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Neuroscience 167 (2010) 1160–1167

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HANGES OF AMPA RECEPTORS IN MPTP MONKEYS WITH

EVODOPA-INDUCED DYSKINESIAS

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. OUATTARA,a D. HOYER,b L. GRÉGOIRE,a

. MORISSETTE,a F. GASPARINI,b

. GOMEZ-MANCILLAb AND T. DI PAOLOa*

Molecular Endocrinology and Genomic Research Center, Centreospitalier Universitaire de Québec (CHUQ), Pavillon CHUL and Fac-lty of Pharmacy, Laval University, QC, Canada

Neuroscience Research, Novartis Institutes for BioMedical Research,002 Basel, Switzerland

bstract—Overactivity of glutamate neurotransmission isuspected to be implicated in Parkinson’s disease and levo-opa-induced dyskinesia. The fast glutamatergic transmission

n the striatum from the cortex is mediated mainly by non-N-ethyl-D-aspartate (non-NMDA) receptors. Animal models ofarkinson’s disease reveal conflicting data concerning striatallutamate AMPA receptors. The present study thus sought tohed light on the relationship of striatal AMPA receptors to theevelopment of levodopa-induced dyskinesia. [3H]Ro 48-8587,highly potent and selective-specific antagonist ligand for

MPA receptors, was used to investigate, by autoradiography,triatal AMPA receptors in 1-methyl-4-phenyl-1,2,3,6-tetrahy-ropyridine (MPTP)-lesioned monkeys treated for 1 month with

evodopa alone, levodopa�CI-1041 (NMDA receptor antagonist)r levodopa�cabergoline (D2 receptor agonist). Levodopa-

reated MPTP monkeys developed dyskinesias while those thateceived levodopa�CI-1041 or levodopa�cabergoline did not.n the anterior caudate nucleus and putamen, specific bindingf [3H]Ro 48-8587 was reduced in all MPTP-treated monkeysompared to control monkeys, but no significant effect of MPTPas measured in the posterior striatum. In dyskinetic monkeys,pecific binding of [3H]Ro 48-8587 was elevated in subregionsf the posterior caudate nucleus and putamen as compared toaline-treated MPTP monkeys. Levodopa�CI-1041 treatment

eft unchanged specific binding of [3H]Ro 48-8587 whereasevodopa�cabergoline treatment reduced it in subregions ofhe posterior caudate nucleus and putamen compared to con-rol and levodopa-treated MPTP monkeys. Specific binding of3H]Ro 48-8587 was low in the globus pallidus and remainednchanged following both lesion and treatments. In conclusion,he elevated values of AMPA receptors in dyskinetic monkeysand their prevention through treatments) were only observed inubregions of the striatum. © 2010 IBRO. Published by Elseviertd. All rights reserved.

ey words: glutamate, motor complications, Parkinson, cabergo-ine, CI-1041, dopamine.

Corresponding author. Tel: �1-418-654-2296; fax: �1-418-654-2761.-mail address: therese.dipaolo@crchul.ulaval.ca (T. Di Paolo).bbreviations: DA, dopamine; DL, dorso–lateral; DM, dorso–medial;-DOPA, levodopa; LTD, long-term depression; LTP, long-term poten-iation; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NMDA,

w-methyl-D-aspartate; PD, Parkinson’s disease; SEM, standard errorf the mean; VL, ventro–lateral; VM, ventro–medial.

