Dysfunction of the cortico-basal ganglia-cortical loop in a ratmodel of early parkinsonism is reversed by metabotropicglutamate receptor 5 antagonism

Abid Oueslati,1 Nathalie Breysse,2 Marianne Amalric,2 Lydia Kerkerian-Le Goff,1 and Pascal Salin11Interactions Cellulaires, Neurodegenerescence et Neuroplasticite, UMR 6186, CNRS-Universite de la Mediterranee, 31 cheminJoseph Aiguier, 13402 Marseille Cedex 20, France2Neurobiologie de la Cognition, CNRS UMR 6155, Marseille, France

Keywords: akinesia, basal ganglia, cytochrome oxidase, MPEP, neuropeptides, Wistar rats

Abstract

This study examined the cellular correlates of the akinetic deficits produced in Wistar rats by discrete bilateral 6-hydroxydopamine(6-OHDA) striatal infusions in the dorsolateral striatum, mimicking the preferential denervation of the motor striatal territory in earlysymptomatic stage of Parkinson’s disease (PD). Intraneuronal gene expression of cytochrome oxidase subunit I (COI), a metabolicindex of neuronal activity, was increased in the subthalamic nucleus, substantia nigra pars reticulata and decreased in frontal corticalareas, but paradoxically unchanged in the striatum, globus pallidus, entopeduncular nucleus and ventrolateral thalamic nucleus.Neither preproenkephalin A nor preprotachykinin mRNA expression, markers of striatal projection neurons, were modified in thedenervated striatal area despite 90% loss of dopamine (DA) terminals. Preproenkephalin A mRNA expression was however,decreased in the nondepleted striatal region, suggesting compensatory increase of dopamine tone from those spared areas. Achronic treatment with the metabotropic glutamate receptor 5 (mGluR5) antagonist 2-methyl-6-(phenylethylnyl)-pyridine (MPEP),which alleviated the akinetic disorders produced by the lesion, reversed the lesion-induced variations of COI gene expression,moderately increased this marker in the structures unaffected by the lesion and did not modify the striatal neuropeptides geneexpression. These data suggest that the expression of akinetic deficits in early parkinsonism is associated with focused metabolicchanges in the cortico-basal ganglia-cortical loop downstream of the striatum and pallidal complex.

Introduction

Parkinson’s disease (PD) is a neurodegenerative disorder characterizedby the progressive loss of the dopaminergic neurons in the substantianigra pars compacta (SNc). Besides dopamine (DA) depletion,reactive increase in glutamatergic drive in the basal ganglia (BG)has been suggested to be central to the expression of PD motorsymptoms. Changes in glutamatergic systems include overactivecorticostriatal transmission (Lindefors & Ungerstedt, 1990; Calabresiet al., 1993; Meshul et al., 1999) and abnormal ‘bursting’ pattern ofneuronal activity in the subthalamic nucleus (STN; Bevan et al.,2002). Accordingly, the success of the surgical treatment of advancedPD by STN high frequency stimulation has demonstrated thetherapeutic value of strategies bypassing the dopamine system torestore balance in the BG circuitry (Benabid et al., 2000). Currentefforts are devoted to find pharmacological tools to counteractglutamate overactivity, with considerable attention to selective ligandsor modulators of metabotropic glutamate receptor (mGluR) subtypes(Rouse et al., 2000; Gasparini et al., 2002; Marino et al., 2002a,2003). Recent studies in animal models indicate that antagonists ofgroup I mGluRs, especially of mGluR5, as well as agonists ofgroup II and III mGluRs hold promise as a therapeutic approach to PD(Senkowska & Ossowska, 2003; Marino et al., 2003).

Our knowledge on the pathophysiological functioning of BG in PDis mostly derived from experimental models of late stages of thedisease, based on extensive lesion of the dopaminergic neurons.Discrete 6-hydroxydopamine (6-OHDA)-induced lesions of thedorsolateral part of the striatum produce consistent deficits in reactiontime tasks in rats that are related to the akinesia observed in PDpatients (Amalric et al., 1995b). We have shown that these akineticdeficits are alleviated by different pharmacological or surgicaltreatments aimed at counteracting glutamate overactivity (Amalricet al., 1995a; Baunez et al., 1995; Breysse et al., 2002). Although thismodel does not reproduce the asymmetrical character of the dener-vation in the early stage of PD, it mimics the preponderant denervationof the dorsal and caudal putamen at such stage and allows fineassessment of motor deficits linked to topographically restricted lossof dopamine afferents. We here used this model of early parkinsonismto investigate the neural substrates of akinesia and identify the sites ofaction of a chronic systemic treatment with a selective antagonist ofmGluR5, 2-methyl-6-(phenylethylnyl)-pyridine (MPEP). As a firststep, we recently showed that expression of akinetic deficits correlateswith increased gene expression of cytochrome oxidase subunit I (COI,a marker of oxidative neuronal activity) in the subthalamic nucleusand of GAD67 (isoform of the GABA-synthesizing enzyme) in thesubstantia nigra pars reticulata (SNr) but not in the entopeduncularnucleus (EP) (Breysse et al., 2003). Whereas a correlation betweenmRNA levels and neuronal activity has been established for COI(Hevner & Wong-Riley, 1993; Vila et al., 2000), it is still debated for

