Modulation of l -DOPA-induced abnormal involuntary movements by clinically tested compounds: Further validation of the rat dyskinesia model

Behavioural Brain Research 179 (2007) 76–89

Research report

Modulation of l-DOPA-induced abnormal involuntary movements by clinically tested compounds: Further validation of the rat dyskinesia model夽 Andrzej Dekundy a,b,∗ , Martin Lundblad b , Wojciech Danysz a , M. Angela Cenci b,∗∗ a

In vivo Pharmacology, Preclinical Research and Development, Merz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, D-60318 Frankfurt am Main, Germany b Basal Ganglia Pathophysiology Unit, Department of Experimental Medical Science, Lund University, BMC F11, S-221 84 Lund, Sweden Received 19 November 2006; received in revised form 15 January 2007; accepted 19 January 2007 Available online 23 January 2007

Abstract l-DOPA-induced dyskinesia (LID) is a major complication of the pharmacotherapy of Parkinson’s Disease. A model of LID has recently been described in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. In the present study, the model was used in order to compare the efficacies of some clinically available compounds that have shown antidyskinetic effects in nonhuman primate models of LID and/or in patients, namely, amantadine (20 and 40 mg/kg), buspirone (1, 2 and 4 mg/kg), clonidine (0.01, 0.1 and 1 mg/kg), clozapine (4 and 8 mg/kg), fluoxetine (2.5 and 5 mg/kg), propranolol (5, 10 and 20 mg/kg), riluzole (2 and 4 mg/kg), and yohimbine (2 and 10 mg/kg). Rats were treated for 3 weeks with l-DOPA for an induction and monitoring of abnormal involuntary movements (AIMs) prior to the drug screening experiments. The antidyskinetic drugs or their vehicles were administered together with l-DOPA, and their effects were evaluated according to a randomized cross-over design both on the AIM rating scale and on the rotarod test. Most of the compounds under investigation attenuated the l-DOPA-induced axial, limb and orolingual AIM scores. However, the highest doses of many of these substances (but for amantadine and riluzole) had also detrimental motor effects, producing a reduction in rotarod performance and locomotor scores. Since the present results correspond well to existing clinical and experimental data, this study indicates that axial, limb and orolingual AIMs possess predictive validity for the preclinical screening of novel antidyskinetic treatments. Combining tests of general motor performance with AIMs ratings in the same experiment allows for selecting drugs that specifically reduce dyskinesia without diminishing the anti-akinetic effect of l-DOPA. © 2007 Elsevier B.V. All rights reserved. Keywords: Levodopa; Motor complications; Glutamate; Serotonin; 5HT1A; Nicotinic; Adrenergic; Receptor

1. Introduction Pharmacological dopamine (DA) replacement by l-DOPA remains the “gold standard” in the treatment of Parkinson’s disease (PD). However, in up to 80% of patients, this treatment leads to the development of abnormal involuntary movements

夽 Presented in preliminary form at Society for Neuroscience 33rd Annual Meeting, New Orleans, USA, 8–12 November 2003. ∗ Corresponding author at: Preclinical R&D, Merz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, 60318 Frankfurt, Germany. Tel.: +49 69 15 03 661; fax: +49 69 15 03 795. ∗∗ Corresponding author. Tel.: +46 46 2221431; fax: +46 46 2224546. E-mail addresses: [email protected] (A. Dekundy), Angela.Cenci [email protected] (M.A. Cenci).

0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2007.01.013

(AIMs), referred to as l-DOPA-induced dyskinesia (LID; for review, see refs. [37,79,80]). Dyskinesia may affect all parts of the body in an idiosyncratic mixture of chorea (i.e., rapid movements that seem to flow from one body part to another), dystonia (slow twisting movements) and myoclonus (jerky muscle contractions); for review, see refs. [37,90]. These movements are most common and most severe at the time when l-DOPA is producing the maximal relief of parkinsonian motor symptoms (hence the term “on” or “peak-of-dose dyskinesia”) [37,79,90]. The two main contributing factors underlying the development of LID are l-DOPA treatment and DA denervation (for review, see refs. [10,51]). These two factors in combination produce plastic changes that prime the brain for a dyskinetic response to DA receptor stimulation [25,92]. LID has a negative impact on physiological motor activities, worsening the patients’ quality of

