.0001, REGION main effect; Fa, = 2221, p c 0.0001, ANTISENSE X REGION interaction effect) but remallied unaffected by either missense or saline treatment (Fig.
11). Chronic fluphenazine did not affect striatd dopamine Dlreceptor bindllig (FI, =
0.81,~= 0.3776, FLUPHENAZINE main effect). Figure 10
Chronic Fluphenazine-lnduced Mouth Movements Follow ing lntrastriatal Infusion of an Oligonucleotide Anüsense to Dopamine DIA Receptor mRNA
vehicle
" saline missense antisense
Treatment
Chrorricffuphertazine-induced vacuous chewing movements. Chronic fluphenazint (25 mg/kg/3 weeks, 18 weeks) signiRcant1y enhanced spontaneous vacuous chew ing movements which were significantty attenuated by intrastriatai infusion of 01igonucIeotide antisenst to dopamine DIA receptor mRNA (7 nmoYday, 3 days) but remained unaffected by either saline or missense matment, Each bar represents the mean (t SEM,) (n=5) duration (s) scored over IO blocks of 3 mm. ** sig. diff. from chronic vehicIe treatment, p c 0.00 1 ## sig, diff. from mtrastriatal saline infiision, p c 0.001 Saline Mikense Antikense G~OO~UE 44.1 5 U3.24 35.81 219.09 52.08 528.62 Locomotion 4-75 k4.75 3.76 I232 17.80 k11.86 Rearina 3-99 kû-88 3.49 13.03 4.53 fi.11 Sniffine 286.07 f84.66 22852 f37.07 273.30 fi5.26
- -- -- e Saline Missense Antkense Groomine 50.82 ~8-62 43.72 S8.50 60.6 ~9.73 Locomotion 6.97 *6.58 4.43 e.02 3.43 s.43 Rearing 6.01 k4.55 4.93 t1.42 5-18 tta Sdffhg 245.20 kS5.18 276.50 k74.98 2 17.74 240.66
Egects of oiiganucleotide antkense to dopamine&, receptor on befiaviours other tfiarr vacuous chewing movements fuflowifig chronicflupfienazine Chronic fluphenazine treatment (25 rng/kg/3 weeks.
18 weeks) did not significantly affect spontaneous grooming, Iocomotion, rearhg, or miffig. As well, these behaviours were not affected by intrastriatal &ion of saline, missense or oligonucleotide aatisense to dopamine DIAreceptor mRNA (7 nrnoüday, 3 days). Each number reptesents the mean ($rS.E.M.) (n=j) duration (s) scored over 10 blocks of 3 min. Figure 11
Dopamine DqReceptor Binding Following Infrastriahl Infusion of an Oligonucleonide Antisense to Dopamine DIA RBcepfor mRNA in Rats Treated Chronically Wtf~ Fluphenazine
Osaline misense Vehicle antisense
Fluphenazine
Striatal Region
Densitom& tneusutement of autoradiograpks representing ~HI~CH23390 Ohtdhg in the sm'a~unr. Dopamine Dl receptor binding was significantIy reduced m the dorsolaterd, ventrotaterai, and ventromedial regions of the &atum following meastriata1 hfusion of oligonucleotide antisense to dopamine DIArecptor mRNA (7 nrnouday, 3 days) m both vehicIe- and fluphmazoie-treated animais. Dopamine DEreceptor bmding was unaffected by either saline or missense treatment- Each bar represmts the mean (&
SEM)(IFS) optical density (nCümg). (DM = dorsornediai, DL = dorsolateraL VL = ventrolaterai, VM = vemtromediai) ** sig. di& fiom Ïn-atal sahe infision, p < 0.00 I ;* p c 0.0 1 Dopamine 4 receptor bbding: Dopamine D, receptor binduig, as assessed using [%Jraclopride,was not significantiy affected by intrastriatal infusion of dopamine
D ,, receptor antisense, missense, or saline (Fu4 = 2.36, p = O. 1163, ANTISENSE main effect) treatment (Fig. 12). Chronic fluphenazine did not significantly elevate dopamine
4 receptor bindmg m the striatum (FIJ4= 1.53, p = 0.2286, EXUPHENAZINE main effect). Regionai ciifferences in dopamine D2 receptor binding were evident (FJvgO=
3 I -42, p c 0.000 1, REGION main effect).
Dopamine D3 receptor binding: Dopamine D, receptor bindmg, as assessed using [w7-OH-DPAT, was not significantly af'Eected by any of the treatments used (FI
= 1.37, p = 0.2536, FLUPHENAZWE main effect; FZt4 = 0.84, p = 0.4444,
MSENSEmain effect) (Fig. 13).
3.2.4 Summury
intrastriatai infusion of oügonucIeotide antisense to dopamine DIA receptor mRNA signifcantiy and selectively reduced dopamine D, receptor binding. This reduction in binding was accompauied by attenuation of chronic fluphenazine-induced
VCMs, a rodent mode1 of TD. These data suggest that the dopamine DIAreceptor plays a critical role in the expression ofchronic neuroleptic-hduced VCMs in a rodent mode1 of TD- Figure 12
Dopamine D2Receptor Binding Follow ing IntraaiistalInfusion of an Oltgonucleotide Antisense to Dopamine DfAReceptor mRNA in Rats Treated Chronically WRh Fluphenazfne
Veh ide
Fluphenazine
Densitonr etric aeasurenr ent of au forodiagruphs repruurtfirg f~/-racto~ridebinding in the striatum. Dopamine D? receptor binding was not affectcd by satine, missense. or arrtistnse to dopamine DrAreceptor mRNA (7 nmollday, 7 days) in either vehicfe- or fluphenaziue-treatcd animais. Each bar ccprcscnts the mean (k S.E.M.) (n=5) optical density (nCilmg)- (DM = dorsorncdiai, DL = dorsdateral, Vt = vcntroInteral, VM = ventramedial) Figure 13
Dopamine D3 Receptor Bhding Folkw hg IntrastriataI lnfuslon of an Olt(lonucieotide Antisense to Dopamine DIA ReceptOr mRNA in Rats Treated Chronicaliy With Fluphenazine
Densirometrie measurentent of autoradiographs representiisg HI-7-OH-DPAT biirdng in the striiot~~nt.Striata1 dopamine D3 receptor binding was not significantIy affected by either chronic fiuphenazine treament or intrastnata1 infusion of oligonucIeotide antisensc to dopamine Dr, receptor mRNA (7 nrnopday, 3 days). Each bar represents the mean (* S.E.M.) (n=5) optical density (nCi/mg). (DM = dorsornedial, DL = dorsolateral, VL = ventrolateral, VM = ventrornedial) 3.3 Effects of Oiigonucleotide Antisense to Dopamine DIAReceptor mRNA on Sensitization of Apomorphine-Induced Rotations by Chronic
Levodopa in Hemiparkinsonian Rats
3.3.1 Introductiin
Treatment with Ievodopa remains the most effective therapy for Parkinson's disease (KolIer & Hubble, 1990). However, its long-term use is often associated with the induction of various motor complications including dyskinesias (Barbeau, 1980). Whik the pathophysiology underlying this complication remahs unclear, it has been suggested that heightened activity of dopamine DIreceptor-bearing hatonigral neurons rnay play a key role (Crossman, 1990; DeLong, 1990). It has also been proposed that a similar neural mechanisrn rnay underly various forrns of dyskinesia including both TD and LID
(Crossman, IWO). Thus, the importance OFthe dopamine DIAreccptor in the expression of behaviours m a rodent model of TD, discussed earlier, fuaher highlights the potential role of this receptor in LID. In the study descnbed here, I examine the effects of in vivo oligonudeotide antisense to dopamine DIA receptor mRNA on sensitization of apomorphine-induced rotations by chroaic pulsatile levodopa in hemparkinsonian rats, a rodent model of LID.
3.3.2 Desi&
Anmiah (N=46) received daterai Iesions of the Ieft media1 forebrain bundle by infusion of the neurotoxin 6-hydrolcydopamine (8 pg/4pI). FoUowing two weeks of recovery, animais were injected twice daiIy with either benserazide (10 m@g, i-p.) done, or a combination of benserazide and levodopa methyl ester HCI (50 mgkg, i.pJ for three weeks. Upon termination of Ievodopa treatment, osmotic minipumps (mode1 1OO3D;
&a, Califo~a)were surgically implanted with attached cannula positioned to target the left striatum (AP +0.50, ML +2.50, DV -6.20) in an area thought to be involved in dopamine Dlreceptor-mediated rotational responses (Fletcher & Starr, 1987). Each purnp detivered either an antisense oligonucleotide complementary to mRNA encoding the initiation site of the rat dopamine DIAreceptor (5'-AGG-AGC-CAT-CTCCA-GA-3')
(7 nmouday), a scrambled missense sequence (5'-CGC-GGT-AAT-CCA-GAT-CA-3')' or saline for 3 days. Animds were then tested for apomorphine (0.3 mgkg, s.c.)-induced rotations. Six hours foIIowing testing, brains were removed and rapidly fiozen. Once the brains were sliced, sections were tested for dopamine D, and D2receptor binding, as well as DAT binding. Behaviourai data were analyzed using a two-way anaiysis of variance
(LEVODOPA X ANTISENSE), while binding data were andyzed ushg a three-way dysis of variance (LEVODOPA X ANTISENSE X HEMISPHERE), with repeated measures on one factor (HEMISPHERE). Where significant F values were round, planned pairwise cornparisons were made using a Newman-Keds test.