Correspondence: Dr Pascal Salin, as above.E-mail: salin@ibdm.univ-mrs.fr

Received 30 June 2005, revised 12 September 2005, accepted 19 October 2005

European Journal of Neuroscience, Vol. 22, pp. 2765–2774, 2005 ª Federation of European Neuroscience Societies

doi:10.1111/j.1460-9568.2005.04498.x

GAD67, which limits the interpretation of these data. The presentstudy therefore investigated the differential responsiveness of the BGoutput structures using COI mRNA levels and extended this analysisto the complete cortico-BG-cortical motor circuitry. In addition,striatal mRNA levels of the neuropeptide precursors, preproenkeph-alin A and preprotachykinin, were measured to segregate the two mainpopulations of striatal output neurons.

Materials and methods

Animals and behavioural testing

Male Wistar rats, weighing 300–350 g at the time of surgery, werehoused in groups of two per cage and maintained under a12-h light : 12-h dark cycle (07:00 h to 19:00 h, lights off). As thebehavioural task involves food reinforcement, rats were food-restric-ted over the entire testing period to maintain a steady level ofmotivation. They received 15–17 g food supply per day, to keep themat approximately 85% of the free-feeding weight. Water was providedad libitum. The experimental protocols, involving animals and theircare, strictly conformed to the guidelines of the French Agricultureand Forestry Ministry (decree 87–848); October 19, 1987.Rats were tested in a behavioural task measuring the performance in

an operant reaction time (RT) task before and after 6-OHDA lesionsfollowed or not by MPEP chronic treatment. As previously shown(Amalric et al., 1995b), the RT task has been designed to assess thedegree of akinesia in rats bearing partial dopamine lesion. In brief, ratswere trained to release a lever quickly at the onset of a cue-light thatwas presented after four randomly generated intervals. RT wasmeasured as the time elapsing between the onset of the light and therelease of the lever. Each correct response, corresponding to RTsbelow 600 ms, was rewarded by a food pellet. After stabilization ofoptimal baseline (2–3 months training), performance was recorded forsix consecutive preoperative days, each daily session ending after100 trials (lasting between 15 and 30 min). After a 1-week postop-erative period, they were then tested daily for 24 sessions up to32 days postsurgery (six consecutive sessions between postoperativedays 9 and 14 to establish the effect of the lesion, and 18 sessions fromdays 15–32 to assess the effects of the MPEP treatment). The index ofakinesia was given by the number of delayed responses with RTslonger than 600 ms and the increase of RTs. The behavioural datahave been reported previously (Breysse et al., 2002, 2003) and willnot be further detailed except for a correlation measured between thelevel of akinesia (measured on the last day of testing) and the cellularreactivity assessed by measuring COI mRNA levels at the level of theSNr and the frontal cortex. All cellular analyses were performed on thesame animals at completion of their behavioural test.

6-OHDA lesions and drug treatment

Animals were anaesthetized with an i.m. injection of xylazine(15 mg ⁄ kg) ⁄ ketamine (100 mg ⁄ kg) and secured in a Kopf stereotaxicapparatus with the incisor bar positioned )3.0 mm under the interauralline for surgical procedures based on the coordinates of Paxinos &Watson (1986). Twenty-one animals received a bilateral infusion of 6-OHDA (Sigma-Aldrich, Lyon, France; 4 lg ⁄ lL, 3 lL per side over9 min) and six animals received infusion of vehicle (ascorbic acidsolution, 0.1 mg ⁄mL in 0.9% saline) in the dorsal striatum at thefollowing coordinates: AP, +9.2 mm; L, ± 3.5 mm; according tointeraural coordinates and DV, )4.8 mm from the skull. Animals didnot show aphagia and adipsia, contrary to that reported after extensivelesion. After a postlesional period of 14 days, the lesion group was

then divided into three groups treated either with saline (vehicle-treated, n ¼ 7) or MPEP at two doses (1.5 mg ⁄ kg n ¼ 7 or 3 mg ⁄ kgn ¼ 7). MPEP hydrochloride (Novartis, Basel, Switzerland) wasdissolved in distilled water and injected i.p., in a volume of 1 mL ⁄ kg,daily for 18 days (days 15–32 after surgery).