306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2010.03.022

1160

xcitatory synapses contain AMPA-type receptors toransmit signals and N-methyl-D-aspartate (NMDA)-typeeceptors to trigger long-term potentiation (LTP) and long-erm depression (LTD); both phenomena appear to beommon mechanisms controlling the postsynaptic expres-ion of AMPA receptors (Barry and Ziff, 2002). LTP iseported to involve the addition and LTD the removal ofMPA receptors (Bredt and Nicoll, 2003). Long-term treat-ent with the dopamine (DA) precursor levodopa (L-OPA) induces dyskinesias in Parkinson’s disease (PD)atients (Picconi et al., 2008). The loss of bidirectionaltriatal synaptic plasticity (LTP and LTD) is believed tonderlie L-DOPA induced-dyskinesias (Picconi et al.,003). Increasing evidence suggests that blockade ofMPA receptors improves L-DOPA-induced dyskinesia inPTP monkeys (Konitsiotis et al., 2000; Bibbiani et al.,005). Further evaluation of the contribution of AMPA re-eptor-mediated mechanisms to motor behaviour haveeen limited in pathologic conditions in animal models ofD. Moreover, these studies reveal conflicting data con-erning the status of the striatal AMPA receptor or itsubunits following lesions that model PD conditions, re-orting increases (Betarbet et al., 2000), decreases (Zavit-anou et al., 1996; Lai et al., 2003) or no change (Porter etl., 1994; Bernard et al., 1996; Silverdale et al., 2002; Lait al., 2003). CI-1041 (PD196860), a selective NMDAR1A/NR2B receptor assembly antagonist (Hadj Tahar etl., 2004), and cabergoline, a long lasting DA D2 receptorgonist (Belanger et al., 2003), were shown to block theevelopment of dyskinesias in 1-methyl-4-phenyl-1,2,3,6-etrahydropyridine (MPTP) monkeys when co-adminis-ered with L-DOPA. The role of NMDA receptors (Ouattarat al., 2009) and mGluR5 (Samadi et al., 2008b) in L-OPA-induced dyskinesia has been investigated in our

aboratory; we hypothesize that AMPA receptors also con-ribute to this motor complication. DA activation of G-pro-ein-coupled D1 and D2 receptors, respectively, excites ornhibits medium spiny neurons by modulating the gatingnd trafficking of voltage-dependent and ligand-gatedMPA and NMDA receptor channels in dendrites of theasal ganglia (reviewed by Surmeier et al., 2007). There isvidence that DA D2 receptors can modulate AMPA re-eptor-mediated neurotoxicity (Zou et al., 2005). Thus,locking NMDA receptors or activating D2 receptors couldttenuate AMPA receptor activity. We investigated thisossibility in the present study.

The reported experiments examined the effect of anMDA receptor antagonist and the activation of D2 recep-

ors on striatal AMPA receptor-specific binding in relation

ith the appearance of motor complications. For this pur-

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B. Ouattara et al. / Neuroscience 167 (2010) 1160–1167 1161

ose, we used [3H]Ro 48-8587, a specific AMPA receptorntagonist ligand. We investigated AMPA receptors in thebove two groups of MPTP treated monkeys in compari-on to intact, MPTP and MPTP�L-DOPA alone treatedonkeys. Thus the hypothesis of this study was thatMPA receptor changes would be associated with L-DO-A-induced dyskinesias and that acting on NMDA or D2

eceptors and receptor-receptor interactions would affecteurotransmission and AMPA receptors.

EXPERIMENTAL PROCEDURES

nimals and drug treatments

xperiments were carried out using 20 drug-naive female ovari-ctomized monkeys (Macaca fascicularis) in accordance with thetandards of the Canadian Council on Animal Care. Animals wereaintained in a temperature-controlled environment lit with artifi-

ial daylight (lights on 6.00–18.00 h). The Laval University com-ittee for the protection of animals approved this study.

