Effect of Repeatedl-DOPA, Bromocriptine, or Lisuride Administration on Preproenkephalin-A and Preproenkephalin-B mRNA Levels in the Striatum of the 6-Hydroxydopamine-Lesioned Rat

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Experimental Neurology 155, 204–220 (1999)Article ID exnr.1998.6996, available online at http://www.idealibrary.com on

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Effect of Repeated L-DOPA, Bromocriptine, or Lisuride Administrationon Preproenkephalin-A and Preproenkephalin-B mRNA Levels

in the Striatum of the 6-Hydroxydopamine-Lesioned Rat

Brian Henry, Alan R. Crossman, and Jonathan M. BrotchieManchester Movement Disorder Laboratory, Division of Neuroscience, School of Biological Sciences, University of Manchester,

1.124 Stopford Building, Manchester, M13 9PT, United Kingdom

Received August 6, 1998; accepted December 4, 1998

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Abnormal involuntary movements, or dyskinesias,lague current symptomatic approaches to the treat-ent of Parkinson’s disease. The neural mechanismsnderlying the generation of dyskinesia following re-eated L-3,4-dihydroxyphenylalanine (L-DOPA) or do-amine agonist administration in Parkinson’s diseaseemain unknown. However, de novo administration ofromocriptine or lisuride to either l-methyl-4-phenyl-,2,3,6-tetrahydropyridine-lesioned primates or pa-ients can alleviate parkinsonian symptoms withouthe development of dyskinesia. In this study, we havenvestigated behavioral responses and alterations inhe expression of opioid neuropeptide precursors pre-roenkephalin-A (PPE-A, encoding methionine- and

eucine-enkephalin) and preproenkephalin-B (PPE-B),he precursor encoding dynorphins (dynorphin A1–17

nd B1–13, leucine-enkephalin, and a-neoendorphin) intriatal output pathways of the 6-hydroxydopamine6-OHDA)-lesioned rat model of Parkinson’s disease.xpression was assessed following repeated L-DOPA,romocriptine, or lisuride administration. Given theunctional organization of basal ganglia circuitry intonatomically discrete parallel circuits, we investi-ated alterations in peptide expression with referenceo the detailed topography of the striatum. Followingepeated L-DOPA administration (6.5 mg/kg, b.d., 21ays) in the 6-OHDA-lesioned rat a rotational responseas observed. This became markedly enhanced with

epeated treatment. We have previously characterizedhe pharmacology of this enhanced response and haveuggested that it is a useful model for the elucidationf the cellular and molecular mechanisms underlying-DOPA- and dopamine agonist-induced dyskinesia. Inontrast to L-DOPA, de novo administration of bromo-riptine (1 or 5 mg/kg, b.d., 21 days) or lisuride (0.01 or.1 mg/kg, b.d., 21 days) did not lead to an enhancedehavioral response. In vehicle-treated, 6-OHDA-le-ioned animals, PPE-A expression was elevated ros-rally and dorsally, while PPE-B expression was re-uced in the striatum at all rostrocaudal levels.
epeated L-DOPA administration was accompanied by U
204014-4886/99 $30.00opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

levations in striatal PPE-B mRNA levels and a furtherlevation, above lesion-induced levels, in PPE-Aexpres-ion. This further elevation was restricted to the dorso-ateral striatum. However, following repeated bromo-riptine or lisuride administration no increase inPE-B expression was observed and the lesion-in-uced increase in PPE-A expression was normalized torelesion levels. Increased PPE-A and PPE-B levelsay, through decreasing GABA and glutamate release,

espectively, in output nuclei of the basal ganglia, playrole in the development of L-DOPA- and dopamine-

gonist induced dyskinesia in Parkinson’s disease.hese studies suggest that anti-parkinsonian treat-ents which are not associated with an elevation inPE-B and/or normalize elevated PPE-A precursorxpression, such as NMDA-receptor antagonists orong-acting dopamine D2 receptor agonists, e.g., caber-oline or ropinirole, may reduce dyskinesia in Parkin-on’s disease. r 1999 Academic Press

Key Words: Parkinson’s disease; L-DOPA-induced dys-inesia; 6-hydroxydopamine; opioids; enkephalin;ynorphin; in situ hybridization.

INTRODUCTION

Dopamine-replacing agents such as the dopaminerecursor L-3,4-dihydroxyphenylalanine (L-DOPA) haveeen widely used in the treatment of Parkinson’sisease for more than 30 years (6, 22, 102). However,ong-term dopamine-replacement therapy is associatedith many disabling side effects, most notably abnor-al involuntary movements, i.e., L-DOPA- and dopa-ine agonist-induced dyskinesia (46, 93). While L-DOPA

s the most common treatment for the relief of symp-oms in Parkinson’s disease, other treatment regimesave incorporated various direct-acting dopamine recep-or agonists, for example, apomorphine (2, 24), per-olide (60) cabergoline (47, 63), lisuride (81), ropinirole10), pramipexole (45), and bromocriptine (66, 67, 83).
nfortunately, the actions of all these agents are

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205PPE-A AND PPE-B EXPRESSION AFTER ANTI-PARKINSONIAN TREATMENTS

ompromised as they also elicit dyskinesia when givenither with low doses of L-DOPA or when given alone toatients previously treated with L-DOPA. However, inonhuman primates rendered parkinsonian with-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),e novo treatment of parkinsonian symptoms withromocriptine does not elicit dyskinesia (7). Similarly,n the clinic, bromocriptine can alleviate parkinsonianymptoms, in previously untreated patients, with auch reduced incidence of dyskinesia (12, 53, 61, 81).nfortunately, only about 30% of patients respond to

he anti-parkinsonian effects of bromocriptine (62).urthermore, following repeated bromocriptine treat-ent patients can suffer from side effects including

onfusion, visual hallucinations, and paranoia (11, 42).he majority of patients, therefore, require eitherromocriptine in combination with L-DOPA or L-DOPAherapy alone, both regimes eventually resulting inyskinesia (87). An understanding of the means byhich bromocriptine is able to alleviate parkinsonismithout eliciting dyskinesia would potentially lead to

he development of novel treatments for Parkinson’sisease.The neural mechanisms underlying treatment-re-

ated dyskinesia remain largely unknown, although inommon with other forms of dyskinesia, e.g., chorea (inuntington’s disease) and hemiballism (following le-

ions of the subthalamic nucleus), they have beenuggested to include underactivity of the medial seg-ent of the globus pallidus (GPm) and other outputuclei of the basal ganglia (24, 72). Lesions or inactiva-ion of the subthalamic nucleus in both humans andonhuman primates elicits hemiballism or chorea (37,1, 101). Furthermore, in experimental chorea andardive dyskinesia in nonhuman primates, the subtha-amic nucleus is thought to be underactive (73, 74, 75).