Tissue preparation

Animals were killed by decapitation 1 h after the last injection. Thebrains were quickly removed, frozen in dry ice, and kept at )80 �Cuntil cryostat sectioning. Coronal (10-lm thick) tissue sections werecut at )20 �C at the level of the striatum (between interauralcoordinates AP, 10.2–9.2 mm; based on the stereotaxic atlas ofPaxinos & Watson (1986), globus pallidus (GP, 8.2–7.20), EP andthalamic nuclei (AP, 6.44–6.00), SNr (AP, 4.20–3.20), STN (AP, 5.40–4.70). The sections were then mounted onto SuperFrost plus glassslides (Fisher Scientific, Elancourt, France). Tissue sections werestored at )80 �C until specific treatment.

3H-mazindol binding to dopamine uptake sites

The loss of DA terminals in the striatum was assessed as an index ofthe extent of the DA denervation by analysis of 3H-mazindol bindingto DA uptake sites, as described previously (Javitch et al., 1985; Salinet al., 2002). Briefly, brain sections were air-dried and rinsed for 5 minin 50 mm Tris buffer with 120 mm NaCl and 5 mm KCl. They werethen incubated for 40 min with 15 nm [3H]-mazindol (NEN DuPont;specific activity, 17 Ci ⁄mm) in 50 mm Tris buffer containing 300 mm

NaCl and 5 mm KCl added with 0.3 mm desipramine to block thenoradrenaline uptake sites. Sections were rinsed twice for 3 min in theTris incubation buffer and for 10 s in distilled water and were air-dried. Autoradiograms were generated by apposing the sections to 3H-sensitive screen (Raytest, Courbevoie, France) for 21 days and werefurther quantified with a beta imager (Fuji-Bas 5000).

In situ hybridization

Radiolabelled antisense synthetic DNA probes (43–48 mer) were usedfor preprotachykinin, preproenkephalin A in situ hybridization, aspreviously described (Salin et al., 2002). Briefly, the oligoprobes were3¢ end-labelled by terminal deoxynucleotide transferase (Roche,Meylan, France) with 35S-dATP (1300 Ci ⁄mmol, NEN Life ScienceProducts, MA, USA). The probes were then purified from unincor-porated nucleotides on sephadex mini spin columns (Roche). Aradiolabelled antisense RNA probe was used for COI mRNAdetection. The cDNA, corresponding to nucleotides 5308–6218 withinthe gene coding for COI of the rat mitochondrial genome (EMBOdatabank, ref MIRNXX) was generously provided by Dr E. Hirsch(Vila et al., 2000). The antisense RNA probe was transcribed from1 lg of linearized plasmid containing the cDNA fragment, using a T3polymerase, 2.5 lL 35S-UTP (1200 Ci ⁄mmol) with ATP, CTP andGTP in excess.Slide-mounted sections were postfixed for 5 min in 3% parafor-

maldehyde, incubated for 30 min in prehybridization buffer[2 · standard saline citrate (SSC) and 1 · Denhardt’s solution] andacetylated for 10 min with 0.25% acetic anhydride in 0.1 m trietha-nolamine. Then, the tissue was treated for 30 min with 0.1 m Tris-glycine, dehydrated and air dried. When using oligoprobes, eachsection was covered with 35 lL of hybridization buffer (50%formamide, 1 · Denhardt’s, 1% yeast tRNA, 1% sheared salmonsperm DNA, 10% dextran sulphate, 4 · SSC) containing 20 nmol of

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radiolabelled probe (�400 000 cpm) and incubated for 12–14 h at45 �C in humid chambers. After hybridization, the sections werewashed sequentially in 1 · SSC for 1 h at room temperature, 1 · SSCfor 1 h at 45 �C, 0.1 · SSC for 1 h at 45 �C. For riboprobe, eachsection was covered with 20 lL of the same hybridization buffercontaining 6 ng of COI probe (�2.5 · 106 cpm) and incubated for 4 hat 50 �C. Posthybridization treatments included washes at 52 �C with50% formamide in 2 · SSC and incubation with RNAse A(100 mg ⁄mL) in 2 · SSC at 37 �C for 30 min. In both cases, thesections were then dehydrated and apposed to Kodak BioMax MRfilms in light-tight cassettes for 2–10 days. Sections processed forcellular analysis were thereafter coated with Amersham LM1 autora-diographic emulsion and exposed at 4 �C for 5 h for COI. Exposedslides were developed in Kodak D-19 for 4 min at 13 �C andcounterstained with toluidine blue. Brain sections from the fourexperimental groups of animals (three sections per individual) wererun in the same in situ hybridization session.