Monkeys weighing 3.0–4.3 kg and aged 4–11 years wereivided into five experimental groups. One non-parkinsonianroup (n�4) remained untreated and served as healthy controlscontrol group), while four groups (each n�4) were renderedarkinsonian with the neurotoxin MPTP (Sigma-Aldrich, Canadatd., Oakville, ON), administered continuously using subcutane-us Alzet minipumps (0.5 mg/24 h in saline solution) until sus-ained parkinsonian features were achieved. This method ofPTP delivery is well tolerated and allows for a more constantdministration of MPTP (Belanger et al., 2003; Hadj Tahar et al.,004). In general, 2–4 weeks are needed to induce sustainedarkinsonian features (i.e., unchanged disability score of eight orore over the course of 1 month). Recovery was seen in someonkeys, in this case, 2 mg of MPTP was administered once aeek for an additional 1–3 weeks. The cumulative dose and the

ime to achieve this goal were on average 15.6�2.4 mg and.2�1 months, respectively. The animals were scored severalimes a week using a disability scale described previously (Be-anger et al., 2003; Hadj Tahar et al., 2004), where the normaltate extends from 0 to 2 and maximal disability is 16 points.

Treatment was started after the bilateral parkinsonian syn-rome had stabilized (after 1–6 months). The MPTP monkeysere then divided into four groups (n�4 in each group), with eachroup having a similar baseline parkinsonian score (Belanger etl., 2003; Hadj Tahar et al., 2004). One of these groups of fourPTP-exposed monkeys received saline while another group of

our received chronic daily oral treatment with L-DOPA/bensera-ide alone (100/25 mg; Prolopa®) (Hoffmann–La Roche limited,ississauga, ON, Canada) and a third group of four was treated

imultaneously with L-DOPA (same dosage) plus CI-1041 (Pfizer,nn Arbor, MI, USA; 10 mg/kg), a highly selective antagonist forR2B-containing NMDA receptors. CI-1041 was dissolved anddministered per os at 9:30 AM and the spontaneous behaviour of

he animals was observed until the effect of the drug was termi-ated. The last group of four monkeys was treated with a combi-ation of oral L-DOPA and a threshold dose of cabergoline ad-inistered by subcutaneous injection. The threshold dose of

abergoline was the minimum dose of cabergoline producing amall stimulatory effect, but this change was not sufficient toodify the antiparkinsonian score obtained using the disability

cale. Cabergoline (Adria Laboratories, Columbus, OH) was dis-olved in 0.9% sterile saline (1 ml) acidified with 2% phosphoriccid (5 �l) and administered at doses ranging from 0.015 to 0.035g/kg. Drugs were administered in the morning (at 9:30 AM) for 4eeks, and spontaneous behaviours were observed until the end

f the antiparkinsonian response (Belanger et al., 2003). t

Treatments lasted for 28 consecutive days; behavioural as-essment of these monkeys was previously reported (Belanger etl., 2003; Hadj Tahar et al., 2004). The spontaneous behaviour ofhe monkeys was assessed through a one-way screen over theotal period of the behavioral effect. Antiparkinsonian effects of-DOPA were similar in all monkeys. All four animals treated with-DOPA alone developed dyskinesias, while CI-1041 and caber-oline completely prevented the induction of dyskinesias in threeonkeys in each of the latter two treatment groups. Only oneonkey in each of these groups developed mild dyskinesias

Belanger et al., 2003; Hadj Tahar et al., 2004).

issue preparation

nimals were killed 24 h after the end of the chronic treatments byn overdose of sodium pentobarbital. The brains were then im-ersed in isopentane (�40 °C) and stored at �80 °C. Hemisectedrains were cut into coronal sections of 12 �m on a cryostat�18 °C) at two different levels of the striatum correspondingpproximately to level A18-A22 (rostral striatum) and level A15-18 (caudal striatum) in the atlas of Szabo and Cowan (Szabond Cowan, 1984). Sections were mounted onto Super Frost PlusFisher, Canada) slides, desiccated overnight at 4 °C and storedt �80 °C.

