Neurobiology of Disease 34 (2009) 340–350

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Neurobiology of Disease

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De novo and long-term L-Dopa induce both common and distinct striatal geneprofiles in the hemiparkinsonian rat

Michèle El Atifi-Borel a,b, Virginie Buggia-Prevot a, Nadine Platet a, Alim-Louis Benabid a,c,François Berger a,b,d, Véronique Sgambato-Faure a,e,⁎a Institut National de la Santé et de la Recherche Médicale, Unité 318, Grenoble F-38043 cedex 09, Franceb Institut National de la Santé et de la Recherche Médicale, Unité 836, Grenoble Institut des Neurosciences, Equipe Nanomédecine et Cerveau, Grenoble F-38043 cedex 09, Francec CEA-MINATEC-LETI, Clinatec, Grenoble F-38054, Franced Centre Hospitalier Universitaire de Grenoble, Grenoble F-38043 cedex 09, Francee Centre National de la Recherche Scientifique UMR 5229 Université de Lyon I, Centre de Neuroscience Cognitive, Institut des Sciences Cognitives, Bron F-69675 cedex, France

⁎ Corresponding author. Centre deNeuroscience CognitLyon I, Institut des Sciences Cognitives, 67 Bd Pinel, Bron

E-mail address: veronique.sgambato-faure@isc.cnrs.Available online on ScienceDirect (www.scienced

0969-9961/$ – see front matter © 2009 Elsevier Inc. Adoi:10.1016/j.nbd.2009.02.002

a b s t r a c t

a r t i c l e i n f o

Article history:

We compared for the first ti Received 16 November 2008Revised 3 February 2009Accepted 6 February 2009Available online 20 February 2009

Keywords:Parkinson's disease6-hydroxydopamineDyskinesiaTranscriptomeMicroarrayStriatum

me the effects of de novo versus long-term L-Dopa treatment inducing abnormalinvoluntary movement on striatal gene profiles and related bio-associations in the 6-hydroxydopamine ratmodel of Parkinson's disease. We examined the pattern of striatal messenger RNA expression over 4854genes in hemiparkinsonian rats treated acutely or chronically with L-Dopa, and subsequently verified some ofthe gene alterations by in situ hybridization or real-time quantitative PCR. We found that de novo and long-term L-Dopa share common gene regulation features involving phosphorylation, signal transduction,secretion, transcription, translation, homeostasis, exocytosis and synaptic transmission processes. We alsofound that the transcriptomic response is enhanced by long-term L-Dopa and that specific biologicalalterations are underlying abnormal motor behavior. Processes such as growth, synaptogenesis, neurogenesisand cell proliferation may be particularly relevant to the long-term action of L-Dopa.

© 2009 Elsevier Inc. All rights reserved.

Introduction

Abnormal involuntary movements or dyskinesias represent adebilitating complication of L-3,4-dihydroxyphenylalanine (L-Dopa)therapy for Parkinson's disease (PD) (Barbeau, 1969; for review,Defebvre, 2004). The genesis of L-Dopa-induced dyskinesias (LIDs) isnot fully understood but major factors include nigral denervation,sensitization of dopaminergic receptors, onset, dose and manner ofadministration of L-Dopa (Jenner, 2008; Obeso et al., 2000, 2004).Consequently, LIDs are associated to an alteration of the neuronaltransmissionwithin the basal ganglia network (for reviews, see Filion,2000; Bezard et al., 2001). Molecular mechanisms underlying primingto L-Dopa are poorly known (Blanchet et al., 2004). Dyskinesias startwithin days to week of L-Dopa treatment initiation, follow a peak-dosepattern but end-of-dose has been observed. Once triggered, dyskine-sias will persist to become a constant feature of any effective L-Dopadose. All dopamine agonists display the capacity to reproduce LIDs to avariable extent once L-Dopa priming has occurred. The molecularadaptive events responsible for the new functional state underlying

ive, CNRSUMR5229UniversitéF-69675 cedex, France.fr (V. Sgambato-Faure).irect.com).

ll rights reserved.

dyskinesias remain misunderstood. Dysregulation of dopaminereceptors and gene expression has been expected and shown onanimal models (for reviews, see Cenci; 2007; Chase, 2004; Jenner,2008). However, most of these studies did analyze the effect of acuteor chronic dopaminergic stimulation on one or two particular genes ofinterest (for example, see Morelli et al., 1993; Cenci et al., 1998). Whatare the global molecular alterations taking place in the striatum afternigral lesion and dopamine stimulation?

The DNA microarray technology (for review, see Mandel et al.,2003; Mirnics and Pevsner, 2004) involves large-scale monitoring ofrelative differences in RNA abundance between samples, which arethought to represent changes in function. Such approach has yieldedinsights into the mechanisms linked to the degenerative process(Bassilana et al., 2005) or to the chronic L-Dopa treatment (Ferrario etal., 2004; Konradi et al., 2004) associated to PD. However, none of thestudies performed in the 6-hydroxydopamine (6-OHDA) rat model ofPD did address the essential question of whether acute versus chronicL-Dopa have the same impact on striatal global gene alterations or leadeach to a specific molecular fingerprint. In this regard, the effectsdriven by an acute dose of L-Dopa on striatal gene regulation have notbeen studied at the transcriptomic level in the 6-OHDA-lesioned rat.

In the present study, combining large-scale gene expressionapproaches, bioinformatics analysis, biochemistry, in situ hybridiza-tion and quantitative PCR analysis, we provide for the first time a

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comparison between the effects of de novo versus long-term L-Dopatreatment on gene expression profiling in the dopamine-depletedstriatum in the rat. We found that i) acute and chronic L-Dopa drive acommon set of biological modifications ii) chronic L-Dopa affects agreater number of genes into this common set of alterations iii) andfinally that chronic L-Dopa leading to dyskinesia also alters a distinctset of genes and bio-associations.

Materials and methods

Experiments were performed in the laboratory INSERM U318authorized by the French Ministry of Environment (Agreement N°B3851610003) and further carried out in accordance with EuropeanCommunities Council Directive of 24 November 1986 (86/609/EEC)for care of laboratory animals.

Lesion surgery

Male adult Wistar rats, weighing 260–300 g were maintained on a12-h light/dark cycle, with food and tap water available ad libitum. Allanimals were anaesthetized with chloral hydrate (400 mg/kg) andsubjected to a unilateral 6-OHDA lesion in the left SNc according toSgambato-Faure et al. (2005). The effectiveness of the lesion wasfurther confirmed post-mortem by means of tyrosine hydroxylaseimmunohistochemistry.

