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patiotemporal Pattern of Striatal ERK1/2hosphorylation in a Rat Model of L-DOPA–Inducedyskinesia and the Role of Dopamine D1 Receptors
enny E. Westin, Linda Vercammen, Elissa M. Strome, Christine Konradi, and M. Angela Cenci
ackground: We examined the activation pattern of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and its dependence on D1ersus D2 dopamine receptors in hemiparkinsonian rats treated with 3,4-dihydroxyphenyl-L-alanine (L-DOPA).
ethods: 6-Hydroxydopamine-lesioned rats were treated acutely or chronically with L-DOPA in combination with antagonists for D1 or D2eceptors. Development of dyskinesia was monitored in animals receiving chronic drug treatment. Phosphorylation of ERK1/2, mitogen- andtress-activated protein kinase-1 (MSK-1), and the levels of FosB/�FosB expression were examined immunohistochemically.
esults: L-DOPA treatment caused phosphorylation of ERK1/2 in the dopamine-denervated striatum after acute and chronic administra-ion. Similar levels were observed in matrix and striosomes, and in enkephalin-positive and dynorphin-positive neurons. The severity ofyskinesia was positively correlated with phospho-ERK1/2 levels. Phosphorylation of ERK1/2 and MSK-1 was dose-dependently blocked byCH23390, but not by raclopride. SCH23390 also inhibited the development of dyskinesia and the induction of FosB/�FosB.
onclusions: L-DOPA produces pronounced activation of ERK1/2 signaling in the dopamine-denervated striatum through a D1-receptor-ependent mechanism. This effect is associated with the development of dyskinesia. Phosphorylated ERK1/2 is localized to both dynorphin-
rgic and enkephalinergic striatal neurons, suggesting a general role of ERK1/2 as a plasticity molecule during L-DOPA treatment.ey Words: Gene transcription, motor complications, Parkinson’sisease, rodent, signaling pathways, therapy
he dopamine (DA) precursor 3,4-dihydroxyphenyl-L-ala-nine (L-DOPA) is still the most effective and commonlyused treatment for Parkinson’s disease (PD), although its
ong-term use is hampered by serious complications, such asbnormal involuntary movements (dyskinesia), in a majority ofatients (Marsden 1994; Nutt 1990). The primary cause of-DOPA–induced dyskinesia is not known, but chronic dyskine-iogenic treatment with L-DOPA is closely paralleled by largehanges in striatal gene expression in both rodent and primateodels of Parkinson’s disease (Aubert et al. 2006; Brotchie et al.998; Cenci et al. 1998; Doucet et al. 1996; Konradi et al. 2004).he intracellular signaling pathways mediating this aberrant geneesponse have yet to be determined.
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) areritical mediators of activity-dependent plasticity as demon-trated by their role in long-term potentiation (English and Sweatt996; Impey et al. 1998; Mazzucchelli et al. 2002), classicalonditioning (Crow et al. 1998), and memory formation (Atkinst al. 1998; Brambilla et al. 1997). Phosphorylation of ERK1/2eflects a balance between the activities of upstream kinases andnactivating phosphatases (Keyse 2000). Active ERK1/2 mediatehanges in gene expression via phosphorylation of histoneinase proteins, such as mitogen- and stress-activated protein
rom the Basal Ganglia Pathophysiology Unit (JEW, LV, EMS, MAC), Depart-ment of Experimental Medical Science, Lund University, Sweden; Labo-ratory of Neurobiology and Gene Therapy (LV), Leuven, Belgium; Depart-ment of Psychiatry (CK), Vanderbilt University, Nashville, Tennessee.
ddress reprint requests to M. Angela Cenci, M.D., Ph.D., Basal Ganglia Patho-physiology Unit, Department of Experimental Medical Science, Lund Uni-versity, BMC F11, 221 84 Lund, Sweden; E-mail: [email protected].
eceived May 4, 2006; revised October 24, 2006; accepted November 21,
2006.006-3223/07/$32.00oi:10.1016/j.biopsych.2006.11.032
kinase-1 (MSK-1; Deak et al. 1998; Dunn et al. 2005) andactivation of nuclear transcription factors, including Elk-1 andCREB (Marshall 1995).
Previous studies reported that selective agonists for D2-typeDA receptors enhanced ERK1/2 phosphorylation in the DA-denervated striatum (Cai et al. 2000; Zhen et al. 2002). Bycontrast, Gerfen and collaborators (2002) showed that agonists ofD1- but not D2 receptors induced ERK1/2 phosphorylation instriatal medium-sized neurons of hemiparkinsonian rats. Bothreports suggested that increased ERK1/2 activity in striatal neu-rons is a possible mechanism of L-DOPA–induced dyskinesia(LID).
Here we examine the DA receptor-dependence of ERK1/2phosphorylation in hemiparkinsonian rats treated with L-DOPA,and we compare it with �FosB, an established molecular markerof dyskinesia in this model (Andersson et al. 1999; Cenci et al.1999; Westin et al. 2001). The fosB gene contains a SRE regula-tory element in its promotor sequence (Lazo et al. 1992), whichis a potential target for ERK1/2-dependent regulation. We pro-vide the first evidence of an association between the striatallevels of ERK1/2 phosphorylation and the severity of the dyski-netic motor response evoked by L-DOPA. Furthermore, we showthat the striatal activation of ERK1/2-dependent signaling byL-DOPA relies on D1- and not D2-type DA receptors and that thisclass of receptors is also essential for the development ofdyskinesia and for the induction of �FosB-like proteins. Finally,we describe a broad striatal distribution of ERK1/2 activation byL-DOPA, encompassing both the dynorphinergic and the en-kephalinergic striatal efferent populations.