eceptor autoradiography

utoradiography of the binding of [3H]Ro 48-8587 to monkey brainlices was performed according to experimental conditions re-orted for rat brain (Mutel et al., 1998a; Allison et al., 2005) withlight modifications. Thus, [3H]Ro 48-8587 autoradiography waserformed as follows: after 20 min of preincubation at room tem-erature in a buffer containing 50 mM Tris–HCl pH 7.0, the sec-ions were incubated for 60 min at 4 °C in a same buffer, supple-ented with 5 nM [3H]Ro 48-8587 (52 Ci/mmol from Novartis,witzerland). Non-specific binding was determined in a set ofdjacent slides through incubation in the presence of 50 �M of theMPA receptor antagonist (1,4-dihydro-6-(1H-imidazol-1-yl)-7-ni-

ro-2,3-quinoxalinedione hydrochloride, YM90K) (Tocris Bio-ciences, USA). After incubation, washing of the labeled sectionsonsisted of two 30 s washes in an ice-cold buffer followed by arief dipping in bi-distilled water (4 °C) to remove salts. Theections were then left to dry at room temperature. Autoradio-rams were generated by apposing the labeled tissues to BioMaxR Films (Eastman Kodak Company, Rochester, NY, USA) with

ritium standards ([3H]-microscales, Amersham) for 14 days or 2onths (for weakly labeled regions such as the globus pallidus) at

oom temperature. For each rostral and caudal brain regionsnvestigated, we used two glass slides bearing 2–3 brain slicesrom each animal for autoradiographic analyses.

mage, data and statistical analysis

uantification of all autoradiograms was performed on a G4acintosh connected to a Sony video camera (model XC-77) andconstant illumination light table using computerized densitome-

ry with the software package NIH Image 1.63 (developed at theSA. National Institutes of Health and available on the Internet atttp://rsb.info.nih.gov/nih-image/). Optical grey densities wereransformed into nCi/mg of tissue equivalent using a standardurve generated with the tritium standards and then converted intomol/mg of tissue using the specific activity of the radioligand. Thehite matter was used to estimate non-specific binding for the firstxposure (14 days) to avoid a possible influence of the back-round of the films. After a long film exposure of 2 months, weere able to measure weak binding in the globus pallidus. Aon-specific slide was exposed with two brain slices for eachonkey on each film and then used in the analysis of binding in

he globus pallidus. The analysis of the striatum and the globus

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allidus were carried out using the 14-day and the 2-month auto-adiograms, respectively.

For analysis, the caudate nucleus and putamen were dividednto four subregions (medial-lateral and dorsal-ventral) at twoostrocaudal coordinates because in our previous studies (Samadit al., 2008a,b; Ouattara et al., 2009), evaluated according tohese subdivisions, changes of NMDA, mGluR5 and mGluR2/3eceptors appeared in some of these subregions. The normality ofur results was tested using the Schapiro-Wilk test and all dataresented was found to have a normal distribution. Therefore,esults were analyzed with a parametric test. Statistical compari-ons of data were performed using a one-way analysis of varianceANOVA) with subregions and treatment as variables, for eachutoradiographic assay, followed by post-hoc pairwise compari-ons with Fisher’s probability of least significant difference testPLSD). Pearson Correlation analyses were performed using Stat-iew software. A P-value �0.05 was required for the results to beonsidered statistically significant.

RESULTS

ehavioural assessment

arkinsonian and dyskinesia scores of these monkeysere previously reported (Belanger et al., 2003; Hadjahar et al., 2004). L-DOPA alone or with CI-1041 orabergoline lowered the parkinsonian scores as comparedo controls. L-DOPA-treated monkeys developed dyskine-ias that were mainly choreic in nature. Co-administration

ig. 1. Examples of autoradiography of the binding of [3H]Ro 48-8587

onkeys treated with saline, L-DOPA, L-DOPA�CI-1041 and L-DOPA�caberorso–medial (DM), ventro–lateral (VL) and ventro–medial (VM) subregions an

f CI-1041 (Hadj Tahar et al., 2004), or cabergoline (Be-anger et al., 2003) completely prevented the developmentf dyskinesia in three monkeys in each respective group,ith no dyskinesia noted until the end of the study; a fourthonkey in each group developed mild choreic movementsf the lower limbs at the end of the fourth week of treat-ent.

enervation assessment

he extent of denervation of these monkeys was previ-usly reported (Samadi et al., 2008b). MPTP lesioningesulted in depletion of DA concentration in the caudatend putamen of all MPTP-lesioned monkeys with an aver-ge decrease of 99% compared to intact monkeys as wells a similarly extensive decrease in DA transporter specificinding as labeled with [125I]RTI-121 in all the groups ofPTP monkeys (Samadi et al., 2008b).