Two pathways originating from the striatum influ-nce the activity of the GPm. These are known as theirect pathway and the indirect pathway. The indirectathway utilizes the inhibitory transmitter GABA,ogether with opioid neuropeptides derived from prepro-nkephalin-A (PPE-A, proenkephalin), including met-nkephalin, as cotransmitters, and projects to theateral segment of the globus pallidus (GPI). The directfferent projection from the striatum to the GPmtilizes opioid neuropeptides derived from the large-olecular-weight precursor preproenkephalin-B

PPE-B, prodynorphin), including dynorphin or leu-nkephalin, as cotransmitters with GABA. The GPl,ia an inhibitory GABAergic pathway, projects to theubthalamic nucleus (STN). The STN in turn projects,tilizing an excitatory amino acid pathway, to the GPm89). The GPl also projects directly to the GPm (55, 92).ecently, we and others have demonstrated that en-ephalin, acting via d-opioid receptors, acts to decrease
he release of GABA in the GPl (41, 69). We have also o
ound that the endogenous ligand for the k-opioideceptor dynorphin and synthetic kappa opioid receptorgonists act to decrease the release of glutamate inutput regions of the basal ganglia (40, 68).Previous studies in rodent models of Parkinson’s

isease have demonstrated alterations in the expres-ion of both PPE-A and PPE-B following experimentalanipulation of the dopaminergic innervation to the

triatum. Blockade of dopaminergic transmission utiliz-ng dopamine receptor antagonists, catecholamineepletion following systemic reserpine administration,r lesion of the dopamine-containing cells of the substan-ia nigra pars compacta with 6-hydroxydopamine (6-HDA) all increase PPE-A mRNA expression levelsnd enkephalin peptide levels (3, 4, 28, 31, 43, 49–51,4, 99, 103). Conversely, following a 6-OHDA lesion,everal authors have described decreased PPE-B mRNAxpression and dynorphin peptide levels (32, 52, 64).However, following repeated dopamine receptor

timulation PPE-A mRNA expression and enkephalineptide levels are either further increased or remainnaltered (26, 28, 31). All studies agree on a markedlevation in PPE-B mRNA expression and dynorphineptide levels (27, 28, 31, 52, 64, 65).It has been suggested that the basal ganglia are

rganized into several functionally and anatomicallyistinct parallel circuits that, broadly speaking, sub-erve motor, associative, and limbic functions (79).ecently, it has been shown that these elevations inPE-A and PPE-B expression following repeated apo-orphine administration are organized in a topographi-

al manner (being more pronounced in motor compo-ents of the circuitry) and correlate temporally withhe development of an enhanced behavioral response toreatment (26, 27). Such an enhanced behavioral re-ponse is also seen following repeated L-DOPA adminis-ration and has pharmacological characteristics identi-al to those of L-DOPA-induced dyskinesias inonhuman primates and man (38).In this study, in situ hybridization utilizing 35S-

abeled oligonucleotide probes was employed to investi-ate levels of PPE-A and PPE-B mRNA in the 6-OHDA-esioned rat model of Parkinson’s disease followingepeated treatment with clinically relevant doses of-DOPA or the ergot dopamine receptor agonists bromo-riptine or lisuride. We assessed the topographicalrganization of changes in PPE-A and PPE-B. Behavioras also analyzed following the various drug treat-ents.

METHODS

nilateral 6-Hydroxydopamine Lesion of the MedialForebrain Bundle

Thirty-six male Sprague–Dawley rats (250–300 g,
btained from Charles River, UK) were maintained

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206 HENRY, CROSSMAN, AND BROTCHIE

nder controlled housing conditions with constant tem-erature (22 6 1°C), humidity (relative, 30%), 12-hight/dark cycles (light period 8 AM/8 PM) and werellowed free access to food (Standard pellets, B & Kniversal) and water. Thirty minutes prior to surgerynimals were injected intraperitoneally (ip) with pargy-ine (5 mg/kg, Sigma) (to inhibit monoamine oxidase-Bctivity and thereby enhance the dopamine depletiony 6-hydroxydopamine) and desipramine (25 mg/kg,igma) (a noradrenaline uptake inhibitor to minimizeamage to noradrenergic neurons), both dissolved interile 0.9% (w/v) sodium chloride (Braun Medical) andnjected at a volume of 1 ml/kg body wt. Rats werelaced in a stereotactic frame, under sodium pentobar-itone anaethesia (60 mg/kg, ip, sagatal, Rhone-erieux). Each animal received a unilateral injection

f 2.5 µl 6-OHDA (Sigma, 5 mg/ml in sterile waterBraun Medical)) with 0.1% ascorbic acid (Sigma) intohe right medial forebrain bundle at co-ordinates 22.8m from bregma, 2 mm lateral to the midline, and 9m below the skull according to the atlas of Paxinos

nd Watson, 84). The 6-OHDA injection was made, byand, over a 5-min period using a 5-µl Hamiltonyringe. Postoperatively, all animals were placed oneated pads and received a subcutaneous injection of2 ml sterile glucose–saline (0.18% (w/v) sodium chlo-ide, 4% (w/v) glucose; Intraven, IVEX Pharmaceuti-als) to prevent postoperative dehydration.

rug Treatment and Behavioral Analysis

Sterile water injection. Twenty-one days postoperationday 0), the 6-OHDA-lesioned rats were removed fromheir home cage, injected with sterile water (1 mg/ml, ip,raun Medical) at 9 AM, and placed immediately in a0-cm-diameter stainless-steel bowl. Behavior was re-orded on videotape. Videotapes were analyzed, by anbserver blinded to the treatment, and the number ofomplete 360° rotations ipsiversive and contraversive tohe lesion was counted over a 2-h period, immediatelyostinjection. 6-OHDA-lesioned animals showing a netpsiversive rotation greater than 30 rotations in 2 h werencluded in the experimental groups. This level of rotationas previously been shown to correspond to a greater than5% depletion in dopamine uptake sites as demonstratedy [3H]mazindol binding autoradiography (27, 38).Repeated dopamine receptor stimulation. Rats were

njected intraperitoneally with either L-DOPA (Sigma,.5 mg/kg) and benserazide (1.5 mg/kg, Sigma), bromo-riptine (2-bromo-a-ergocryptine (Sigma), 5 or 1 mg/g), lisuride (R(1)-lisuride hydrogen maleate, Re-earch Biochemicals International, 0.1 or 0.01 mg/kg),r vehicle (1 ml/kg) twice daily at 9 AM and 5 PM.The methyl ester form of L-DOPA was administered,

s it is more stable and soluble than nonesterified-DOPA. L-DOPA methyl ester is rapidly deesterified in
ivo by nonspecific esterases to form L-DOPA (21). In all l
xperiments, L-DOPA was administered with 1.5 mg/kgenserazide (Sigma). All drugs were dissolved in 0.05%thanol (BDH) and 0.1% ascorbic acid (Sigma) in sterileater (Braun Medical) and injected at a volume of 1l/kg of body weight. Vehicle consisted of 0.05% etha-

ol (BDH) and 0.1% ascorbic acid (Sigma) in sterileater (Braun Medical) and was injected at a volume ofml/kg body wt. Analysis of rotational behavior, for 3 hostinjection, was carried out as described above imme-iately following the 9 AM injection on days 1, 3, 5, 7, 10,4, 17, and 21. Following behavioral analysis on day 21ll animals were killed by stunning and cervical disloca-ion. The brains were removed and rapidly frozen insopentane cooled to 245°C. Brains were stored desic-ated at 270°C until further processing.