Methods and Materials
Detailed protocols can be found in Supplement 1.
SubjectsFemale Sprague–Dawley rats (225 g; Harlan, Zeist, The Neth-
erlands) were housed under a 12-hour light–dark cycle, with ad
libitum access to food and water. Animal care and treatmentBIOL PSYCHIATRY 2007;62:800–810© 2007 Society of Biological Psychiatry
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aradigms were approved by the Malmö-Lund Ethical Committeen Animal Research.
opamine Denervating Lesions6-Hydroxydopamine (6-OHDA-HCl; Sigma-Aldrich, Stock-
olm, Sweden) was injected into the right-ascending DA fiberundle, as previously described (Cenci et al. 1998). Two weeksostlesion, striatal denervation was evaluated using amphet-mine-induced rotation (2.5 mg/kg D-amphetamine intraperito-eal injection [IP]). Rats showing greater than 5 full turns/minpsilateral to the lesion, corresponding to � 90% striatal dopa-ine depletion (Carta et al., 2006; Winkler et al. 2002), were
elected for this study. The extent of the lesion was verifiedhrough immunohistochemical staining for tyrosine hydroxylaseTH) of DA axon terminals in the striatum and DA cell bodies inhe substantia nigra.
rug TreatmentAdministration of L-DOPA methyl ester (10 mg/kg; Sigma-
ldrich) and the peripheral DOPA-decarboxylase inhibitorenserazide-hydrochloride (15 mg/kg; Sigma-Aldrich) was initi-ted 3 weeks postamphetamine screening, as described previ-usly (Andersson et al. 2001; Cenci et al. 1999). The D1 receptorntagonist SCH23390 (.25 mg/kg or .05 mg/kg in .9% NaCl, IP;igma-Aldrich) or the D2 receptor antagonist raclopride (2.0g/kg in .9% NaCl IP; Sigma-Aldrich) were administered IP 30in before L-DOPA.
xperimental DesignAnimals with 6-OHDA lesions were randomly assigned to
cute L-DOPA (12 days of saline followed by L-DOPA on Day3), chronic L-DOPA (13 daily L-DOPA injections), or salinereatment (13 daily saline injections). Chronically treated animalsere tested as previously described (Cenci et al. 1998; Cenci andundblad 2005; Lundblad et al. 2002) and labeled as “dyskinetic”r “nondyskinetic” (see Supplement 1). Animals defined asondyskinetic exhibited either no or only occasional abnormalnvoluntary movements (AIMs) of minimal severity. The devel-pment of AIMs during chronic L-DOPA treatment in animalslassified as dyskinetic or nondyskinetic is shown in Supplement 2.
Twenty-two animals (n � 6 dyskinetic, 4 nondyskinetic, 8cute, and 4 saline control animals) were prepared for Westernmmunoblotting 20 min or 3 hours after the last L-DOPA injec-ion, and 49 animals were processed for immunohistochemistryIHC) at 20 min (n � 8 chronic and 7 acute), 60 min (n � 8hronic and 8 acute), 120 min (n � 4 chronic and 3 acute), or 24ours (n � 8 chronic and 3 acute) after the last L-DOPA injection.
For the studies on DA receptor subtype involvement, ratsere treated with either the D1 receptor antagonist SCH23390 or
he D2 receptor antagonist raclopride 30 min before an acute-DOPA challenge (n � 8 per group). As a reference group, eightats received saline 30 min prior to L-DOPA. Control animalseceived injections of saline (n � 4), SCH23990 only (n � 4), oraclopride only (n � 6). All animals were killed 30 min after thehallenge injection, and the brains were processed for IHC. Aroup of animals (n � 8) received an acute dose of thentiakinetic drug, bromocriptine (5 mg/kg IP), which mainly actss a D2 agonist with some D1 antagonistic properties (Bedard etl. 1986; Lieberman and Goldstein 1985). Bromocriptine-treatedats were killed 90 min (n � 4) or 180 min (n � 4) after the drugnjection, which represent the beginning and the plateau phase,espectively, in the pharmacodynamic curve in rats (Lundblad et
l. 2002).In a chronic coadministration experiment, animals receivedSCH23390 (.25 mg/kg, n � 8; or .05 mg/kg, n � 8), 30 min beforeL-DOPA for 13 days. Control groups received saline 30 minbefore L-DOPA (n � 8) or SCH23390 (.05 mg/kg, n � 3; or .25mg/kg, n � 3) followed by an injection of saline. All rats werekilled 30 min after the last injection and the brains wereprocessed for immunohistochemistry (IHC).
To verify the occurrence of L-DOPA–induced ERK1/2 activa-tion in enkephalinergic neurons, three dyskinetic and threesaline-treated rats were prepared for the combined detection ofphospho-ERK1/2 by IHC and enkephalin mRNA by in situhybridization.
Tissue Preparation for IHCAll animals were anesthetized with sodiumpentabarbitone
(240 mg/kg body weight IP; Apoteksbolaget, Uppsala, Sweden)and transcardially perfused with 4% ice-cold, buffered (pH 7.4)paraformaldehyde (PFA; Merck, Stockholm, Sweden). Brainswere rapidly extracted, postfixed for 2 hour in PFA, and trans-ferred to 20% buffered sucrose for 24 hours; coronal sectionswere cut on a freezing microtome at 40-�m thickness or 16 �mfor IHC in combination with in situ hybridization histochemistry(ISHH). The brains were cut between the level of nucleusaccumbens (bregma �1.70) to caudal to substantia nigra(bregma –7.0; (Paxinos and Watson 1998). Free-floating sectionswere stored in cryoprotective solution at –20°C until furtherprocessing.