3.3.3 Resufis
Rotational behaviour: Apomorphine (0.3 mg/kg, s.c.) Uiduced a robust
contrdaterai rotationai response which was signincantly greater m those animais treated
chronicaLiy with Ievodopa (50 mgkg, i.p.; twice daiIy for 3 wks) (F146= 11 1-36,p c
0.0001; LEVODOPA main effect) (Fig. 14). The potdation of apomorphine-induced Figure 14
Effects of lntrastriatal Infusion of Oligonucleotide Antisense to Dopamine DIA Receptor mRNA on Apomorphine4nduced Rotations in a Rodent Model of LID
ve hicle Idopa
saline misense antisense
Treatment
Apomorpkine-induced conha~aterafrotations. Chronic Ievodopa (2X 50 mg/kg/day, t 1 days) hduced a sensituation of apomorphine (0.3 mg/kg)-induced rotations, This sensitization was significant Iy attenuated fo IIowing intrastriatal in fusion of oligonudeotide antisense to dopamine D receptor &NA (7 nmo Vday, 3 days) but was unaffected by either salme or missense treatment, Each bar represents the mean (+ S.E.M.) (~10)number ofcontralateral rotations recorded over a 1 hour period. ## sig. diff fiom chronic vehick treatment, p < 0.00 t ** sig. diff. fiom nitrastriata1 saline mfusion, p c 0.00 1 rotation by chronic levodopa was significantly attenuated by dopamine DlA receptor antkense (neIo) (F~46= 8-03, p = 0.001; ANTISEbJSE main effect) but remallied unaf5ected by either missense or saline treatment (F2,46= 3.92, p = 0.0268; LEVODOPA
X ANTISENSE interaction effect). Apomorphhe-induced rotations in chronic vehicle- treated animals remained unaffected by either saline, missense or antisense treatment.
Dopamine transporter binding: The efficacy of 6-hydroxydopamhe lesions was assessed by dopamine transporter binding using ['HIWIN35,428. Only those animak with greater than 80 % reduction in DAT binding in the Ieft striatum were included in the study.
Dopamine D, receptor binding: Dopamine DI receptor binding, as assessed using ['H]SCH 23390, was significantly reduced (41 %) in the left striahm of those animds treated with dopamine DIA receptor antisense (Fig. 15,I6) but no significant difference in binding was seen following either mksense or saline treatments (Fins =
28.83, p < 0.0001; KEMISPHERE main effect; F242 = 24.98, p c 0.0001, ANTEENSE main effect; F&48 = 34.92, p c 0.0001, HEMISPHERE X ANTISENSE interaction effect). Dopamine DIA receptor antisense significantiy reduced Dl receptor binduig in both vehicle- and Ievodopa-treated animais wÏth no dinerences between these two groups
(Fi 42 = 1.99, p = 0.1657; LEVOWPA main effect).
Dopamine 4 receptor binding: Dopamine D2 receptor binduig, as assessed with
['~ra~lo~ride,was not affected by either saline, missense, or antisense treatment (F2,42
= 0.09.p = 0.9 119, ANnSENSE main effect; FI ,48 = 0.95, p = 03365, HEMISPHERE Figure 15
Striatal Dopamine Dl Receptor Binding Following Unilateral Intrastriatal Infusion of Oligonucleotide Antisense to Dopamine DIA Receptor mRNA in a Rodent Model of LID
-- Y saline missense anüsense
saline missense antisense
Treatment
Densitometric M easuremenr of anturadiographs representing 'H- 23390 bindi~tg rir the striatum. Dopamine DLreceptor binding was reduced in the lefi stnaturn of both chronic vehicle- and Ievodopa-treated anünals fotlowmg in-atal infüsion of oligonucleotide antisense CO dopamine DIA receptor mRNA (7 nmoilday, 3 days) but was unaffected by either saline or missense treatrnent. Each bar represents the mean (+ S.E.M.) (n= t 0) optical densky (nCi/rng). ** sis diE fiom saline treatmerrt, p c 0.00 1 Figure 16
Autoradiograph represen ting ~"H]~cH23390 bindiiig. Autoradiograp h of
[3~-~~~23390 binding in a section taken through the saiatum foilowing unilaterd infusion of oligonucleotide antisense to dopamine DIA receptor mRNA (7 nmoUday, 3 days) into the left striatum. main effect; 448= 0.27, p = 0.7655, ANnSENSE X HEMISPHERE interaction effect)
(Fig. 17,18). Dopamine D2 receptor binding in the left striaturn was srightIy, but significantly, reduced following chronic levodopa compared to that seen following vehicle treatment (F1,42= 0.37, p = 0.5482, LEVODOPA main eEect; F2.48 = 4.26, p =
0.0452, LEVODOPA X HEMISPHERE interaction effect). However. post-hoc tests fàiled to detect significant differences.
Dopamine 4 receptor binding: Dopamine Dj receptor binding, as assessed using [%J~-oH-DPAT, was signincantiy elevated in the leA hernisphere following chronic levodopa (Fi,is= 12.66, p = 0.0023, LEVODOPA main eflect; FI,, = 6.90. p =
0.0171, HEMISPHERE main effect; FIl4 = 29.3 1, p < 0.0001, LEVODOPA X
HEMISPHERE interaction effect) and this elevation in binding was significantiy reduced to control levels following antisense treatment (F2,is= 3.73, p = 0.0441, ANTTSENSE main effect; F,, = 5.97, p = 0.0103, ANnSENSE X KEMISPHERE interaction effect;
F2, = 3.99, p = 0.0369, LEVODOPA X ANnSENSE X HEMISPHERE interaction effect) (Fig. 19).
3.34 Sumrnary
intrastriatai infusion of oligonucleotide antisense to dopamine DIA receptor mRNA effectively and selectively reduced dopamine D, receptor binding. msreduction in btnding was accompanied by attenuation of chronic pulsatile Ievodopa-induced behaviourai sefzsitization of the rotational respoase to apomorphine. Thus, the results reported here, suggest that the dopamine DIA receptor plays a critical roIe m the expression of behaviod sensitization in Uiis rodent mode1 of LID. Further investigation Figure 17
Striatal Dopamine D2 Receptor Binding Followhg Unilateral lntmrtriatal Infusion of Oligonucfeotide Antisense to Dopamine DfAReceptor mRNA in a Rodent Mode1 of CID
-.- saline missense anüsense
Densitom& measurement of artoradiograpAs represe~ting['HI-ritctoeridc binding iir the striatum. Dopamine Dz rcceptor binding was not affected by saline, missense, or antisense to dopamine D 1A rectptor mRNA (7 nrnollday, 7 days) in eithet vehicle- or Ievodopa-treated animals- Each bar represents the mean (f S.E.M.) (F IO) optical density (ncilmg). Arrioradiogruph representing t&hdopride binding. Autoradiograph of [JH]- radopride binding m a section taken through the striatum following unilateral infusion of oligonucleotide antisense to dopamine DIA receptor MA(7 moUday, 3 days) into the Ieft striatum. Figure 19 Striatal Dopamine DJReceptor Binding Following lntrastriatal Infusion of Oligonudeotide Antlsense to Dopamine DfA Receptor mRNA in a Rodent Model of LID
Levodopa ** T
saline
üensitometri~ neusurcrnent of avmrudCo~raphs rcprescnthg ~H~-~-oH-DPAT binding Ln rlte srrianrnr, Suiatat dopamine D, receptor binding was signifiantly devatcd ipsiIatcral to the 6-hydroxydopamine lesion following chmnic revodopa matmcnt (2 .Y 50 mglkglday, 21 days). This elcvation in D, binding was reduccd to convol levers following in-ara1 infÿsion of oligonuctcotidc antiscnse to dopamine DIA ccceptor mRNA (7 nmollday, 3 days). Dopamine D, receptor binding cemained unat'cted fottowing saline or missense marnent in both vchicfe- and Ievodopa-tmated animais. Each bar reprcscnu the mcan (k S.E.M.) (II=%optical dcnsity (nCi/mg). ** sig. diff, from right hemisphcre. p c 0.00 L f#C sig. diff from intnstnatal saline infusion, p -= 0.00 t of how the dopamine DIreceptor may contriiute to the development of LID in humans may have important implications for the treatment of PD. 3.4 Effects of OlïgonucIeotide Antisense to Dopamine D, Receptor
mRNA on Chronic Levodopa-Induced Behaviouraï Sensitization
3.4.1 Introduction
The dopamine D3receptor was discovered less than a decade ago, and much is yet to be Iearned about its functional significance. However, study of the D, receptor is made difncdt by its extremely low level of expression (Sokoloff et ai., 1992) and the absence of appropriately selective Iigands (De Boer et ai., 1998). Thus, the fiuiction of the dopamine
D3 receptor mains speculative. This receptor has been proposed to play a role in
Iocomotor activity (Accili et al., 1996; Ekman et al., 1998; Kling-Petersen et ai., 1994), reward (Chaperon & Thiebot, 1996), and deveIopment (Demotes-Mainard et al.. 1996).
Recently, the dopamine D3 receptor has been proposed to play a role in a rodent mode1 of
levodopa-uiduced dyskinesias. Elevations in D, receptor MAand binding were seen in
the denervated striaturn of hemiparkmsonian rats treated chronically with ievodopa (Bordet et ai., 1997).