3H]Mazindol Radioligand Binding Autoradiography

To assess the extent of 6-OHDA lesion, dopamineptake sites were assessed using [3H]mazindol, essen-ially as we and others have previously described (24,7, 47). Sections were obtained from the rostral stria-um (level 2), as described below. Sections were pro-essed in triplicate. Sections were freeze-dried over-ight and were then preincubated in 50 mM Tris–HCl

pH 7.9) for 5 min at 4°C followed by 60 min incubationt 4°C in 50 mM Tris–HCl containing 300 mM NaCl, 5M KCl, 10 nM [3H]mazindol (15–30 Ci/mmol; NEN/uPont, UK), and 50 nM desipramine (RBI, U.S.A.).pecific binding was defined as that displaced by 100M nomifensine (RBI). Following incubation, sectionsere washed in ice-cold Tris–HCl (23 1 min), dip-

insed in ice-cold distilled water, dried in a stream ofold air, and finally opposed to 3H-sensitive film (Hyper-lm), alongside a 3H microscale standard (Amersham),or 14 days at 4°C. Films were developed using Kodak-19 and fixed using Kodak Unifix as described above.

mage Analysis

Mazindol radioligand binding. Image analysis wasarried out using Seescan Image Analysis SystemSeescan, Inc). Average optical density was measuredrom both lesioned and nonlesioned striata and con-erted to fmol/mg protein by comparison to the micros-ale standard. Nonspecific binding was subtracted fromotal binding to provide a measure of specific binding.or each animal, the percentage reduction in the

esioned striatum compared to the nonlesioned stria-um was determined. Only 6-OHDA-lesioned rats with

reduction in specific mazindol ligand binding ofreater than 95% were further processed for in situybridization studies.

ynthetic Oligonucleotide Probes

Synthetic oligonucleotide probes (30–45 bases in
ength) were synthesised (Gibco BRL) so as to be

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omplementary to the mRNA encoding rat PPE-A andPE-B. The probe targeted against PPE-A mRNA wasomplementary to the sequence coding bases 343–384f the rat PPE-A gene (Accession No. M28263; 44),equence 58-CTT CAT GAA GCC TCC ATA CCG TTTAT GAA CCC TCC ATA CTT-38. The probe targetedgainst PPE-B mRNA was complementary to the se-uence coding bases 754–798 of the rat PPE-B geneAccession No. RNEKB M10088; 17), sequence 58-GCTCT CTT GGG GTA TTT GCG CAA AAA GCC GCCTA GAG TTT GGC-38. A probe for glyceraldehyde-phosphate dehydrogenase (G3PDH, Clontech) wassed as a control probe in all in situ hybridizations.his probe is complementary to nucleotides 751–780 ofhe G3PDH gene, sequence 58-CAC GGA AGG CCAGC CAG TGA GCT TCC CGT-38 (5). Specificity ofhese probes has been previously described by ouraboratory (27).

adiolabeling of Oligonucleotide Probes

38-End labeling of each oligonucleotide probe was carriedut using 35S-dATP. One microliter of the oligonucleotide20 ng/µl) was tailed by the isotope at an incubationemperature of 37°C in a mixture containing 10 µl sterileater, 12.5 µl reaction buffer (sodium cocodylate (120 mM),H 7.2, and dithiothreitol (100 mM)), 2.5 µl cobalt chloride2 mM), and 2 µl terminal deoxynucleotide transferase, pH.2 (all reagents DuPont/NEN) with 7 µl of 35S-dATP (82.5Ci, NEN). Following 60 min incubation, the reaction wastopped by addition of 50 µl of ice-cold sterile water. Theabeled probe was subsequently purified utilizing Bio-spinhromatography columns (Bio-Rad) centrifuged at 1100gavor 4 min (Z382K, Hermel). The volume of 35S-dATP-abeled probes eluted from the column was measured and a3 volume of elute of 1 M dithiothreitol was added. Oneicroliter of the probe was removed and counted by a

iquid scintillation counter (Tricarb, 1500 Packard)-counter to assess the efficiency of radioactive labeling.

n Situ Hybridization

Sections were cryostat cut (219°C) at 15 µm, thaw-ounted onto gelatin/chrome-alum-coated slides, and

tored desiccated at 270°C until further processing.ections were obtained at four rostrocaudal levels suchhat level 1 represented most rostral striatum (1.7 mmnterior from bregma), level 2 represented rostraltriatum (1 mm anterior to bregma), level 3 repre-ented the intermediate striatum (0.26 mm posterior toregma), and level 4 represented the caudal striatum0.92 mm posterior to bregma as defined by Paxinosnd Watson (84); Fig. 1). Before hybridization waserformed, sections were thawed at room temperatureor 30 min, fixed in a 4% paraformaldehyde solution for0 min, rinsed in diethylpyrocarbonate treated water,
nd incubated in 0.25% solution of acetic anhydride in i
.1 M triethalolamine and 0.9% saline (pH 8.0) for aurther 10 min. Sections were then dehydrated in aeries of ascending concentrations of ethanol (1 min in0%, 1 min in 80%, 2 min in 90%, and 1 min in 100%),efatted for 5 min in 100% chloroform, rehydrated for 1in in 100% ethanol and 1 min in 95% ethanol, and

nally air-dried at room temperature. Slides were thenlaced in an air-tight hybridization box containingaper towels soaked in 50% formamide. Labeled probesere added to hybridization solution (50% formamide,3 standard sodium citrate (SCC), 10% dextran sul-ate, and 10 mM dithiothreitol) to a final concentrationf 3 3 106 cpm per milliliter of hybridization solution.ifty milliliters of this solution was added to each of theections, which were then covered with NescofilmBando Chemical Ltd) and incubated in an incubationven (Mini 10, Hybaid) at 42°C for 18 h. Followingncubation, Nescofilm coverslips were floated off with3 SSC and the slides were returned to racks forurther processing. Stringent washes were carried outor 30 min at room temperature in 13 SSC, 30 min at5°C in 13 SSC, and 10 min at 55°C in 0.13 SSC.ections were then dehydrated for 2 min in 70 and 95%thanol and allowed to air-dry. Once dry, the sectionsere placed in a cassette and exposed to film sensitive

o b-emitting isotopes (b-max Hyperfilm, Amersham)or 14 days at 4°C. An autoradiographic 14C microscaletandard (range, 31–833 nCi/g; Amersham) was alsolaced in each cassette. The film was developed inodak D-19 developer for 2–3 min, rinsed in cold water,nd then fixed in Kodak Unifix for 15 min, given a finalinse in water, and left to dry at room temperature.

