Effect of chronic L-DOPA treatment on 5-HT1A receptors in parkinsonianmonkey brain

Golnasim Riahi a,b,f, Marc Morissette b, Daniel Lévesque d, Claude Rouillard c,e, Pershia Samadi c,e,Martin Parent e,f, Thérèse Di Paolo a,b,!a Faculty of Pharmacy, Université Laval, Quebec City, Canada G1K 7P4b Axe Endocrinologie et Génomique, Centre de Recherche du CHUQ-CHUL, Quebec City, Canada G1V 4G2c Axe Neuroscience, Centre de Recherche du CHUQ-CHUL, Quebec City, Canada G1V 4G2d Faculty of Pharmacy, Université de Montréal, Montreal, Canada H3C 3J7e Department of Psychiatry and Neuroscience, Université Laval, Faculty of Medicine, Quebec City, Canada G1K 7P4f Centre de Recherche de l’institut universitaire en santé mentale de Québec, Quebec City, Canada G1J 2G3

a r t i c l e i n f o

Article history:Received 5 April 2012Received in revised form 9 August 2012Accepted 15 August 2012Available online 24 August 2012

Keywords:Parkinson’s diseaseL-DOPA-induced dyskinesiaSerotoninBasal ganglia8-OH-DPATPrimate

a b s t r a c t

After chronic use of L-3,4-dihydroxyphenylalanine (L-DOPA), most Parkinson’s disease (PD) patients sufferfrom its side effects, especially motor complications called L-DOPA-induced dyskinesia (LID). 5-HT1A ago-nists were tested to treat LID but many were reported to worsen parkinsonism. In this study, we evalu-ated changes in concentration of serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) and of5-HT1A receptors in control monkeys, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys,dyskinetic MPTP monkeys treated chronically with L-DOPA, low dyskinetic MPTP monkeys treated withL-DOPA and drugs of various pharmacological activities: Ro 61-8048 (an inhibitor of kynurenine hydrox-ylase) or docosahexaenoic acid (DHA) and dyskinetic MPTP monkeys treated with L-DOPA + naltrexone(an opioid receptor antagonist). Striatal serotonin concentrations were reduced in MPTP monkeys com-pared to controls. Higher striatal 5-HIAA/serotonin concentration ratios in L-DOPA-treated monkeys com-pared to untreated monkeys suggest an intense activity of serotonin axon terminals but this value wassimilar in dyskinetic and nondyskinetic animals treated with or without adjunct treatment with L-DOPA.As measured by autoradiography with [3H]8-hydroxy-2-(di-n-propyl) aminotetralin (8-OH-DPAT), adecrease of 5-HT1A receptor specific binding was observed in the posterior/dorsal region of the anteriorcingulate gyrus and posterior/ventral area of the superior frontal gyrus of MPTP monkeys compared tocontrols. An increase of 5-HT1A receptor specific binding was observed in the hippocampus of MPTPmonkeys treated with L-DOPA regardless to their adjunct treatment. Cortical 5-HT1A receptor specificbinding was increased in the L-DOPA-treated MPTP monkeys alone or with DHA or naltrexone and thisincrease was prevented in low dyskinetic MPTP monkeys treated with L-DOPA and Ro 61-8048. Theseresults highlight the importance of 5-HT1A receptor alterations in treatment of PD with L-DOPA.

! 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Parkinson’s disease (PD) affects one percent of the populationover the age of sixty and the number of cases is rising due to the

increasing life expectancy (Olanow et al., 2001). The dopamine pre-cursor, L-3,4-dihydroxyphenylalanine (L-DOPA), is the gold stan-dard drug treatment of PD. However, after chronic use of L-DOPA,motor complications such as L-DOPA-induced dyskinesia (LID) ap-pear in at least half of PD patients and limit its use (Olanow et al.,2001). The mechanisms involved in LID are not fully understood.

Pharmacological studies in animal models of PD suggest thatdopamine released from serotonin (5-hydroxytryptamine, 5-HT)axon terminals acts as a false neurotransmitter and is the mainpre-synaptic determinant of LID (Carta et al., 2007; Cenci andLindgren, 2007). Being devoid of dopamine transporter andautoreceptors, 5-HT axon terminals would release dopamine in anon-physiological manner, leading to excessive swings in extracel-lular dopamine and to dyskinesia after L-DOPA treatment (Cartaet al., 2007). These findings make the 5-HT system a target for

0197-0186/$ - see front matter ! 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.neuint.2012.08.009

Abbreviations: 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, 5-hydroxytryptamine(serotonin); 6-OHDA, 6-hydroxydopamine; 8-OH-DPAT, 8-hydroxy-2-(di-n-propyl)aminotetraline; ac, anterior commissure; AcgG, anterior cingulate gyrus; CA, cornuammonis; DG, dentate gyrus; DHA, docosahexaenoic acid; Hip, hippocampus; L-DOPA, L-3,4-dihydroxyphenylalanine; LID, L-DOPA-induced dyskinesia; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’s disease; SFG, superiorfrontal gyrus.! Corresponding author at: Axe Endocrinologie et Génomique, Centre de Recher-

che du CHUQ-CHUL, 2705 Laurier Boulevard, Quebec City, QC, Canada G1V 4G2.Tel.: +1 (418) 654 2296; fax: +1 (418) 654 2761.

E-mail address: therese.dipaolo@crchul.ulaval.ca (T.D. Paolo).

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anti-dyskinetic therapies. Indeed, specific 5-HT receptor drugswere shown to reduce LID in PD (for review see Fox et al., 2009)but changes in 5-HT activity in the brain caused by this neurode-generative disease and its treatments are not well documented.Up to now, 5-HT1A receptors were investigated more than other5-HT receptor subtypes in LID treatment. Besides 5-HT1A autore-ceptors in the raphe nucleus, there is evidence from immunocyto-chemistry, in situ hybridization, autoradiography and lesion ofstriatal axonal projections (Francis et al., 1992) of the post-synapticlocalization of 5-HT1A receptors in the hippocampus and the cere-bral cortex (Kia et al., 1996; Pompeiano et al., 1992; Francis et al.,1992; Hajos et al., 1999).