MPA receptors

epresentative autoradiograms of basal ganglia binding of3H]Ro 48-8587 are shown in Fig. 1. The schematic brainegions analyzed are also shown, along with the quantifiedrain subregions. The caudate and putamen showed bind-

ng of [3H]Ro 48-8587 uniformly distributed in their subre-ions and similar binding was observed in the caudate

receptors in the striatum of an intact control monkey as well as MPTP

to AMPA goline (CAB). Schematic representations of the dorso–lateral (DL),alyzed are also shown.

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B. Ouattara et al. / Neuroscience 167 (2010) 1160–1167 1163

ucleus compared to the putamen. Nonspecific bindingas low (�8% of total binding) even with a film exposure ofmonths (data not shown). The internal and external

lobus pallidus showed weak binding of [3H]Ro 48-8587nd no effect of lesion and treatments were measured inny of the experimental groups studied (data not shown).

In the anterior caudate nucleus and putamen, specificinding of [3H]Ro 48-8587 was reduced in all MPTP mon-eys, reaching statistical significance in the DM, VL and DLf the putamen (Fig. 2). No significant effect of lesion waseasured for specific binding of [3H]Ro 48-8587 in theosterior striatum (Fig. 3).

L-DOPA-treated MPTP monkeys exhibited significantlyeduced specific binding of [3H]Ro 48-8587 in the anteriorL caudate nucleus and DM putamen compared to intactontrol monkeys (Fig. 2). Maximal dyskinesia scores forhese monkeys as previously reported (Belanger et al.,003; Hadj Tahar et al., 2004) did not correlate with spe-ific binding of [3H]Ro 48-8587 in the subregions of thenterior caudate nucleus and putamen (data not shown).n the posterior striatum of dyskinetic monkeys, specificinding of [3H]Ro 48-8587 in both the DM caudate nucleusnd the VM putamen was elevated compared to saline-reated MPTP monkeys (Fig. 3).

In the anterior DL caudate nucleus and DM and VLutamen of MPTP monkeys treated with L-DOPA�CI-041, specific binding of [3H]Ro 48-8587 was reducedompared to controls (Fig. 2). There was no significantifference in specific binding of [3H]Ro 48-8587 in MPTPonkeys treated with L-DOPA�CI-1041 as compared to

ig. 2. Specific binding of [3H]Ro 48-8587 to AMPA receptors in antePTP monkeys (n�4), L-DOPA-treated MPTP monkeys (n�4), MPTPith L-DOPA�cabergoline (CAB) (n�3). Results are expressed as thene hundred percent control values in fmol/mg of tissue are anterioredial (VM)�757�58, ventro–lateral (VL)�677�51; anterior putameucleus, DM: F4,13�1.9, P�0.15; VM: F4,13�1.7, P�0.20; DL: F4,13��0.04; VM: F4,13�2.1, P�0.1; DL: F4,13�3.6, P�0.03; VL: F4,13�4.3,s. L-DOPA-treated MPTP monkeys.

PTP monkeys treated with L-DOPA alone. In the poste- d

ior striatum, L-DOPA�CI-1041 treatment did not changepecific binding of [3H]Ro 48-8587 compared to intactontrols or saline-treated MPTP monkeys (Fig. 3).