nalysis of In Situ Hybridization Signal

Densitometric analysis of the PPE-A, PPE-B, and3PDH autoradiographs was performed using a See-

can Image Analysis system. Optical density (OD)easurements were obtained from 18 regions from

oth the lesioned and nonlesioned neostriatum. Thesereas are illustrated in Fig. 1 (modified from Paxinosnd Watson, 84). In striatal levels 1 and 2 the striatumas divided into 6 regions as shown in Figs. 1A and 1B.

n the rostral striatum, levels 1 and 2, the limbic-ssociated areas are located in the ventral striatum,ncluding the core and shell regions of the nucleusccumbens. Sensorimotor-associated areas are locatedn the most dorsal and lateral areas of the striatum,ith the associative areas located in more ventral andedial areas of the striatum. Level 3, the intermediate

triatum, was divided into four regions as shown in Fig.C. Level 4, the caudal striatum, was divided into twoegions as shown in Fig. 1D. Striatal levels 3 and 4ontain virtually no limbic-associated areas, with senso-imotor-associated areas located in the ventral andateral areas of the striatum and the remainder compris-
ng associative input. The nonspecific hybridization

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tatistical Analyses

Data for [3H]mazindol radioligand binding and theatios of PPE-A:G3PDH or PPE-B:G3PDH in in situybridization studies are expressed as means 6 SEM

3

FIG. 1. Diagrammatic representation of the four levels throughndicated. In the most rostral striatum, level 1 (A), and the rostaudate-putamen (dorsolateral (DL), dorsomedial (DM), ventrolateivided into core (NAcc) and shell (NAcs) regions. In the intermedaudate-putamen (dorsolateral (DL), dorsomedial (DM), ventrolateratriatum is divided into two regions of caudate-putamen (dorsal (D) a

alues. Specific reductions in [ H]mazindol radioligand g

inding between all drug-treatment groups were ana-yzed by one-way analysis of variance (ANOVA). For initu hybridization studies the ratio of PPE:A:G3PDH orPE-B:G3PDH within a single region of interest, at

ndividual rostrocaudal levels throughout the striatum,or all drug-treatment groups was analyzed by one-wayNOVA followed by Student–Newman–Keuls post hocnalysis. Combined rotational behavioral data for allrug-treatment groups, over the 21-day period, werenalyzed by two-way ANOVA. Rotational behavioralata for individual drug-treatment groups were ana-yzed, with respect to time, by one-way ANOVA, fol-owed by Student–Newman–Keuls post hoc analysis. Inll tests significance was assigned when P , 0.05.

RESULTS

esion Assessment

All brains were processed for [3H]mazindol radioli-

e rostrocaudal extent of the striatum with regions of measurementstriatal, level 2 (B), the striatum is divided into four regions of(VL), and ventromedial (VM)) with the nucleus accumbens (NAc)striatum, level 3 (C), the striatum is divided into four regions of

L), and ventromedial (VM)). In the caudal striatum, level 4 (D), theentral (V)).

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ehavioral Analysis

In all animals included in the study, injection of 1l/kg sterile water produced spontaneous rotation

TABLE 1

Percentage Reduction of Specific [3H]Mazindol Radioligandinding in the Striatum Following 6-OHDA Lesion andubsequent Vehicle, L-DOPA, Bromocriptine, or Lisuridereatment (n 5 6, P . 0.05, one-way ANOVA)

6-OHDA-lesioned group

% Reduction in specific[3H]mazindol binding

(means 6 SEM)

ehicle 98.61 6 0.83-DOPA (6.5 mg/kg group) 98.32 6 1.02romocriptine (1 mg/kg group) 96.45 6 1.04romocriptine (5 mg/kg group) 96.63 6 1.12isuride (0.01 mg/kg group) 97.25 6 1.46isuride (0.1 mg/kg group) 97.42 6 0.80

FIG. 2. Rotational behavior in the 6-OHDA-lesioned rat followin-OHDA-lesioned rats were treated twice daily with L-DOPA, bromoco the 6-OHDA lesion, in the 2 h following vehicle (h), L-DOPA (6.5romocriptine (1 mg/kg) (r), lisuride (0.1 mg/kg (s), or lisuride (0.01 misuride-treated rats 2 h immediately following the 9 AM ip injectionollowing the 9 AM ip injection. This 1-h period following injection wasean (6SEM) complete rotations contraversive to the lesion (n 5 6;, 0.0001; two-way ANOVA, **P , 0.01, ***P , 0.001 compared to
euls test).
psiversive to the 6-OHDA lesion immediately followingnjection. There was no significant difference in thealine-induced ipsiversive rotation between any of theroups (vehicle, 33 6 6; L-DOPA (6.5 mg/kg), 36 6 4;romocriptine (5 mg/kg), 35 6 8; bromocriptine (1 mg/g), 37 6 5; lisuride (0.1 mg/kg), 39 6 5; lisuride (0.01g/kg), 38 6 4; ipsiversive rotations/2 h, n 5 36,. 0.05, ANOVA).Two-way analysis of variance using drug treatment

nd time as a factor showed significant effects of drugdministration and time on the rotational responsebserved (effect of drug, F5,270 5 198, P , 0.0001; effectf time, F5,270 5 46.97, P , 0.0001; two-way ANOVA).Following a single injection of vehicle, rotation ipsi-

ersive to the 6-OHDA-lesion was observed (33 6 5psiversive rotations/2 h; Fig. 2). With repeated treat-

ent there was no significant alteration in the rota-ional response to vehicle-administration (F7,47 5 0.48,

. 0.05, ANOVA).Following a single injection of L-DOPA (6.5 mg/kg)

nd benserazide (1.5 mg/kg), no significant rotationontraversive to the 6-OHDA lesion was observed22 6 8, ipsiversive rotations/2 h). Rotational behaviorontraversive to the 6-OHDA lesion began on day 518 6 4 contraversive rotations/2 h) with a robust re-ponse developing by day 7 (289 6 39). However, follow-

epeated vehicle, L-DOPA, bromocriptine, or lisuride administration.ine, lisuride, or vehicle. Data shown are net rotations, contraversive/kg) and benserazide (1.5 mg/kg) (j), bromocriptine (5 mg/kg) (m),g) (n) treatment. Locomotion was assessed in vehicle-, L-DOPA-, andcomotion was assessed in bromocriptine-treated groups for 2 h, 1 hllow for differences in drug pharmacokinetics. Data are expressed asct of drug, F5,270 5 198.92, P , 0.0001; effect of time, F5,270 5 46.97,1 administration, one-way ANOVA followed by Student–Newman–