Data analysis

Analysis of striatal preproenkephalin A and preprotachykinin mRNAlabelling was performed inside and around the area showing dopaminedenervation on sections adjacent to those used for 3H-mazindol binding.The levels of labelling were quantified by digitized image analysis fromfilm autoradiograms using a BIOCOM analysis system (Densirag,BIOCOM, Les Ulis, France). Gray levels were converted to opticaldensities (OD) using external standards (calibrated density step tablet,Kodak). The mean OD value was determined from three sections peranimal after subtracting the background signalmeasured on each sectionby scanning a cortex area that is known to lack substance P andenkephalin neurons. Analysis of COImRNA labelling was performed atthe cellular level on emulsion-coated sections in layer V of frontalcortical areas (Fr1, Fr2, FL; anteroposterior 9.48–9.20 mm according tothe atlas of Paxinos &Watson, 1986), GP (8.08–7.60), EP (6.44–6.20),SNr (3.8–3.20), STN (5.40–4.84), ventrolateral thalamic nucleus (VL)and in the ventral posterolateral (VPL) thalamic nucleus (A 6.44–6.20 mm). Sections were observed under dark-field epilumination withan immersion 20· objective of a microscope connected to a COHUcamera and the digitized images were transferred to the screen of a videomonitor with a resulting magnification of 1000·. Using the Visioscanimage analysis software from BIOCOM, the number of silver grains percell was estimated under polarized light by measuring OD with respectto a standard curve of a defined number of silver grains. In the presentexperiments, autoradiographic background was very low, so that thecorresponding value was not subtracted. A random sampling of 50labelled neurons (more than 10 grains) in the whole surface of thestructure per section and per brain side was quantified in three sectionsfrom each animal and the mean number of silver grains per neurondetermined. The data from the number (n) animals per condition werethen averaged and expressed as means ± SEM. Results were presentedas percentage of the corresponding mean control value. Statisticalcomparisons were performed for each anatomical region using a one-way analysis of variance (anova) followed by Scheffe’s test formultiple group comparison. A significance of P < 0.05 was required forrejection of the null hypothesis.

Results

Striatal dopamine denervation

Examination of autoradiograms from 3H-mazindol-labelled frontalstriatal sections showed that dopamine denervation was quite restricted

to a circular area in the dorsolateral part of the striatum (Fig. 1A), whichwas observed in all the anteroposterior levels examined from A 9.7 mmto A 8.74 mm. The denervation area showed a maximal surface of2.73 ± 0.32 mm2 on sections close to the injection site (A 9.2 mm),representing 31% of the whole striatal surface at this level, in theuntreated animals with the dopamine denervation. Similar extension ofthe lesion was measured in the MPEP-treated animals. Quantitativeanalysis of 3H-mazindol labelling within the denervation core showed85–92% decrease in the three groups of lesioned animals, with orwithout MPEP treatment, and a nonsignificant 15–18% decrease in thesurrounding striatum (Fig. 1B). A possible neuroprotective mechanismis therefore unlikely to be involved in the effects of MPEP in ourexperimental condition where the treatment is started 14 days after thedopamine lesion.

Preproenkephalin A and preprotachykinin mRNA labellingin the striatum

Quantitative analysis for striatal preproenkephalin gene expressionrevealed significant differences between experimental groups in thespared striatal area (F3,18 ¼ 4.86, P < 0.05), but not in the denervatedstriatal region (F3,18 ¼ 1.46, NS). No significant difference betweengroups is observed for preprotachykinin gene expression in thedenervated area (F3,19 ¼ 2.14, NS) and in the nondepleted region(F3,19 ¼ 1.01, NS). In the striatal dopamine denervated area, neitherpreproenkephalin A nor preprotachykinin mRNA levels were signi-ficantly modified in the animals with the lesion alone as compared tocontrols (Fig. 1A and B). No significant change in these markers wasmeasured after MPEP treatment at the two doses tested. In therelatively spared striatal area, preproenkephalin A mRNA levels werereduced by 19–23% (P < 0.05) both in the MPEP-treated or untreatedlesioned animals in the absence of significant change in preprotach-ykinin mRNA expression (Fig. 1B).

Intraneuronal COI mRNA levels in the striatum

No significant change in the mRNA levels of COI in striatal neuronswas measured either in the dopamine-denervated area (F3,19 ¼ 0.40,NS) or in the spared region (F3,19 ¼ 0.20, NS) in the differentexperimental conditions tested (Fig. 2).