Further investigation of the involvement of dopamine D3 receptors in LID is
hampered by the lack of seIective ligands. Thus, mterpretation of pharmacologicai studies is
di&cuIt, as the D2Qseiectivity of many drugs varies coosiderably, with none being huIy
selective for dopamine D3receptors. This is emphasized by a ment study using dopamme
D, receptor mutant mice. niese animais exhiited ÎdenticaI Iocomotor responses to
putative D, receptor-seIective agonists as their dd-type cornterparts (Tremblay et al,
1997). Whife icnockout mice provide a level of serectivity which may be lachg in
traditionai pharmacoIogicaI approaches, compensatory adaptatiom must be considaed in the hterpretation of these studies, particuiarly in Light of the important role the dopamine D3 receptor appears to play in neuronal development. Such knockouts aiso Iack matornicd specincity, mahg interpretation ciBicuit. Thus, in v&o antisense may provide an ideal alternative. In the study desmied here, I use in vivo oiignucleotide antisense to dopamine
D3receptor dWAto clan@ the role of the dopamine D, receptor in a rodent mode1 of LID.
3.4.2 Design
Animais (N=42) received unilateral lesions of the left mediai forebrain bundle by infusion of the neurotoxin 6-hydroxydopamine (8 microg/4microI). Following two weeks of recovery, animais were injected twice daily with either benserazide (10 mg/kg, i.p.) alone, or a combination of bensemide and levodopa methyl ester HCI (50 mgkg, i.p.) for three weeks. Two days prior to the termination of levodopa treatment, osmotic minipumps
(mode1 2001; &a, California) were nugicaily irnpIanted with the attached cannula positioned to target the Ieft &atrmi (AP +1.70, ML +090, DV -7.30). Each pmp deiivered either an antisense oiigonucleotide complernentary to mRNA encodïng the initiation site of the rat dopamine 4 receptor (5'-GCT-CAG-AGG-TGC-CAT-GGC-3')(7 nmokhy), a scmbled missense sequence (5'-AGC-CAG-AGT-GTC-GCG-CAT-3'),or saline for 5 days. Animafs were then tested for apomorphine (0.3 mgkg, s.c.)4nduced rotations. TweIve hours foIIowing testmg, brak were ranoved and rapidly kozen. Once brains were sliced, sections were tested for dopamine DI,Dz, and 4 recqtor biading as weiI as DAT bindmg, 3.4.3 Resufts
RotationaI behaviour: Apomorpbe (0.3 mgkg, s.c3 induced a robust contralateral rotational response which was signincantiy greater in those animals rreated chronically with Ievodopa (2 X 50 mgkg, i-p.; 3 weeks) (FIS = 37.06, p < 0.000 1;
LEVODOPA main effect) (Fig. 20). The potentiation of apomorphine-induced rotation b y chronic Ievodopa was significantly attenuated by dopamine D3receptor antisense (n=I 1) but remained unaffected by either mkmeor saline treatment (F2,J4= 3.41, p = 0.042 1,
AMISENSE main effect; FZvd6= 6.63, p = 0.0031, ANTiSENSE X LEVODOPA interaction effect).
Dopamine transporter binding: The efficacy of 6-hydroxydopamllie lesions was assessed by dopamine transporter binding using 3mWIN 35,428. AnimaIs with less than
80 % reduction in DAT binding in the left striatum were eliminated hmthe shidy. 01 the fi@ a.dsoriguially tested, 8 showed greater than 20% ninnvai accompaaied by
Iittle or no rotationai response to apomorpbe.
Dopamine D3 receptor binding: Dopamine D, receptor binding, as assessed using [3~7-~~-~~~~,was significandy elevated in the left striatum lollowuig chronic
Ievodopa (2 X 50 mgkg, i.p., 3 weeks) treittment (Frao= 7.72, p = 0.0083, LEVODOPA main effect; = 34.98, p < 0.0001, LEVODOPA X HEMISPHERE interaction effect)
(Fig. 21). This eIevation in D3 binding was sÏgaincantly attenuated by antisense but rernaiaed tmaffected by missense treatment (F2,*= 19.04, p < 0.0001, ANTISENSE X
HEMISPHERE interaction effect; Fm = 11 -79, p < 0.000 1, LEVODOPA X Figure 20
Effects of Intrastriatal Infusion of Oligonucleotide Antirense to Dopamine D3 Receptor mRNA on Apomorphine-lnduced Rotations in a Rodent Model of LID
.)
O* m~ehid8 levoda pa
Apomorpkine-induced contralateral rotations, Chronic Ievodopa (2 X 50 rng/kg/day, 2 1 days) induced a sensitization of apo morp hme (0.3 mg/kg)-mduced rotations. This sensitization was significantty attenuated folbwing intrastriatal infusion of ofigonucleotide antisense to dopamine Dg receptor mRNA (7 nmoVday, 5 days) but was unaffected by either satine or missense treatment. Each bar represents the mean (+- S.E.M.) (n=EO) number of contralateral rotations recorded over a 1 hour period. ** sig. diff. fiom chronic vehicle tceatrnent, p < 0.001 ## sig, diff, fiom mtrastriatat saline infirsion, p c 0.00 t Figure 21 SMatal Dopamine D3 Receptor Binding Following Intrastriatal Infusion of Oligonucleotfde Antirense to Dopamine D3 Receptor mRNA in a Rodent Model of LID
---- saline missense
Densitantrnic meeruninent of uutoritdiogruphs represenring PHI-7-0 K-DPA T brirdr'ng iir the stnatum. Striata1 dopamine D3 reccptor binding was significantly elevated ipsiiateral to the Iesion fotlowing chronic levodopa treatment (2 X 50 mg/kg/day, 21 days). This elevation in D3 bindhg was reduced to control leveis following intrastriataI intusion of oligonucleotide antisense to dopamine D3 receptor mRNA (7 nrnoVday, 5 days). Dopamine D3 receptor binding remamed unaffected foIIowmg saline or rnissense treamient in both vehicle- and Ievodopa-treattd anhals- Each bar represents the mean (+ S.E.M.) (n= 10) optical density (nCi/mg). ** sig. dE Eom rïght hemispherevp< 0.001 ## sig. dB. fiom mtrastriatal sahe infitsion, p < 0.00 1 ANTISENSE X HEMISPEIERE interaction esect). in chronic vehicle-treated animais, binding remained unafFected by either antisense, missense, or saline.
Dopamine D, receptor binding: Dopamine DI receptor binding? as assessed using [3Hl~~23390, was not signincantiy af3ected by any of the treatments (FI,, =
2.45, p = 0.1252, LEVODOPA main effect; F2,40= 0.29. p = 0.7535, ANTISENSE main effect; Fl,ro= 0.30, p = 0.5868, HEMISPHERE main effect) (Fig. 22).
Dopamine D, receptor binding: Dopamine D, receptor binding, as assessed using ['HJracloptide, was not afYected by either saline, missense, or antisense treatment
(Ft, = 0.08, p = 0.9268, ANTISENSE main effect; FI,, = 0.46, p = 0.4297.
KEMISPIfERE main effect; F2, = 1.60, p = 0.2155, ANTISENSE X HEMISPKERE interaction effect). However, dopamine D2 receptor binding in the Ieft stnatum was slightly, but significantiy, reduced bllowing chronic levodopa compared to that seen lollowing vehicle treatment = 0.64, p = 0.4297, LEVODOPA main effect; F,,, =
12.30,p = 0.001 1, LEVODOPA X HEMISPHERE interaction effect) (Fig. 23). However, post-hoc tests failed to detect significant differences.
3.4.3 Summary
Thus, intrastriatai infusion of oligonucleotide antisense to dopamine D3 receptor mRNA effectively reduced chronk Ievodopa-induced dopamine DI receptor binding in denervated striatum. This reduction in Dj receptor binduig was accompanied by reduced sensitization of apomorphine-induced rotations by chronic pdsatile fevodopa, suggesting that the dopamine D3receptor pIays a criticai rote în the expression of behaviod Figure 22 Sbiatal Dopamine Dl Receptor Bindlng Following Unilateral Intrastriatai Infuston of Oligonucleotide Antisense fo Dopamine D3 Receptor mRNA in a Rodent Model
Veh kle 307
" saline antisense Levodopa 307
saline antisense
De~sitontcirknteasurentent af autoradiographs represettting ~H~~CCH23190 bindhg in the strrirtunt, Dopamine Dlreceptor bhding was not significantly affected by saime, missense, or antisense ro dopamine Dg receptor mRNA (7 nmollday, 3days) in either vehicle- or Ievodopa-treated arrhals. Each bar represents the mean (f S.E.M.) (n= 10) opticat density (nCi/mg). Figure 23
Striatal Dopamine 4Receptor Binding Following Intrastriatal Infusion of ~ligonucleotideAnthense to Dopamine D3 Receptor mRNA in a Rodent Model of LID
W." saline missense antisense
Levodopa 20.07
saline missense antisense Tmatment
Densitonetric meosucement uf autortzdiogrugb representr'ng f~/-racfo~rêdebinding iir the striktrra Dopamine Dz receptor bmding was not significantly affected by saline, missense, or antïsense to dopamine D3 receptor mRNA (7 nmollday, 3dqs) in either vehicle- or Ievodopa-treated mimals. Each bar represcnts the rnearr (* S.E-M-) (n= 1O) opticai density (nCi/mg). sensitization in this rodent mode1 of LID. Further investigation into the possible invoIvement of dopamine D, receptors in human LID is certaidy warranted. 3.5 Effeets of OIigonucIeotide Antisense to Dopamine Transporter
mRNA on Locomotor Responses To Levodopa and Amphetamine
3.5.1 Introduction
The DAT is a plasma membrane protein Iocated on presyuaptic nerve tennuids of dopamine neurons and is responsiile for the termination of dopatninergic neuro~ssionthrough transmitter reuptake (Hitri et al., 1994, Kuhar, 1998). Thus, by regdahg the concentration of dopamine in the synapse, the DAT sets the tone for dopaminergic activity, and chronic alterations in DAT function couid play a significant rote in the development of dyskinesias. Undentanding how changes in the DAT could affect striatal function and subsequent behaviourai responses may shed some Iight on possible mechanisms involved in the pathogenesis of dmg-induced dys kinesias.