5-HT1A receptor agonists are believed to reduce LID through dif-ferent mechanisms: (1) having a higher sensitivity to pharmaco-logical stimulation than other types of 5-HT1A receptors (Eskowet al., 2009), activation of 5-HT1A autoreceptors located on dorsalraphe neurons would decrease their firing rate, reducing dopaminerelease as a false neurotransmitter by their axon terminals(Kannari et al., 2001; Lindgren et al., 2010) and (2) activation of5-HT1A heteroreceptors in the motor cortex may reduce glutamaterelease in the striatum by corticostriatal neurons which are be-lieved to be hyperactive in LID (Antonelli et al., 2005; Dupreet al., 2011). It was shown that injection of a 5-HT1A receptor ago-nist into the primary motor cortex, alleviates motor complicationsin dyskinetic rats (Ostock et al., 2011). The presynaptic 5-HT1A

autoreceptors located on raphe nucleus are the primary target ofpharmacological stimulation that regulates raphestriatal L-DOPA-derived dopamine release involved in LID development (Careyet al., 2004; Eskow et al., 2009). Higher doses of 5-HT1A receptoragonists are needed to stimulate extra-raphe postsynaptic recep-tors that modulate locomotor and hypothermic responses (Bertet al., 2006; Blanchard et al., 1993).

LID treatments with 5-HT1A agonists were reported to reducedyskinesia but some of these treatments were shown to worsenPD symptoms, which represent the major problem in their efficacy(Fox et al., 2009). Striatal 5-HT1A stimulation with 8-hydroxy-2-(di-n-propyl) aminotetralin (8-OH-DPAT) reduced D1 agonist-induced dyskinesia while it increased movements inhemiparkinsonian rats (Dupre et al., 2008a; Mignon and Wolf,2007). In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)marmoset monkeys, a decrease of LID by systemic administrationof (+) 8-OH-DPAT, a more potent 5-HT1A receptor agonistenantiomer, has been associated with an increase of other motordisabilities that may be related to postsynaptic 5-HT1A receptorsstimulation (Iravani et al., 2006). However in another study,administration of the racemic (±)-8-OH-DPAT mixture decreasedLID without worsening PD symptoms in MPTP macaques (Munozet al., 2008). Administration of tandospiron, another 5-HT1A

agonist, is known to reduce LID in PD patients but also to worsenPD symptoms (Kannari et al., 2002). An adjunct treatment with a5-HT1A receptor agent may lead to decreased LID without worsen-ing PD symptoms. For this purpose and to clarify the role of 5-HT1A

receptors, the effects of different pharmacological treatments on5-HT1A receptor levels in L-DOPA-treated monkeys were evaluatedin the present study.

In addition to their 5-HT1A agonist properties, the compoundsmentioned above are known to exert other pharmacologic activi-ties. Sarizotan, a 5-HT1A receptor agonist effective in the treatmentof LID in MPTP monkeys (Grégoire et al., 2009) and PD patients(Bara-Jimenez et al., 2005), acts also as a dopamine antagonist(see Iravani et al., 2006). Tandospirone possesses antagonist activ-ity on adrenergic and dopamine receptors and (±)-8-OH-DPAT, un-der certain conditions, may have affinity for dopamine and 5-HT7A

receptors (see Fox et al., 2009; Iravani et al., 2006). Therefore, theantidyskinetic effect of these drugs as well as worsening of PDsymptoms may also be related to their activity on other receptors.

Indeed, L-stepholidine, a 5-HT1A receptor agonist and a D2 receptorantagonist, was shown to reduce LID in rats via both receptors (Moet al., 2010).

In the present study, we investigated possible alterations ofbrain 5-HT1A receptors in control and MPTP monkeys that devel-oped or not dyskinesia following chronic L-DOPA administrationand adjunct treatments of various pharmacological activities in or-der to clarify their role in PD and LID expression. 5-HT concentra-tions and 5-HT1A receptor levels were measured in different brainareas of MPTP monkeys treated with L-DOPA and Ro 61-8048 (akynurenine hydroxylase inhibitor that inhibits glutamate releaseand blocks glutamate NMDA receptors), docosahexaenoic acid(DHA, a retinoid X receptor agonist) or naltrexone (a nonselectiveopiate receptor antagonist). This is the first report showing altera-tions of 5-HT1A receptors in MPTP monkeys associated with LID ascompared to MPTP monkeys treated with L-DOPA and adjuncttreatments to inhibit dyskinesias. Chronic treatment with L-DOPAmight lead to several changes in the brain, not specifically relatedto dyskinesias. Animals co-treated with L-DOPA and antidyskineticdrugs compared to animals treated with L-DOPA displaying dyski-nesia allow a better assessment of 5-HT1A receptors in LIDexpression.

2. Materials and methods

2.1. Animals

Thirty-one de novo adult female monkeys (Macaca fascicularis,2.5–4.3 kg) were investigated in the present study according tothe standards of the National Institute of Health guide for the careand use of laboratory animals (NIH publications Nos. 80–23, re-vised 1978). They were ovariectomized and used for the presentexperiment one month after ovariectomy. All procedures were de-signed to minimize animal suffering and to reduce the number ofanimals used, and were approved by the Institutional Animal CareCommittee of Université Laval.

Of these 31 monkeys, 4 remained untreated and served as con-trols whereas 27 received MPTP (Sigma–Aldrich Canada Ltd., Oak-ville, ON, Canada) continuously at a dose 0.5 mg/day in salinesolution, via a subcutaneous osmotic mini-pump until stabilizationof bilateral PD syndrome (5–6 months). MPTP monkeys were trea-ted for one month and included six groups: MPTP monkeys treatedwith saline (n = 4), MPTP monkeys treated daily with L-DOPA/Ben-serazide (100 mg/25 mg: Prolopa!: Hoffmann-La Roche, Mississau-ga, ON, Canada) (termed L-DOPA thereafter) (p.o.) that developedLID and were killed 24 h (n = 4) or 4 h (n = 5) after their last doseof L-DOPA, MPTP monkeys treated with the same dose of L-DO-PA + Ro 61-8048 (50 mg/kg/day: Newron Pharmaceuticals, Italy)(by nasogastric gavage) that developed mild LID and were killed24 h after their last dose of L-DOPA (n = 5) (Grégoire et al., 2008),MPTP monkeys treated with the same dose of L-DOPA + DHA(100 mg/kg/day: Cayman Chemical, Ann Arbor, MI, USA) (p.o.) thatdeveloped mild LID (n = 5) (Samadi et al., 2006) and finally, a groupwith L-DOPA + naltrexone (1 mg/kg/day: Sigma, St. Louis, MO, USA)(s.c.) that developed severe LID (n = 4) (Samadi et al., 2005); thetwo latter groups were killed 4 h after their last dose of L-DOPA.Groups of animal brains were analyzed according to their time ofeuthanasia to consider its possible effect on 5-HT turnover andreceptors. Dyskinesias are believed to be associated with long-termchanges in the brain. Hence, animals were killed 24 h and 4 h aftertheir last dose of L-DOPA, that is after behavioral activation in orderto dissociate long-term changes induced by the drug from acute ef-fects in its presence. Indeed, the L-DOPA behavioral antiparkinso-nian activity and dyskinesias returned to baseline within 4 h oftreatment as reported in the behavioral description of these

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monkeys (Samadi et al., 2005, 2006). Moreover, the striatal dopa-mine concentrations are low in MPTP monkeys killed 4 or 24 hafter the last dose of L-DOPA being at levels of vehicle-treatedMPTP monkeys (Tamim et al., 2010). However, L-DOPA concentra-tions were higher in the striatum of animals killed 4 h compared toanimals killed 24 h after last dose of L-DOPA (Ouattara et al., 2010).A summary of the motor behavior of these monkeys is reported inTable 1; their detailed description was previously reported (Grégo-ire et al., 2008; Samadi et al., 2005, 2006).