Intraneuronal COI mRNA levels in STN and target structures,GP, EP and SNr

Quantitative analysis for COI gene expression revealed highlysignificant differences between experimental groups in STN(F3,18 ¼ 8.82, P < 0.001), in EP (F3,18 ¼ 5.53, P < 0.01), in SNr(F3,18 ¼ 19.19, P < 0.0001) but not in GP (F3,19 ¼ 1.94, NS). In STNneurons, expression of COI mRNAwas increased by 48% (P < 0.05)in the animals with the lesion alone as compared to controls (Fig. 3).This effect was counteracted by MPEP treatment at both doses, thevalues being no more different from control and significantly reducedcompared to untreated lesioned rats ()46%, P < 0.01 for MPEP1.5 mg ⁄ kg; )33%, P < 0.05 for MPEP 3 mg ⁄ kg). In SNr, intraneur-onal COI mRNA expression was significantly increased by 45%(P < 0.01) in the animals with the dopamine lesion alone (Fig. 3).This lesion-induced increase was reversed by MPEP treatment, thevalues obtained being no more different from controls and signifi-cantly reduced by 20.1% (P < 0.05) and by 37% (P < 0.01) afterMPEP 1.5 and 3 mg ⁄ kg, respectively, as compared to untreatedlesioned animals. The levels of COI mRNA in GP (Fig. 3) and EP

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neurons (Fig. 3) were not modified in the dopamine lesion condition.These levels tended to be increased by 22–30% after MPEP treatmentin both structures, but this effect reached significance only at thehigher dose of MPEP in the EP (+ 35%; P < 0.05), as compared tocontrol values.

Intraneuronal COI mRNA levels in thalamic nucleiand cerebral cortex

Quantitative analysis revealed significant differences between experi-mental groups for COI gene expression in frontal cortex (F3,19 ¼ 4.86,P < 0.05), but not in VL (F3,19 ¼ 3.37, NS), nor in VPL(F3,19 ¼ 1.12, NS). Intraneuronal COI mRNA levels in the motorthalamic nucleus VL (Fig. 3) did not show significant change inanimals with the dopamine lesion alone. MPEP treatment induced atendency towards increased levels, which however, did not reachstatistical significance. Such tendency was not measured in thenonmotor thalamic nucleus VPL (Fig. 3). In the frontal cortical areasoverlying the striatum (Fig. 4), layer V neurons showed decreased COImRNA levels ()24.5%; P < 0.05) in the animals with the dopaminelesion alone (Fig. 4C). This effect, confirmed by the shift to the left ofthe frequency distribution of labelling per cell, was antagonized byMPEP treatment, the values obtained at both doses being no moresignificantly different from controls (Fig. 4B). However, only MPEP at3 mg ⁄ kg induced significant increase as compared with the valuesdetermined in the lesion condition (+ 31.9%; P < 0.05). No significantchange was measured in the different experimental conditions inneurons of layer III of the frontal cortex (not shown), pointing to thespecificity of the effects measured in layer V.

Fig. 1. (A) Digitized autoradiographic images of frontal sections at striatal level illustrating the effects of the dopamine lesion alone on 3H-mazindol binding andon preproenkephalin A and preprotachykinin mRNA levels, as compared with control labelling. The dotted lines delineate the dopamine denervated area. Scale bar,2 mm. (B) Quantitative analysis of the effects of the lesion alone or combined with MPEP treatment at the doses of 1.5 mg ⁄ kg and 3 mg ⁄ kg on the same markerswithin (solid bars) and around (hatched bars) the striatal area showing marked dopamine denervation (n ¼ 6–7 animals in the different experimental groups). Dataare expressed as percentages ± SEM of the mean control optical density. Statistical comparisons were performed using a one-way anova, followed by Scheffe’s test.*P < 0.05 and **P < 0.01 compared with the control group.

Fig. 2. Effects of partial bilateral dopamine lesion and subsequent chronictreatment with MPEP on COI mRNA levels within (solid bars) and around(hatched bars) the striatal area showing marked dopamine denervation. Thehistograms represent the mean number of silver grains per labelled neuron ineach experimental condition (n ¼ 6 dopamine lesion and six in each MPEP-treated group) expressed as percentage ± SEM of control (n ¼ 5). Statisticalcomparisons by Scheffe’s test did not show significant differences.

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Relationship between the changes in gene expression of COIand the akinetic deficits

We previously reported that MPEP treatment alleviates the akineticdeficit produced by the dopamine lesion in our model of topograph-ically restricted striatal dopamine denervation. The level of akinesia,assessed individually as the percentage change in delayed responses inthe lesioned animals treated or not with MPEP, was positivelycorrelated with the percentage change of COI mRNA levels in theSTN and of GAD67, the marker of GABA neuron activity, in SNr(Breysse et al., 2003). We here confirm the role of the SNr in the

expression of akinesia by showing a positive correlation (R2 ¼ 0.649;P < 0.001) between the changes in gene expression of COI in thisstructure and the level of akinesia (Fig. 5). An inverse correlation wasfurther found between the change in COI in neurons of layer V offrontal cortex and the level akinesia (R2 ¼ 0.568; P < 0.001; Fig. 5).