Drug treatments

All drugs were purchased from Sigma (Saint Quentin Fallavier,France). Four to 5weeks post-lesion, hemiparkinsonian rats received i.p. injection of physiological saline (vehicle-treated rats, n=12) orL-Dopa (L-3,4-Dihydroxyphenylalanine methyl ester hydrochloride)(50 mg/kg) combined with benserazide hydrochloride (10 mg/kg)(L-Dopa-treated rats, n=21) acutely (a single injection, n=15) orchronically (twice injections per day, for 10 days, n=18). As we wereinterested in only rats that developed significant AIM scores, the doseof 50 mg/kg for L-Dopa was chosen as it allows in our model theinduction of dyskinesia in all 6-OHDA-lesioned rats, while a lower dose(10 mg/kg) does not (see in Sgambato-Faure et al., 2005). Of note, ithas been shown that the dose of L-Dopa is a significant risk factor forthe development of LIDs (Putterman et al., 2007). Unlesioned rats(n=12) received acute (n=6) or chronic (n=6) physiological saline.

Motor behavior analysis

Rats were placed back to their home cage after each injection.Abnormal involuntary movements were subdivided into forelimbdyskinesia, orolingual dyskinesia and axial dyskinesia (Anderssonet al., 1999). One hour after each L-Dopa injection, dyskineticmovements were evaluated according to the scale defined by Cenciet al. (1998). Turning sensitizationwas also evaluated by counting thenumber of contralateral turns per minute during 10 min.

Tissue preparation for RNA extraction and protein isolation

Two hours after the L-Dopa injection (last one in case of chronictreatment), animals were anaesthetized with a lethal dose of chloralhydrate (600 mg/kg; i.p.) and killed by decapitation. The brains wereremoved, the dorsal striata were dissected and immediately frozen onliquid nitrogen, while the posterior parts of the brains were immersedin cold paraformaldehyde. Frozen striata were then crushed in RNAse-free conditions in a solubilization buffer (containing 50 mM Tris–HCl[pH 7.4], 100 mM NaCl, 5 mM EDTA, 10 mM NaF, 0.1 mM PMSF,0.1% NP40 and protease inhibitor cocktail [Roche, Meylan France]),2/3 being used for RNA extraction and 1/3 being used for proteinisolation.

RNA extraction and transcriptomic analysis

Total RNA from each dorsal striatum was isolated using a phenol:chloroform extraction (RNAgent® Total RNA isolation system, Pro-mega, Charbonnières-les-Bains, France) and further controlled (Bio-Analyser, Agilent technologies, Massy, France) for quality andconcentration. Total RNAs belonging to a same experimental group[no lesion+vehicle (NLV), lesion+vehicle (LV), or lesion+L-Dopa(LDO)] were then pooled together. The rat library (RATLIB96) waspurchased from Sigma Genosys (Haverhill, England) and consisted of4854 long oligonucleotides (65-mers long each) representing all therat genes associated with public mRNA sequences. Additionally,oligonucleotide probes corresponding to the internal RNA standard(an Arabidopsis thaliana gene) were used for normalization assay.Each oligonucleotide (diluted to 100 μM in 1× SSC) was spotted twiceonto nylon membranes (Nytran+; Schleicher & Schuell, Mantes laVille, France) using an Affymetrix® 417 microarrayer (MWG, Ebers-berg, Germany) and cross-linked by UV radiation. Of note, the samespotting batch of membranes was used in this study as we have shownthat probe amounts are reproducible between membranes spotted inthe same run (El Atifi et al., 2002, 2003). The transcriptomic analysiswas performed for both acute and chronic conditions. For eachcondition, the 3 experimental groups were represented [no lesion+vehicle (NLV), lesion+vehicle (LV), and lesion+L-Dopa (LDO)] bysynthesizing three complex cDNA targets. Complex cDNA targets werelabeled (using dT25 primers andα[33-P]dCTP; Amersham Biosciences,Orsay, France) and purified. Each membrane was prehybridized at55 °C for at least 6 h in hybridization buffer (5× SSC, 5× Denhardt'ssolution, sperm salmon DNA at 1 mg/ml and 0.5% SDS) before addinglabeled cDNA for 5 days of hybridization. Then, the membranes werewashed three times 1 h in 2× SSC, 0.1% SDS buffer at 45 °C. Afterdrying, membranes were exposed 72 h on phosphorus image platesbefore scanning (Phosphor Imager BAS 5000, Fuji, France).

Arrays quantification and normalization

The hybridization signals were quantified using the Array GaugeSoftware (Fuji-Film™; Raytest, Paris la Defense, France). Any spot withintensity lower than the mean of the background plus 3 standarddeviations was ranked as 0. Linear regression curves were establishedbetween the arrays. All genes with invariant expression (RNA stan-dard and others housekeeping genes) were used to determine theslope coefficient that was used as normalization factor. The expressionlevels are reported in arbitrary units. The genes were identified asdifferentially expressed if first, they had a fold difference≥1.4, second,if the p value (Student t-test) for comparisons between replicates wasat least b0.05 and third if the ratio datawere validated for the differentsamples with the non-parametric Mann–Whitney test. These com-bined criteria further increased the reliability of the data by elimi-nation of significant but very small expression changes that may havea marginal biological effect (Mirnics and Pevsner, 2004).

Protein isolation and western blot analysis

Proteins were isolated from the 1/3 part left from striatal crushes.Briefly, samples were lysed in the solubilization buffer for 30 min onice. Insoluble material was removed by centrifugation at 14000 rpmfor 30 min. Supernatants were then aliquoted and frozen at −80 °Cuntil they were processed. Tissue lysates were boiled before electro-phoresis (20 μg per lane) through a SDS 10% polyacrylamide gel.After transfer to nitrocellulose membranes and blocking in TBS-T(TBS+0.1% Tween 20) buffer with 5% milk, primary antibodies wereadded in TBS-T with 2% milk at the following concentrations: rabbitpolyclonal anti-TH (from Jacques Boy, France) at 1/2000, rabbit poly-clonal anti-c-Fos (Ab-5 from Calbiochem, distributed by VWR inter-nationals, Fontenay-sous-Bois, France) at 1/1000, rabbit polyclonal

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anti-FosB (102, from Santa Cruz, distributed by Tebu-Bio SA, Le Perrayen Yvelines, France) at 1/500, overnight at 4 °C. The use of suchantibodies allowed us to assess the specificity of the lesion (anti-TH)and of the L-Dopa treatment (acute or chronic with an anti-c-Fos oranti-FosB, respectively). After multiple washes in TBS-T, membraneswere incubated with appropriate HRP-conjugated secondary anti-bodies (1/10000, Sigma, Saint Quentin Fallavier, France) and washedagain prior to visualization using Super Signal (Pierce, Perbio Science,Brebières, France) and film exposure (X-OMAT, Kodak, Sigma, SaintQuentin Fallavier, France). Then the blots were stripped (Re-Blot Plus,Chemicon, distributed by Euromedex, Mundolsheim, France), re-saturated and incubated with the antibodies directed against alpha-tubulin (mouse monoclonal antibody used at 1/20000 from Calbio-chem distributed by VWR internationals, Fontenay-sous-Bois, France).