2.2. Tissue preparation

All monkeys were killed by an overdose of pentobarbital. Thebrains were then immersed in isopentane (!40 !C) and stored at!80 !C. Hemisected brains were cut into 12 lm-thick coronal sec-tions on a cryostat (!18 !C) at three rostro-caudal levels. Accordingto the atlas of Martin and Bowden (2000), the anterior level ex-tends from +4 mm to 0 mm relative to the anterior commissure(ac), the medial level corresponds to 0 mm to !4 mm and theposterior level extends from !4 mm to !7 mm. Sections weremounted onto Super Frost Plus slides (Brain Research laboratories,Newton, USA), desiccated overnight at 4 !C and stored at !80 !C.

2.3. Biogenic amine assay

Small frozen pieces of the striatum were homogenized in250 lL of 0.1 M HClO4 at 4 !C. The homogenate was centrifugedat 10,000g for 20 min and the supernatants were kept at !80 !C.The pellets were dissolved in 100 lL of 0.1 M NaOH for assay ofprotein contents. The concentrations of biogenic amines were mea-sured by high-performance liquid chromatography under chro-matographic conditions we have previously used to assaybiogenic amines (Di Paolo et al., 1986; Tamim et al., 2010).

2.4. Autoradiography of 5-HT1A receptors

Autoradiography with [3H](R)-(+)-8-OH-DPAT (187 Ci/mmol:Perkin Elmer, Boston, MA, USA) was used to label 5-HT1A receptorsaccording to a previously published procedure (Stockmeier et al.,1996) which was shown to be highly selective for 5-HT1A receptorsand to lack affinity for dopamine receptors (Iravani et al., 2006). Inbrief, four to six slide-mounted brain sections per animal for eachlevel were pre-incubated for 30 min in buffer: 170 mM Tris–HCl(Bioshop: ON, Canada), 4 mM CaCl2 and 0.01% ascorbic acid (BakerChemical Co. Philipsburg, NJ, USA), pH 7.7. The slides were thenincubated with 2 nM [3H](R)-(+)-8-OH-DPAT for 1 h at room tem-perature. At this concentration, [3H](R)-(+)-8-OH-DPAT has noaffinity for 5-HT7 receptors with pKi = 6.6 (Wood et al., 2000).Hence, the equilibrium dissociation constant Ki of 8-OHDPAT for5-HT7 receptors is 10!6.6 M (0.25 lM) compared to 0.47 nM for5-HT1A receptors (Lejeune et al., 1997) that is almost a thousand

fold difference supporting the specificity of [3H](R)-(+)-8-OH-DPATfor 5-HT1A receptors. To block 5-HT transporter binding sites, 1 lMCitalopram-hydrobromide (Tocris Bioscience, Park Ellisville, MO,USA) was also added in the incubation buffer. To determine non-specific binding, 10 lM 5-HT (Sigma, St-Louis, MO, USA) was addedin the incubation buffer. Slides were then washed twice for 5 minin a 170 mM Tris–HCl, 4 mM CaCl2 buffer, pH 7.7 at 4 !C. They weredipped in distilled water at 4 !C for a few seconds and allowed toair-dry overnight. Sections were then exposed to [3H]-sensitivefilms (Kodak BIOMAX MR, USA) along with tritium standards([3H]-Microscales, Amersham, UK) for 7 weeks at room tempera-ture. Films were developed and autoradiograms were analyzedby densitometry.

2.5. Data analysis

Quantification of autoradiograms was performed on a computerconnected to a Sony video camera (model XC-77) with a constantillumination light table using computerized densitometry withthe software package NIH Image 1.63 (developed at U.S. NationalInstitutes of Health). [3H]8-OH-DPAT specific binding was mea-sured in the basal ganglia, the hippocampus, the frontal and cingu-late cortex. Based on the organization of its cortical afferents, thedorsal striatum of primates can be subdivided into main associa-tive and sensorimotor territories (Parent and Hazrati, 1995). Theassociative territory comprises most of the head of the caudate nu-cleus and a large portion of the pre-commissural putamen whereasthe sensorimotor territory includes most of the post-commissuralputamen and the dorsolateral part of the caudate nucleus (Parentand Hazrati, 1995). To achieve a more precise description of thedistribution of 5-HT1A receptor in the striatum, the caudate nu-cleus and the putamen were each divided into four subregions:medial, lateral, dorsal and ventral. The cerebral cortex was also di-vided into four subregions: dorsal and ventral superior frontalgyrus (SFG) and dorsal and ventral anterior cingulate gyrus (AcgG).The hippocampus (Hip) was divided into three subregions: cornuammonis (CA)1, CA2/CA3 and dentate gyrus (DG). During the anal-ysis, the investigator (G.R.) was blind to group treatments. Statisti-cal comparisons of [3H]8-OH-DPAT specific binding andindolamine concentrations between groups were performed usingthe Statview 4.51 program with a one way analysis of variance fol-lowed by post hoc pair wise comparisons with Fisher’s probabilityof least significant difference test. A P 6 0.05 was consideredsignificant.

3. Results

3.1. Behavioral assessments

A detailed description of the parkinsonian and dyskinesia scoresof the monkeys used in the present study was previously reported

Table 1Summary of Dyskinesia and basal PD scores of the MPTP monkeys.