In the anterior striatum of MPTP monkeys treated with-DOPA�cabergoline, specific binding of [3H]Ro 48-8587n the DL caudate nucleus and the DM, VL and DL puta-

en was reduced compared to controls. In the lateral (VLnd DL) anterior putamen of MPTP monkeys treated with-DOPA�cabergoline, specific binding of [3H]Ro 48-8587as reduced compared to those treated with L-DOPAlone (Fig. 2). L-DOPA�cabergoline treatment induced aeduction in the posterior medial (DM and VM) caudateucleus and in the posterior medial putamen (DM and VM)ompared to control and L-DOPA-treated MPTP monkeysFig. 3). L-DOPA�cabergoline treatment also induced aeduction in the posterior DM caudate nucleus and in theM putamen compared to L-DOPA�CI-1041 treatedPTP monkeys (Fig. 3).

DISCUSSION

MPA receptors mediate most of the excitatory neuro-ransmission in the mammalian CNS and also participaten synaptic plasticity thought to underlie memory and learn-ng, as well as the formation of neural networks duringevelopment (O’Neill et al., 2004). Furthermore, AMPAeceptor potentiators alter downstream signaling pathwaysO’Neill et al., 2004) and this may suggest a role for AMPAeceptors in CNS disorders.

The present study showed that the MPTP lesion re-

gions of the striatum of control monkeys (intact, n�4), saline-treateds treated with L-DOPA� CI-1041 (n�3) and MPTP monkeys treatedercentage of the mean of four control monkeys � SEM (bars) values.dorso–medial (DM)�694�40, dorso–lateral (DL)�650�26, ventro–50�57, DL�657�74, VM�659�63, VL�701�73. Anterior caudate0.05; VL: F4,13�2.2, P�0.12 and anterior putamen, DM: F4,13�3.4,* P�0.05; ** P�0.01 and *** P�0.005 vs. control monkeys; $ P�0.05

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uced specific binding of [3H]Ro 48-8587 in the anterior

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B. Ouattara et al. / Neuroscience 167 (2010) 1160–11671164

utamen. L-DOPA-treated MPTP monkeys developed dys-inesias and had elevated specific binding of [3H]Ro 48-587 in the posterior DM caudate nucleus and VM puta-en compared to saline-treated MPTP monkeys. Prevent-

ng dyskinesias with L-DOPA�CI-1041 treatment leftpecific binding of [3H]Ro 48-8587 at MPTP saline-treatedalues whereas L-DOPA�cabergoline treatment induced aeduction in the medial caudate nucleus and putamenompared to control and L-DOPA-treated MPTP monkeys.e previously reported, with [3H]AMPA as a radioligand,

n upregulation of AMPA receptors in the development ofyskinesias following DA D1 agonist treatment in MPTPonkeys (Calon et al., 2002). In addition, specific bindingf [3H]AMPA was elevated in the lateral putamen of PDatients with motor complications compared to those with-ut (Calon et al., 2003). Silverdale et al. suggested anveractivity of AMPA receptor-mediated transmission in

he pathogenesis of L-DOPA-induced dyskinesias (Silver-ale et al., 2005), but in a previous study, they observed nohange in MPTP and dyskinetic monkeys compared toontrols (Silverdale et al., 2002).

The inconsistent AMPA receptor results reported in theiterature may have diverse causes. The radioligand used

ay be a factor. The present study used [3H]Ro 48-8587,potent and selective AMPA receptor antagonist ligandith high affinity, selectivity and specific binding (Mutel etl., 1998b). The AMPA agonist ligand [3H]-(S)-5-fluorowil-

ardiine labels two sites and the high affinity site waseported to remain unchanged in nondyskinetic and dyski-etic MPTP monkeys (Silverdale et al., 2002). Anotherxplanation of the discrepancies may be the mechanism of

ig. 3. Specific binding of [3H]Ro 48-8587 to AMPA receptors in postePTP monkeys (n�4), L-DOPA-treated MPTP monkeys (n�4), MPTith L-DOPA�cabergoline (CAB) (n�3). Results are expressed as thene hundred percent control values in fmol/mg of tissue are: poentro–medial (VM)�575�96, ventro–lateral (VL)�494�95; posterioraudate nucleus, DM: F4,13�3.8, P�0.02; VM: F4,13�3.7, P�0.03;