While various DAT Ligands do exist, their actions are varied and they ofien bind to other monoamine transporters or nonspecificaily to other brah proteins (Rudnick &
Wall, 1991) even though striatai dopamine uptake is almost solely attributed to DAT
(Gùos et al., 1996). Furthemore, repeated use of such agents may be associated with regdatory attenuation. A more selective approach rnay be provided by transgenic knockout of the DAT in mice. Such studies have hdicated an important role of the DAT m regdating gene expression and striatd hction (Guos et ai., 1996). However, while such hockouts do aIIow a more specinc targeting of the DAT without many of the complications often associated with pharmacological interventions, there are concems regarding possibie compensatory changes which may occur during development in these animais. In fact, a substantid percentage of mice Iacking the DAT die before adulthood (Bezard et al., I999), suggesting that these animais may be compromised in some other way. ThusYrestricting experiments to surviving animais may create a biased sample. As weU, this approach reçults in the global absence of DAT expression, rather than selectively targeting the region of interest. Thus, the molecular and matornicd specificity characteristic of oligonucIeotide antisense may provide an effective akemative to pharmacoIogicd or transgenic approaches. in the study desded here, I examined the effects of in vivo antisense to dopamine transporter mRNA on behaviourai responses to dopaminomimetic dnigs.
3.5.2 Design
Animais (N=48)were anaesthetized uing ketamine and a unilateral cannula was implanted stereotaxicalIy into the left substantia nigra pars compacta (Ai? -5.30, ML
+2.20, DV -7.80). the primary site of striata1 DAT production (Hitri et al., 1994).
Attached to the cannula, by poiyethylene tubmg, was an osmotic minipump (mode1 100 L ;
Aiza, California) which delivered either oligonucleotide antisense complementary to mRNA encoding the initiation site of the rat dopamine transporter protein (5" AGA-TTC-
AGT-GGA-TCC-AT-)'), a scrambled missense sequence (5'-AGC-ATT-GM-CU-
GCC-ATJ'), or saiine at a rate of 1 pI/hr or I nrnoVday for 7 days. Following antisense infiision, animds were injected with either methylamphetamine HCI (2 rng/kgy s.c.), a combination of bensenide HCI (15 mgkg, i.p.) and Ievodopa methyl ester (50 mglkg, i-p.), or their vehick (0.9% saInie) and tested for rotation. Twelve hours Iater, animais were decapitated and the braius removed and flash frozen. Once the brains were sliced? sections were tested for dopamine transporter and nemonai vesicular monoamine transporter binding- Data were anaIyzed using a two-way analysis of variance
[(ANTISENSE X TREATMENT) or (ANTISENSE X HEMISPHERE)] one factor
(TREATMENT or HEMISPHERE). Where significant F vaiues were found, planned pairwise cornparisons were made ushg a Newman-Keds test.
3.5.3 Results
Dopamine transporter binding: Dopamine transporter binding, as assessed using [3Hl~35-428, was significantiy reduced (59%) in the stria- ipsiiateral to antisense infusion compared to the mtreated side, while no significant difference was seen in either saIine- or missense-treated controls (FlJ4= 35.33, p < 0.0001.
HEMISPHERE main effect; F,,, = L 7.57, p c 0.000 1, AN'MSENSE main effect; F,, =
19.74, p < 0.0001, HEMISPHERE X ANTISENSE interaction effect) (Fig. 2425).
Binding was ais0 slightly, but significantly, lower in the right, untreated stnatum of antisense-treated animals compared to that of saline-treated controls.
Vesicular monoamine transporter binding: Neuronal vesicular monoamine transporter (VMAT2), as assessed using [~TBZ,was not significantly affected by any of the treatments described (FU,= 1-04,p = 0.366 1, ANnSENSE main effect; F,;,
= 2.84, p = 0.10, HEMISPHERE main effect) (Fig. 26).
Levodopa-hduced rotations: The combination of benserazide (15 mgkg) and levodopa (50 mgkg) hduced a significant rotational response, contralaterai to the side of intranÎgral infizsion, in antisense-treated animais, with no signincant effect in either saline- or missense-treated anmiais (F124= 5.33, p = 0.03 1, TREATMENT main effect;
Fu, = 5.54, p = 0.0 12, ANnSENSE main eEect; Fm = 6.0 1, p = O.OO9, TREATMENT Figure 24
Striatal Dopamine Transporter Binding Following Unilateral Infusion of Oligonucleotide Antisense to Dopamine Transporter mRNA
" saline antisense
Derrsittmetric measurement of autoradiographs representing ['HI- W~V35-428 binditrg rir the sîriotuns. Dopamine transporter binding was significantly reduced in the Ieft versus right striatuni folIowing unÎiateral mfasion of oiigonucIeotide antisense to DAT mRNA (1 nmaVday, 7 days) mto the Ieft substantia nigra, No nich ciifferencc was seen m either saline- or missense-treated controis. DAT bindmg was also siightIy, but significantly, Iower on the untreated side of antiscnse-treated animais compared to saline- treated controIs. Each bar rqresents the mean (f SEM.) (n=8) opticai density (nCi/rng). ** sig. dEfiom right hemisphere, p < 0.001 # sig. dEf?om intmnigrd saline infusion, p < 0 .O I Figure 25
Autoradiograph representing PHI-WZN 35,428 &&de. Autoradiograph of [3m-WIN 35-428 binding in a section taken through the stnatum folIowing unilateral infusion of oligonucleotide antisense to dopamine transporter mRNA ( I nmoi/day, 7 days) into the left substantia nigm Figure 26
Strfatal Vesicular Monoamine Transporter Binding Following Unilateml lntranigtal Infusion of Oligonucleotide Antisense to Dopamine Transporter mRNA
Densitout etrfc memurem ent of auzoroPIographs representhg ['HI-MT.6inding in the striatum. Vesicuiar monoamine transporter binding was not affected by intranigral infusion of either saline, missense, or antisense to DAT mRNA (I nmollday, 7 days). Each bar represents the mean (k SEM.) (~8)optical density (nCümg). X MSENSE interaction effect) (Fig. 27). No baseüne rotation was seen in any of the groups studied.
Amphetamine-induced rotations: Amphetamine (2 mgkg) induced a significant rotationd response, contralataal to the side of indgrai infision, in antisense-treated animals, with no significant effect in either saline- or missense-treated anirnals (F,, =
25.56, p < 0.000 1, TREATMENT main effect; Fu, = 9-80,p = 0.001, ANTISENSE main effect; FU, = 9.56, p = 0.001, TREATMENT X MSENSE interaction effect) (Fig.
28). No baseline rotation was seen in any of the groups studied.
3-54 Summury
Intranigral infiision of O tigonucleotide antisense to dopamine transporter mRNA effectively and selectively reduced dopamine transporter binding in ipsilateral striatum.
This antisense 'hockdown' of the DAT resuited in an asymmetricai Iocomotor response both to levodopa and to amphetamuie, seen as contralaterd rotations. The reduction in
DAT expression, by reducing dopamine reuptake, may have enhanced dopamine overflow, thereby increasing dopaminergic neurotransmission on the antisense-treated side. Thus, in vivo antisense is an effective means of studying the significance of the
DAT in basai gangIia fiinction. Figure 27
Effects of Unilateral lntranlgral lnfusion of Oligonucleotide Antisense to Dopamine Transporter mRNA on Locomotor Response ta Levodopa
missense antisense
Levodopa-induced contrataterai rotations* tcvodo pa (50 mgkg) mduced a significant rotational response directed away fiom the side of intranigrd infusion of oIigonucleotide antisense to DAT mRNA (1 nmoilday, 7 days), Levodopa faiied to elicit significant rotations in either sahe- or missense-treated controis. Each bar represents the mean (k S-E-M.) (n=6) number of contralateral rotations recorded over a one hour period, ** sig. diff- fiom ihtranigral saIme intùsion, p e 0.00 1 Figure 28
Effects of Unilateral lntranigral Infusion of Oligonucleotide Antisense to Dopamine Tansporter mRNA on Locomotor Response to Amphetamine
vehide lam phetamine
Treatment
Ampketanrhte-induced contralateral rotations. Amphetamme (2 mgkg) tnduced a significant rotational response directed away fiom the side of mtranigral infusion of oligonucteotide antisensc to DAT mRNA (1 nmoYday, 7 days). Amphetamine failed to eficit significant rotations m either sarme- or missmse-treatcd controts. Each bar represents the mean (k S.E.M.) (n=8) number of contraIatera1 rotations recorded over a one hour period. * * sig. diff, fiom mtranigral saline mfusion, p < 0.00 C 3.6 Effects of OligonucIeotide Antisense to Dopamine Transporter
mRNA on the Nearotoxicity of MPP~and 6-OHDA
3.6.1 Introduction
As well as regdahg synaptic dopamine, the DAT may contribute to the
pathogenesis of Parkinson's disease. Dopamine transporter expression correlates well
with susceptiibiiity to neuronal degeneration in I-methyl4phenyI- l,2,3,6-
tetrahydropyrkiine (MPTP)-induced parkinsonism. Recent studies have implicated the
DAT in the uptake of both this neurotoxin and its metabolite MPP' as welI as another experimental neuro toxin, 6-hydroxydopamine (Gainetdinov et ai., 1997; Giros & Caron,
1993; Kitayama et ai., 1998; Pifl et ai., 1993; Simantov et ai., 1996; Uhl, 1998).