Treatmenta Mean LID scoreb Max LID scoreb (group mean, range) Basal PD scoreb

Control 0 0 0MPTP 0 0 10.25 (6.8–12.6)L-Dopa/benserazide (24 h) 5.44 (3.7–7.5) 8.69 (7.0–11.0) 9.73 (6.5–12.5)

L-Dopa/benserazide + Ro 61-8048 (24 h) 3.46 (1.7–5.5) 7.38 (4.0–10.0) 10.24 (7.7–12.0)

L-Dopa/benserazide (4 h) 2.09 (1.4–3.0) 5.80 (2.0–9.0) 10.88 (9.9–13.7)

L-Dopa/benserazide + DHA (4 h) 1.25 (0.4–2.7) 2.90 (0–6.0) 11.56 (9.6–13.5)

L-Dopa/benserazide + naltrexone (4 h) 4.03 (2.4–5.5) 7.0 (4.0, 9.0) 11.48 (9.3–13.7)

a Monkeys were killed 24 or 4 h after the last dose of their chronic treatment.b A detailed description of the behavior of these monkeys was previously reported (Grégoire et al., 2008; Samadi et al., 2005, 2006). Mean LID and maximal (Max) scores

from the two last days of L-DOPA treatment are presented.

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(Grégoire et al., 2008; Samadi et al., 2005, 2006) and is summarizedin Table 1. In animals killed 24 h after last dose of L-DOPA, all mon-keys treated with L-DOPA alone developed dyskinesia while thosethat received adjunct treatments with Ro 61-8048 developedsignificantly less dyskinesia (Grégoire et al., 2008; Samadi et al.,2005, 2006). In monkeys killed 4 h after last dose of L-DOPA, allmonkeys treated with L-DOPA alone developed dyskinesias. Thosetreated with naltrexone developed severe dyskinesia while mon-keys that received adjunct treatments with DHA developed milddyskinesia (Grégoire et al., 2008; Samadi et al., 2005, 2006).

3.2. Assessment of extent of denervation

3.2.1. Measure of dopamine denervationThe extent of striatal dopamine denervation in all MPTP mon-

keys used in the present study was 95% or more compared to con-trol monkeys, as indicated by measurement of [125I] RTI-121(3beta-(4-125I-iodophenyl)tropane-2beta-carboxylic acid isopropylester) specific binding to the dopamine transporter and dopamineconcentrations (Tamim et al., 2010).

3.2.2. Measure of serotonin denervationStriatal concentrations of 5-HT and its major brain metabolite,

5-hydroxyindoleacetic acid (5-HIAA) (Figs. 1 and 2) were de-creased in the MPTP monkeys compared to controls but to a lesserextent than dopamine.

3.2.2.1. Caudate nucleus. In the caudate nucleus of MPTP monkeyskilled 24 h after the last dose of L-DOPA, no significant differenceof 5-HT and 5-HIAA concentrations or the 5-HIAA/5-HT ratio wasobserved compared to control monkeys (Fig. 1A and B).

In L-DOPA treated MPTP monkeys killed 4 h after their last doseof L-DOPA, caudate nucleus 5-HT concentrations were decreased(F4,17 = 3.22, P = 0.039) compared to control monkeys with no dif-ference of 5-HIAA concentrations (Fig. 1C). The 5-HIAA/5-HT ratiowas increased in the caudate nucleus of L-DOPA treated MPTPmonkeys killed 4 h after their last dose of L-DOPA compared to con-trols and to MPTP saline-treated monkeys (F4,17 = 4.40, P = 0.013)(Fig. 1D).

3.2.2.2. Putamen. In the putamen of saline-treated MPTP monkeysand L-DOPA-treated alone MPTP monkeys killed 24 h after the lastdose of L-DOPA, 5-HT concentration were decreased compared tocontrol monkeys (F3,13 = 4.65, P = 0.027) (Fig. 2A) and 5-HIAA con-centrations (Fig. 2A) or the 5-HIAA/5-HT ratio (Fig. 2B) wereunchanged.

In MPTP monkeys killed 4 h after their last dose of L-DOPA, a de-crease of 5-HT concentrations was observed in the putamen of allMPTP-treated monkeys (F4,17 = 4.18, P = 0.015) compared tocontrol monkeys and 5-HIAA concentrations were unchanged(Fig. 2C). An increase of 5-HIAA/5-HT ratio was measured in theputamen of L-DOPA-treated monkeys compared to controls andto saline-treated MPTP monkeys (F4,17 = 5.08, P = 0.007) (Fig. 2D).

3.3. Distribution of 5-HT1A receptors in control monkeys

At the anterior brain level (ac +4 mm to ac 0 mm) of controlmonkeys, 5-HT1A receptor specific binding were highest in theAcgG, high in the SFG and the amygdala, intermediate in the nu-cleus accumbens and low in the caudate nucleus and the putamen(F5,17 = 69.44, P < 0.0001) (Fig. 3A, Tables 2 and 3). At the medial le-vel (ac 0 mm to ac !4 mm), 5-HT1A receptor specific binding washighest in the hippocampus, high in the frontal cortex (AcgG and

Fig. 1. The indolamines serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), concentrations as well as the 5-HIAA/5-HT ratio in the caudate nucleus of control, MPTPmonkeys treated with saline, L-DOPA, L-DOPA + Ro 61-8048, L-DOPA + DHA or L-DOPA + naltrexone. Monkeys were grouped according to their time of euthanasia: 24 h aftertheir last dose of L-DOPA (A and B) and 4 h after their last dose of L-DOPA (C and D). ⁄P < 0.05 and ⁄⁄P < 0.01 vs. control monkeys; £P < 0.05 and ££P < 0.01 vs. saline-treatedMPTP monkeys.

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SFG), low in the striatum and not detectable in the pallidum(F5,17 = 31.57, P < 0.0001) (Fig. 3B, Tables 2 and 3). The hippocam-pus and the frontal cortex showed respectively the highest andhigh levels of [3H]8-OH-DPAT specific binding in posterior brain re-gion (ac !4 mm to ac !7 mm) analyzed whereas specific bindingwas low in the substantia nigra, the subthalamic nucleus, the stri-atum and non detectable in the pallidum at this rostro-caudal level(F5,17 = 189.81, P < 0.0001) (Fig. 3C, Tables 2 and 3).

3.4. 5-HT1A receptors in MPTP-treated lesioned monkeys

Examples of autoradiograms of [3H]8-OH-DPAT binding of con-trol monkeys and MPTP-treated monkeys are shown in Fig. 4.

3.4.1. HippocampusIn MPTP monkeys killed 24 h after the last dose of L-DOPA, the

anterior part of the hippocampus (ac 0 mm to ac !4 mm) showedan increase of [3H]8-OH-DPAT specific binding for L-DOPA-treatedmonkeys compared to control monkeys, when L-DOPA-treatedmonkeys were grouped together regardless to their adjunct ther-apy. This effect was significant in the CA2/CA3 (F2,14 = 4.18,P = 0.04) and did not reach statistical significance in the CA1 orin the DG (Fig. 5 A). [3H]8-OH-DPAT specific binding was un-changed in the posterior hippocampus (levels ac !4 mm to ac!7 mm) of monkeys killed 24 h after last dose of L-DOPA (Fig. 5 B).