4,13�3.2, P�0.04; VM: F4,13�5.3, P�0.01; DL: F4,13�2.3, P�0.11;reated-MPTP monkeys; $$ P�0.01 vs. L-DOPA-treated MPTP monke

egulation of these glutamate receptor channels. Native f

MPA receptors are composed of heteromeric subunitsGasic and Hollmann, 1992; Nakanishi, 1992; Nakanishi etl., 1998). Four different genes (GluR1, GluR2, GluR3, andluR4) encode AMPA receptor subunits (Wisden and See-urg, 1993; Hollmann and Heinemann, 1994) and wereound to be differently modulated in the 6-OHDA-lesionedtriatum (Bernard et al., 1996; Lai et al., 2003). GluR1,luR4, and GluR2L (a long splice form of GluR2) have a

ong cytoplasmic carboxy-terminal, while GluR2, GluR3,nd GluR4c (a short splice form of GluR4) have short andtructurally similar ones (Wisden and Seeburg, 1993; Holl-ann and Heinemann, 1994). The mechanisms for syn-ptic AMPA receptor trafficking are hypothesized to de-end on subunit composition. As reviewed by Kessels andalinow, synaptic strengthening involves an activity-de-endent addition of long-tailed (e.g., GluR1-containing)MPA receptors to synapses, and synaptic weakeningccurs through activity-dependent endocytosis of either

ong-tailed or short-tailed AMPA receptors from synapses.owever, short-tailed AMPA receptors constitutively move

nto synapses independent of activity and without changingynaptic strength (Kessels and Malinow, 2009).

The functional properties of AMPA receptors are re-ated to the expression of the GluR2 subunit in the chan-els (Pellegrini-Giampietro et al., 1997). GluR1 over-ex-ression produces homomeric GluR1/1 receptors, unlikehe normal assembly containing both GluR1 and GluR2ubunits. Mediators of LTP rectify the GluR1/1 imbalancey inserting GluR2 and removing GluR1 (Hayashi et al.,000; Kakegawa et al., 2004). This leads to the formationf heteromeric GluR1/2 receptors (Shi et al., 2001) which

egions of the striatum of control monkeys (intact, n�4), saline-treatedys treated with L-DOPA�CI-1041 (n�3) and MPTP monkeys treatedercentage of the mean of four control monkeys � SEM (bars) values.udate: dorso–medial (DM)�535�75, dorso–lateral (DL)�505�96,: DM�461�88, DL�411�94, VM�482�88, VL�491�90. Posterior

�1.7, P�0.37; VL: F4,13�3.2, P�0.05 and posterior putamen, DM:�1.5, P�0.24. ** P�0.01 vs. control monkeys; # P�0.05 vs. saline0.05 vs. MPTP monkeys treated with L-DOPA�CI-1041.

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ctivity-induced synaptic trafficking (Kessels and Malinow,009). Thus, a relevant issue arises regarding the extent tohich synaptic AMPA receptors are affected in an overex-ression context such as PD or L-DOPA-induced dyskine-ia. The effect is probably not large, based on the lack of orodest effects reported in the literature and our overall

esults from the total anterior to posterior striatum ob-erved in all experimental groups.

Movement of AMPA receptors from dendrites to syn-pses is associated with an exchange of subunit compo-ition (Plant et al., 2006; Guire et al., 2008) and mayequire Ca2� permeability of GluR2-lacking receptors.owever, phosphorylation events may play an important

ole in AMPA receptor activation (Song and Huganir, 2002;steban et al., 2003; Boehm et al., 2006). Moreover, in thebsence of neural activity, AMPA receptors can move intoynapses without changing the magnitude of synapticransmission (Shi et al., 2001; Kakegawa et al., 2004)uggesting a mechanism for one-for-one exchange of re-eptors from extrasynaptic to synaptic sites (Kessels andalinow, 2009).