Western ImmunoblottingStriatal protein extracts were prepared from left and right
striatum, and 2.3 �g protein from each rat was used for Westernimmunoblotting (protocol in Supplement 1).
The primary antibodies used were rabbit polyclonal antiphos-pho(Thr202/Tyr204) p44/42MAPK, 1:1000; and rabbit polyclonalanti-p44/42MAPK,1:2000 (both from Cell Signaling Technology,Beverley, Massachusetts). All blots were stripped and reprobedwith 1:20,000 anti-�actin (Sigma).
Bright-Field ImmunohistochemistryImmunohistochemistry was performed as in Cenci et al.
(1999) using a peroxidase-based detection method and 3-3=-diaminobenzidine (DAB; Sigma-Aldrich) as the chromogen. Thefollowing primary antisera were used: antiphospho(Thr202/Tyr204) p44/42MAPK (1:250 for 36 hours; Cell Signaling Tech-nologies), rabbit monoclonal antiphospho(Ser376) MSK-1 (1:200for 48 hours; Epitomics, Burlingame, California), or goat anti-FosB/�FosB (1:10000 for 16 hours; Santa Cruz Biotechnology,Santa Cruz, California).
Two-Color Dual Antigen Fluorescence ImmunohistochemistryFor dual-antigen immunofluorescence, sections were first
stained for phospho-ERK1/2 using a mouse monoclonal anti-body (1:100; Cell Signaling Technologies) followed by one of thefollowing antisera: rabbit polyclonal against met-enkephalin(1:1000; BioSorin, Saluggia, Italy), rabbit polyclonal antidynor-phin (1:2000, kindly provided by Dr. L. Terenius, Uppsala,Sweden), or rabbit monoclonal against phospho(Ser376) MSK-1(1:100; protocol in Supplement 1).
Combined Immunohistochemistry and In Situ HybridizationHistochemistry
To verify the occurrence of L-DOPA–induced phospho-ERK1/2 in enkephalinergic striatal neurons, bright-field IHC
staining for phospho-ERK1/2 was combined with the detectionwww.sobp.org/journal
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f enkephalin mRNA by radioactive in situ hybridization (proto-ol in Supplement 1). Sections were stained for phospho-ERK1/2ccording to the standard protocol, except that all buffers usedere autoclaved to minimize RNA degradation, blocking solu-
ion was made up of 3% BSA (Fraction V, Sigma-Aldrich), andlocking time was reduced to 20 min.
mage Analysis and Cell CountsWestern immunoblots were developed on autoradiographic
ilms (Kodak, Rochester, New York), scanned, and analyzed withhe Image J software (http://rsb.info.nih.gov/ij/index.html). Theptical density was determined on each band of interest after themages had been calibrated using a Kodak photographic stepablet according to the software instructions. Optical density dataere expressed as a percentage of saline control values within
he same experiment, unless otherwise stated.Cells immunoreactive for phospho-ERK1/2 and phospho-
SK-1 were counted by a blinded investigator using the Image Joftware. Sample areas (.410 � .328 mm) were visualized under20� objective in a Nikon Eclipse 80i microscope (Nikon,
okyo, Japan) and digitized through a Nikon DMX 1200F videoamera. Analysis was carried out in both the medial and lateralart of the caudate–putamen (CPu) in 16 sample areas pernimal, distributed in two sections through the mid-rostrocaudalevels of the structure (see Figure 4E). Data are expressed as theumber of immunoreactive cells per square mm.
igure 1. Western immunoblots and bar histograms of phospho-extracelluopamine-denervated striata after treatment with 3,4-dihydroxyphenyl-L
thicker band) were upregulated in chronically L-DOPA-treated dyskinetic ahronically L-DOPA–treated rats were killed 20 min after the last injection. At 20 min (group a), but not 3 hours postinjection (group A). Data are exprep � .05 versus saline-treated control group, # p � .05 versus chronic, nondyfter one-factor analysis of variance, where group effect � .002). (B and C) Throups. (D) Simple regression of phospho-ERK1/2 O.D. levels on the cumu
reated rats (groups d and n) shows that ERK1/2 phosphorylation was posit
he probability value of the regression (p) are given in the bottom right corner.ww.sobp.org/journal
Antigen colocalization on immunofluorescent sections wereanalyzed using a Zeiss Confocal laser scanning microscope andthe LSM 5 PASCAL software (Carl Zeiss, Heidelberg, Germany).Sampling was done on two sections per animal (480 �m apart)from mid-rostrocaudal CPu. Sample areas were selected using a40� objective and magnified threefold before scanning. Thesample areas were systematically moved along the dorsoventralaxis of the section at two mediolateral coordinates (approxi-mately 1.25 and 2.25 mm lateral to the midline, respectively),randomly covering both striosomal and matrix compartments.Cells were evaluated in the z dimension with a minimum of fiveconsecutive optical sections. One hundred cells were sampledfrom the medial part and the lateral part of the striatum. Sectionsimmunostained for phospho-ERK1/2 and subsequently hybrid-ized for PPE mRNA were examined under bright-field optics.