In MPTP monkeys killed 4 h after the last dose of L-DOPA, an in-crease of 5-HT1A receptor specific binding was also found in theanterior hippocampus of L-DOPA-treated monkeys compared tocontrol monkeys when L-DOPA-treated monkeys were grouped to-

gether regardless to their adjunct therapy. The increase of 5-HT1A

receptor specific binding was observed in the CA2/CA3 of allL-DOPA-treated monkeys compared to control monkeys (F2,19 =3.92, P = 0.04) and not in the CA1 nor in the DG (Fig. 5C). Anincrease of specific binding was observed in the posteriorCA2/CA3 hippocampus of all L-DOPA-treated monkeys killed 4 hafter last dose of L-DOPA compared to control monkeys (F2,19 =3.72, P = 0.04) (Fig. 5D).

3.4.2. Frontal cortexIn the anterior (ac +4 mm to ac 0 mm) cortical AcgG and SFG re-

gions of monkeys, no significant change of [3H]8-OH-DPAT specificbinding was observed (Tables 2 and 3).

In animals killed 24 h after the last dose of L-DOPA, in the med-ial (ac 0 mm to ac !4 mm) cortical AcgG and SFG, no significantdifference of 5-HT1A receptor specific binding was observed(Fig. 6A). More caudally, a decrease of 5-HT1A receptor specificbinding of saline-treated MPTP monkeys compared to controlmonkeys and an increase of [3H]8-OH-DPAT specific binding of L-DOPA-treated alone MPTP monkeys compared to saline-treatedMPTP monkeys was observed in the posterior (ac !4 mm to ac!7 mm) dorsal cortical AcgG (F3,13 = 3.37, P = 0.05) (Fig. 6B). More-over, there was a decrease of 5-HT1A receptor specific binding inthe posterior ventral SFG of saline-treated MPTP monkeys com-pared to control monkeys and this binding was increased in MPTPmonkeys treated with L-DOPA alone (F3,13 = 5.59, P = 0.01), this wasnot significant in the dorsal SFG (Fig. 6B).

In MPTP monkeys killed 4 h after last dose of L-DOPA, 5-HT1A

specific binding in the medial region of dorsal AcgG was increased

Fig. 2. The indolamines, serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations as well as the 5-HIAA/5-HT ratio in the putamen of control, MPTP monkeystreated with saline, L-DOPA, L-DOPA + Ro 61-8048, L-DOPA + DHA or L-DOPA + naltrexone. Monkeys were grouped according to their time of euthanasia: 24 h after their lastdose of L-DOPA (A and B) and 4 h after their last dose of L-DOPA (C and D). ⁄P < 0.05 and ⁄⁄P < 0.01 vs. control monkeys; £P < 0.05 and ££P < 0.01 vs. saline-treated MPTPmonkeys.

1164 G. Riahi et al. / Neurochemistry International 61 (2012) 1160–1171

in all L-DOPA-treated MPTP monkeys compared to saline-treatedMPTP monkeys (F2,19 = 3.21, P = 0.05), but not in the ventral AcgGnor the SFG (Fig. 6C). In the posterior (ac !4 mm to ac !7 mm)

cortex of these animals, a decrease of [3H]8-OH-DPAT specificbinding of saline-treated MPTP monkeys compared to controlmonkeys and an increase of 5-HT1A specific binding of all

(A) (B) (C)

Fig. 3. Distribution of [3H]8-OH-DPAT specific binding to 5-HT1A receptors in control monkey brains with representative autoradiograms and schematic frontal hemi-sections, adapted from the macaque brain maps (Martin and Bowden, 2000), at the anterior level (A, ac + 4 mm to ac 0 mm), the medial level (B, ac 0 mm to ac – 4 mm) andthe posterior level (C, ac – 4 mm to ac – 7 mm). Acb, nucleus accumbens; AcgG, anterior cingulated gyrus; Amg, amygdala; CD, caudate nucleus; D, dorsal; DG, dentate gyrus;DL, dorsolateral; DM, dorsomedial; GPe and GPi, external and internal segments of the globus pallidus; Hip, Hippocampus; PUT, putamen; SFG, superior frontal gyrus; SI,substantia innominata; SNr, substantia nigra reticulata; STN, subthalamic nucleus; V, Ventral; VL, ventrolateral; VM, ventromedial. ⁄P < 0.01, vs. caudate nucleus; £P < 0.01, vs.putamen; ¤P < 0.01 vs. Acb; §P < 0.01 vs. AcgG; #P < 0.01 vs. SFG; !P < 0.01 vs. SNr; ¥P < 0.01 vs. STN; ND, non detectable.

Table 2[3H] 8-OH-DPAT specific binding to 5-HT1A receptors in different brain regions of animals killed 24 h after their last dose of L-DOPA.

Brain region Control MPTP MPTP + L-DOPA MPTP + L-DOPA + Ro 61-8048

Caudate nucleusAnterior 3.08 ± 0.81 3.53 ± 1.51 8.60 ± 5.80 4.79 ± 1.16Medial 8.34 ± 1.85 8.18 ± 1.11 8.65 ± 1.64 7.56 ± 2.08Posterior 10.81 ± 2.40 8.10 ± 2.83 10.40 ± 3.00 10.06 ± 0.29

PutamenAnterior 2.63 ± 0.86 2.69 ± 1.04 7.55 ± 5.59 3.86 ± 1.11Medial 2.97 ± 0.58 3.67 ± 0.81 4.21 ± 1.22 3.44 ± 1.03Posterior 11.67 ± 3.42 6.38 ± 1.56 4.70 ± 1.48 10.28 ± 1.11

Anterior cingulate gyrusVentral 118.38 ± 11.33 113.04 ± 8.67 136.35 ± 18.18 106.95 ± 11.84Dorsal 121.33 ± 3.13 108.26 ± 10.19 142.28 ± 18.57 119.75 ± 14.24

Anterior superior frontal gyrusVentral 88.72 ± 6.23 83.68 ± 12.23 106.18 ± 16.09 87.80 ± 12.02Dorsal 68.14 ± 9.26 57.81 ± 8.84 76.69 ± 11.63 59.49 ± 8.73

Nucleus accumbens 14.73 ± 2.27 13.41 ± 1.94 22.87 ± 9.43 16.75 ± 5.06

Amygdala 75.42 ± 12.72 72.08 ± 7.59 84.50 ± 13.87 96.79 ± 14.58

Subthalamic nucleus 15.87 ± 3.96 10.28 ± 4.28 5.69 ± 2.02 12.26 ± 3.44

Substantia nigra 16.57 ± 4.11 10.63 ± 3.60 7.734 ± 1.94 14.49 ± 2.55

Results are shown as the mean ± SEM expressed in fmol/mg of tissue.