Regulation of AMPA receptor trafficking results inhanges in receptor number at the postsynaptic mem-rane, and hence changes in synaptic strength (Malinownd Malenka, 2002; Bredt and Nicoll, 2003; Hernandez-cheagaray et al., 2004). This activity, as all physiologic

esponses to glutamate, requires Ca2� influx throughMPA and NMDA receptors (Lu and Mattson, 2001; Guiret al., 2008). NMDA receptor-mediated postsynaptic Ca2�

nflux (Liao et al., 2001) enhances AMPA internalizationHanley and Henley, 2005). Preventing L-DOPA-inducedyskinesias with a NMDA/NR2B receptor antagonist re-uced specific binding to NMDA/NR2B receptors (Ouatt-ra et al., 2009). Because of the functional interactionetween NMDA and AMPA receptors, blocking one mayffect the other. This supports the suggestion that attenu-tion of the activity of these receptors could improve dys-inesias. Moreover, DA activation of G-protein-coupledeceptors modulates the gating and trafficking of voltage-ependent and ligand-gated (ionotropic) channels embed-ed in the dendritic membrane (Surmeier et al., 2007;ernandez-Lopez et al., 2000), DA D2 receptors can

herefore modulate AMPA receptor-mediated activity (Zout al., 2005). The activation of D2 receptors decreasesMPA receptor currents in medium spiny neurons in tis-ues slices (Cepeda et al., 1993) and also diminishesresynaptic release of glutamate (Bamford et al., 2004).hus, activating D2 receptors could attenuate AMPA re-eptor activity (Liu et al., 2004; Braithwaite et al., 2006). Inupport of this, cabergoline reduced specific binding toMPA receptors in both the anterior and posterior striatumf MPTP-treated monkeys. The subsequent diminution ofhe glutamate receptors prevented L-DOPA-induced dys-inesia in that experimental group (Belanger et al., 2003).

CONCLUSION

n conclusion, the present experiments showed a signifi-

ant reduction in the specific binding to striatal AMPA

eceptors in the anterior putamen following the MPTP le-ion. L-DOPA in combination with cabergoline, preventingyskinesias, reduced the specific binding to striatal AMPAeceptors compared to elevated values in dyskinetic MPTPonkeys treated with L-DOPA alone, while L-DOPA withI-1041 prevented an increase specific binding to these

eceptors. Some differences in AMPA receptor specificinding were measured between the two non-dyskineticroups (treated with either cabergoline or CI-1041). Con-inuous stimulation of D2 receptors reduces both NMDAnd AMPA receptor activity (Liu et al., 2004; Braithwaite etl., 2006). Since activation of NMDA receptors has a pos-

tive effect on AMPA receptors (Hanley and Henley, 2005),2 activation may induce a pronounced inactivation ofMPA receptors. By contrast, CI-1041 antagonizes onlyR2B/NMDA receptors. The lack of sustained action on2 receptors in the CI-1041 experimental group may ex-lain the difference in AMPA receptor levels. In spite of thecarcity of data concerning AMPA receptors in PD and-DOPA-induced dyskinesias, the above results and otherre-clinical studies provide evidence that decreasingMPA receptor stimulation may improve PD symptomsnd L-DOPA induced-dyskinesias and warrant furtheronsideration.

cknowledgments—This research was funded by a grant from theanadian Institutes of Health Research to TDP. The radioligand

3H]Ro 48-8587 was provided by Novartis, Switzerland. B.O. holdsstudentship from the Ivory Coast government. We thank Dr. F.alon for his contribution to earlier studies with this group ofonkeys.

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(Accepted 10 March 2010)(Available online 18 March 2010)