Immunohistochemistry

The posterior part of the brain immersed in cold 4% PFA wasincubated overnight in 0.1M phosphate buffer containing 15% sucrose,before freezing for further TH immunohistochemistry. Coronalsections (20 μm) were cut on a cryostat and kept in a cryoprotectivesolution at 20 °C until proceeding for TH immunohistochemistry (seedetailed protocol in Sgambato-Faure et al., 2005).

In situ hybridization

Additional animals were sacrificed as previously (Sgambato-Faureet al., 2005) by intracardiac perfusion of ringer followed by 4% PFAunder normal conditions. Brains were removed, post-fixed 2 h,washed overnight in 0.1 M phosphate buffer containing 15% sucroseand frozen. Rostro-caudal series of coronal sections (20 μm) were cuton a cryostat and then kept in a cryoprotective solution at 20 °C untilproceeding. Oligonucleotidic probes, obtained from Sigma Genosys,were labeled with [35 S]dATP (1000 Ci/mmol, NEN Life Science) by 3′terminal deoxynucleotide transferase (Amersham) and purified onmicrocon YM30 (Millipore, France). Radioactive probes were dis-solved (specific activity 1–2×109 c.p.m./μg) to a final concentration of2.5 ng/ml in hybridization buffer (50% v/v deionized formamide,saline sodium citrate (SSC)×4 (trisodium citrate 0.06 M, NaCl 0.6 M),1×Denhardt's solution, 1% N-lauroyl-sarcosine, 0.5 mg/ml denatu-rated salmon sperm DNA, 0.25 mg/ml yeast t-RNA, 0.25 mg/mlpolyadenosine and 0.1 M dithiothreitol). After three 10 min washes inPBS, sections were acetylated for 10 min (SSC ×4, 0.1 M triethano-lamine, 0.25% acetic anhydride). The labeled oligonucleotide solution(80 μl/slide) was dropped on sections, which were coverslipped andincubated for 16 h in a humid chamber at 48 °C. After hybridization,slides were briefly immersed in SSC ×4 at room temperature toremove the coverslips and then washed at 48 °C in successivelySSC ×2, SSC ×1 twice and SSC ×0.5. After a quick rinse at roomtemperature in SSC ×0.5, sections were progressively dehydrated ingraded alcohol, air dried and exposed. The sequences (5′–3′) of theoligonucleotidic probes that were used are available in the supple-mentary material (Supplementary Table S1).

Real time quantitative polymerase chain reaction (RT-qPCR)

RT-qPCR was performed to confirm some of the gene alterationevidenced by the transcriptomic analysis in case of chronic treatment.For that purpose, we used the pool of total RNAs extracted for thetranscriptomic study. For each used experimental condition (LV andLDO), 2 μg of total RNA was reverse transcribed for 60 min at 42 °Cusing M-MLV Reverse Transcriptase (Promega, Charbonnières-les-Bains, France) with random hexamers. Real-time qPCRs were carriedout with SYBR Green PCR master mix (Qiagen, Courtaboeuf, France)and performed in 22 μl reactions using 1/25 of the cDNA produced byreverse transcription. The following conditions were used for PCR:

95 °C for 10min and in 50 cycles, 95 °C for 15 s, 55 °C for 25 s, and 72 °Cfor 30 s. The analysis of RT-qPCR output data followed theΔCtmethod,using the relationship: ΔCt=Ln(fold change)/Ln(2). The 100%efficiency of PCR was checked for each target gene using the standardcurve method. For each sample, the gene expressions were normal-ized by the relative expression of housekeeping ribosomal gene Rps27(accession number X59375) that showed a stable expression in anycondition. RT-qPCR was done three times and the mean of ratiocalculated. Khi-deux test was performed to confirm significant foldchange of expression between samples. Finally the rat sequence-specific primers are available in the supplementary material (Supple-mentary Table S2).

Expression pattern and gene network analysis

The cluster Treeview software was used for samples clustering(available on http://bonsai.ims.u-tokyo.ac.jp/∼mdehoon/software/cluster/software.htm) based on Eisen et al. (1998). For the principalcomponents analysis (PCA), we used the multiple array viewersoftware analysis in data reduction (http://www.tm4.org). Biologicalinterpretation of the DNA microarray data was performed usingPubGene (http://www.pubgene.org).

Results

Characterization of the unilateral 6-hydroxydopamine lesion

Dopaminergic lesion was evaluated post-mortem by a histochem-ical analysis (Fig. 1). A complete loss of tyrosine hydroxylase (TH)immunostaining was observed ipsilaterally in the substantia nigrapars compacta (SNc) of 6-OHDA-injected rats (panel B) compared tovehicle-injected rats (panel A). Accordingly, a total loss of TH proteinwas also observed by western blotting in the ipsilateral striatumfrom 6-OHDA-injected rats (panel 1D) compared to the contralateralside or both striata from vehicle-injected rats (panel C). As expected,the alpha-tubulin protein, which was chosen as an internal controlin all the western blotting experiments, remained at constant levelsin both striata for each experimental condition, i.e. unlesioned- orlesioned-animals.

Effectiveness of the L-Dopa treatment

The effectiveness of acute and chronic L-Dopa treatment wasevaluated at both biochemical and behavioral levels. We first analyzedby western blotting the expression of c-Fos and ΔFosB, respectively(Fig. 2). As expected, while alpha-tubulin levels remained constant, c-Fos was induced in the ipsilateral striatum in response to acute L-Dopa(Fig. 2A) and not to vehicle (data not shown) after 6-OHDA lesion, aresult in agreement with a previous study (Morelli et al., 1993).Similarly FosB-like proteins were specifically induced in the ipsilateraldepleted striatum in response to chronic L-Dopa (Fig. 2B) and not tovehicle (data not shown), as already demonstrated (Andersson et al.,1999; Sgambato-Faure et al., 2005).

While no abnormal motor activity was observed after chronicadministration of vehicle, L-Dopa-treated hemiparkinsonian ratsdeveloped locomotor sensitization (Fig. 2C, linear regression coeffi-cient R2=0.9262, pb0,001) as well as the different subtypes ofdyskinesia (axial, orolingual and forelimb) (Fig. 2D), a result inagreement with previous data (Sgambato-Faure et al., 2005).

Gene profiling and related bio-associations induced by de novo L-Dopa

The transcriptomic analysis was performed using total RNApurified from the same striatal samples for each experimentalcondition. An unsupervised and hierarchical clustering of the striatalsamples was performed with all the data obtained from the DNA

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microarray chips relative to the acute experiments (Fig. 3A) to test ifvariations of gene expression levels could be explained by the lesionor the treatment. Interestingly the dendrogram (Fig. 3A) shows thatLDO highly differs from LV and NLV which are also distinguishable.These results are further confirmed by the PCA analysis presentedin three dimensions (Fig. 3B). These data demonstrate that de novoL-Dopa after DA lesion drives a unique gene expression profile.Moreover, this clustering illustrates some specific transcripts poten-tially induced or repressed in response to a specific condition, such asthe lesion or the L-Dopa treatment itself (data not shown).