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L-DOPA-treated MPTP monkeys compared to saline-treated MPTPmonkeys was found in the posterior region of dorsal AcgG(F2,19 = 3.67, P = 0.04) (Fig. 6D). There was a decrease of 5-HT1A

receptor binding in the ventral SFG of saline-treated MPTP mon-keys compared to control monkeys when L-DOPA-treated monkeyswere grouped regardless to their adjunct therapy (F2,19 = 4.59,P = 0.02) (Fig. 6D).

3.4.3. Other brain regionsNo significant change of [3H]8-OH-DPAT specific binding was

observed in the anterior, medial and posterior region of the cau-

date nucleus, the putamen, the nucleus accumbens, the amygdala,the substantia nigra and the subthalamic nucleus of the investi-gated monkeys (Tables 2 and 3).

3.5. Correlation of dyskinesias with 5-HT1A receptor specific binding

In MPTP monkeys killed 24 h after their last dose of L-DOPA,there were significant positive correlations between the maximumdyskinesia scores of monkeys at the end of one month treatmentand 5-HT1A receptor specific binding in the medial dorsal of AcgG

Table 3[3H] 8-OH-DPAT specific binding to 5-HT1A receptors in different brain regions of animals killed 4 h after their last dose of L-DOPA.

Brain region Control MPTP MPTP + L-DOPA MPTP + L-DOPA + DHA MPTP + L-DOPA + naltrexone

Caudate nucleusAnterior 3.08 ± 0.81 3.53 ± 1.51 4.44 ± 1.43 2.76 ± 1.33 5.96 ± 2.75Medial 8.34 ± 1.85 8.18 ± 1.11 9.99 ± 2.53 8.30 ± 2.15 9.48 ± 0.97Posterior 10.81 ± 2.40 8.10 ± 2.83 7.37 ± 0.68 8.50 ± 0.77 6.80 ± 1.20

PutamenAnterior 2.63 ± 0.86 2.69 ± 1.04 3.74 ± 1.14 3.14 ± 1.57 4.90 ± 2.65Medial 2.97 ± 0.58 3.67 ± 0.81 4.04 ± 1.27 3.43 ± 0.88 4.41 ± 1.21Posterior 11.67 ± 3.42 6.38 ± 1.56 8.91 ± 1.97 7.09 ± 0.77 6.75 ± 1.93

Anterior cingulate gyrusVentral 118.38 ± 11.33 113.04 ± 8.67 134.60 ± 7.20 129.16 ± 11.75 119.14 ± 12.41Dorsal 121.33 ± 3.13 108.26 ± 10.19 130.81 ± 5.30 139.89 ± 19.27 139.42 ± 16.44

Anterior superior frontal gyrusVentral 88.72 ± 6.23 83.68 ± 12.23 95.03 ± 3.14 105.75 ± 12.78 97.85 ± 15.88Dorsal 68.14 ± 9.26 57.81 ± 8.84 75.44 ± 4.90 76.21 ± 11.05 74.98 ± 11.27

Nucleus accumbens 14.73 ± 2.27 13.41 ± 1.94 17.15 ± 4.76 14.44 ± 6.58 15.87 ± 6.62

Amygdala 75.42 ± 12.72 72.08 ± 7.59 102.75 ± 16.15 86.71 ± 21.18 103.28 ± 10.11

Subthalamic nucleus 15.87 ± 3.96 10.28 ± 4.28 12.27 ± 0.93 8.80 ± 1.02 7.47 ± 2.29

Substantia nigra 16.57 ± 4.11 10.63 ± 3.60 13.48 ± 1.63 12.09 ± 1.90 10.63 ± 1.49

Results are shown as the mean ± SEM expressed in fmol/mg of tissue.

Fig. 4. Representative autoradiograms at the medial level (ac 0 mm to ac !4 mm) showing the effect of MPTP and chronic treatment with L-DOPA alone or in combinationwith adjunct treatments (Ro 61-8048, DHA and naltrexone) on [3H]8-OH-DPAT binding to 5-HT1A receptors along with nonspecific binding in monkey brain.

1166 G. Riahi et al. / Neurochemistry International 61 (2012) 1160–1171

(R = 0.569, P = 0.034), medial ventral dorsal of AcgG (R = 0.549,P = 0.042) and posterior ventral of AcgG (R = 0.498, P = 0.040).

In MPTP monkeys killed 4 h after their last dose of L-DOPA,there were significant positive correlation between the maximumdyskinesia scores of monkeys at the end of one month treatmentand 5-HT1A receptor specific binding in the medial dorsal of AcgG(R = 0.577, P = 0.015).

4. Discussion

This is the first report of brain 5-HT1A receptor modulation inMPTP monkeys chronically treated with L-DOPA and displayingdyskinesia or with adjunct treatment alleviating dyskinesia com-pared to control and to saline-treated MPTP monkeys. Increase 5-HT activity was observed in animals treated with L-DOPA that werekilled 4 h after the last dose of L-DOPA. The regional distributionsof [3H]8-OH-DPAT specific binding to 5-HT1A receptor measuredhere were similar to previous reports in non-human (Saigal et al.,2006) and human (Moller et al., 2009) primate brains. We observeda decrease of 5-HT1A receptor specific binding in the posterior cor-tex by the MPTP lesion and an increase with the L-DOPA treatmentalone or with DHA or naltrexone but no increase with Ro 61-8048,the latter treatment giving low dyskinesias. In the hippocampus ofall L-DOPA-treated MPTP monkeys, regardless to their dyskinetic

behavior, an increase of 5-HT1A receptor specific binding was ob-served compared to control and to saline-treated MPTP monkeys.