Studies using transgenic DAT knockout mice have indicated an important role of the DAT in MPTP toxicity (Bezard et al., 1999; Gainetdinov et ai., 1997). However, the issue of possible compensatory changes makes interpretation of these hduigs difficult.
Furthermore, the neurodegeneration which occurs in Parkinson's disease appears after neuronal development is comptete. In this context, thetefore. embryonic interventions rnay be inappropriate. Here, in vivo oligonucleotide antisense to DAT mRNA, described earlier, provides an alternative means of studying the roIe of the DAT. In these shidies, I examine the effects of indgrd mfusion of oiigonucIeotide antisense to DAT mRNA on the neurotoxicity of MW' and 6-hydroxydopamhe- 3.6.2 Design
Anmiais (N=t 7) were anaesthetized using ketamine and a unilaterd cannula was impIanted stereotaxicdy hto the Ieft substantia nigra pars compacta (AP -5.30, ML
+2.20, DV -7.80). Attached to the cannula, by polyethytene tubing, was an osmotic minipump (mode1 2001; Alza, California) which delivered either DAT antisense (5'-
AGA-TTC-AGT-GGA-TCC-AT-3'). missense (5'-AGC-AIT-GAA-CAA-GCC-AT-3'), or saline at a rate of 1 pVhr or 1 nmoVday for 7 days. At that the, bilateral cannulae were aiso implanted into the Ieft and right striata (AP 4.20, ML +3.00, DV 4.00). FolIowing antisense infusion, animals received bilaterd infiision of either 6-hydroxydopamine hydrobromide (8 pg/4pI) or I -rnethyi-4-p heny1pyridiniu.m iodide (MPP') (8 pg/3 pl). Two weeks later, animals were tested for apomorphuie (0.3 mgkg, SC.)-induced rotations.
Twelve hours later, animais were decapitated and the brains removed and flash bzen.
Those braIns enposed to 6-hydroxydopamine were siiced and sections were tested for dopamine transporter and neuronal vesicuiar monoamine transporter binding. Those brains exposed to MPP' were dissected to remove striatd tissue, which was subsequentiy assayed for dopamine concentration by HPLC. Data were andyzed ushg a one-way analysis of variance with repeated measures.
DAT antisense and 6-hydro~ydoparninetoxicity
Dopamine transporter bhding: Dopamine transporter binding, as assessed ushg [m35-428, was significantiy (35%) Iower in the right versus left sniatum of those anirnals treated with unilateral DAT antisense in the Iefi SN, prior to bilateral
Vesicular monoamine transporter binding: Neuronal vesicular monoamine transporter binding, as assessed usuig [~JMTBZ,was significantly (57%) Iower in the nght versus Iefi striatan of those animais treated with unilateral DAT antisense in the left
Apomorphine-induced rotations: Apomorphine (0.3 mgkg) induced a significant rotational response ipsilatd to the side of intranigral infiision in those animais treated with DAT antisense prior to bilateral intrastriatal infusion of 6- hydroxydopamine = 7.66, p = 0.0394) (Fig. 32).
DAT antisenre and MPP+ toxicity
Striata1 dopamine content: Striatal dopamine content, as assessed using HPLC. was significantly Iower in the right venus left stn'attnn of those animds treated with unilateral DAT aatisense prior to bilaterd infusion of MPP' (F,,, , = 7.89, p = 0.01 08)
(Fig. 33).
Apomorphine-induced rotations: Apomorphine (0.3 mgkg) induced a significant rotational response $daterai to the side of htranigraI infusion in those animais treated with DAT antisense pnor to biIateral infusion of MPP' (FI,,,= 5.80. p =
0.0368) (Fig. 34).
3.6.4 Surnmmy
Thus, the dopamine transporter qpears to play a critical roIe m detennining susceptibiIity to the experimental nemtoxins MPP+ and 6-hydroxydopamine. The DAT Figure 29
Strirtll Dopamine Transporter Binding Following Bilateral Intrastrïatal Infiision of the Neurotoxin 6-Hydroxydopamine in Animals Pretreated W it h Unilateral lnfuslon of Oligonulcleotide Antisense to Dopamine Transporter mRNA
left rfght
Densitometrie nicosurenrent of autoradiogropls represemtimg ~H~~WBY35.428 binding in the strr'arunr. Foliowing pretreatment with unilateral oiigonucIeotide antisense to DAT mRNA (1 nmollday, 7 days) infusion into the Ieft substantia nigra, bilaterai intrastnatal infusion of 6-hydraxydopamine resuked in a significant disparity in dopamine transporter bindmg, with significantly lower binding evident m the right versus Iefi striatum, Each bar represents the mean (& SEM.) (~6) opticai derrsity (nCümg). ** sig, diff, fiom Ieft hemisphere, p < 0.00 1 Figure 30
Autoradiograph representing tE+WlV 35,428 binding. A~uradiograph of [3~-WIN35-428 binding in a section taken through the strianmi followiag biIaterai intras~atdinfusion of 6-hydroxydopamùie in animais pretreated with unilaterai Uifunon of oiigonucleotide antisense to dopamine transporter mRNA (1 moilday, 7 &YS) Uito the Ieft substantia nigm Figure 31
Striatal Vesicular Monoamine Transporter Binding Following Bilateral infuision of the Neurotoxin 6-Hydroxydopamine in Animab Pretreated With Unilateral Infusion of Oligonucfeotide Antisense to Dopamine Transporter mRNA
Densitonetrie nreasurenrcnt of ~utoradiogruphs reprcsentimg [3~/-~~~~ bittdr'rrg in the strtatunr, FolIowing prctreatment w ith uniIateraf O ligonucleotide ancisense to DAT mRNA (1 nmovday, irdays) infision tnto the teft substantia nigra, bilateral inuasniataI infusion of 6-hydroxydopamfne resuhed in a significant disparity in vesicular manoaminc transporter binding, with significantly Iower binding evident in the right versus teR striatum, Each bar represents the mean (f S.E.MJ (n=6) optical density (nCf/mg). *+ sig. diff. from Ieft hemisphere,p c 0.001 Figure 32
Apomorphine-lnduced Rotations in Animals lnfused Bilaterally W lh6-OHDA Following Pretreatment With Unilateral DAT Antisense Oligonucleotides
vehicle apormrphine
Apomorphke-iirduceti ipsiiaterat rotations. Apomorphine (03 rng/kg) induced a significant ipsilateral rotationai response two weeks following btlateral infusion of 6- hydroxydopamine into the media1 forebrain bundle of animals pretreated with unilateral intranigral ~Cigonucteotide antisense to dopamine transporter mRNA (1 nmoliday, 7days). Each bar represents the mean (n=6) (k SEM.) number of ipsdateral rotations recorded over a C hour period- sig. diff. hmvehicle rreatrnent, p c 0.0 1 Figure 33
Striatal Dopam'ne Content Following Bilateral MPP+ Infusion in Rats Pretreated Wth Unilateral DAT Antisense Oligonucleotides
left right
Strtutut dopamine content as measured by HPLC. Prior treatment with undateral oIigonucIeotide antisense to dopamine transporter mRNA (1 nmollday, 7 days) resulted in a significant disparity in striatal dopamine content in the ri-ght versus leR striaturn follow ing intrahatai infusion of M PP*(8 pg13 pl). Each bar represents the mean (n= 1 1 ) (+ S.E.M.) striatal tissue dopamine content. ** sinnificantiv different frorn pretreated Ieft side (p< 0.00 f 1 Figure 34
Apomorphine-lnduced Rotations in Animals lnfused Bilaterally With MPP+ Following Pretreatment Mth Unilateral DAT Antisense Oligonucleotides
* mvehicle T apomrphine
Apomorphlne-tnduced @sifateru&rotations, Two wce ks fo llow ing bilaterai inrrasniatal infusion of MPP' (8pg/3$) . apomorphine (OJmgkg. ss.) induced a signiticant rotational response directed towards the side of intranigral mfusion of oifgonucIeotide antisense to dopamine transporter mRNA (1nmoVday. fdays). Each bar represenu the mean (SEM) (n = L i ) nurnber of ipdateral rotations recorded over a one hour period.
* significantly différent fiom vehicIe injection (p c 0.0 1) may similady serve as an uptake site for as yet unidentified neurotoxic substances which rnay contribute to Parkinson's. In Light of this, the dopamine transporter rnay prove usefiil, both as a marker for susceptiiiIity to Parkinson's disease, and as a target hr therapeutic intervention. Chapter 4
GENERAI., DISCUSSION
4.1 Discussion
Antisense oligonucIeotides provide a novel and highly specific tool for studying the expression and hction of a diverse range of proteins. The main advantage of antisense technoIogy is its specificity, characterized by selective hybridization of the oligonucleotide to a specific part of a single mRNA species and inhibition of the expression of a single target protein. in these studies, antisense oligonucleotides provided a Ievel of specificity which might not have been achieved with a traditional pharmacologicai approach. This is particularly important in light of recent evidence for the existence of novel subtypes of the dopamine D, receptor, the lack of appropriately selective Ligands for the dopamine D3receptor, and the variable actions of DAT ligands.