Statistical AnalysesCounts of phospho-ERK1/2 and phospho-MSK1 immunore-
active cells in the intact and lesioned striatum were analyzed bytwo-factor analysis of variance (ANOVA; group � side), followedby Tukey’s Honestly Significant Difference (HSD) test. Westernblots and counts of immunoreactive cells on the DA-denervatedside of the striatum were analyzed by one-factor ANOVA followedby post hoc Newman–Keuls test. Global AIMs scores were analyzedby repeated-measures ANOVA, and relevant differences within or
gnal-regulated kinases 1 and 2 (ERK1/2), total ERK1/2 and �-actin levels inne (L-DOPA) or saline. (A) Phospho-ERK1 (thin band) and phospho-ERK2s (d) compared with nondyskinetic cases (n) and saline-injected controls (s).cute L-DOPA treatment, ERK1/2 phosphorylation was significantly elevateds a percentage of the optical density (O.D.) levels of saline control animals.ic L-DOPA group, § p � .05 versus acute L-DOPA group (Newman–Keuls testl levels of ERK1/2 and �-actin did not differ between any of the experimentalaxial, limb, and orolingual AIM scores recorded from chronically L-DOPA
orrelated with dyskinesia severity. Pearson’s correlation coefficient (R) and
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etween groups were analyzed pairwise by Tukey’s HSD test. Theull hypothesis was rejected when p � .05.
esults
triatal ERK1/2 Activation after Acute or Chronic-DOPA Treatment
Levels of phosphorylated (Figure 1A) and total ERK1/2 (Fig-re 1B) were assessed in tissue lysates from the DA-denervatedtriatum from rats treated with L-DOPA either chronically orcutely. Both antibodies revealed two bands with the expectedolecular weights of ERK1 (44 kDa) and ERK2 (42 kDa), withRK2 as the prominent isoform (Mazzucchelli et al. 2002).
Acute administration of L-DOPA significantly increasedRK1/2 phosphorylation in the lesioned striatum 20 min, but not hours postinjection (Figure 1A). Among chronically L-DOPAreated rats (killed 20 min after the last drug injection), dyskineticnimals had 50% higher levels of ERK1/2 phosphorylation thanon-dyskinetic animals (Figure 1A). No significant differences inRK1/2 protein levels (Figure 1B) or �-actin levels (Figure 1C)ere found. Accordingly, a similar pattern of group differencesas found when phospho-ERK1/2 levels were expressed as a
atio over �-actin (170 � 10 for dyskinetic rats; 124 � 15 forondyskinetic rats; 138 � 19 for acute 20 min; 117 � 12 for acutehours; and 101 � 8 for saline-treated control rats; p � .002,
ne-factor ANOVA).A positive linear relationship was observed between the
everity of dyskinesia and the amount of phospho-ERK1/2 in theesioned striatum in chronically L-DOPA treated rats (Figure 1D).n the intact hemisphere, striatal levels of phospho-ERK1/2,otal ERK1/2, and �-actin were unaffected by acute or chronic
L-DOPA treatment and/or by the occurrence of dyskinesia (datanot shown).
Time Course and Distribution of L-DOPA–Induced ERK1/2Phosphorylation
The distribution and amount of phospho-ERK1/2 expressionwere evaluated semiquantitatively at the onset (20 min), the peak(60 min), and the decline phase (120 min) of the L-DOPA dosingcycle in rats treated with the drug either acutely or chronicallyand compared with the pattern in animals killed 24 hours afterthe last (or only) injection of L-DOPA.
The distribution of phospho-ERK1/2 did not show any con-sistent difference between the medial and the lateral part of thestriatum after either acute or chronic L-DOPA administration(Figure 2; see also Figures 3 and 4). In rats treated acutely withL-DOPA, phospho-ERK1/2 immunoreactivity was induced at 20and 60 min post– drug injection (Figure 2 A, 2A=, 2B, and 2B=),and decreased visibly by 120 min (Figure 2C and 2C=). In thechronically L-DOPA–treated group, levels of phospho-ERK1/2immunoreactivity were upregulated 20 and 60 min after theinjection (Figure 2E–2F=) and remained elevated also at the 120min time point (Figure 2G and 2G=). One animal per time pointin the chronic L-DOPA group did not develop dyskinesia, andthese rats displayed much fewer phospho-ERK1/2 immunoreac-tive cells. Twenty-four hours after the final L-DOPA injection,phospho-ERK1/2 was no longer detectable in either the acute orthe chronic L-DOPA groups (Figure 2D and 2D= and 2H and 2H=).
Effects of SCH23390 and RacloprideThe effect of selective antagonists for D1- or D2-type recep-
tors was examined with SCH23390 and raclopride, respectively.
Figure 2. Photomicrographs of phospho-extracellularsignal-regulated kinases 1 and 2 (ERK1/2) immunoreac-tive cells in the medial and lateral part of the caudate-putamen at 20, 60, and 120 min, and 24 hours after acute(A–D and A=–D=) or chronic 3,4-dihydroxyphenyl-L-ala-nine (L-DOPA) treatment with dyskinesia (E–H andE=–H=). Photos were taken on the side of the striatumipsilateral to the 6-hydroxydopamine lesions. Scale �100�m.
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s a marker of signaling pathway activation downstream ofRK1/2, we studied phospho(Ser376) MSK-1 (Deak et al.998), a histone H3 kinase that plays an essential role inRK1/2-dependent chromatin remodeling and transcriptionalhanges (Dunn et al. 2005). Although MSK-1 can also behosphorylated by p38MAPK kinase (Dunn et al. 2005), thisathway is not recruited by L-DOPA under our experimentalonditions (data not shown). Confocal microscopy showed thathospho-ERK1/2 and phospho-MSK-1 were colocalized (seeigure 7H–7H==).
The dose of .25 mg/kg SCH23390 was used to yield a maximal1 receptor occupancy without losing selectivity (Bischoff et al.986), and 2.0 mg/kg raclopride was chosen because it has beenroven sufficient to block D2 receptor-mediated cellular re-ponses in the striatum (Paul et al. 1992). The behavioral effectsf the selected doses were evaluated in a preliminary experimentsee Supplement 1).