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ng a further 7 days of treatment, a further increase inhe rotational response was observed following subse-uent injections. This potentiated behavioral responseeached a plateau by day 14, and by the end of the1-day treatment period, a 4861% increase in theotational response was observed (875 6 98; Fig. 2)hen compared to day 5. The rotational response to

-DOPA was significantly different to day 1 on days 7,0, 14, 17, and 21 (day 7, P , 0.01; in all other cases, 0.001; Student–Newman–Keuls test).Following a single injection of bromocriptine (5 mg/

g), rotation contraversive to the 6-OHDA lesion wasbserved (14 6 8 contraversive rotations/2 h; Fig. 2).ith repeated treatment there was no significant differ-

nce in the rotational response to bromocriptine admin-stration (5 mg/kg, F7,47 5 0.27, P . 0.05, ANOVA).

Following a single injection of bromocriptine (1 mg/g), rotation contraversive to the 6-OHDA lesion wasbserved (4 6 14 contraversive rotations/2 h; Fig. 2).ith repeated treatment there was no significant differ-

nce in the rotational response to bromocriptine admin-stration (1 mg/kg, F7,47 5 0.48, P . 0.05, ANOVA).

Following a single injection of lisuride (0.1 mg/kg),otation contraversive to the 6-OHDA lesion was ob-erved (2 6 4 contraversive rotations/2 h; Fig. 2). Withepeated lisuride treatment there was no significantlteration in the rotational response to lisuride-dministration (F7,47 5 0.18, P . 0.05, ANOVA).

opographical Organization of StriatalPreproenkephalin-A mRNA

PPE-A expression is represented as a ratio of theybridization signal of PPE-A:G3PDH, as detailed un-er Materials and Methods. Mean values (6SEM) forhe ratio PPE-A:G3PDH in L-DOPA, bromocriptine,isuride, or vehicle treatment groups are documented inable II. In each region, at each level, three statisticalomparisons of PPE-A expression levels are made: (i)etween lesioned and nonlesioned striatum in eachroup, (ii) between the lesioned striata of drug-treatedroups compared to the vehicle-treated group, and (iii)etween drug-treated or vehicle-treated lesioned striatand that of the L-DOPA-treated lesioned striata.

PE-A Expression Following Vehicle Administration

Following 6-OHDA lesion and subsequent 21-dayehicle administration no significant elevations in theatio of PPE-A:G3PDH were observed in any region ofhe most rostral striatum (level 1, P . 0.05, ANOVA;able II). In the rostral striatum (level 2) the ratio ofPE-A:G3PDH expression was elevated in the dorsolat-ral, dorsomedial, and ventrolateral regions of theaudate-putamen when compared to the nonlesionedide (DL, DM, P , 0.05; VL, P , 0.001; Fig. 3, Table II).
n the intermediate (level 3) and caudal striatum (level s
), the ratio of PPE-A:G3PDH was not elevatedP . 0.05; Table II).

PE-A Expression Following L-DOPA Administration

Following 21-day L-DOPA (6.5 mg/kg) and bensera-ide (1.5 mg/kg) administration, the ratio PPE-A:3PDH was increased compared with the vehicle-

reated group at all rostrocaudal levels, in all regions ofhe caudate-putamen (except dorsal, level 4) but not inhe nucleus accumbens. The ratio of PPE-A:G3PDHxpression was significantly increased (compared tohe nonlesioned striata) in the dorsolateral, dorsome-ial, ventrolateral, and ventromedial regions of theaudate-putamen in the most rostral striatum (level 1,L, DM, and VL, P , 0.001; VM, P , 0.05). The ratio ofPE-A:G3PDH was further increased (over lesion fol-

owed by vehicle treatment) in dorsolateral and dorso-edial regions of the caudate-putamen in the most

ostral striatum (level 1, DL, P , 0.001; DM, P , 0.05).n the rostral striatum (level 2) the ratio of PPE-A:3PDH was significantly increased over the nonle-

ioned striata in dorsolateral, dorsomedial, and ventro-ateral areas of the caudate-putamen (DL and VL,

, 0.01; DM, P , 0.001). No alterations in the ratio ofPE-A:G3PDH was observed in the nucleus accumbensore or shell in the lesioned striata of the rostraltriatum (level 2) when compared to the nonlesionedtriata (P . 0.05). The ratio of PPE-A:G3PDH in theesioned striata of the rostral striatum (level 2) was notignificantly different from that of the lesioned striatan the vehicle treated group in any area of the caudate-utamen or the nucleus accumbens (P . 0.05). In thentermediate striatum (level 3) the ratio of PPE-A:3PDH was increased over the nonlesioned striata in

he dorsolateral, dorsomedial, and ventrolateral cau-ate-putamen (DL, DM, and VL, P , 0.05), with noignificant increases observed in any other areas of theaudate-putamen or nucleus accumbens (P . 0.05).urthermore, no significant differences between theehicle-treated and L-DOPA-treated groups were ob-erved in any areas of the intermediate striatum (level, P . 0.05). In the caudal striatum (level 4) followingepeated L-DOPA administration no significant differ-nce between lesioned and nonlesioned striata wasbserved (P . 0.05). Furthermore, no significant differ-nces between the lesioned striata of vehicle-treated or-DOPA-treated groups were observed (P . 0.05).

PE-A Expression Following BromocriptineAdministration

Following 21-day high-dose bromocriptine adminis-ration (5 mg/kg, b.d.) lesion-induced increases in theatio of PPE-A:G3PDH were decreased and were notignificantly different from those of the nonlesioned
ide in any region throughout the striatum (P . 0.05;

TAB

Ratio of Preproenkephalin-A (PPE-A) to GlycemRNA Hybridization Signal in the 6-OHDA-LesL-DOPA (6.5 mg/kg) and Benserazide (1.5 mg/kg0.01 mg/kg) Administrtion

Note. Data represent mean (6SEM) optical density racompared to nonlesioned striatum in same group; b*ipsilateral striatum of vehicle group; and c* P , 0.05,

LE 2

raldehyde 3-Phosphate Dehydrogenase (G3PDH)ioned Rat Following 21-Day, Twice Daily, Vehicle,), Bromocriptine (5 or 1 mg/kg), or Lisuride (0.1 or

tios (n 5 6; a* P , 0.05, a** P , 0.01, and a*** P , 0.001P , 0.05, b** P , 0.01, and b*** P , 0.001 compared toc** P , 0.01, and c*** P , 0.001 compared to ipsilateral

striatum of L-DOPAgroup; one-wayANOVAfollowed by Student–Newman–Keuls post hoc analysis).