While it is true gene knockouts also aord a high degree of rnolecuiar specificicity, the possibility of compensatory changes occurring during development in transgenic animds must be taken Uito consideration when interpreting these studies. As well, antisense ailows a measure of anatornicd specificity not seen in transgenic studies. Direct infusion mto a specific region limits 'knockdown' to the area of mterest, leaWig expression m most other areas of the CNS tmchanged. Shldies incorporahg antisense technology may work in concert with pharmacologicd and transgenic studies to fom a more compIete picmof the functionaf role of various striatai protek. One of the main concerns associated with the use of antisense oligonucIeotides as an experimental tool is the difnculty designhg a sequence that wiiI provide the necessary
Ievel of specificity and stability while keeping nonspecific toxicity to a minimum.
UafominateIy, even a well-designed oligonucleotide nms the rkk of exerûng nonspecific effects, incIuding nonspecinc binding to prote&, inhibition of various polymerases, and inhibition of RNA splicing. The use of in vivo antisense has aiso been associated with nonspecific toxicity (Robinson et d., 1997). Aithough the cause is not clear, toxicity may result hma number of sources, including secondary effects of such nonspecific actions noted above, cytokine release, or actions of metabolites formed following degradation
(Crooke & Bennett, 1996). Unmodified oligonucleotides are relatively unstable both iri vitro and in vivo, and are rapidy degraded by cellular nucleases. While the phosphorothioate modification used in these studies enhances their resistance to nuclease activity, it also inmeases nonspecific effects including toxicity (Brysch &
Schluigensiepen, 1994). By ushg partialIy modified oIigonucIeotides, we were able to achieve a baiance, enhancing stability while miniminne toxicity
Because of the large nmber of nonspecitic effects which rnay &se, it is important to mclude the proper controls. The studies reported here dl incorporated an appropriate missense sequence consisting of the same bases in scrambled sequence and with the same degree of phosphorothioate modification. These control sequences were designed to controI for any nonsequence-specinc effects which might be atûicbutable to oligonucleotide mfiision. The missense sequences had no signincant effect on either radiofigand binding or behavioural responses in any of these studies, snggesting that the effects of autisme were sequence specific and not the result of nonspecific actions or toxiciiy.
The possibility of nonspecinc damage can ais0 be examined by Iooking at the expression of other proteins in the target region. In these studies, oligonucleotide antisense to dopamine DI, receptor mRNA reduced dopamine DIreceptor binding in the striatum but failed to aiter dopamine D2 receptor binding, suggesting that striatal neurons remained intact. uI our Iab, earlier work with dopamine DIA receptor antisense raised concems regarding nonspecinc damage (unpublished data). However, by shortening the sequence, reducing the degm of phosphorothioate modificatioa, changing the mode of administration (nom pulsatile to contuiuous inhision), and shortening the duration OF infusion, we were able to reduce these non-specific effects. Hebb & Robertson (1997) have reported that a singIe phosphorothioate modification on either end of an oligonucleotide antisense sequence is sufficient to produce effective inhibition of gene expression in vivo while miniminne toxic effects. As weII, while specificity is associated with Ionger sequences in vitro, the reverse is true in YIYO, with longer sequences finding homologies in umelated genes (WahIstedt, 1994). The mode of administration cm also affect toxicity. The use of osmotic minipumps for a sIow continuous infusion, rather than twice-daily bolus infusions, may produce Iess non-specific damage (WhiteseII et al.,
1993). FinaiIy, the Ionger the duration of infusion, the greater the chance of toxicity
(Pilowsky et ai., 1994). Smce the dopamine Direceptor has a sIightly shorter turnover rate than other dopamine receptors such as the dopamine D2 receptor (Fuxe et al., 1987;
Leff et aI., 1984; Qian et ai., I993), we reduced antisense &on time fiom 5 days to 3 days. Together, these saategies have he1ped to minimize non-spdc toxicity. SimiIar strategies were subsequentiy employed for the use of dopamine D, receptor antisense, using a continuous mode of deIivery and minimizhg oligonucleotide length and modification. In this study, O tigonucleotide antisense to dopamine D, receptor mRNA blocked the increase in striatal dopamine D3receptor binding seen in dopamine- denervated animais treated with pulsatile levodopa, but failed to alter either dopamine D, or D2 receptor binding in this region, again niggesting the absence of nonspecific damage. The missense oligonucleotide also failed to alter any of the receptor binding performed and had no effect on behaviourd responses. However, eariier work with a different missense sequence was suggestive of nonspecific actions. The missense sequence resulted in a partiai reduction in behaviour and significantiy reduced doparnine
Di,D, and DI receptor binding. These reductions mom Likely reflected what appeared to be a considerable degree of nonspecific toxicity in the striatum. Once the missense sequence was aitered these problems were no longer encountered. It is important to check al1 oligonucleotide sequences against a gene database. However, much of the genome remains yet to be sequenced Ieaving the chance for newly created sequences to hybndize to unintended mRNA.
For DAT studies, examining possible toxicity is not as simple. [n these shidies, adsense ~Iigonucleotideswere not uifused into the area of interest, but were, rather, infused into the ceiI body region of the nigrostriatai pathway, where the DAT is synthesized. Thus, any nonspecinc damage would be expected to occur in nigrostriaid neuons, affecthg presynaptic termioals in the stria- rather than postsynaptic striatai neurons. Dopamine receptor binding wodd, therefore, not be a good mdicator in this case. Instead, we used methoxytetrabenazine bindhg to the centrai vesicular monoamine transporter (VMAT2) as an indicator of dopamine terminal integrity in the striatum. Unilateral infùsion of oligonucIeotide antisense to DAT mRNA reduced DAT binding in the striaturn but fded to alter MTBZ binding, indicating that the dopamine terminais remahed intact.
Intrastriatai nifusion of dopamine DIAreceptor antisense reduced dopamine DI agonist-induced VCMs and grooming, connnning the view that this receptor does indeed play a vital role in these behaviours, despite recent suggestions that another, unknown DI receptor subtype rnay be involved (Deveney & Waddington, 1997). hterestingly, in con- to SKF 38393-induced VCMs, grooming was ody partiaily affected by dopamine DIAreceptor "knockdown". This is sornewhat consistent with pharmacological findings which indicate that grooming can be eiicited by a wide variety of dopamine DI selective agonists associated with varying modes of action @eveney & Waddington,
1997). It rnay be that, whiIe the data presented here suggest that grooming is mediated by dopamine DIAreceptors, other D,-Iike receptor mbtypes may also be involved.
Similady, VCMs induced by chronic neurolqtic treatment were also attenuated by intrastriatai infusion of oligonucIeotide antisense to dopamine DIA receptor mRNA. suggesting that mouth movernents in this mode1 may also be mediated by the DlA receptor. In the pas& supersensitivity of dopamine D2 receptors has been proposed as the principle basis for TD. This theory was based primarily on hdings of increased D2 receptor den* in rat stnatum following chronic administration of antipsychotics (Btnt et al., 1977). However, there is poor tempord and spatial correlation between the deveIopment of dopamine receptor supenensitivity and tardive dyshesia (Fibiger and
LIoyd, 1984), and there is no direct post-mortem (Komhuber et aI., 1989) or positron emission tomographie (Andersson et al., 1990) evidence for increased D2 doparnine receptor binding in patients with TD. As weII, changes in D2 receptors are not well correlated with behaviourai indices in rodent models of this disorder (Knable et al., 1994;
Waddington et ai., 1983; Waddington, f99O).
The hdings reported here are in agreement with suggestions that TD may involve a disrupiion in the balance between dopamine D, and D2 receptor bcbon favoring activity in dopamine DIreceptor-bearing striatonigral neurons. A relative increase in striatonigral activity would result in inhibition of GABAergic efferent neurons Iocalized in SN, and a consequent disinhibition of thalamic output. Indeed, depressed GABAergic activity in the SN, has been conelated with mouth movements in rats and dyskinesia in monkeys following chronic netnoleptic ûeatrnent (Gunne & Haggstrom, L983; Gume et al., L984; Kaneda et ai., 1992).
The hdings desrribed here suggest that the dopamine DlA receptor aiso plays a critical role in the expression of behavioural sensitization in a rodent mode1 of UD. The role of the dopamine D, receptor in LID has been somewhat controversial to date. The D, receptor was nrst implicated when it was found that, udike Levodopa, comparably effective doses of bromocriptine did not induce dyskinesias either in MPTP monkeys or
PD patients (Bédard et ai., 1986; Lees & Stem, 1981). Bromocriptine is a dopamine Dr like receptor agonist with DI antagonist properties. SirniMy, clozapine, whose behaviourai actions are mediateci, at Ieast in part, by dopamine DIreceptor antagonism, exerts a dosedependent suppression of LID in PD patients (Bennett et al., 1994) and
MPTP-treated monkeys (Grondin et al., 1999). A~cordingiy~dyskinesias can be elicited m these animds by chronic treatment with selective dopamine DI reccptor agonists. leading to suggestions that dopamine Dlreceptor stimulation might be invoIved in the
development of LID (Boyce et al., 1990; Falardeau et ai., 1988; Nomoto & Fukuda,
1993). Other studies in MPTP monkeys, however, suggest that repeated treatment with a
Dlreceptor agonist cm deviate parkinsonian symptoms with fewer dyskinesias than
seen with Ievodopa (Bédard et al., 1993; BIanchet et al-, 1993). or may even reduce
dyskuiesias in Ievodopa-primed animals (Pearce et al., 1995). This disph& cm be
reconciled by taking into consideration the duration of action of the compounds used in
these studies. It has been shown that long-acting DLagonists produce a more continuous
stimulation of the receptor resulting in the rapid deveIopment of behavioural toIerance
(Blanchet et al., 1996). This is in agreement with studies suggesting that fewer dyskinesias are associated with continuous than with pulsatile forms of treatment (Chase.