In all the experimental animals, the antagonists were given 30in before an acute injection of L-DOPA, and the rats were killed
0 min later to study striatal ERK1/2 and MSK-1 activation by
HC. Counts of phospho-ERK1/2 and phospho-MSK-1 immuno-ww.sobp.org/journal
reactive cells were carried out in the medial and the lateral partof the CPu on both sides of the brain. When administered alone,L-DOPA induced a highly significant 10- to 20-fold increase in thenumber of phospho-ERK1/2 and MSK-1 immunoreactive cells inall aspects of the DA-denervated CPu (Figure 3A–A==; phospho-MSK-1 is shown in photomicrographs in Supplement 3, panelIA–A==) whereas the intact side was not affected at all (Figure4A– 4D). The induction of phospho-ERK1/2 and phospho-MSK-1by L-DOPA was completely inhibited in animals pretreated withSCH23390 (Figures 3 and Supplement 3, panels B= and B==),whereas raclopride had no effect (Figures 3 and Supplement 3,panels C= and C==). Either alone or combined with L-DOPA,raclopride induced a significant increase in the number ofphospho-ERK1/2 immunoreactive cells in the intact but not thedenervated striatum (Figure 4A and 4B), which is in agreementwith the report by Gerfen and coworkers (2002) that D2 receptorantagonists require an intact nigrostriatal system to activateERK1/2 in striatal neurons. Raclopride alone did not however,produce a significant change in the number of phospho-MSK-1positive cells on either the intact or the DA-denervated side of the
Figure 3. Photomicrographs illustrating the effects of D1- andD2-receptor antagonist pretreatment on extracellular signal-regulatedkinases1and2(ERK1/2)phosphorylationasassessed30 min after an acute challenge injection of 3,4-dihydroxyphe-nyl-L-alanine (L-DOPA) (Panel I). Each animal was given twoinjections in sequence, which are specified by the cap-tions on left-hand side (first injection) and on the top ofthe panel (second injection). In both the lateral and themedial striatum, administration of SCH23390 30 min be-fore L-DOPA completely inhibited phospho-ERK1/2 im-munoreactivity (B= and B==), whereas raclopride adminis-tration had no effect (C= and C==, cf. with L-DOPA alone inA= and A==). Photomicrographs in Panel II illustrate theeffects of bromocriptine, an antiparkinsonian drug thatdoes not induce dyskinesia. Although having only a weakstimulatory effect on phospho-ERK1/2 in the striatum (D),bromocriptine induced strong neuronal immunoreactiv-ity for phospho-ERK1/2 in the globus pallidus (GP) (E), astructure where L-DOPA did not have any effect (F). Thebromocriptine-treated animal illustrated in this picturewas euthanized 90 min postinjection, a time point atwhich the motor effects of the drug in this rat model startto become appreciable. All pictures were taken on theside of the striatum ipsilateral to the 6-hydroxydopaminelesion. Scale � 100 �m.
striatum (Figure 4C and 4D). SCH23390 alone had no effect on
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he number of phospho-ERK1/2 or phospho-MSK-1 positive cellsFigure 4A and 4D).
Striatal ERK1/2 and MSK-1 phosphorylation was also exam-ned after treatment with bromocriptine, an antiparkinsonianrug that produces intense motor activation without dyskinesiaLundblad et al. 2002). A high dose of bromocriptine (5 mg/kg;undblad et al. 2002) induced some weak cellular staining forhospho-ERK1/2 (Figure 3D) in both the intact and the dener-ated striatum, and the effect reached significance in the lateralPu on the intact side (Figure 4B). Interestingly, bromocriptineroduced strong ERK1/2 activation in neurons in the globusallidus (GP; Figure 3E), a structure where neither L-DOPAFigure 3F) nor any of the other treatments tested had inducedhospho-ERK1/2. Bromocriptine did not raise the number ofhospho-MSK-1 immunoreactive cells above control levels inither the CPu (Figures 4C and 4D, and Supplement 3) or the GPSupplement 3).
hronic Treatment with SCH23390 in Combination with-DOPA Inhibited the Development of Dyskinesia
To examine the effects of D1-antagonist treatment on dyski-
esia, animals were injected with either of two doses ofSCH23390 (.05 or .25 mg/kg/day) or with saline, 30 min beforetheir daily L-DOPA injection over 13 days. Ratings of AIMs werecarried out daily. The D1 antagonist suppressed the developmentof dyskinesia in a dose-dependent manner (Figure 5A). Thereduction in global AIMs per session by SCH23390 was due todecreased severity of dyskinesia (lower AIMs score per observa-tion point) as well as to a shortening of dyskinesia duration(Figure 5B). The effect of SCH23390 cannot be ascribed togeneral motor suppression, which lasted for only 1 hour in ourpreliminary experiments (cf. Supplement 1). Moreover, the an-tagonist was injected 50 min before the first observation point inthe session and could thus have affected only the initial score.