Discussion

This study shows that restricted lesion of striatal dopamine afferents tothe dorsolateral striatal territory produces focused changes in activity-

Fig. 3. Partial bilateral dopamine lesion and subsequent chronic MPEP treatment differentially affect the levels of COI mRNA expression in the STN, SNr, GP, EP,VL and VPL. Histograms representing the mean number of silver grains per labelled neuron in each experimental condition (n ¼ 5–6 controls, 5–7 dopamine lesionand 5–7 in each MPEP-treated group), expressed as percentage ± SEM of control. Statistical comparisons were performed using a one-way anova, followed bySheffe’s test. *P < 0.05 and **P < 0.01 compared with the control group; #P < 0.05 and ##P < 0.01 compared with values obtained in animals with the 6-OHDAlesion alone.

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related gene expression in the cortico-BG cortical loop; it decreasedstriatal preproenkephalin A mRNA expression in the nondenervatedareas, increased COI mRNA expression in the STN and the SNr and

decreased COI in cortical layer V. Except for the decrease inpreproenkephalin A, these changes were prevented by mGluR5blockade with chronic MPEP treatment. Paradoxically, MPEP

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treatment tended to increase the COI mRNA levels in the pallidalcomplex as well as in motor thalamic nucleus VL, which were notmodified after the dopamine lesion alone.

Cellular changes produced in the basal ganglia by restricteddopamine denervation of the dorsolateral striatum

Expression of parkinsonian motor symptoms is classically viewed asthe result of a cascade of changes in the activity of BG structures

initiated by striatal dopamine denervation and leading to overactivityof the output structures, SNr and EP. The explanation for thisoveractivity is two fold: (i) reduced activity of the ‘direct’ pathwayconnecting the striatum to the output structures, formed by theGABA ⁄ substance P-expressing striatal neurons, and (ii) increasedactivity of the striatal neurons expressing GABA ⁄ enkephalin at theorigin of the ‘indirect’ pathway, subsequent reduction of the inhibitoryinfluence of GP onto STN and hyperactivity of excitatory STNprojection to output structures. Data obtained under extensivedopamine denervation conditions mostly fit with the model predictionsby showing decreased preprotachykinin and increased preproenkeph-alin A gene expression in the striatum and increased GAD67 or COImRNA in SNr and EP (Gerfen et al., 1991; Vila et al., 1999; Salinet al., 2002). Converging data also show abnormal STN activation(see Hirsch et al., 2000), which is however, unlikely to be mediated bydecreased GP activity as pallidal gene expression of GAD67 or COI isincreased rather than decreased (see Chesselet & Delfs., 1996; Hirschet al., 2000).The present study shows that a markedly different pattern of

metabolic changes in the BG network is associated with akineticdeficits in rats with dopamine denervation restricted to thedorsolateral striatal territory, the preferential target of sensorimotorneocortical projections (McGeorge & Faull, 1989). Neitherpreproenkephalin A and preprotachykinin mRNA expression norCOI intraneuronal mRNA levels are modified in the denervatedzone, despite 90% loss in dopamine terminals, suggesting thatactivity of the striatal projection neurons are maintained therewithin normal limits. In line with our results, striatal preproenkeph-alin A gene expression is not modified after 6-OHDA lesion of thelateral substantia nigra pars compacta producing preferential den-ervation of the dorsolateral striatum (Christin et al., 1996). Giventhe abundant literature showing compensatory increase in dopaminefunction in partial lesion conditions (see Zigmond, 1997), increaseddopamine tone in the nondenervated area could provide dopamineto the denervated area through volume transmission (see Bezard &Gross, 1998; Zoli et al., 1998) and account for the maintenance ofpeptides and COI expression within the denervated site. Thishypothesis is further supported by the finding that preproenkeph-alin A mRNA levels, which are negatively regulated by dopamine,are decreased in the spared striatal territories. Therefore, evenimportant but topographically restricted loss of dopamine innerva-tion may not be sufficient to affect striatal neuron activity.We further show the absence of significant change in COI mRNA

expression in GP and EP neurons, suggesting that the activity of thepallidal complex is also maintained within normal limits. No change inthis marker was measured in the motor thalamic nucleus VL. The lackof change in VL is in line with the recent data from a primate model ofPD showing unchanged firing rate and pattern of thalamic neuronsreceiving projection from BG in both the asymptomatic and symp-tomatic stages after MPTP intoxication, the major dysfunction being aloss of information segregation (Pessiglione et al., 2005). Finally, weconfirm the hyperactivity of both STN and SNr neurons and providenew evidence for motor cortex hypoactivity by showing decrease inCOI mRNA levels in neurons of layer V of frontal cortical areas. A

Fig. 4. Chronic treatment with MPEP reverses the dopamine lesion-induced decrease in COI gene expression in layer V of frontal cortical areas. (A)Photomicrographs taken under dark-field epi-illumination and (B) histograms of frequency distribution of labelling, showing the effects of the dopamine lesionwithout or with subsequent treatment with MPEP. The lesion (n ¼ 6) induces a decrease in intraneuronal labelling as compared with controls (n ¼ 5), confirmed bya shift to the left of the distribution curve (toward the lower levels), that is no longer observed after MPEP treatment at the doses of 1.5 mg ⁄ kg (n ¼ 5) and 3 mg ⁄ kg(n ¼ 7). (C), Histograms representing the mean number of silver grains per labelled neuron in each experimental condition expressed as percentage ± SEM ofcontrol. Statistical comparisons were performed using a one-way anova, followed by Scheffe’s test. *P < 0.05 compared with the control group; #P < 0.05compared with values obtained in animals with the DA lesion alone.