To finally identify the most promising genes altered in response tode novo L-Dopa treatment, the ratios LDO/LV and LV/NLV and theircorresponding significances (Student t-test between replicates) werecalculated for each detected gene. Only genes having an LDO/LV foldratio ≤0.6 (and therefore potentially repressed) or ≥1.4 (andtherefore potentially induced) with significant statistical differencewere selected. The ratio LV/NLV was calculated but not used forfurther selection since genes unaltered by the lesion itself (fold ratiocomprised between 0.7 and 1.6) can be modified by the L-Dopatreatment after lesion. Supplementary Table S3 shows the list ofselected genes that are significantly altered in response to de novoL-Dopa after the dopaminergic nigrostriatal lesion. Using in situhybridization, we have further confirmed the induction of some ofthem in the depleted striatum after acute L-Dopa (Fig. 3C), such as theparathyroid hormone related sequence (pthrs) (X95094), Krox-20(AB032419) and the beta subunit of the calcium and calmodulinkinase II (CaMKIIb) (M16112), besides homer-1a (AJ276327) (whichwas not included in the rat library but used as a positive control; Berkeet al., 1998). Of note, these genes are not induced after acute NLV or LV(data not shown).

To retrieve information in a biological context, which is thestrength of gene arrays experiments, we have analyzed the bio-associations issued from genes of Supplementary Table S3 usingPubGene (http://www.pubgene.org) (Table 1). The bio-associationsare divided in the following categories: process, function andcomponent. Phosphorylation, signal transduction, secretion, trans-lation and regulation of transcription are the first most relevant

Fig. 1. Characterization of the unilateral 6-hydroxydopamine lesion. (A, B) Representative phand ventral tegmental areas after unilateral vehicle (panel A) or 6-OHDA (panel B) injection (extracted striata of vehicle- (panel C) or 6-OHDA- (panel D) injected rats; left and right lanblots were first probedwith TH antibody (upper parts on C and D), then stripped and reprobein the ipsilateral striatum of 6-OHDA-injected rats. Abbreviations are: 6-OHDA, 6-hydroxydostriatum; TH, tyrosine hydroxylase; VTA, ventral tegmental area.

processes (Table 1). Regarding components, the most represented aremembrane, plasma membrane, extracellular matrix, protein complexand focal adhesion. MAP kinase activity and RNA-directed DNApolymerase activity have the highest score for function. Finally, it isinteresting to note that a very small number of these bio-associationsappear to be specific to de novo L-Dopa.

Gene profiling and related bio-associations induced by long-term L-Dopa

The transcriptomic analysis was performed using total RNApurified from the same striatal samples for each experimentalcondition. As for the acute condition, a hierarchical clustering of thestriatal samples was performed with all the data obtained from theDNA microarray chips relative to chronic experiments (Fig. 4A). Thisclustering allowed us to identify the tendency of each experimentalcondition. From the dendrogram, it appears that LDO highly differsfrom LV and NLV, these two being also distinguishable. These resultsare further confirmed by the PCA analysis presented in threedimensions (Fig. 4B). Long-term L-Dopa has then a strong impact onthe transcriptomic response in the striatum. Moreover, this clusteringillustrates some specific transcripts potentially altered in response to aspecific condition (data not shown).

To finally identify the most promising genes altered in response tochronic L-Dopa treatment after lesion, the ratios LDO/LV and LV/NLVand their corresponding significances (student t-test between repli-cates) were calculated for each detected gene. Only genes having anLDO/LV fold ratio ≤0.6 (and therefore potentially repressed) or ≥1.4(and therefore potentially induced) with significant statistical differ-ence were selected. The ratio LV/NLV was calculated but not used forfurther selection since genes unaltered by the lesion itself (fold ratiocomprised between 0.7 and 1.6) can be modified by the L-Dopatreatment after lesion. Supplementary Table S4 lists the selected genesthat are altered in the DA-depleted striatum in response to chronicL-Dopa.Wehave further confirmed the alteration of someof themby insitu hybridization, such as neuroserpin (AF193014), Irp94 (AF077354)and calcineurin subunit A alpha (NM_017041), besides the expectedinduction of homer-1a (AJ276327) (Sgambato-Faure et al., 2005)

otomicrographs of brain sections showing TH immunoreactivity in the substantia nigrathe lesioned side is indicated with an asterisk). (C and D)Western blots of proteins fromes represent proteins from ipsilateral and contralateral striatum, respectively. Westerndwith theα-tubulin antibody (lower parts on C and D). Note that TH proteins are absentpamine; contra, contralateral; ipsi, ipsilateral; SNc, substantia nigra pars compacta; str,

Fig. 2. Characterization of the effectiveness of the L-Dopa treatment. (A, B) Western blot of proteins from extracted striata of acutely (A) and chronically (B) L-Dopa-treatedhemiparkinsonian rats; left and right lanes represent proteins from ipsilateral and contralateral striatum, respectively. Western blot was first probed with c-Fos or FosB antibody(upper parts of panel A or B, respectively), then stripped and reprobed with theα-tubulin antibody (lower parts on A and B). Note the induction of c-Fos and FosB-like proteins in theipsilateral striatum in response to acute and chronic L-Dopa respectively. (C, D) 6-OHDA-lesioned rats were chronically treated with L-Dopa (50 mg/kg; n=6) for 10 days, a periodduring which locomotor sensitization (panel C; average standard error of the mean comprises between 0, 9 and 3,1) as well as the different subtypes of dyskinesia (panel D) wereanalyzed. Abbreviations are: str, striatum; ipsi, ipsilateral; contra, contralateral; kDa, kilodaltons; dysk, dyskinesia.

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(Fig. 4B). Of note, these genes remained stable after chronic NLV or LV(data not shown). Finally, using real time quantitative PCR (Table 2),we have further confirmed the inhibition of synaptosomal-associatedprotein 25 (AF245227), cofilin (NM_017147), neuronal pentraxinreceptor (AF005099), Adenylyl cyclase (M80550), heterogeneous

Fig. 3. Gene regulation in the lesioned-striatum after acute L-Dopa treatment. (A) Cluster of texperimental conditions: no lesion+acute vehicle (NLV), lesion+acute vehicle (LV) and lcondition. Note that LDO is distinguished from LV and NLV. (B) Principal componentsRepresentative photomicrographs of brain sections showing in situ hybridization of specifiIdentity of each indicated gene is given by its accession number. Abbreviations are: LDO, le

nuclear ribonucleoprotein L (AF260436), cathepsin B (X82396) andconfirmed the induction of thyrotropin releasing hormone(NM_013046) and of homer-1a (AJ276327), following chronic L-Dopa treatment in the depleted striatum. The reference gene (theribosomal protein S27 — X59375) remained constant (Table 2).

he normalized expression levels of detected genes in the acute study with the followingesion+acute L-Dopa (LDO). Each arm of the dendrogram represents an experimentalanalysis in three dimensions. (C) Gene changes validated by in situ hybridization.c transcripts induced in the lesioned striatum in response to acute L-Dopa treatment.sion+L-Dopa; LV, lesion+vehicle; NLV, no lesion+vehicle.