211

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ig. 3, Table II). In contrast, following 21-day low-doseromocriptine administration (1 mg/kg, b.d.), the reduc-ion in the lesion-induced increase of PPE-A:G3PDHas not observed, with significantly increased levels ofPE-A expression in all regions of the most rostraltriatum (level 1, DL, P , 0.001; DM, P , 0.01; VL andM, P . 0.05), with no change in the core or shellegions of the nucleus accumbens (P . 0.05; Fig. 3,able II). Similarly, in the rostral striatum (level 2)ollowing 21-day low-dose bromocriptine administra-ion (1 mg/kg, b.d.), the ratio of PPE-A:G3PDH wasignificantly increased in all regions of the caudate-utamen (DL, P , 0.001; DM, P , 0.05; VL, P , 0.01;M, P , 0.05) with no changes in the core or shellegions of the nucleus accumbens (P . 0.05; Table II).n the intermediate striatum (level 3) following low-ose bromocriptine (1 mg/kg, b.d., 21 days), the ratio ofPE-A:G3PDH was significantly increased in dorsolat-

FIG. 3. Pseudocolour transformations of autoradiographs of inreproenkephalin-A (PPE-A) or preproenkephalin-B (PPE-B) in theection) following 21-day, twice daily, vehicle, L-DOPA (6.5 mg/kg) and.01 mg/kg) treatment in the 6-OHDA-lesioned rat model of Parkinso

ral, dorsomedial, and ventrolateral regions of theaudate-putamen (all regions, P , 0.05). In the caudaltriatum (level 4) no significant difference in the ratio ofPE-A:G3PDH between lesioned and nonlesionedtriata were apparent following low-dose bromocriptinedministration (Table II). The ratio of PPE-A:G3PDHas further increased (over lesion followed by vehicle

reatment) in all areas of the most rostral striatumlevel 1, DL, P , 0.01; DM, VL, and VM, P , 0.05).

PE-A Expression Following Lisuride Administration

Following both high-dose (0.1 mg/kg, b.d., 21 days)nd low-dose (0.01 mg/kg, b.d., 21 days) lisuride treat-ent there was no significant difference between the

esioned and nonlesioned striata in any region through-ut the caudate-putamen or nucleus accumbensP . 0.05; Table II).

hybridization utilizing an oligonucleotide probe targeted againststral striatum (level 2, the lesioned striata are on the right of theserazide (1.5 mg/kg), bromocriptine (5 or 1 mg/kg), or lisuride (0.1 or

disease.

situroben

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opographical Organisation of StriatalPreproenkephalin-B mRNA

PPE-B expression is represented as a ratio of theybridization signal of PPE-B:G3PDH, as detailed un-er Materials and Methods. Mean values (6SEM) forhe ratio PPE-B:G3PDH in L-DOPA, bromocriptine,isuride, or vehicle treatment groups are documented inable III. In each region, at each level, three statisticalomparisons of PPE-B expression levels are made (i)etween lesioned and nonlesioned striatum in eachroup, (ii) between the lesioned striata of drug-treatedroups compared to the vehicle-treated group, and (iii)etween drug-treated or vehicle-treated lesioned striatand that of the L-DOPA-treated lesioned striata.

PE-A Expression Following Vehicle, Bromocriptine,or Lisuride Administration

Following 6-OHDA lesion and subsequent 21-dayehicle administration, there was no significant alter-tion in the ratio of PPE-B:G3PDH throughout theaudate-putamen or core or shell regions of the nucleusccumbens in the most rostral, rostral, or intermediatetriatum (levels 1, 2, and 3, respectively; P . 0.05;able III, Fig. 3). Similarly, no significant alteration inhe ratio of PPE-B:G3PDH between the lesioned striataf both high- and low-dose bromocriptine or lisurideere observed (Table III).

PE-A Expression Following L-DOPA Administration

In contrast to vehicle, bromocriptine, or lisuridedministration, following repeated L-DOPA treatmentmarked significant increase in the ratio of PPE-B:3PDH was observed in the dorsolateral, dorsomedial,nd ventrolateral regions of the caudate-putamen inhe most rostral striatum (level 1, P , 0.01, all re-ions). In the most rostral striatum (level 1) no signifi-ant differences in the ratio of PPE-B:G3PDH werebserved between lesioned and nonlesioned striata inhe ventromedial region of the caudate-putamen or inhe core or shell of the nucleus accumbens (P . 0.05,able III).In the rostral striatum (level 2) a similar pattern of

PE-B:G3PDH was observed when compared to theost rostral striatum (level 1) following L-DOPA treat-ent. In the dorsolateral, dorsomedial, and ventrolat-

ral regions of the rostral striatum (level 2; Fig. 3, TableII) a significant increase in the ratio of PPE-B:G3PDHas observed in the lesioned striata when compared to

he nonlesioned (P , 0.001, all regions). No significantifference between lesioned and nonlesioned caudate-utamen was apparent in the ventromedial caudate-utamen or the core or shell of the nucleus accumbensn the rostral striatum (level 2, P . 0.05, all regions).

In the intermediate striatum (level 3), significant (

ncreases in the ratio of PPE-B:G3PDH were observedn the dorsolateral, dorsomedial, and ventrolateralegions of the caudate-putamen (P , 0.001, all re-ions), with no significant difference observed in theentromedial region (P . 0.05). In the caudal striatumlevel 4), a significant increase in the ratio of PPE-B:3PDH was observed in both the dorsal and ventralreas of the caudal caudate-putamen in the lesionedtriata when compared to the nonlesioned striataP , 0.001, both regions, Table 3).