SCH23390 Inhibited ERK1/2 and MSK-1 Phosphorylation andthe Induction of FosB/�FosB by Chronic L-DOPA
Striatal levels of phospho-ERK1/2, phospho-MSK-1, andFosB/�FosB were analyzed by IHC in the animals pretreatedwith SCH23390 during chronic L-DOPA administration, whichwere euthanized 30 min after the final drug injection. SCH23390suppressed the activation of ERK1/2 and MSK-1 by L-DOPA in adose-dependent manner in both the medial and the lateral part of
Figure 4. Quantitative analysis of phospho-extracellularsignal-regulated kinases 1 and 2 (ERK1/2) (A, B) and phos-pho-mitogen- and stress-activated protein kinase-1(MSK-1) (C, D) immunoreactivity in the medial (A, C) andthe lateral part (B, D) of the caudate-putamen (CPu). Ani-mals were euthanized 30 min after an acute injection of3,4-dihydroxyphenyl-L-alanine (L-DOPA), alone or com-bined with the D1-antagonist, SCH23390, or the D2-an-tagonist, raclopride. The pair of bars farthest to the right ineach diagram illustrate the effects of bromocriptine. Greybars represent the lesioned hemisphere and white barsthe intact hemisphere in each group. Data are expressedas number of immunoreactive cells per square millimeter.Cell counting was carried out at the two rostrocaudallevels represented by the drawings (E), where hatchedareas indicate the medial and lateral regions sampled. p �.05 versus the lesioned side of, § vehicle treatment (nodrug); # SCH23390 only; SCH23390 � L-DOPA; raclo-pride only, � Bromocriptine. p � .05 versus the intact sideof � L-DOPA only; � Raclopride � L-DOPA; � SCH23390only; SCH23390 � L-DOPA; Bromocriptine (HonestlySignificant Difference test after two-factor analysis of vari-ance, where p � .001 for the effects of group, side, and thegroup-side interaction in each A, B, C, and D).
the CPu (photomicrographs in Supplement 4; quantification in
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igure 6A–D). In animals cotreated with L-DOPA and the higherose of the antagonist, levels of phospho-ERK1/2 and phospho-SK-1 did not differ from control values (Figure 6A– 6D). The
ower dose of SCH23390 could not fully inhibit L-DOPA–inducedctivation of phospho-ERK1/2 in the striatum (Supplement 4 andigures 6A and 6B). No correlation was found between thisesidual number of phospho-ERK1/2 immunoreactive cells andhe severity of dyskinesia (p � .38, regression analysis of AIMscore and number of phospho-ERK1/2 immunoreactive cells inhe low-dose SCH23390 group).
FosB/�FosB immunoreactivity was increased in dyskineticnimals in the patchy striosomes of the medial striatum and alonghe rim of the lateral CPu, as reported earlier (Andersson et al.999; Cenci et al. 1999). The distribution of FosB/�FosB immu-oreactivity thus was different from phospho-ERK1/2 and phos-ho-MSK-1, which were rather uniform across striatal compart-ents and regions (Supplement 4, compare B and B=, F and F=,
nd J and J=). Interestingly, the induction of FosB/�FosB byhronic L-DOPA treatment was completely inhibited by bothoses of SCH23390 (Supplement 4; Figure 6E and 6F).
n Dyskinetic Rats, Phospho-ERK1/2 Was Present inynorphinergic and Enkephalinergic Neurons,
n Striosomes, and in the MatrixL-DOPA–induced dyskinesia has been suggested to require a
referential activation of the striosome versus the matrix com-artment (Graybiel et al. 2000) and of the dynorphinergic versushe enkephalinergic striatal efferent pathway (Andersson et al.999; Sgambato-Faure et al. 2005; St-Hilaire et al. 2005). We thusxamined the compartmental distribution and cellular phenotypef striatal ERK1/2 activation in four chronically L-DOPA–treatedyskinetic rats. Dual-antigen immunofluorescence for phospho-RK1/2 and the matrix marker, Calbindin D28 (Kincaid andilson 1996; Moratalla et al. 1996), revealed no difference in
igure 5. Effects of SCH23390 coadministration on 3,4-dihydroxyphenyl-L-lanine (L-DOPA)–induced abnormal involuntary movements (AIMs). Thevolution of AIM scores during 12 days of L-DOPA treatment is shown in A.nimals receiving saline instead of SCH23390 before the daily L-DOPA dose
howed a rapid development of dyskinesia (filled circles). Pretreatment with05 mg/kg SCH23390 partially inhibited the development of L-DOPA–in-uced AIMs, whereas .25 mg/kg SCH23390 completely suppressed dyskine-ia during the first 8 days of treatment. The time plot in B shows the AIMcores recorded at each observation point (20 –180 min) during test session1. SCH23390 dose-dependently reduced both the severity of the AIMs atach observation point and their duration. p � .05 versus *SCH23390 only,
.25 mg SCH23390 � L-DOPA, § .05 mg/kg SCH23390 � L-DOPA (Honestlyignificant Difference test after repeated-measures analysis of variance, inhich p � .001 for the effects of group, time, and the group–time interaction
n both A and B).
ither the density or the staining intensity of phospho-ERK1/2
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Figure 6. Quantitative analysis of phospho-extracellular signal-regulatedkinases 1 and 2 (ERK1/2) (A, B), phospho-mitogen- and stress-activatedprotein kinase-1 (MSK-1) (C, D) and FosB/�FosB immunoreactivity (E, F) inthe DA-denervated caudate–putamen (CPu) after chronic treatment with3,4-dihydroxyphenyl-L-alanine (L-DOPA) and SCH23390. Note that the scal-ing on the y axes differs among markers to match their different absolutelevels of expression. p � .05 versus *SCH23390 only, � .25mg SCH23390 �L-DOPA, § .05 mg/kg SCH23390 � L-DOPA (Newman–Keuls test after one-
in
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mmunoreactive cells between calbindin-rich (matrix) and calbi-din-poor (striosomes) areas (Figure 7A–E==).