Fig. 5. Correlations between the akinetic deficit, expressed as the number ofdelayed responses, and the changes in the expression of COI mRNA in neuronsof the layer V of the frontal cortex and of the SNr. Each point represents thedifference in the number of delayed responses between experimental andcontrol scores, expressed as percentage of control, with respect to percentage ofchange in mRNA levels in individual animals from the different experimentalgroups.

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positive correlation is shown between COI mRNA expression and thelevel of akinesia in the SNr, as reported previously for GAD67 in thisstructure and for COI in the STN (Breysse et al., 2003), whereas aninverse correlation is found in the cortex. This correlation betweenreduced cortical activity and akinesia is consistent with recentelectrophysiological data showing reduced baseline and movement-related firing rates of motor cortical neurons in rats showingbradykinesia induced by dopamine D2 receptor blockade (Parr-Brownlie & Hyland, 2005) and in akinetic MPTP-treated primates(Escola et al., 2003). Altogether our data show for the first time thatoveractivity of STN and SNr and hypoactivity of motor corticalneurons are associated with akinetic deficits under partial striataldenervation condition insufficient to produce the changes in theindirect pathway upstream STN and in the direct pathway character-istic of late PD state. They emphasize a primary role of STN in theonset of parkinsonian motor symptoms and provide strong support tothe emerging hypothesis that STN hyperactivity in the parkinsonianstate may be related to changes in the activity of STN extrinsicafferents rather than to changes in intrinsic BG loops (see Hirsch et al.,2000). Contrary to the present finding, a recent study in MPTP-treatedmice has shown that partial loss of nigrostriatal neurons inducesmodifications in striatal neuropeptide gene expression in the absenceof metabolic change in GP or SNr (Meissner et al., 2003). A possibleexplanation may be that different adaptive mechanisms are involveddepending on the pattern rather than severity of the denervation;uniform striatal denervation, although partial, may produce compen-satory changes in striatal output neurons whereas topographicallyrestricted denervation, even severe, may not produce striatal changesbut lead to STN overactivity. Obeso et al. (2004) have proposed thatSTN hyperactivity may occur in the presymptomatic PD stage beforesignificant change in striatal neuron activity, due to insufficient andasymmetrical striatal dopamine loss, and contribute to an internalcompensatory process through STN ⁄GPe ⁄GPi circuit. Our data areconsistent with this view and further suggest that STN overactivitymay not depend primarily on the symmetrical ⁄ asymmetrical characterof the dopamine denervation, and may produce, in de-compensatedearly stage, a hyperactivity of the SNr, selectively.The dissociation between the responsiveness of SNr and EP in our

PD model is another intriguing finding compared to the overactivity ofboth output structures occurring in extensive lesion models. Aschanges in COI mRNA levels in SNr parallel those in STN, it could bethat our lesion condition unmasks the existence of a preferentialfunctional link between STN and SNr activity in the parkinsonianstate. In support for such a differential link, there is accumulatingevidence for a preferential impact of the manipulation of STN activityon the SNr vs. the pallidal complex in rodent (Robledo & Feger, 1991;Salin et al., 2002; Bacci et al., 2004) or primate models of PD (Guridiet al., 1996) as well as in PD patients (Su et al., 2001).

Effects of MPEP treatment on the pathophysiologicalfunctioning of basal ganglia

mGluR5 is expressed at moderate to high levels in all the BGstructures (Testa et al., 1994, 1995; Shigemoto & Mizuno, 2000).Earlier reports have shown that treatment with the mGluR5 antagonistMPEP alleviates parkinsonian-like symptoms produced by extensivedopamine lesion or blockade of DA receptors (Gasparini et al., 1999;Spooren et al., 2000; Ossowska et al., 2001), but the neural substratesof these beneficial effects remained unexplored. Here we found thatthe antiakinetic effect of chronic MPEP treatment in our PD model isassociated with reversal of the dopamine lesion-induced changes inCOI mRNA expression in STN, SNr and cerebral cortex. Such

reversal is likely to involve direct blockade of mGluR5 in STN andSNr, as local activation of these receptors produces direct excitationand potentiation of NMDA receptor currents in STN neurons (Awadet al., 2000) and depolarizes SNr neurons in PD-like state (Marinoet al., 2002b). Reversal of STN and SNr overactivity could in turnsuppress cortical hypoactivity. Inversely, normalization of corticalmetabolic activity by MPEP treatment may directly modulate theaberrant neuronal activities downstream of the striatum. This sugges-tion follows recent electrophysiological and PET imaging datashowing a drastic functional recovery in a primate model of PD aftermotor cortex activation (Drouot et al., 2004).Despite major expression of mGluR5 in striatal enkephalin neurons