Table 1Bio-associations driven by acute and chronic L-Dopa.

(continued on next page)

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Bio-associations are listed first for acute L-Dopa, then for chronic L-Dopa and finally for both. For each part, processes (in grey), functions (in white) and components (in dark grey)are successively indicated. Bio-associations are listed according to the percentage of tested genes. Regarding the part common to both, left percentages correspond to acute and rightones to chronic L-Dopa.

Table 1 (continued)

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Fig. 4. Gene regulation in the lesioned-striatum after chronic L-Dopa treatment. (A) Cluster of the normalized expression levels of detected genes in the chronic study with thefollowing experimental conditions: no lesion+chronic vehicle (NLV), lesion+chronic vehicle (LV) and lesion+chronic L-Dopa (LDO). Each arm of the dendrogram represents anexperimental condition. Note that LDO is greatly distinguished from LV and NLV, closely related. (B) Principal component analysis in three dimensions. (C): Gene changes validatedby in situ hybridization. Representative photomicrographs of brain sections showing in situ hybridization of specific transcripts induced in the lesioned striatum in response tochronic L-Dopa treatment. Identity of each indicated gene is given by its accession number. Abbreviations are: LDO, lesion+L-Dopa; LV, lesion+vehicle; NLV, no lesion+vehicle.

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To again retrieve information in a biological context, we haveanalyzed the bio-associations issued from genes of SupplementaryTable S4 using PubGene (Table 1). Localization, growth, phosphoryla-tion, signal transduction, catabolism and secretion are the first mostrelevant processes that are related to chronic L-Dopa (Table 1).Regarding components, plasma membrane, cell, cytoplasm, extra-cellular region, cytoskeleton and synapse are the most represented.Diverse functions are also found, some of them being highlyrepresented. Noteworthy, an important number of these bio-associa-tions specifically correlate to long-term L-Dopa, suggesting that newfunctional pathways are taking place with repeated administration ofthe drug.

Comparison between de novo and long-term L-Dopa-induced geneprofiles and related bio-associations

To further analyze the transcriptomic data, we compared the datasets obtained from both acute and chronic L-Dopa treatments. While asimilar number of genes were detected, a higher number of geneswere altered in response to the long-term L-Dopa treatment (about 3times more, see Fig. 5 and Fig. 6). This is particularly noticeable forsome categories of gene ontology such as metabolism, signaltransduction and transport, transcription and translation. Moreover,we found a relative small number of genes commonly regulated byacute and chronic treatment (Table 3 and Fig. 6). Interestingly, some ofthem are inversely regulated such as SNAP-25 (synaptosomal-associated protein 25), VDAC3 (voltage dependent anion channel 3),MAP1LC3 (light chain 3 of MAP1A and MAP1B) and CLDN5 (claudin5), which are up-regulated after acute L-Dopa, while down-regulatedafter chronic L-Dopa treatment.

Table 2Gene changes validated by quantitative PCR.

Gene name (and symbol) Accession number

Synaptosomal-associated protein 25 (Snap25) AF245227Cofilin 1 (Cfl1) NM_017147Neuronal pentraxin receptor (Nptxr) AF005099Adenylyl cyclase (Adcy) M80550Heterogeneous nuclear ribonucleoprotein L (HnRNP-L) AF260436Cathepsin B (Ctsb) X82396Tryrotropin releasing hormone (Trh) NM_013046Ribosomal protein S27 (Rps27) X59375Homer-1a AJ276327

A comparison with the ratio obtained by microarray is given for each gene. Abbreviationsvehicle; ND, not determined.

Finally, we have searched for the bio-associations commonlydriven by acute and chronic L-Dopa (Table 1). Among them, the mostrepresented processes are phosphorylation, signal transduction,secretion, translation, protein biosynthesis, homeostasis, exocytosisand synaptic transmission. Regarding functions, glutathione transfer-ase activity, MAP kinase activity, RNA-directed DNA polymeraseactivity and DNA binding showed the highest percentages of testedgenes. For components, the highest percentages concerned plasmamembrane/membrane and actin cytoskeleton/cytoskeleton. Butothers listed components are also very interesting.

Discussion

Microarray analysis enables an examination of large numbers ofmRNAs expressed in different biological systems and provides avaluable tool in a discovery process intended to lead to novelhypothesis. The main goal of the present study, performed in theclassical 6-OHDA rat model of PD, was to investigate and comparethe transcriptomic modulation related to de novo versus long-termL-Dopa treatment in the dopamine-depleted striatum. We found thatde novo and long-term L-Dopa (LIDs) drive a common set of biologicalalterations as well as distinct gene expression profiling and relatedbio-associations. Furthermore, this work strongly suggests that LIDsmight be underlined by the implication of novel processes and cellularcomponents.

There is a plethora of studies having analyzed the effects of acute orchronic dopaminergic stimulation on gene regulation in the striatalneurons of hemiparkinsonian rats. For example, acute L-Dopa or D1agonist administration leads to the transient up-regulation of variousimmediate early genes in the dopamine depleted striatum (Morelli et

Ratio LDO/LV Khi-deux test — 1 if true

Microarray qPCR

0.3 0.4 0.990.2 0.3 0.950.2 0.1 0.840.2 0.3 0.990.2 0.3 0.80.2 0.1 0.556 8.2 0.751 0.92 0.99ND 2.5 ND

are: qPCR, quantitative polymerase chain reaction; LDO, lesion+L-Dopa; LV, lesion+

Fig. 5. Comparison between acute or chronic L-Dopa-induced gene profiles. Chartillustrating the number of regulated genes in response to both acute and chronic L-Dopatreatments. Genes are classified according to their ontology. Note that a greater numberof genes are altered by chronic L-Dopa.

Table 3Genes that are commonly regulated by acute and chronic L-Dopa.