1998). Thus, whiIe repeated administration of the dopamine DIreceptor-selective agonist
A-77636 appears to relieve dyskinesias in Ievodopa-primed MPTP monkeys, this compotmd is a Iong-acting agonist and its Iong-term use rnay, therefore, remit in desensitization of DIreceptor responses.
The nature of the dopamine DI receptor involvement in this mode1 remains
unclear. However, ment work with striatd neuropeptides suggests that dynorphin and preprodynorphin mRNA, fomd in dopamine Di receptor-bearing sûiatonigral neurons,
are overexpressed m hemiparkinsonian rats foIIowing chronic treatment with Ievodopa
(Cenci et ai., 1998; Engber et ai., 1991; Gerfen et aI., I990), an effect mediated by
dopamine Di receptor activity (Steiner & Gerfen, 1998), and that their IeveIs are strongIy
coneIated with measures of behaviourai sensitization (Cenci et al., 1998; Steiner &
Gerfen, 1998). Dynorphm, released in the substantia nigra, mhibits GABAergic neurons of the pars reticdata (Matsumoto et al, 1988) and may aiso inhribit glutamate release fkom subthalamo-nigrai projections (Maneuf et al., 1995), thereby disinhiiitùig thaiamocortical projections and stllnulating or enhancing movement. Dynorphin acts at the kappa opioid receptor and pharmacological blockade of this receptor attenuates sensitized rotation in a rodent model of LID (Newman et al., 1997). Elevations of striatai
Nos& typicdy seen in animal models of LJD (Doucet et ai., L996), are aiso dependent upon dopamine Di receptor activity and central infiision of antisense O Iigonucleotides to
Afos B BAhas recentiy been shown to attenuate the induction of FosB in a rodent model, accompanied by a reduction in upreguiated preprodynorphin mRNA levels and associated behaviod indices (Andersson & Cenci, 1999). Thus, the dopamine Di receptor may indirectly affect behaviour in this model through its influence on dynorphin expression, an effect which may depend upon induction of FosB.
Striatal neuropeptide expression is aiso regulated by activity at dopamine D, recepton (Tremblay et ai., 19981, which are colocaiized with DIrecepton (Anano &
SibIey, 1994) and are themselves regulated by DI receptor activity (Schwartz et al.,
1998). The dopamine D3mceptor is of parûcular interest in this rodent mode1 of LID due to the ectopic induction of striatai D3receptor binding and mRNA seen in these anÏmaIs folLowing chronic Ievodopa treatment and the paraIIel in the courses between this induction and the expression of behaviod sensitization. The data reported here are consistent with these hduigs, as two separate studies found elevated dopamine D3 receptor binding in the denervated following chronic Ievodopa Intmtriatai mfusion of ~Iigonttcleotideantisense to dopamine D3 receptor mRNA signincmtly attemated this induction without affecthg either dopamine DI or D2 receptor bhding- This change in 4 biuding was accompanied by a reduction in behaviornal sensitization, further underlining the importance of the dopamine 4 receptor in this rodent mode1 of
Lm.
Whether such hdùigs can be readily generalized to primates remains an issue for debate. Although initia1 studies failed to hdalterations in stnatal dopamine D, receptor expression (HurIey et al., 1996a,b), more recent hidies have demonstrated reductions in dopamine D, receptor bindiag in the stria- of Parkinson's disease (Piggott et ai., 1999:
Ryoo et ai., 1998) and MPTP-treated monkeys. in MPTP-treated monkeys, these depressed levels of D3 receptor expression are reversed by chronic treatment with either levodopa (Police et al., 1999) or a selective dopamine Dlreceptor agonist (Morissette et ai., 1998). However, there is no evidence for the overexpression or ectopic induction that has been demonstrated in rodent models. This may be related to slight species differences in basal expression. While the dopamine D3 receptor demonstrates a very similar disnibution pattern in rodents and primates, basal expression in the striaturn does tend to be more prominent in both monkeys and hmans. Regardless, striatal dopamine D, receptoa are cledy af5iected by dopaminerpic tone in a manner reminiscent of that seen in rodents (Bordet et al., 1997). Aiso, as with rodent rnodels, 'selective' dopamine D3 receptor antagonists are effective antidyskinetic agents in primate mode1s of LID (Bédard et al., 1999; Blanchet et ai., 1997; Tahar et aI., 1999). Unforttmately, no mch data are yet available Eom humstudies.
The IeveI of dopamine D3 receptor expression is regttiated, not only by its own activation, but dso by that of the dopamine DIreceptor. Thus, stimtdatioo of the D3 receptor results in opregdation of D3 receptor transcripts, whüe stimdation of the Dl receptor upreguiates bath DIand 4 receptor transcripts (LevaviSivan et ai., 1998). To date, no evidence exists for dopamine D3 receptor regulation of Dl receptor expression.
The ectopic induction of dopamine D3 receptor expression seen folIowing chronic levodopa in hemiparkinsonian rats is readily reproduced by treatment with a selective dopamine DI receptor agonist and blocked by a D, antagonist (Bordet et al., 1997). This is consistent with the hdùigs reported here. IntrasûiataI infusion of oligonucleotide antisense to dopamine DlA receptor mRNA significantly attenuated both dopamine DI and D3 receptor buidhg in the striatum. Thus, these data firrthcr support a role for the dopamine DIreceptor in the regulation of dopamine D3receptor expression.
Dopamine DJD3 receptor interaction extends beyond CO-regulation. The dopamine DIand D3 receptors have been shown to have both opposite and synergistic functional interactions dependhg on the brain region (Ridray et al., 1998). It has been suggested that in the striahim, synergistic interaction between these two recepton may serve to decrease the response threshold to dopamine in order to maintain a hi@ level of activity in striatonigral neurons (Ridray et al., 1998). Administration of a 'selective' dopamine D3 receptor agonist potentiates SKT 38393-induced rotations following sensituation in a rodent mode1 of Lm (Schwartz et al., 1998). Therefore, dopamine Dt receptors may enhance dopamine Dl receptor activation of striatonigral neurons, which contniutes to the generation of rotational behaviour in these animais (Accili et ai., L 996;
Gerfen et aI., 1990) and possibIy dyskinesias in humans @e Long, 1990). Alth0ug.h the exact mechanism through which the dopamine D3 receptor facilitates activation of stnatonigral neurons remains unclear, aiterations in neuropeptide expression may be a key factor. In the rodent mode1 of LID desmbed here, dopamine Dz receptor binding was not af3ected by eitha dopamine Di or D, receptor antisense treatment. However, binding in the left striatum, ipsilateral to the lesion, was reduced in those anirnals chronically treated with levodopa Although this was a rnodest effect, it is consistent with clinical data indicating an upregulation of dopamine D2 teceptors in the caudate-putamen of
Parkinson's patients, an effect which is revened by chrooic levodopa therapy (Bokobza et al., 1984; Gutûnan & Seeman, 1985). Thus, the slight relative reduction in dopamine Dî receptor binding seen here may reflect levodopa-induced normaiization of elevated levels typicdly seen in the of 6-OHDA-lesioned rats (Angulo et ai., 1991 ; Lisovoski et al., 1992; Neve et al.. 1991). Similar hdhgs have been reported with MPTP-Iesioned monkeys (Alexander et al., 1993; Falardeau et al., 1988; Gagnon et al., 1990; Herrero et ai., 1996).
Determination of changes in dopamine D, receptor expression following long-term neuroleptic exposure has yieIded mixed resuits. Chronic neuroleptic treatment results in up to a five-fold Uicrease in dopamine D3receptor mRNA expression in whole brah (Buckiand et al., 1992). More modest inmeases of only 40-60 % are seen m the nucleus accumbens and olfactory tuberde following subchronic treaûnent (Wang et al., 1996). While no such induction of dopamine D3receptor expression in the caudatdputamen has yet been reporte& subchronic nemieptic administratioa may be insufficÏentto mduce signincant changes. As weii, these -dies fded to inchde behaviourd correIates, which tend to requk extreme1y
Iong perîods of aeuroleptic treatment (Ellison et aL, 1988). A ment study ceports Ïncreased suscepb'biiay to TD in schizophraiic patients with a variant fom of D, receptor (Segman et al, 1999; Steen et al., 1997). The data reported here establish an miportant roie for haronigral dopamine DIreceptors in a rodent mode1 of TD. Ifthe dopamine Djreceptor does interact with the DI receptor to enhance activation of striatonigrai neurons, then it may also influence chronic neuroleptic-hduced behaviours. Thus, ui Light of the importance of the dopamine D3receptor in LlD demoastrated here, it might be of interest in the future to examine the role of this receptor in a rodent mode1 of TD. While no statistically significant changes in D3binding were seen following chronic ffuphenazine in these experiments, variability was high and numbers were Iow making fitrther examination necessary before definitive concIusions can be reached.