(Testa et al., 1995), striatal preproenkephalin A gene expression isunaffected by dopamine denervation and by subsequent MPEPtreatment in our model. This lack of effect is in line with recentdata reporting that MPEP treatment has no effect on basal striatalexpression of preproenkephalin A, whereas it efficiently counteractsthe overexpression induced by D2 dopamine receptor blockade(Parelkar & Wang, 2003; Wardas et al., 2003). The mGluR5-mediatedcontrol of enkephalin neurons can be then assumed to depend oncorticostriatal glutamatergic drive, consistent with the primarilyextrasynaptic localization of this receptor in the striatum (Paquet &Smith, 2003) and permissive role on NMDA-mediated responses(Pisani et al., 2001). Contrasting with the reduction of both STN andSNr hyperactivity, which fits with removal of excitatory influence, weshow that MPEP treatment tends to increase COI mRNA expression inboth GP and EP as well as VL, this effect being however, significantonly in EP at the highest dose of MPEP tested. Although reduction ofSNr inhibitory outflow may contribute to the increase in VL neuronactivity and account for reversal of cortical hypoactivity, it is difficultto explain the increased activity of GP and EP neurons throughintrinsic BG circuit, so that these responses may rather involve localeffects. Interestingly, recent evidence shows that in the GP, wheremGluR5 and mGluR1 are coexpressed by most efferent neurons,MPEP potentiates the mGluR1-mediated depolarization and preventsdesensitization of mGluR1 (Poisik et al., 2003). It then could be thatMPEP treatment reduces or increases neuronal activity among BGpopulations depending on both the direct control exerted by mGluR5and the interaction with other glutamate receptor subtypes.Recently, MPEP has been identified as a positive allosteric

modulator for mGluR4 (Mathiesen et al., 2003). This group IIImGluR subtype is abundantly expressed in the BG (Corti et al., 2002),notably at striatal and GP level, and its activation has been shown toproduce benefit in rodent models of PD (Marino et al., 2003; Valentiet al., 2003). Therefore, one cannot exclude an implication of mGluR4in the effects of MPEP reported here, which could account, inparticular for the slight increase in COI mRNA in the pallidalcomplex. However, such a contribution might be minimized by thelow MPEP doses used.In conclusion, this study provides the first cellular evidence that

hyperactivity of STN and SNr can occur in the symptomatic stage ofPD in the absence of significant change in neuronal activity instriatum, GP and EP, and lead to reduced cortical motor outflow at theorigin of akinesia. We also demonstrate that the beneficial effects ofMPEP treatment on akinetic deficits are associated with the reversal ofthe abnormal activation of STN and SNr, and increased thalamocor-tical activity. MPEP treatment has only moderate effects on theactivity of the BG populations that are unaffected by the dopaminedenervation, which functional implications remain to be determined.Summing up, our findings point to the subthalamo-nigro-corticalsubcircuit as an essential therapeutic target, and support the strongpotential of mGluR5 antagonists, such as MPEP or the highly potent

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and bioavailable compound 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyr-idine (MTEP; Cosford et al., 2003), as a promising antiparkinsonianstrategy.

Acknowledgements

This research was supported by the Centre National de la RechercheScientifique, the Universities of Aix-Marseille I and II and by a grant fromthe Fondation de France and the French Ministry of Education and Research(ACI ‘Neurosciences Integratives et Computationnelles’, NIC 0057). Theauthors are grateful to Dr Fabrizio Gasparini (Novartis Pharma AG, Basel,Switzerland) for the generous gift of MPEP. A. OUESLATI was supported by agrant from the ‘Comite d’Entente et de Coordination des Associations deParkinsoniens’ (CECAP).

Abbreviations

6-OHDA, 6-hydroxydopamine; BG, basal ganglia; COI, cytochrome oxidasesubunit I; DA, dopamine; EP, entopeduncular nucleus; GAD67, isoform of theGABA-synthesizing enzyme; GP, globus pallidus; mGluR, metabolic glutamatereceptor; MPEP, 2-methyl-6-(phenylethylnyl)-pyridine; OD, optical densities;PD, Parkinson’s disease; RT, reaction time; SNr, substantia nigra pars reticulata;SSC, standard saline citrate; STN, subthalamic nucleus; VL, ventrolateralthalamic nucleus; VPL, ventral posterolateral thalamic nucleus.

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