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Table 1 Clinically approved drugs shown to reduce the severity of levodopa-induced dyskinesia in MPTP-intoxicated monkeys and/or in Parkinson’s disease patients Drug name

Pharmacological mechanism

Studies in monkeys

Studies in humans

Amantadine

NMDA and neuronal nicotinic receptor antagonist, sigma receptor blocker, aromatic l-amino acid decarboxylase inhibitor Serotonin 5-HT1A receptor agonist, partial agonist at dopamine D2 receptors, it’s major metabolite 1-(2-pyrimidinyl)-piperazine is an alpha-2 adrenoreceptor antagonist Alpha2-adrenergic agonist Atypical neuroleptic (broad-spectrum antagonist with high affinity for 5-HT receptors including 5-HT2 , 5-HT3 , 5-HT6 and 5-HT7 subtypes, but also an alpha1-adrenergic, muscarinic 1-5 acetylcholine, and weak D2-like dopamine receptor antagonist) Serotonin (5-HT) selective uptake inhibitor Non-selective nicotinic receptor antagonist Non-selective beta-adrenergic antagonist, 5-HT1 receptor antagonist Glutamate release inhibitor Alpha2-adrenergic antagonist

[12]

[30,62,75,84,91,99]

Not tested

[16,33,40,53]

[41] [42]

[78] [8,9,35,34]

Not tested Not tested [41]

[36,33,40] Not tested [21]

Not tested [41]

[74] Not tested

Buspirone

Clonidine Clozapine

Fluoxetine Mecamylamine Propranolol Riluzole Yohimbine

life and increasing healthcare costs [32]. Despite recent advances in the management of PD, LID continues to be a clinical and therapeutic challenge [82]. The wide range of neurotransmitters and neuromodulators present in the basal ganglia has prompted a search for novel and alternative, non-dopaminergic therapeutic targets for LID. Particular efforts have been devoted to identifying compounds that can reduce the incidence and severity of dyskinesia when coadministered with l-DOPA [10,18,92]. We have advocated the utility of 6-OHDA-lesioned rats for a preclinical screening of potential antidyskinetic drugs (reviewed by Cenci and Lundblad [24]). We have also emphasized the importance of developing behavioural testing methods that can ensure a correct interpretation of the results obtained in rodents [24,26]. Dyskinesia can be attenuated by treatments that have motor depressant effects or otherwise interfere with the anti-akinetic action of l-DOPA, but these treatments are unlikely to lead to clinical therapeutic improvements. It is therefore essential to be able to detect and quantify treatment-dependent effects both on abnormal movements and on measures of physiological motor performance in the same study. As a part of our ongoing efforts towards the optimization of rodent models, we have here compared the effects of various non-dopaminergic compounds on both l-DOPA-induced AIMs and l-DOPA-induced motor improvement in 6-OHDA lesioned rats. Physiological motor function was assessed using the rotarod test [96]. We have previously shown that this test allows for detecting lesion-induced motor deficits and l-DOPA-induced motor improvements in the rat [65,88]. In our behavioural screening study, we have attempted to reproduce as closely as possible the randomized, double-blind, placebo (vehicle)controlled design that is recommended for clinical trials of antidyskinetic drugs in PD [92]. In each animal, motor performance was compared after treatment with the compound of interest plus l-DOPA, versus l-DOPA plus vehicle, through sequential testing (“cross-over design”). Most of the compounds