AIthough the results of these studies emphasize the importance of the direct striatonigrd pathway in dnig-induced dyskinesias, they do not preclude the involvement of the indirect stnatopdlidai pathway. Rather, basal ganglia hction is based in Iarge part on balance. Thus, the expression of dyskinesia may reflect an imbaiance of activity resulting nom aiterations in either pathway. Aithough TD appears to involve heightened activity at the dopamine DIreceptor, other mechanisms may be invohed as wel1. For example, increased activity of the subthafamic nucleus (resulting fiom dopamine Dz receptor blockade) may aiso contn7ute to the pathogenesis of TD (Tmgrnan et al.. 1994).
Indeed, chronic neuroleptic-induced VCMs are significantly reduced foIIowing bilateral excitotoxic Iesions of the mbthdamic nucleus (Stoessl & Rajakurnar, I 996). S unilariy, while elevation of dynorphin levels in striatonigral neurons is a key feature of LID, the expression of Lm is aiso correlated with the expression of enkephdin in striatopaIlidal neuioos (Lee et ai, 1997). Although enkephalui is relatively unaffected by chronic
Ievodopa treatment, the initiai doparnine denervation dramatically increases enkephalin
IeveIs in the stria- (Engber et ai., 1992). Since denervation is necessary for chronic Ievodopa treatment to elicit dyshesias (Boyce et ai, 1990), aiterations in striatopaIlidal enkephalin might be a necessary permissive feature for the development of Lm. Thus, dismpted activity in the indirect striatopaIlidal pathway may figure promuiently in both
TD and LID.
ui these studies, unilateral infusion of oiigonucIeotide antisense to DAT mRNA into the SNc resuited in a significant reduction in striataI %I-WIN35-428 binding on the treated sîde. This reduction in DAT expression was dso evidenced behaviourdly in the fom of drug-induced contralaterai rotations. Both the dopamine precursor levodopa and the dopamine-releasing agent amphetamine resuited in signincant contralateral rotations in antisense-treated animds. These effects appear to be selective for antkense treatment and not the result of mechanicd damage or nonspecific toxicity resulting fiom
01igonucIeotide infusion, as no changes were seen following either saline or missense treatment- Furthennore, nonspeci fic damage to the nigrostriatd dopamine projection would be expected to resdt in ipsiiateral not contralateral rotation in response to amphetamine. An asymmetncaf reduction of DAT expression following antisense treatment would have resdted m increased synaptic dopamine in the striatum of the antisense-treated side in response to dopamine releasing agents (Jones et al., 1998; Silvia et al., 1997), thus creating an imbaiance of activity leadhg to the observed rotational respouse. WhiIe Nnilar work by SiIvia et al. has akeady examined the effects of DAT knockdown on respoases to amphetamme, the response to Ievodopa, a more physioIogicai measure of DAT bction, has not been previoudy tested. As weII, our group was able to achieve a much greater reduction m DAT expression, which may aiIow a more accurate determinatÏon of the fimctÏond roIe of the DAT. No spontaneous rotation was observed in these animais. This is consistent with prevïous work involving DAT antisense oligonucteotides (Silvia et al., L997). Gene knockout studies have reported spontaneous hyperlocomotion in mice Iacking the DAT
(Giros et al., 1996). However, heterozygote mice exhibithg ody a 50% reduction in
DAT expression showed no increase in spontaneous locomotion. In the studies reported here, DAT expression was reduced by only approximately 60% in antisense-treated anirnals. As weil, the complete absence of DAT in knockout mice has been associated with vansous other changes including a reduction in mRNA encoding dopamine D2 autoreceptors- While no rneasure of actud dopamine release was made here, compensatory mechanisrns may be at work such that activation of dopamine D? autoreceptors by excessive synaptic dopamine can attenuate basal dopamine release.
Thus, residuai DAT may be suflicient to accommodate the reuptake of basai dopamine efflwc, making a pharmacological challenge necessary for an observabte behavioural effect.
The ability of amphetamine to evoke rotation contraiateral to the side of uitmnigrai antisense uifusion might initiaily seem sucp8sing. Some controversy ai11 surrounds the action of amphetamine on dopamine systems. Amphetamine is thought to act as a substcate for the DAT, causing an increase in extracellular dopamine as a result of two actions: release of dopamine Rom presynaptic nerve terminais and inhibition of reuptake (Liang & RutIedge, 1982; Seiden et ai., 1993). The degree of DAT mvoivement in these actions, however, remains controversiaf. Mice Iacking DAT faiI to exhibit m increase in locomotion iu response to amphetamine with no evidence of dopamine effIux in stnatd siices nom these animds (Giros et ai., 1996; Jones et aI., 1998) suggestkg that the DAT is required for amphetamine action. Based on these observations, the reduction of DAT expression seen in antisense-treated animais here might have been expected to reduce the response to amphetamine, perhaps eliciting rotation towards the side of antisense treatment. However, when DAT expression is ody partiaily reduced, amphetamine-induced locomotion is not blocked (Giros et al., L996; Itzhak et al., 1997;
Silvia et al., 1997). Thus, residual DAT appears to be sficient to maintain amphetamine-induced dopamine release and Iocomotion. if amphetamine occupies the residuai DAT to evoke release of cytoplasmic dopamine, the number of DAT sites available for dopamine reuptake would be Merreduced. Thus, amphetamine may, in fact, be able to elicit the reIease of dopamine, whose subsequent reuptake is inhriited by the antisense knockdown. It is intereshg to note that amphetamuie induced a much more mbun rotationai response than Ievodopa
Having demonstnited the effectiveness of oligonucleotide antisense treatment in reducing both the expression and funetion of the DAT, we went on to examine the role of the DAT in the uptake of the experimentd neurotoxins MPP' and 6-OHDA. Unilateral infusion of oligonucleotide antisense to DAT MAprior to bilaterai intrastriatal fision of either MPP' or 6-OHDAresdted in significant rotations ipsilaterd to the side of antisense infusion in response to the nonseIective dopamine receptor agonist apomorphine. This suggests a highIy asymmetnc IesÎon and suggests that the antisense treated side was re1ativeIy protected fkom the effects of the neurotoxins by DAT knockdown. This was confhned by the highiy asymmetnc reduction of striacal 'H-WIN
35-428 binding and dopamine content on the untreated side. Together, this evidence suggests that antisense treatment provides some form of neuroprotection. By reducing DAT expresgon, we were able to reduce the uptake of these two neurotoxins, thereby preventing cell death.
Parkinson's disease involves the selective loss of specific populations of dopaminergic neurons. Understanding the pathogenesis of this disease requires an understanding of the basis for this selectivity. Just as the DAT plays a roIe in the lrptake of experimentd neurotoxins, it may aiso serve a sùnilar function in the deveIopment of
PD. Levels of DAT expression, or more specifically, the ratio of DAT to vesicular monoamine transporter2 expression, could serve as a marker for vuInerability to PD.
Neurotoxins taken up by the DAT may be subsequently sequestered into vesicles via vesicular monoamine transporter 2 (Reinhard et ai., 1987). thereby reducing its toxic potentid (Liu et al.. 1992; Liu et ai., 1994). Thus, the DAT and the vesicular monoamine transporter 2 rnay work together to regulate cytoplasmic Ievels of these neurotoxins and any alteration in the expression of either may result in dtered susceptibility to their toxic effects. ALterations in DAT or species differences in DAT can affect DAT expression
(Wang et af., 1995) and fiinction (Buck & Amara, 1994; Kitayama et al., I992; Lee et al.,
1996). PoIymorphisms in the DAT gene (Sano et al.. 1993) could determine mdividuai variations in DAT expression or its a£finity for neurotoxins (Le Couteur et al.. 1997).
Identification of gene variants codd dso provide a marker for susceptibility. As well, fii~rrework couid Iead to therapeutic approaches targehg DAT, possibly slowuig the progress of this disease.
It may be interestmg to use the techniques developed in these studies to examine the possible role of the DAT in the development of dnig-hduced dyskinesias. As discussed, the DAT is respons~bIefor regufating ~ynapticdopade, imparhg bath tempod and spatial coI1SfLaints on dopaminergic neurotransmission. Aiterations in DAT ftuictim, such as the loss of DAT in PD (Horstink et al., 1990) or the inhicbition of DAT- mediated dopamine uptake by neuroIeptics (Lee et ai., 1997; Rothblat & Schneider,
1997), codd alter normal dopaminergic transmission, possibIy increasing the relative stimulation of extrasynaptic dopamine Dl receptors (Gonon, 1997) and triggenng downstream neuropeptide changes (Giros et al., 1996). This is of particular interest in
Iight of the present hdings Unpikathg the D, receptor in both LID and TD. Thus, a closer study of DAT fùnction in these models would be of great interest. 4.2 Conclusions
1. SKI? 38393-induced VCMs and grooming are rnediated by striatai dopamine DIA
receptors.
2. The expression of chronic neuroleptic-induced VCMs in a rodent mode1 of human TD
is dependent on striatai dopamine DIAreceptors.
3. Striata1 dopamine DlA receptors are aiso Uivoived in the expression of sensitized
rotational responses lollowing chronic levodopa in a rodent model of human Lm.
4. The dopamine D, receptor aiso pIays a rote in chronic Ievodopa-induced sensitization
of apomorphine-induced rotations in a rodent model of LID.
5. The DAT mediates the neurotoxic effects of the experimentd neurotoxins 6-
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