DISCUSSION

In the 6-OHDA-lesioned rat, repeated L-DOPAadmin-stration leads to a potentiation in the behavioralesponse to L-DOPA. A similar increase in behavioralesponse has been previously reported following re-eated L-DOPA, apomorphine, or bromocriptine admin-stration following a single ‘‘priming’’ dose of apomor-hine (8, 9, 14, 26, 27, 30, 39). We have previouslyharacterized the pharmacology of this response anduggested that it may be a useful model for the elucida-ion of the cellular and molecular mechanisms underly-ng L-DOPA- and dopamine agonist-induced dyskinesia38). In contrast to L-DOPA de novo treatment withromocriptine or lisuride is not associated with aotentiation in response.In this study ‘‘low-dose’’ bromocriptine and lisuride

dministration were calculated to reflect ‘‘clinicallyelevant’’ doses which fall within the range of dosesemonstrated, in the clinical situation, to be associatedith a lower incidence of dyskinesia. Thus, the dose ofromocriptine used in this study, 2 mg/kg/day, is compa-able to a dose of 140 mg/day in man. This falls withinhe range used in man, 2.5–300 mg/day (60, 61, 80, 83).imilarly, the dose of lisuride used in this study of 0.02g/kg/day is comparable to a dose of 1.4 mg/day inan. This falls within the range used in man, 1.2–10g/day (59, 88). The ‘‘high-dose’’ administration re-

ects, for comparison, doses of bromocriptine and li-uride previously utilized in rodent models of Parkin-on’s disease to elicit behavioral responses (38, 39, 58,6).The neural mechanisms underlying the development

f L-DOPA- and dopamine agonist-induced dyskinesian parkinsonism involve abnormalities in the directnd indirect striatal efferent pathways, such that theedial segment of the globus pallidus becomes underac-

ive (24). Several studies have demonstrated that thebnormalities observed in the striatal output pathwaysre not simply due to alterations in striatal dopamine1 or D2 receptor number or affinity (16, 31, 95),lthough recent studies have suggested that dopamine3 receptor abnormality may play a role in dyskinesia

9). However, it is clear that dopamine receptors control

TABLE 3

Ratio of Preproenkephalin-B (PPE-B) to Glyceraldehyde 3-Phosphate Dehydrogenase (G3PDH)mRNA Hybridization Signal in the 6-OHDA-Lesioned Rat Following 21-Day, Twice Daily, Vehicle,L-DOPA (6.5 mg/kg) and Benserazide (1.5 mg/kg), Bromocriptine (5 or 1 mg/kg), or Lisuride (0.1 or0.01 mg/kg) Administration

Note. Data represent mean (6SEM) optical density ratios (n 5 6; a* P , 0.05, a** P , 0.01, and a*** P , 0.001compared to nonlesioned striatum in same group; b* P , 0.05, b** P , 0.01, and b*** P , 0.001 compared toipsilateral striatum of vehicle group; and c* P , 0.05, c** P , 0.01, and c*** P , 0.001 compared to ipsilateralstriatum of L-DOPAgroup; one-wayANOVAfollowed by Student–Newman–Keuls post hoc analysis).

214

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he synthesis of neuropeptides utilised as cotransmit-ers in the efferent pathways of the striatum. There-ore, we hypothesized that repeated stimulation ofopamine receptors leads to neuropeptide alterationshat may influence the activity of the direct and indi-ect striatal pathways, resulting in decreased activityf the medial segment of the globus pallidus andltimately dyskinesia. Underpinning this hypothesisre findings that enkephalin and dynorphin modulateABA and glutamate release, respectively, in the palli-al complex (40, 41, 68, 69). A potential role of peptidebnormalities in dyskinesia is suggested by the abilityf opioid antagonists to reduce the behavioral enhance-ent following repeated L-DOPA administration in the

-OHDA-lesioned rat (14, 39, 77).In this study, we demonstrate that PPE-A expression

s increased and PPE-B decreased following 6-OHDAesion in animals receiving vehicle treatment. Thesendings are in keeping with many previous reports (28,1, 32, 99, 103). However, the topographical analysis ofhese changes is of interest. The changes are onlybserved in dorsal regions of the striatum that wouldorm part of the sensorimotor circuit. The widely de-cribed elevation in PPE-A is only observed in theostral caudate-putamen. This is in keeping with otherontopographical studies (e.g., 31) that have focusedheir attention upon this region. Repeated administra-ion of L-DOPA elicits an enhanced behavioral responsend is associated with increased PPE-A and PPE-BRNA expression. These findings are similar to those

reviously demonstrated with apomorphine (27). Theopographical organization of these increases furtherupports the involvement of opioid peptides in theehavioral enhancement observed following repeated-DOPA or apomorphine administration as increasedPE-A and PPE-B mRNA expression is observed inreas of the striatum associated with the control ofotor activity, i.e., the dorsolateral areas of the rostral

audate-putamen (27). However, following repeateddministration of anti-parkinsonian agents that do notause a behavioral enhancement, and are not associ-ted with dyskinesia when administered de novo in thelinical setting, i.e., bromocriptine and lisuride, noncrease in PPE-B mRNA expression is observed andPE-A mRNA expression levels are normalized torelesion levels. However, it is of interest to note that,n certain regions of the rostral striatum, low-doseromocriptine failed to reduce the lesion-induced in-rease in PPE-A expression to prelesion levels and wasot associated with an increased behavioral response toepeated treatment, perhaps indicating a more promi-ent role for PPE-B in the enhanced behavioral re-ponse as has been previously described (15, 26).Thus, increased dynorphinergic and, to a lesser ex-

ent, enkephalinergic transmission, generated from
nhanced synthesis of PPE-A and PPE-B precursors, i
rovides a potential mechanism for the appearance ofyskinesia following repeated L-DOPA or dopaminegonist administration in the treatment of Parkinson’sisease. It has been suggested that dyskinesias ineneral arise due to underactivity of the medial seg-ent of the globus pallidus and other output nuclei of

he basal ganglia (24, 72). We have previously proposedhat increased enkephalinergic transmission, actingia delta opioid receptors, would decrease GABA re-ease in the GPl (41, 69). This would, indirectly, lead toncreased inhibition of GPm by disinhibiting both thePl to GPm pathway and the inhibitory GPl to subtha-

amic nucleus pathway, leading to a reduced subtha-amic excitatory drive to the GPm. Similarly, increasedynorphinergic transmission in the GPm, acting onresynaptic k-opioid receptors on glutamatergic effer-nts from the subthalamic nucleus, would decrease thelutamatergic excitation of the GPm (see Ref. 39 foreview). To support this hypothesis, positron emissionomography has demonstrated increased opioid neuro-eptide transmission in the basal ganglia of Parkin-on’s disease patients displaying L-DOPA-inducedyskinesia when compared to nondyskinetic L-DOPA-reated parkinsonian patients (85).

The factors underlying the increase in PPE-A andPE-B mRNA expression following repeated L-DOPAdministration, in contrast to the decrease in PPE-A torelesion levels and lack of an increase in PPE-Bollowing repeated bromocriptine or lisuride administra-ion, may include the fact that bromocriptine andisuride have longer half-lives than L-DOPA or thathese dopamine agonists are selective for specific dopa-ine receptor subtypes.Bromocriptine and lisuride are dopamine receptor

gonists with long half-lives of approximately 8 and 3, respectively, compared to 1.5 h for L-DOPA (1, 70, 82).he longer acting nature of both bromocriptine and

isuride may play a role in the lack of behavioralotentiation and the normalization of neuropeptiderecursors. In support of this, previous studies haveemonstrated that the lesion-induced increase in PPE-ARNA expression and enkephalin peptide levels can be

ifferentially modulated by dopamine receptor ago-ists, depending on the mode of administration. In the-OHDA-lesioned rat, continuous administration, vian osmotic minipump (for 21 days), of the dopamine2-like receptor agonist quinpirole reduced levels ofPE-A mRNA to prelesion levels. However, intermit-

ent treatment (i.e., single daily injections for 21 days)ith quinpirole, caused no alteration in the lesion-

nduced increase in PPE-A mRNA expression (31).imilar studies in the MPTP-lesioned nonhuman pri-ate, utilizing the dopamine D2 receptor agonist U

1356A, have demonstrated a decrease in the lesion-
nduced elevation of PPE-Afollowing continuous admin-

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stration when compared to intermittent administra-ion (76).