tested in this study have already been approved for clinical use, and have been previously shown to alleviate dyskinesia in nonhuman primate models of LID and/or PD patients (Table 1). Although the compounds under investigation may potentially interact with several receptor systems (Table 1), they can be grouped in three main categories based on the putative target of their antidyskinetic action. Thus, two of the tested compounds can interfere with glutamate neurotransmission by either exerting noncompetitive antagonism at NMDA receptors (amantadine) or by inhibiting glutamate release (riluzole). Three compounds affect serotonin neurotransmission by either interacting with specific 5-HT receptors (buspirone, 5-HT1A; clozapine, 5-HT2A/C, 5-HT6 and 5-HT7) or by blocking serotonin reuptake (fluoxetine). Three of the tested compounds target noradrenaline neurotransmission by interacting with either alpha 2 (clonidine, yohimbine) or beta (propranolol) adrenoceptors. We have also tested one compound that specifically blocks nicotinic receptors (mecamylamine) in order to rule out that the antidyskinetic effect of amantadine is due to its antagonist action at nicotinic receptors (see Table 1). The study was performed in rats that had been rendered dyskinetic by a prior course of lDOPA treatment. This approach addresses the acute palliative effects of the tested compounds on the expression of LID, and not their potential prophylactic efficacy in preventing the dyskinesia priming process (the latter is defined in refs. [18] and [25]). 2. Methods 2.1. Subjects The study was performed on male Sprague–Dawley rats (220–225 g body weight at the beginning of the experiment; N = 78; Elevage–Janvier, Strasbourg, France). The animals were housed under 12-h light:12-h dark cycle, with free access to standard laboratory food (chow pellets) and tap water. The animals were housed at least 1 week before surgery. The treatment of the animals and the experimental procedures had been approved by local ethical

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Committees (Regierungspr¨asidium Darmstadt, Germany; and Malm¨o/Lunds Djurf¨ors¨oksetiska n¨amnden; Sweden).

2.2. 6-OHDA lesions DA-denervating lesions were performed by unilateral injection of 6-OHDA in the ascending nigrostriatal pathway as described previously [23]. Rats were anaesthetised with 100 mg/ml ketamine and 20 mg/ml xylazine (5:1, 1 ml/kg bodyweight). 6-OHDA was injected into the right ascending DA bundle at the following coordinates (in mm) relative to bregma and dural surface: (1) toothbar position −2.3, A = −4.4, L = 1.2, V = 7.8, (7.5 ␮g 6-OHDA), and (2) toothbar position +3.4, A = −4.0, L = 0.8 and V = 8.0 mm (6 ␮g 6-OHDA). The neurotoxin injections were performed at a rate of 1 ␮l/min, and the injection cannula was left in place for an additional 2–3 min thereafter. Two weeks after surgery rats with nearly complete (>90%) lesions were selected by means of an amphetamine-induced rotation test The animals were placed in plastic Perspex bowls (30 cm in diameter) and the rotational behaviour (360◦ turns) was recorded by an automated rotometer for 90 min after the i.p. injection of 2.5 mg/kg d-amphetamine sulphate. Fifty-one rats showing an average of ≥5 full turns/min in the direction ipsilateral to the lesion were selected for further experiments. We have previously shown that this rotational rate corresponds to levels of striatal DA depletion > 90%, as determined by DA transporter autoradiography, counts of DA neurons in the substantia nigra, or tissue levels of DA and its metabolites [3,22,57,103].

2.3. Drugs and treatment regimens The doses of compounds to be tested were chosen based on the results of our previous studies [63,64] or literature from experimental studies in rodents [4,48,94]. The choice was guided by considerations on the drugs’ pharmacokinetic–pharmacodynamic profiles, and lack of reported motor alterations and/or toxic effects of the given doses. All drugs tested were dissolved in a volume of 1 ml vehicle per kg. Unless otherwise stated, the vehicle was physiological saline solution. l-DOPA methyl ester (Sigma–Aldrich, Germany) was given at the dose of 6 mg/kg/day, combined with 12–15 mg/kg/day of benserazide HCl (Sigma–Aldrich, Germany). This dose of benserazide is required to effectively block activity of the peripheral aromatic l-amino acid decarboxylase in the rat, and previous experience from our group has shown that by lower doses of benserazide, l-DOPA dose–action curve is too short. Chronic treatment with this dose of l-DOPA and benserazide was given for 3 weeks to all the rats with good lesions in order to induce a gradual development of dyskinetic-like movements [3,63,103]. Thereafter, 3 rats that had not developed dyskinesia were excluded from the study, and 48 rats with a cumulative AIM score ≥ 28 points over five testing sessions (dyskinesia severity ≥ grade 2 on each axial, limb and orolingual scores) were kept on a drug treatment regimen of at least two injections of l-DOPA/benserazide per week in order to maintain stable AIM scores [59,63]. The selected rats were allocated to 5 groups of 9–10 animals each, which were balanced with the respect to AIM severity. Up to seven substances were tested in each of the experimental groups over a period of 7 months. A time lag of 1–3 weeks was allowed between the testing of different drugs. In the drug screening experiments, the compounds were evaluated using a blind, randomized, cross-over design, whereby half of the rats were tested with l-DOPA (6 mg/kg combined with 15 mg/kg benserazide) plus a drug of interest, or l-DOPA plus vehicle on 1 day, and then the treatment allocation was switched on the second day of testing. The drugs were administered prior to l-DOPA, at time intervals matching their pharmacodynamic and pharmacokinetic profiles. Amantadine (1-aminoadamantane hydrochloride, Aldrich, Germany) was administered s.c. at the doses of 20 and 40 mg/kg, 100 min before l-DOPA. Riluzole (2-amino-6-(trifluoromethoxy)benzothiazole, Sigma–Aldrich Sweden AB, Sweden) was mixed with 45 ␮l of 20% acetic acid, dissolved in 38 ◦ C-warm, bidistilled water and given i.p. at 2 and 4 mg/kg. Fluoxetine (±)N-methyl-[4-(trifluoromethyl)phenoxy] benzenepropanamine hydrochloride, Sigma–Aldrich, Germany) was dissolved in 5% DMSO-saline and administered i.p. at the doses of 2.5 and 5 mg/kg, 30 min before l-DOPA. Buspirone (N[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione hydrochloride, Sigma–Aldrich, Germany) was administered i.p., at the doses of