Gene name (and symbol) Accessionnumber

Activity regulated cytoskeletal-associated protein (Arc) NM_019361ATPase isoform 2, Na+K+ transporting, beta polypeptide 2 (Atp2b2) NM_012508Claudin-5 (Cldn5) AF241260Cofilin 1 (Cfl1) NM_017147Enolase 1, alpha (Eno1) NM_012554Glutathione-S-transferase, mu type 2 (Yb2) (Gstm2) NM_017014Groucho (Aes) NM_019220Homer-1a AJ276327Ischemia responsive protein 94 kDa (Irp94) (Hspa4) AF077354Laminin receptor 1 (Lamr1) NM_017138Lin-7-A AF090133Lysosomal membrane glycoprotein (Lamp1) U75406MAP1A and 1B light chain 3 (Map1lc3b) U05784Mitochondrial voltage dependent anion channel (Vdac3) AB039664Synaptosomal-associated protein 25 (Snap25) AF245227"Zinc-finger transcription factor NGFI-C (Egr4) NM_019137

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al., 1993). By contrast, the chronic stimulation of dopamine receptors(by repeated administration of L-Dopa or psychostimulants) triggerslong-lasting alteration of other genes such as the chronic Fos-relatedantigens ΔFosB (Andersson et al., 1999), the striatal neuropeptidesdynorphin and enkephalin (Cenci et al., 1998), as well as variousstructural- and plasticity-related genes (Sgambato-Faure et al., 2005).Interestingly, it has been shown from experimental studies usinganimalmodels of LIDs that persistent molecular alterations, noticeablyrelated to both dopamine and glutamate signaling, are taking place inthe striatum and are critically involved in both the occurrence and theseverity of LIDs (see for reviews, Chase, 2004; Jenner, 2008). However,most of these studies were looking at one or two particular genes ofinterest and did not allow having a complete picture of biochemicaland molecular alterations underlying priming to L-Dopa and LIDs.

Biotechnological tools such as cDNA microarrays or protein chipsallow global change investigations and therefore the potentialemergence of families of genes and biological pathways. In the fieldof PD, transcriptomic studies analyzing tissues from MPTP-treatedmice (Kühn et al., 2003), MPTP-intoxicated monkeys (Bassilana et al.,2005) or parkinsonian patients (Grünblatt et al., 2004) have shownthat the pathogenesis of neurodegeneration correlates to alterations ofgenes involved in diverse functions such as oxidative stress, energymetabolism, protein folding/degradation, dopamine, glutamate andneurotrophic factors pathways. Similar findings were obtained fromproteomic studies investigating tissues from animal models (De Iuliiset al., 2005) or from parkinsonian patients (Basso et al., 2004). In thefield of movement-related disorders, both transcriptomic (Ferrarioet al., 2004; Konradi et al., 2004) and proteomic (Valastro et al., 2007)

Fig. 6. Venn diagram. 137 genes are regulated by chronic (C) L-Dopa, 55 by acute (A)L-Dopa treatment and 16 by both treatments in the dopaminergic depleted striatum.

studies have been performed on striatal samples from LIDs rats.Altogether, these studies have shown that chronic L-Dopa treatment isassociated to an increase of transcriptional activity, structural andsynaptic plasticity, calcium signaling,microvascular remodeling and toa decrease of capacity of energy production and of protein degradation.However, due to time of sacrifice (18 h after the last L-Dopa dose)(Konradi et al., 2004; Valastro et al., 2007) and way of administration(L-Dopa in tap water) (Ferrario et al., 2004), the global changesoccurring during abnormal motor manifestation itself have not yetbeen investigated. Furthermore, none of these studies did compare theeffects of de novo versus long-term L-Dopa on striatal global altera-tions. Such a comparison has been investigated in the MPTP-treatedmonkey at the proteomic level using two-dimensional difference in gelelectrophoresis and mass spectrometry (Kultima et al., 2006; Scholzet al., 2008). Although the classical priming concept posits that LIDsresults from a progressive sensitization through repeated L-Dopaadministration, it has been suggested that thefirst L-Dopa dose equatesto chronic exposure (Scholz et al., 2008). Noticeably, de novo L-Dopawould induce irreversible post-translational modification-based pro-teomic changes (such as phosphorylation or proteolytic cleavage) thatremain after chronic L-Dopa treatment. More in agreement with theclassical priming view, shift in protein expression in several metabolicpathwayswould also correlate to LIDs (Scholz et al., 2008). Our presentrat transcriptomic study brings very complementary data. Indeed,we show that acute and chronic L-Dopa treatments share commongene regulation features. But we also found that the transcriptomicresponse is enhanced by chronic L-Dopa and that specific biologicalalterations are underlying abnormal motor behavior driven by therepeated treatment.

Among the common genes regulated by acute or chronic L-Dopaare found arc and homer-1a. Both of them were already shown to beinduced in the dyskinetic rat (Sgambato-Faure et al., 2005). Highlyenriched in neuronal dendrites where it localizes in a distribution thatparallels that of F-actin, Arc interacts with cytoskeletal proteins suchas MAP-2 and promotes CaM kinase II-dependent neurite extension.Its fast dendritic translation is involved in a protein synthesis-dependent form of plasticity. Arc regulates AMPA receptor traffickingand synaptic plasticity, and is implicated in memory mechanisms(Tzingounis and Nicoll, 2006). It may thus play an important role inmediating experience-induced reorganization and/or development ofsynaptic connections. Homer proteins regulate signal transduction,synaptogenesis and receptor trafficking, in addition to maintainingand regulating extracellular glutamate levels in certain brain regions(Szumlinski et al., 2008). Interestingly, potential role of this proteinhas been evidenced in behavioral pathologies associated with neuro-psychiatric disorders (Szumlinski et al., 2006). Other genes might be

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very interesting such as SNAP-25, the synaptosomal associatedprotein, crucial for neurotransmitter release. Deficiency of SNAP-25expression seen in the Coloboma mice, a model of attention deficithyperactive disorder, correlates to hyperactivity and implicatesSNAP-25 in behavioral regulation (Hess et al., 1996). Alterations ofSNAP-25 levels have been found in several pathologies such asHuntington's disease (Smith et al., 2007) and bipolar disorder (Scarret al., 2006). Alterations of such genes as soon as the first L-Dopadose strongly support the critical involvement of phosphorylation,signal transduction, homeostasis, exocytosis and synaptic transmis-sion processes, comforting previous data obtained in the MPTP ma-caque model of PD and LIDs (Scholz et al., 2008). Of note, previousstudies already pointed out the relevance of phosphorylation-, signaltransduction- and synapse-related mechanisms (Gerfen et al., 2002;Picconi et al., 2003). Similarly, altered expression of cofilin andMAP1LC3 strongly suggest the early requirement for cytoarchitec-tural modifications. Cofilin family proteins are key regulators of actinfilament turnover and cytoskeleton reorganization (Meng et al.,2003). MAP1LC3 is a microtubule-associated protein (MAP) thatinteracts with MAP1A and MAP1B, involved in microtubule stabilityrequired for formation and development of axons and dendrites, aswell as axonal transport. Altogether, these data are comforted by theappearance of membrane and cytoskeleton as the most importantcomponents common to both acute and chronic L-Dopa. Noteworthy,alterations of dendritic morphology has been shown in prefrontalcortical and striatal neurons after DA depletion (Solis et al., 2007). Areduction of spine density and of glutamatergic synapses have beendemonstrated in indirect-pathway striatal neurons in hemiparkinso-nian rats (Day et al., 2006). The impact of L-Dopa on such morpho-logical changes remains to be elucidated.