Dual-antigen immunofluorence for phospho-ERK1/2 and ei-her dynorphin or met-enkephalin was used to determine theistribution of activated ERK1/2 in the two main efferent popu-ations of striatal neurons (Gerfen 1992). In agreement withrevious observations (Cenci et al. 1999), the distribution ofynorphin immunoreactivity in the DA-denervated striatum ofyskinetic animals was patchy in the medial striatum but ex-eeded the striosomal borders in the lateral part of the CPu. Theistribution of enkephalin immunoreactivity was even in alltriatal subregions (data not shown). Analysis of antigen colocal-zation was carried out by random selection of 200 phospho-ERKmmunoreactive cells per peptide marker per rat along theorsoventral coordinate in both the medial and the lateral partsf the CPu. Of the phospho-ERK1/2 positive cells sampled in thisay, similar numbers stained positively for enkephalin (43.2% �.3%; Figure 7F–F==) and for dynorphin (52.0% � 5.5%; Figure
G–G==).The occurrence of phospho-ERK1/2 in enkephalinergic neu-rons was verified in a group of 4 dyskinetic animals in whichphospho-ERK1/2 was detected by IHC and preproenkephalin(PPE) mRNA by radioactive in situ hybridization. Positive immu-nostaining for phospho-ERK1/2 occurred in a large number ofneurons that contained dense clusters of 35S-generated silvergrains (� 30 grains per cell), indicating labeling by the PPE probe(Figure 7I).
Discussion
Phosphorylation of ERK1/2 was induced in DA-denervatedbut not intact striatal neurons after both acute and chronictreatment with L-DOPA. Phosphorylated ERK1/2 was detectablefor as long as 2 hours but not at 24 hours after a drug dose. Thisinduction kinetics rules out a possible accumulation of phospho-ERK1/2 in striatal neurons during the course of chronic L-DOPAtreatment and implies de novo phosphorylation after each single
Figure 7. Distribution of phospho-extracellular signal-regu-lated kinases 1 and 2 (ERK1/2) immunoreactive neurons in adyskinetic rat relative to the striosome and matrix compart-ment of the striatum. The matrix is identified by high levels ofcalbindin expression. Photomicrograph of a striatal sectiondouble-stained for phospho-ERK1/2 (A), and calbindin (A=).The merged image is shown in A==. Boxed insets (B–E; B=–E=,and B==–E==) are shown at higher magnification below. Co-localization of phospho-ERK1/2 with the peptide markersenkephalin and dynorphin is illustrated by confocal three-dimensional pictures in F–F= and G–G==, respectively. Colo-calization of phospho-ERK1/2 with preproenkephalin (PPE)mRNA is shown in the bright-field photomicrograph in I.Positive labeling for PPE mRNA appears as a dense cluster ofblack (silver) grains, and arrowheads show examples of dou-ble-labeled cells. Confocal photomicrographs in H–H==show cells colabeled for phospho-MSK-1 and phospho-ERK1/2 under the confocal microscope. All phospho-MSK-1immunoreactive cells stained positively also for phospho-ERK1/2.
drug injection. However, the temporal pattern of L-DOPA–
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nduced ERK1/2 activation was prolonged compared with DAgonist-induced phospho-ERK1/2 in the intact striatum, whicheaks after 5–15 min, declines at 30 min, and returns to baseliney 60 min (Gerfen et al. 2002; Sgambato et al. 1998a; Sgambatot al. 1998b; Valjent et al. 2000). The sustained activation ofRK1/2 by L-DOPA likely reflects the supersensitivity of DAeceptor-dependent signaling that is produced by severe DA-enervating lesions.
In chronically L-DOPA treated rats, the extent of striatalRK1/2 phosphorylation produced by the last drug dose wasositively and strongly correlated with the AIMs scores recordeduring the treatment period. Levels of ERK1/2 activation inhronically L-DOPA treated, nondyskinetic animals did not differignificantly from baseline values. Thus, the pronounced activa-ion of ERK1/2 in DA-denervated striatal neurons provided aolecular counterpart to the induction of AIMs by L-DOPA.ecause an increased phosphorylation of ERK1/2 was alsoroduced by acute L-DOPA treatment (which does not elicitignificant AIMs), our data suggest that the core signaling alter-tion associated with dyskinesia consists in an inability toesensitize the phospho-ERK1/2 response with repeated expo-ure to L-DOPA.
Both D1 and D2 receptor agonists have been reported totimulate ERK1/2 phosphorylation (Cai et al. 2000; Gerfen et al.002; Zhen et al. 2002). In our study, the D2 receptor antagonistaclopride did not have an effect on L-DOPA–induced ERK1/2ctivation. Similarly, L-DOPA–mediated induction of phos-ho(Ser376) MSK-1, a histone kinase that plays an essential role
n the transcriptional changes brought about by ERK1/2 (Deak etl. 1998; Dunn et al. 2005), was not affected by raclopride. Inontrast, the D1 receptor antagonist SCH 23390 completelylocked phosphorylation of both kinases. We further showedhat SCH 23390 was able to inhibit ERK1/2 and MSK-1 activationfter a chronic course of L-DOPA treatment, which leads tonternalization and possible desensitization of D1 receptorsFiorentini et al. 2006). Bromocriptine, an antiparkinsonian drugith strong motor-stimulating properties but no dyskinesiogenicotential (Lundblad et al. 2002), exerting agonistic activity at D2ut not D1 receptors (Bedard et al. 1993; De Keyser et al. 1995),id not produce a significant activation of ERK1/2 and MSK-1 inhe DA-denervated striatum. Taken together, these data indicatehat D2 receptors are not implicated in the induction of ERK1/2-ependent signaling by L-DOPA, and support an associationetween ERK1/2 activation and the supersensitivity of D1 recep-or-mediated signaling that follows DA denervation (Gerfen et al.002). Increased signaling through D1 receptors is implicated inhe abnormal regulation of molecular and synaptic responses intriatal neurons at the heart of L-DOPA–induced dyskinesiaAubert et al. 2005; Konradi et al. 2004; Picconi et al. 2003). Ourata suggest an involvement of ERK1/2 in these abnormal striatalesponses. Indeed, while blocking ERK1/2 and MSK-1 phosphor-lation, SCH 23390 produced a similar dose-dependent suppres-ion of L-DOPA–induced AIMs and prevented the striatal upregu-ation of FosB/�FosB proteins, which is causally linked withyskinesia development in this rat model (Andersson et al. 1999,001).