1, 2 and 4 mg/kg, 30 min before l-DOPA. Clozapine (8-chloro-11-(4-methyl1-piperazinyl)-5H-dibenzo[b,e][1,4]-diazepine, Sigma–Aldrich, Germany) was dissolved in a drop of 1N HCl and subsequently diluted with saline and administered i.p. at the doses of 4 and 8 mg/kg, 30 min before l-DOPA. Propranolol (±)-1-isopropylamino-3-(1-naphthyloxy)-2-propanol hydrochloride, Sigma–Aldrich, Germany) was administered i.p. at 5, 10 and 20 mg/kg, 20 min before l-DOPA. Clonidine (2-(2,6-dichloroanilino)-2-imidazoline hydrochloride, Tocris-Cookson, United Kingdom) was administered i.p. at the doses of 0.01, 0.1 and 1 mg/kg, 30 min before l-DOPA. Yohimbine (methyl 17alpha-hydroxy-yohimban-16alpha-carboxylate hydrochloride, Sigma–Aldrich Sweden AB, Sweden) was mixed with 45 ␮l of 20% acetic acid and dissolved in 38 ◦ C-warm bidistilled water and given i.p. at 2 and 10 mg/kg. Mecamylamine (N,2,3,3-tetramethylbicyclo[2.2.1]heptan-2-amine hydrochloride, Sigma–Aldrich, Germany) was administered i.p. at the doses of 2 and 4 mg/kg, 30 min before l-DOPA.

2.4. AIMs ratings AIMs ratings were performed by an investigator who was kept unaware of the pharmacological treatment administered to each rat (experimentally blinded). In order to quantify the severity of the AIMs, rats were observed individually in their standard cages every 20th minute at 20–180 min after an injection of lDOPA. As described previously [3,23,59,63,103], rats’ AIMs were classified into four subtypes: (1) axial AIMs, i.e., dystonic or choreiform torsion of the trunk and neck towards the side contralateral to the lesion; (2) limb AIMs, i.e., jerky and/or dystonic movements of the forelimb contralateral to the lesion; (3) orolingual AIMs, i.e., twitching of orofacial muscles, and bursts of empty masticatory movements with protrusion of the tongue towards the side contralateral to the lesion; (4) locomotive AIMs, i.e., increased locomotion with contralateral side bias. The latter AIM subtype was recorded in conformity with the original description of the rat AIM scale [23], although it was later established that locomotive AIMs do not provide a specific measure of dyskinesia [63,64], but rather provide a correlate of contralateral turning behaviour in rodents with unilateral 6-OHDA lesions [63]. Each of the four subtypes was scored on a severity scale from 0 to 4, where 0 = absent, 1 = present during less than half of the observation time, 2 = present for more than half of the observation time, 3 = present all the time but suppressible by external stimuli, and 4 = present all the time and not suppressible by external stimuli. Axial, limb and orolingual AIMs were found to be modulated in a similar way by all the tested substances. Therefore, scores from these three AIM subtypes were summed. The sum of either locomotive or axial, limb and orolingual AIM scores per testing session were used for statistical analyses (see below).