Among the genes specifically regulated by long-term L-Dopafigure neuroserpin and calcineurin A alpha. Neuroserpin is a memberof the serine proteinase inhibitor (serpin) superfamily that is secretedfrom growth cones of neurons and inhibits the enzyme tissue-typeplasminogen activator (tPA) (Miranda and Lomas, 2006). Its temporaland spatial pattern of expression suggests a role in synaptogenesisand is most prominent in brain areas of the brain that participate inlearning, memory and behavior. Neuroserpin is released fromneurons in response to neuronal depolarization and plays animportant role in the development of synaptic plasticity. It alsoprovides neuronal protection in cerebral ischemia and epilepsy.Calcineurin is a critical protein phosphatase whose main function isto negatively modulate learning, memory and plasticity. In pre- andpostsynaptic compartments, calcineurin has a role in exocytosis,endocytosis and signaling. In the cytoplasm, it regulates calciumhomeostasis. It also has an impact on regulation of gene expression inthe nucleus. Finally, calcineurin has been associated with many braindisorders including for example Huntington's and Alzheimer'sdiseases, amyotrophic lateral sclerosis, dementia (Mansuy andShenolikar, 2006). The specific alteration of these two genes bylong-term L-Dopa strongly suggests that molecular and cellularmodifications involving complex processes are taking place in thedifferent part of the neuron. This is corroborated by our bio-associations results showing the potential implication of variouscomponents after long-term L-Dopa.

In conclusion, our data provide the first large-scale transcriptomiccomparison between acute and chronic L-Dopa treatments in the6-OHDA rat model of Parkinson's disease and evidence that both com-mon and distinct biological responses are induced in striatal neurons.

Acknowledgments

The authors thank Drs. Lydia Kerkerian-Le Goff and Erwan Bezardfor critical reading of the manuscript. The authors thank also FrédéricMantel for technical assistance. This study was supported by theInstitut National de la Santé et de la Recherche Médicale.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.nbd.2009.02.002.

References

Andersson, M., Hilbertson, A., Cenci, M.A., 1999. Striatal fosB expression is causallylinked with L-DOPA-induced abnormal involuntary movements and the associatedupregulation of striatal prodynorphin mRNA in a rat model of Parkinson's disease.Neurobiol. Dis. 6, 461–474.

Barbeau, A., 1969. L-Dopa therapy in Parkinson's disease: a critical review of nine yearsexperience. Can. Med. Assoc. J. 101, 791–800.

Bassilana, F., Mace, N., Li, Q., Stutzmann, J.M., Gross, C.E., Pradier, L., Benavides, J.,Ménager, J., Bezard, E., 2005. Unraveling substantia nigra sequential geneexpression in a progressive MPTP-lesioned macaque model of Parkinson's disease.Neurobiol. Dis. 20, 93–103.

Basso, M., Giraudo, S., Corpillo, D., Bergamasco, B., Lopiano, L., Fasano, M., 2004.Proteome analysis of human substantia nigra in Parkinson's disease. Proteomics 4,3943–3952.

Berke, J.D., Paletzki, R.F., Aronson, G.J., Hyman, S.E., Gerfen, C.R., 1998. A complexprogram of striatal gene expression induced by dopaminergic stimulation.J. Neurosci. 18, 5301–5310.

Bezard, E., Brotchie, J.M., Gross, C.E., 2001. Pathophysiology of levodopa-induceddyskinesia: potential for new therapies. Nat. Rev. Neurosci. 2, 577–588.

Blanchet, P.J., Calon, F., Morissette, M., Hadj Tahar, A., Bélanger, N., Samadi, P., Grondin,R., Grégoire, L., Meltzer, L., Di Paolo, T., Bédard, P.J., 2004. Relevance of the MPTPprimate model in the study of dyskinesia primingmechanisms. Parkinsonism Relat.Disord. 10, 297–304.

Cenci, M.A., 2007. Dopamine dysregulation of movement control in L-DOPA-induceddyskinesia. Trends Neurosci. 30, 236–243.

Cenci, M.A., Lee, C.S., Bjorklund, A., 1998. L-DOPA-induced dyskinesia in the rat isassociated with striatal overexpression of prodynorphin- and glutamic aciddecarboxylase mRNA. Eur. J. Neurosci. 10, 2694–2706.

Chase, T.N., 2004. Striatal plasticity and extrapyramidalmotor dysfunction. ParkinsonismRelat. Disord. 10, 305–313.

Day, M., Wang, Z., Ding, J., An, X., Ingham, C.A., Shering, A.F., Wokosin, D., Ilijic, E., Sun, Z.,Sampson, A.R., Mugnaini, E., Deutch, A.Y., Sesack, S.R., Arbuthnott, G.W., Surmeier,D.J., 2006. Selective elimination of glutamatergic synapses on striatopallidalneurons in Parkinson disease models. Nat Neurosci. 9, 251–259.

De Iuliis, A., Grigoletto, J., Recchia, A., Giusti, P., Arslan, P., 2005. A proteomic approach inthe study of an animal model of Parkinson's disease. Clin. Chim. Acta 357, 202–209.

Defebvre, L., 2004. Motor complications in dopa treatment of parkinson disease: clinicaldescription and evaluation. Thérapie 59, 93–96.

Eisen, M.B., Spellman, P.T., Brown, P.O., Botstein, D., 1998. Cluster analysis and display ofgenome-wide expression patterns. Proc. Natl. Acad. Sci. U. S. A. 95, 14863–14868.

El Atifi, M., Dupré, I., Rostaing, B., Chambaz, E.M., Benabid, A.L., Berger, F., 2002. Longoligonucleotide arrays onnylon for large-scale geneexpressionanalysis. Biotechniques33, 612–618.

El Atifi, M., Dupré, I., Rostaing, B., Benabid, A.L., Berger, F., 2003. Quantification of DNAprobes on nylonmicroarrays using T4 polynucleotide kinase labeling. Biotechniques35, 262–266.

Ferrario, J.E., Taravini, I.R., Mourlevat, S., Stefano, A., Delfino, M.A., Raisman-Vozari, R.,Murer, M.G., Ruberg, M., Gershanik, O., 2004. Differential gene expression inducedby chronic levodopa treatment in the striatum of rats with lesions of thenigrostriatal system. J. Neurochem. 90, 1348–1358.

Filion, M., 2000. Physiological basis of dyskinesia. Ann. Neurol. 47, S35–S40.Gerfen, C.R., Miyachi, S., Paletzki, R., Brown, P., 2002. D1 dopamine receptor

supersensitivity in the dopamine-depleted striatum results from a switch in theregulation of ERK1/2/MAP kinase. J. Neurosci. 22, 5042–5054.