Following dopamine denervation and subsequentopamine receptor stimulation in the 6-OHDA-lesionedat, PPE-B mRNA and dynorphin peptide levels areassively upregulated (27, 28, 31, 52, 65). This upregu-

ation of dynorphin synthesis, in a manner similar tohat of enkephalin, can be differentially modulated byopamine receptor agonists depending on the nature ofdministration, i.e., fluctuating or nonfluctuating. Inter-ittent treatment (i.e., single daily injections) with a

opamine D1 receptor agonist (SKF 38393) for 21 daysncreased the level of PPE-B mRNA, while continuousreatment, via an osmotic minipump, did not alterPE-B (31). Therefore, depending on the mode ofdministration, dopamine receptor stimulation in the-OHDA-lesioned rat model of Parkinson’s disease hasontrasting effects on PPE-A and PPE-B mRNA expres-ion levels within the basal ganglia. This may havemportant consequences as clinical and experimentalvidence suggests that dopamine receptor agonistsith long half-lives, continuous treatment through

smotic minipumps, or L-DOPAcontrol-release prepara-ions elicit fewer dyskinetic side effects in both parkin-onian patients and in MPTP-treated nonhuman pri-ates (34, 56, 61, 66, 80).A second mechanism by which bromocriptine and

isuride may cause the normalization of PPE-A and aack of elevation in PPE-B mRNA expression and thusot lead to dyskinesia is that these compounds do nottimulate D1 dopamine receptors (25, 90). If bromocrip-ine and lisuride only stimulate the dopamine D2eceptors on striatal medium spiny neurons, PPE-Axpression, which is modulated by dopamine D2 recep-ors, may be reduced and PPE-B expression wouldemain unaltered. In support of this hypothesis, L-OPA increased activator protein-1 (AP-1) and cyclicMP-responsive element (CRE)-binding protein (CREB)NA-binding activity in the striatum of the 6-OHDA-

esioned rat, whereas bromocriptine did not (54). AP-1nd CREB binding sites have been demonstrated to bef critical importance in promoting neuropeptide genexpression (19, 57). However, since intermittent admin-stration of the D2-like dopamine receptor agonistuinpirole also results in a lack of reduction in PPE-A31) and the D2-like dopamine receptor agonist (1)-4-ropyl-9-hydroxynaphthoxazine (PHNO) elicits dyski-esia following de novo administration in MPTP-reated primates (18, 33), the lack of D1 dopamineeceptor stimulation seems unlikely to be the soleechanism underlying the normalization of PPE-A andPE-B mRNA expression and the lack of dyskinesialicited following de novo administration of bromocrip-ine or lisuride. The lack of dyskinetogenic action ofromocriptine may result from the fact that, unlike
HNO and quinpirole, it is a more potent agonist at D2 p
ather than D3 receptors (29, 100). Alternatively, aombination of pharmacological specificity and pharma-okinetics may determine the propensity of a D2-likeopamine receptor agent to elicit dyskinesia.This proposed role for enkephalin and dynorphin

nderlying dyskinesia suggests the intriguing possibil-ty that antagonists acting at the k- and d-opioideceptors may provide a useful adjunct therapy to-DOPA, and dopamine agonists, in the treatment ofarkinson’s disease. Furthermore, in a manner similar

o that of bromocriptine, novel dopamine D2 receptorgonists with long half-lives, e.g., ropinirole or cabergo-ine, may provide a nonfluctuating response and normal-ze peptide levels in striatal output pathways and thusould not be expected to elicit dyskinesia when admin-

stered de novo. Alternatively, compounds with theapacity to normalize PPE-A expression may be usefuln reducing dyskinetic side effects of long-term dopa-

ine replacement therapy. A maintained cortical inputo the striatum, through activation of NMDA receptors,s required to maintain the elevated PPE-A expressionollowing dopamine denervation (13, 36, 97). Further-

ore, single-photon emission tomography demon-trates that areas of the cerebral cortex projecting tohe striatum, namely, the supplementary motor areand primary motor area, are overactive in parkinso-ian patients displaying L-DOPA-induced dyskinesia

86). Therefore, coadministration of NMDA antagonistsith dopamine replacement therapy may act to de-

rease PPE-A mRNA expression and lead to a reductionn dyskinesia. In keeping with this speculation, coad-

inistration of NMDA antagonists to MPTP-lesionedrimates and small-scale clinical trials in patients witharkinson’s disease have demonstrated a reduction of-DOPA-induced dyskinesia without loss of anti-arkinsonian efficacy (78, 98). However, it is not clearhether the acute nature of the challenge in thePTP-lesioned primate study would be sufficient to

rovide downregulation in striatal neuropeptides. Aurther treatment strategy suggested by the presenttudy is that, in patients already ‘‘primed’’ to elicityskinesia following the administration of L-DOPA oropamine receptor agonists, it may be possible toeduce dyskinesia by normalizing the elevated levels ofPE-A without increasing PPE-B mRNA expression.his could be achieved by administration of high-dosesf nonfluctuating dopamine receptor stimulation withgents with long half-lives such as ropinirole or caber-oline or by pump infusion of shorter acting agentsuch as apomorphine. Indeed, high-dose pergolide ornfusions of apomorphine has been demonstrated to bessociated with a much lower incidence of dyskinesiahen administered to patients previously displaying

evere L-DOPA-induced dyskinesia (20, 91).In conclusion, enhanced neuropeptide precursor ex-

ression in the striatal output pathways may not

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imply reflect overactivity of striatal output pathwaysut may play a pivotal role in the generation of-DOPA- and dopamine agonist-induced dyskinesiasollowing repeated treatment of Parkinson’s disease.

oreover, opioid receptor antagonists, or other meansf reducing opioid transmission, may provide a novelherapeutic approach to the treatment of dyskinesia inarkinson’s disease.

ACKNOWLEDGMENTS

The authors acknowledge the financial support of the Medicalesearch Council (UK).

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Effect of Repeatedl-DOPA, Bromocriptine, or Lisuride Administration on Preproenkephalin-A and Preproenkephalin-B mRNA Levels in the Striatum of the 6-Hydroxydopamine-Lesioned Rat