In animals killed 4 h after the last dose of L-DOPA, 5-HT concen-trations in the putamen were decreased in all MPTP monkeys andin the caudate nucleus of all L-DOPA-treated monkeys. This findingis in accordance with other biochemical measurements of 5-HTconcentration in the striatum (Agid et al., 1989; Kish et al.,2008), hippocampus, frontal and cingulate cortex (Scatton et al.,1983) of post-mortem PD patients and in the striatum (Frechillaet al., 2001; Riahi et al., 2011), hippocampus and raphe nucleusof MPTP monkeys (Frechilla et al., 2001). Serotonin transporter(SERT) levels are also reported to change in PD. A decrease of SERTlevels was found in the striatum of PD patients in both post-mor-tem tissues (Cash et al., 1985; Raisman et al., 1986; Chinagliaet al., 1993) and living humans (Kerenyi et al., 2003; Kim et al.,2003; Guttman et al., 2007; Roselli et al., 2010). Moreover, positronemission tomography image analysis showed varying levels ofSERT binding reduction in the left lesioned side of the striatumwhen compared to the right non-lesioned side of the striatum in6-hydroxydopamine (6-OHDA)-lesioned rat PD model (Wanget al., 2010). In the present study, no difference in 5-HT concentra-tion between dyskinetic and nondyskinetic monkeys was ob-served. This finding is in agreement with a post-mortem humanstudy that reported no change in striatal 5-HT concentration

(A) (B)

(C) (D)

Fig. 5. [3H]8-OH-DPAT specific binding in the anterior (A and C, ac 0 mm to ac – 4 mm) and posterior hippocampus (B and D, ac – 4 mm to ac – 7 mm) to 5-HT1A receptors incornu ammonis (CA)1, CA2/CA3, and dentate gyrus (DG) of control, MPTP monkeys treated with saline, L-DOPA, L-DOPA + Ro 61-8048, L-DOPA + DHA or L-DOPA + naltrexone.Monkeys were grouped according to their time of euthanasia: 24 h after their last dose of L-DOPA (A and B) and 4 h after their last dose of L-DOPA (C and D). ⁄P < 0.05 for L-DOPA-treated monkeys grouped together, regardless to their adjunct therapy vs. control monkeys.

G. Riahi et al. / Neurochemistry International 61 (2012) 1160–1171 1167

between PD patients who developed LID and those that did not(Kish et al., 2008). The 5-HIAA/5-HT ratio is regarded as a measureof 5-HT turnover. The fact that high 5-HT turnover was measuredin the striatum of animals killed 4 h after their last dose of L-DOPAand not in those killed after 24 h supports an increased activity of5-HT axon terminals following L-DOPA administration. This obser-vation is in accordance with previous data showing that L-DOPAtreatment induces a sprouting of the striatal 5-HT axons and ahigher activity-dependent potentiation of dopamine release bythese axon terminals along with an increase in their synaptic inci-dence (Rylander et al., 2010). A high 5-HT turnover was also ob-served in low dyskinetic monkeys that were treated with L-DOPA + DHA suggesting that reduction of dyskinesia mediated bythis compound does not necessarily involve a decrease of the 5-HT turnover.

In the present study, MPTP monkeys killed 24 h after last doseof L-DOPA demonstrated higher dyskinesia score compared to ani-mals killed 4 h after last dose of L-DOPA (Table 1). Moreover, mon-keys treated with L-DOPA and killed 24 h compared to those killed4 h after last dose of L-DOPA displayed higher 5-HT1A receptor spe-cific binding in the medial and the posterior cortex and the medialCA2/3 of hippocampus. This difference is in agreement with our re-sults showing higher 5-HT1A receptor specific binding in the cortexand the hippocampus of dyskinetic animals compared to low dys-kinetic animals treated with Ro-61 8048. Moreover, it was reportedthat activation of 5-HT1A heteroreceptors increases dopamine

release in the cortex and the hippocampus in rats (Li et al.,2004). Therefore, it is proposed that the increase of 5-HT1A hetero-receptors binding in the cortex and the hippocampus of MPTPmonkeys treated with L-DOPA and killed 24 h after last dose of L-DOPA is a compensatory mechanism to release dopamine.

Increased 5-HT turnover were measured in the striatum ofMPTP monkeys killed 4 h compared to 24 h after their last L-DOPAadministration. Although the striatal dopamine concentrationswere similar in all groups of MPTP monkeys (Tamim et al., 2010)those that received L-DOPA 4 h before death had elevated striatalL-DOPA content (Ouattara et al., 2010). To investigate the contribu-tion of 5-HT concentrations or 5-HT1A receptor specific binding todyskinesia, animals were divided in two main groups according tothe time of euthanasia to separate the effect of different endpointsof L-DOPA on 5-HT1A receptor specific binding in dyskinetic ani-mals compared to nondyskinetic animals.

We observed significant alterations of 5-HT1A receptors in supe-rior and posterior frontal gyrus considered as parts of motor andprimary motor cortex innervating the dorsolateral putamen (Par-ent and Hazrati, 1995); this suggests that alterations of 5-HT1A

receptors on glutamatergic cortical neurons innervating the senso-rimotor striatum play an important role in the expression of PDmotor symptoms and in the development of LID. An increase of5-HT1A receptors was found in the posterior SFG of MPTP monkeystreated with L-DOPA alone while it was unchanged in MPTP mon-keys treated with L-DOPA + Ro 61-8048 compared to control and to

(A) (B)

(C) (D)

Fig. 6. [3H]8-OH-DPAT specific binding at the medial cortical level (A and C, ac 0 mm to ac – 4 mm) and the posterior cortical level (B and D, ac – 4 mm to ac – 7 mm) to 5-HT1A receptors in ventral and dorsal parts of the anterior cingulate gyrus (AcgG) and superior frontal gyrus (SFG) of control, MPTP monkeys treated with saline, L-DOPA, L-DOPA + Ro 61-8048, L-DOPA + DHA or L-DOPA + naltrexone. Monkeys were grouped according to their time of euthanasia: 24 h after their last dose of L-DOPA (A and B) and4 h after their last dose of L-DOPA (C and D). ⁄P < 0.05 and ⁄⁄P < 0.01 vs. control monkeys; £P < 0.05 for L-DOPA-treated monkeys grouped together, regardless to their adjuncttherapy vs. saline-treated MPTP monkeys and ££P < 0.01 vs. saline-treated MPTP monkeys.