Unexpectedly, the striatal pattern of phospho-ERK1/2 inyskinetic rats differed greatly from the distribution of FosB/FosB immunoreactivity. Moreover, although FosB/�FosB iselectively expressed in dynorphinergic neurons (Andersson etl. 1999; Cenci et al. 1999), the activation of phospho-ERK1/2 by-DOPA in dyskinetic rats did not show selectivity for dynorphin-
ersus enkephaline-positive cells. The great efficacy of the D1ww.sobp.org/journal
antagonist in blocking all ERK1/2 activation is somewhat difficultto explain when considering that enkephalinergic cells expresspredominantly D2 receptors (Gerfen et al. 1992). Previous stud-ies have shown a positive modulation of gene expression instriatal enkephalin neurons by muscarinic receptor activation(Wang and McGinty 1996). However, it is highly unlikely thattreatment with L-DOPA would lead to an increased activation ofstriatal muscarinic receptors in our model (Jackson et al. 1993).The most likely explanation for the phospho-ERK1/2 response inenkephalin neurons implicates an activation of polysynaptic,corticobasal ganglionic circuits. L-DOPA–induced dyskinesia isindeed associated with an excessive activation of motor andpremotor cortical areas (Rascol et al. 1998), and cortical activa-tion induces immediate-early gene expression (Berretta et al.1997) and ERK1/2 phosphorylation (Gerfen et al. 2002) predom-inantly in “indirect pathway” neurons. The importance of corticalafferents in activating these neurons has been further demon-strated by Ferguson and Robinson (2004). In their study, treat-ment of rats with amphetamine induced c-fos both in enkepha-lin-positive and in enkephalin-negative neurons within thestriatum, but only the response evoked in the enkephalin-positive population was dependent on the integrity of thecorticostriatal pathway. Interestingly, this response was alsodependent on the activation of ERK1/2 (Ferguson and Robinson2004). Supporting a facilitatory role of striatal D1 receptors oncortical function, Steiner and Kitai (2000) have shown that localintrastriatal antagonism of D1 receptors results in blockade ofDA-dependent immediate-early gene expression both in thestriatum and in the cerebral cortex.
The molecular adaptations produced by chronic L-DOPAtreatment in indirect pathway neurons are poorly understood.Some recent studies performed on rats with 6-OHDA lesionshave specifically addressed the relative localization of L-DOPA–induced changes in striatal gene expression to dynorphinergic orenkephalinergic neurons and have consistently found a selectiveupregulation of transcription factors and plasticity genes in theformer population (Carta et al. 2005; Sgambato-Faure et al. 2005;St-Hilaire et al. 2005). Although it activates the transcription ofplasticity genes in direct pathway neurons, chronic treatmentwith L-DOPA can also normalize denervation-induced changesin gene expression in enkephalin-positive neurons (Aubert et al.2006), and it is tempting to propose involvement of ERK1/2 inthese effects. Alternatively, ERK1/2 activation in enkephalin-positive neurons may not result in alterations of gene expression.Studies in several model systems have clearly shown that cellularactivation of ERK1/2 is not in itself sufficient to cause changes atthe nuclear level, but that the duration of and the signalingcontext in which this response occurs are crucial in determiningthe gene transcription outcome (Caunt et al. 2006; Impey et al.1998). After treatment with L-DOPA, a simultaneous activation ofERK1/2 and other positive modulators of nuclear signaling, suchas the PKA-DARPP 32 pathway (Greengard et al. 1998), occurs instriatal neurons that bear D1 but not D2 receptors. This mayexplain the occurrence of large gene expression changes in onestriatal efferent population but not in the other one, despitesimilar levels of ERK1/2 activation.
In conclusion, our results show that treatment with L-DOPA indyskinetic animals produces a pronounced, sustained, and broadstriatal activation of ERK1/2 signaling. This response is depen-dent on D1 receptors but occurs in both dynorphinergic (D1-receptor-rich) and enkephalinergic (D2-receptor-rich) striatalneurons. Activation of ERK1/2 in dynorphinergic neurons is
likely implicated in the abnormal molecular changes associated
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ith LID (Andersson et al. 1999, 2001; Cenci et al. 1998). Theignificance of ERK1/2 activation in enkephalinergic neurons isurrently unknown, but our findings provide the first evidence ofodulation of intracellular signaling processes by L-DOPA in this
ellular population in an animal model of PD.
This work was supported by grants from the Michael J. Foxoundation for Parkinson’s Research, the Elsa and Thorstenegerfalk Foundation, the Johan and Greta Kock Foundations,he King Gustaf V and Queen Victoria Foundation, the Swedishoundation for Parkinson’s Research, the Swedish Nationalesearch Council (MAC), and NIH Grant No. NS048235 (CK).e thank the Gaemzeus Foundation and the Swedish Society foredical Research for their financial support to JEW, the Marieurie Host Fellowship Training Program for its support to LV,nd the Parkinson Society Canada for its support to EMS. We
hank Inga-Lill Bertilsson and Ulla Jarl for their assistance in theistologic part of the study.
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