Grünblatt, E., Mandel, S., Jacob-Hirsch, J., Zeligson, S., Amariglo, N., Rechavi, G., Li, J.,Ravid, R., Roggendorf, W., Riederer, P., Youdim, M.B., 2004. Gene expressionprofiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitin–proteasome, heat shock protein, iron and oxidative stress regulated proteins, celladhesion/cellular matrix and vesicle trafficking genes. J. Neural. Transm. 111,1543–1573.

Hess, E.J., Collins, K.A., Wilson, M.C., 1996. Mouse model of hyperkinesis implicatesSNAP-25 in behavioral regulation. J. Neurosci. 16, 3104–3111.

Jenner, P., 2008. Molecular mechanisms of L-DOPA-induced dyskinesia. Nat. Rev.Neurosci. 9, 665–677.

Konradi, C., Westin, J.E., Carta, M., Eaton, M.E., Kuter, K., Dekundy, A., Lundblad, M.,Cenci, M.A., 2004. Transcriptome analysis in a rat model of L-DOPA-induceddyskinesia. Neurobiol. Dis. 17, 219–236.

Kühn, K., Wellen, J., Link, N., Maskri, L., Lübbert, H., Stichel, C.C., 2003. The mouseMPTP model: gene expression changes in dopaminergic neurons. Eur. J. Neurosci.17, 1–12.

Kultima, K., Scholz, B., Alm, H., Sköld, K., Svensson, M., Crossman, A.R., Bezard, E.,Andrén, P.E., Lönnstedt, I., 2006. Normalization and expression changes inpredefined sets of proteins using 2D gel electrophoresis: a proteomic study ofL-DOPA induced dyskinesia in an animal model of Parkinson's disease using DIGE.BMC Bioinformatics 7, 475.

Mandel, S., Weinreb, O., Youdim, M.B., 2003. Using cDNA microarray to assessParkinson's disease models and the effects of neuroprotective drugs. TrendsPharmacol. Sci. 24, 184–191.

350 M.E. El Atifi-Borel et al. / Neurobiology of Disease 34 (2009) 340–350

Mansuy, I.M., Shenolikar, S., 2006. Protein serine/threonine phosphatases in neuronalplasticity and disorders of learning and memory. Trends Neurosci. 29, 679–686.

Meng, Y., Zhang, Y., Tregoubov, V., Falls, D.L., Jia, Z., 2003. Regulation of spine morphologyand synaptic function by LIMK and the actin cytoskeleton. Rev. Neurosci. 14, 233–240.

Miranda, E., Lomas, D.A., 2006. Neuroserpin: a serpin to think about. Cell. Mol. Life Sci.63, 709–722.

Mirnics, K., Pevsner, J., 2004. Progress in the use of microarray technology to study theneurobiology of disease. Nat. Neurosci. 7, 434–439.

Morelli, M., Cozzolino, A., Pinna, A., Fenu, S., Carta, A., Di Chiara, G., 1993. L-dopastimulates c-fos expression in dopamine denervated striatum by combinedactivation of D-1 and D-2 receptors. Brain Res. 623, 334–336.

Obeso, J.A., Rodríguez-Oroz, M.C., Rodríguez, M., Lanciego, J.L., Artieda, J., Gonzalo, N.,Olanow, C.W., 2000. Pathophysiology of the basal ganglia in Parkinson's disease.Trends Neurosci. 23, S8–S19.

Obeso, J.A., Rodriguez-Oroz, M., Marin, C., Alonso, F., Zamarbide, I., Lanciego, J.L.,Rodriguez-Diaz, M., 2004. The origin of motor fluctuations in Parkinson's disease:importance of dopaminergic innervation and basal ganglia circuits. Neurology 62,S17–S30.

Picconi, B., Centonze, D., Håkansson, K., Bernardi, G., Greengard, P., Fisone, G., Cenci,M.A., Calabresi, P., 2003. Loss of bidirectional striatal synaptic plasticity in L-DOPA-induced dyskinesia. Nat. Neurosci. 6, 501–506.

Putterman, D.B., Munhall, A.C., Kozell, L.B., Belknap, J.K., Johnson, S.W., 2007. Evaluationof levodopa dose and magnitude of dopamine depletion as risk factors forlevodopa-induced dyskinesia in a rat model of Parkinson's disease. J. Pharmacol.Exp. Ther. 323, 277–284.

Scarr, E., Gray, L., Keriakous, D., Robinson, P.J., Dean, B., 2006. Increased levels of SNAP-25 and synaptophysin in the dorsolateral prefrontal cortex in bipolar I disorder.Bipolar Disord. 8, 133–143.

Scholz, B., Svensson, M., Alm, H., Sköld, K., Fälth, M., Kultima, K., Guigoni, C., Doudnikoff,E., Li, Q., Crossman, A.R., Bezard, E., Andrén, P.E., 2008. Striatal proteomic analysissuggests that first L-dopa dose equates to chronic exposure. PLoS ONE 3, e1589.

Sgambato-Faure, V., Buggia, V., Gilbert, F., Lévesque, D., Benabid, A.L., Berger, F., 2005.Coordinated and spatial upregulation of arc in striatonigral neurons correlates withL-dopa-induced behavioral sensitization in dyskinetic rats. J. Neuropathol. Exp.Neurol. 64, 936–947.

Smith, R., Klein, P., Koc-Schmitz, Y., Waldvogel, H.J., Faull, R.L., Brundin, P., Plomann, M.,Li, J.Y., 2007. Loss of SNAP-25 and rabphilin 3a in sensory–motor cortex inHuntington's disease. J. Neurochem. 103, 115–123.

Solis, O., Limón, D.I., Flores-Hernández, J., Flores, G., 2007. Alterations in dendriticmorphology of the prefrontal cortical and striatum neurons in the unilateral6-OHDA-rat model of Parkinson's disease. Synapse 61, 450–458.

Szumlinski, K.K., Kalivas, P.W., Worley, P.F., 2006. Homer proteins: implications forneuropsychiatric disorders. Curr. Opin. Neurobiol. 16, 251–257.

Szumlinski, K.K., Ary, A.W., Lominac, K.D., 2008. Homers regulate drug-inducedneuroplasticity: implications for addiction. Biochem. Pharmacol. 75, 112–133.

Tzingounis, A.V., Nicoll, R.A., 2006. Arc/Arg3.1: linking gene expression to synapticplasticity and memory. Neuron 52, 403–407.

Valastro, B., Dekundy, A., Krogh, M., Lundblad, M., James, P., Danysz,W., Quack, G., Cenci,M.A., 2007. Proteomic analysis of striatal proteins in the rat model of L-DOPA-induced dyskinesia. J. Neurochem. 102, 1395–1409.