1168 G. Riahi et al. / Neurochemistry International 61 (2012) 1160–1171

saline-treated MPTP monkeys. It was demonstrated that 5-HT1A

receptor binding was increased in the motor cortex of 10 PD pa-tients treated chronically with L-DOPA and experiencing motorcomplications (Huot et al., 2012b). Upregulation of 5-HT1A recep-tors in motor cortex might be part of a compensatory mechanismdesigned to decrease the release of glutamate by corticostriatalneurons (Mauler et al., 2001). This mechanism has been proposedas an explanation for the efficacy of 5-HT1A agonists in LID treat-ment (Antonelli et al., 2005). The fact that Ro 61-8048 blocksNMDA receptors and inhibits glutamate release by increasing theformation of kynurenic acid (Grégoire et al., 2008) might explainwhy no upregulation of 5-HT1A receptors was observed in the pos-terior SFG of MPTP monkeys treated with L-DOPA + Ro 61-8048.This theory is also confirmed by a recent study showing that stria-tal and systemic administration of 8-OH-DPAT reduced LID andstriatal glutamate level in 6-OHDA-lesioned rats, while administra-tion of 5-HT1A receptor antagonist reversed these effects (Dupreet al., 2011). Altogether results from the present study and the ref-erences cited above suggest that efficacy of 5-HT1A receptor ago-nists in treatment of dyskinesia could be improved (1) byaddition of an antiglutamatergic agent to the serotonergic thera-peutic regimen (Huot and Brotchie, 2011) or (2) by developingselective 5-HT1A receptor agonists targeting corticostriatal neuro-transmission (Huot et al., 2011). It is suggested that functionalinteractions between 5-HT1A and NMDA receptors rather thanother glutamate receptors might be involved in the reduction ofLID since the antiglutamatergic agent used in this study, Ro 61-8048, prevents the upregulation of 5-HT1A receptors via blockingNMDA receptors. Moreover, the combined administration of alow dose of 8-OH-DPAT (0.03 mg/kg) and MK-801, a NMDA recep-tor blocker, while having no effect alone, reduced abnormal invol-untary movements in 6-OHDA-lesioned rats treated with L-DOPA(Dupre et al., 2008b).

A decrease of 5-HT1A receptors was observed in the dorsal AcgGand the ventral SFG in MPTP monkeys compared to controls. Theseresults are in agreement with previous studies reporting a downregulation of 5-HT1A receptors in the cerebral cortex of PD patients(Agid et al., 1989) and in the prefrontal cortex of MPTP monkeys(Frechilla et al., 2001). Down regulation of 5-HT1A receptors hasalso been shown in the external layers of the motor and premotorcortex but not in the middle layers in MPTP-lesioned monkeys(Huot et al., 2012a). The reduction of 5-HT1A receptors may repre-sent dysfunctions in response to alterations of the 5-HT systemsknown to occur in PD (Agid et al., 1989; Bédard et al., 2011).

The increase of 5-HT1A receptor specific binding in the hippo-campus and the AcgG of all L-DOPA-treated monkeys, regardlessto their dyskinesias compared to control and to saline-treatedMPTP monkeys, may underlie the involvement of 5-HT1A receptorsin non-motor effects of L-DOPA. Indeed, it is reported that L-DOPAhas antidepressant activity in unipolar depressed patients (Good-win et al., 1970) that could be explained by alterations of 5-HT1A

receptors induced by L-DOPA in these brain areas, as reported inthe present study. Moreover, L-DOPA administration is known todecrease the release of 5-HT and to favor the release of dopamineby 5-HT neurons in the prefrontal cortex and the hippocampus,two brain regions involved in the regulation of mood and cognitionthat play a role in the emergence of motor and nonmotor symp-toms of PD (Navailles et al., 2010). Therefore, upregulation of 5-HT1A receptors reported here might be part of a compensatorymechanism induced by low 5-HT concentrations in these brainareas.

In the present study, specific binding of 5-HT1A receptors in thestriatum was low. No significant change was observed in the lowlevel of 5-HT1A receptors in the caudate nucleus and the putamenof MPTP-lesioned and L-DOPA-treated MPTP monkeys with orwithout dyskinesias. The latter observation is in agreement with

previous studies that have reported low level of 5-HT1A receptorsin the striatum of MPTP monkeys (Barnes and Sharp, 1999; Frechil-la et al., 2001) and no change in striatal 5-HT1 receptors in PD pa-tients (Agid et al., 1989; Rinne et al., 1980) whereas in a recentstudy, labeling 5-HT1A receptors with the radioligand [3H]-WAY100635 in intact female monkeys, an increase of 5-HT1A receptorsin the striosomes of the caudate nucleus of MPTP-treated monkeysand in the matrix of chronic L-DOPA-treated monkeys with nochange in putamen was reported (Huot et al., 2012a).

The similar level of 5-HT1A receptors in different brain regionsbetween monkeys treated with L-DOPA alone and monkeys treatedwith L-DOPA + naltrexone or L-DOPA + DHA suggests that the ef-fects of naltrexone (a nonselective opioid receptor antagonist)and DHA are not directly related to 5-HT1A receptor alterations.The link between DHA and 5-HT1A receptors has not yet beenestablished. Although it is known that DHA is required for the nor-mal development and maturation of the rat 5-HT pathways, no sig-nificant change of 5-HT1A receptors was found in the midbrain ofDHA-deficient rats compared to controls (McNamara et al., 2009).As for naltrexone, it has been shown that mu, but not kappa opi-oids receptors are able to modulate the effect of a 5-HT1A receptoragonist (Morgan and Picker, 1995). Therefore, it is suggested thatmechanisms other than those involving alterations of the 5-HT1A

receptors might contribute to the antidyskinetic effect of DHAand dyskinetic effect of naltrexone. Indeed, previous studies havesuggested the modulation of Nur77, an orphan nuclear receptor,to be linked to the reduction of dyskinesia by DHA (Mahmoudiet al., 2009) and the reduction of opioid transmission, a compensa-tory mechanism designed to attenuate changes induced by dopa-mine denervation, to be associated with the dyskinetic effect ofnaltrexone (Samadi et al., 2005).

5. Conclusion

The present study provides new insights to better understandthe role of 5-HT in PD and LID development, especially throughits 5-HT1A receptors. The increase of 5-HT turnover in L-DOPA-trea-ted monkeys killed 4 h after last dose of L-DOPA alone or with ad-junct treatment, underlies the hyperactivity of 5-HT axonterminals in the dopamine-denervated striatum following L-DOPAadministration regardless dyskinetic activity. Although these re-sults are correlative, the decrease of cortical 5-HT1A receptors inMPTP monkeys and the increase of these receptors in the hippo-campus and cerebral cortex of L-DOPA-treated monkeys highlightthe involvement of 5-HT1A receptors in PD and its treatment withL-DOPA. The fact that Ro 61-8048, an antiglutamatergic agent, pre-vents dyskinesia and 5-HT1A receptor changes induced by L-DOPA,confirmed the importance of interaction between 5-HT1A andNMDA receptors in LID treatment without worsening PDsymptoms.

Acknowledgments

This work was supported by grants from the Canadian Instituteof Health Research (CIHR) to T.D.P. and M.P. and by a studentshipfrom the Fonds d’Enseignement et de Recherche (FER) of the Fac-ulty of Pharmacy, Université Laval, to G.R.

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