2.5. Rotarod test The rotarod test was performed after the administration of l-DOPA plus one of the drugs under investigation, or l-DOPA plus vehicle, using the same crossover design that was applied in the AIMs ratings sessions. The rotarod test served the purpose of detecting potential deleterious effects of the compounds studied on the rats’ motor performance and coordination. The rotarod test was performed using a previously described protocol [65]. Shortly, the animals were placed on the accelerating rod apparatus (Ugo Basile, Italy) at an initial speed of 4 rotations per minute (rpm), with the speed increasing gradually and automatically to 40 rpm over 300 s. The animals were pre-trained to reach a stable performance in this test before initiating the drug screening studies. The training consisted of three sessions on 3 consecutive days, and each session included two separate testing trials. Between the testing sessions, the animals were given a shorter “motivational session” where the rod speed was increased from 4 to 14 rpm of 25 s only. Animals could stay on the rod for the entire 25 s in these low-speed sessions, which has been shown to have a positive effect on the animals’ willingness to perform in this test [24]. To maintain the alertness of the animals during all the testing sessions, the animals were tapped on their tails several times by the experimenter. In the drug-screening experiments, the animals were placed on the rod at 45–60 min interval after l-DOPA administration (i.e., at the time when central levels of l-DOPA reach their peak [22,105]. We have previously shown that the performance of 6-OHDA lesioned rats on the rotarod is significantly improved at this interval post l-DOPA injection [65]. The rotarod performance was expressed as total number of seconds spent on the accelerating rod.

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2.6. Statistics Within-animal comparisons of AIM scores were made using Wilcoxon signed rank test, whereby the results recorded after treatment with l-DOPA plus a given drug were compared to the values recorded in the same animals after the administration of l-DOPA and vehicle on the preceding or consecutive testing day. Within-subject comparisons of rotarod performance were carried out using paired t-test, using an identical 2-consecutive-day testing paradigm as used to compare the AIM scores. The null hypothesis was rejected when p < 0.05. In the following text, the effects produced by the drugs under investigation (co-administered with l-DOPA) are expressed as a percentage of the performance recorded after l-DOPA plus vehicle treatment. Axial, limb and orolingual AIM scores are analysed and presented separately from locomotive AIM scores.

3. Results 3.1. Compounds targeting glutamatergic transmission Both doses of amantadine tested in this study (20 and 40 mg/kg) significantly reduced the rats’ axial, limb and orolingual AIMs scores, the maximal reduction (49%) being produced by the higher dose (Fig. 1A). In contrast, locomotive AIMs scores were not significantly reduced by either dose (Fig. 1B). At the doses tested, amantadine did not have any adverse motor effect, and did not worsen the rotarod performance in l-DOPAtreated rats (Fig. 1C). Riluzole was tested at 2 and 4 mg/kg, which significantly reduced the axial, limb and orolingual AIM scores by 18 and 33%, respectively (Fig. 2A), without worsening rotarod performance (Fig. 2C). Neither of the two doses produced any significant change in locomotive AIMs (Fig 2B). 3.2. Compounds targeting serotonergic transmission Fluoxetine (2.5 and 5 mg/kg) did not produce a significant decrease in axial, limb and orolingual AIMs scores. However, at the higher dose tested this drug displayed a strong trend to reduce AIMs (p = 0.058; 37% reduction; Fig. 3A). Locomotive AIMs were not significantly reduced by fluoxetine (Fig. 3B). Neither dose of fluoxetine had any significant negative impact on the rotarod performance of the l-DOPA-treated rats (Fig. 3C). Buspirone dose-dependently attenuated the rats’ axial, limb and orolingual AIMs, producing a decrease by 34, 58, and 83% at the doses of 1, 2, and 4 mg/kg, respectively (p < 0.05 for each dose; Fig. 4A). None of these doses caused a decrease in the l-DOPA-induced locomotive scores (Fig. 4B). The lowest dose of buspirone (1 mg/kg) did not impair the rats’ rotarod performance, but the doses of 2 and 4 mg/kg caused significant 25 and 20% reductions, respectively, in the time spent on the rod (Fig. 4C). Clozapine failed to produce a significant reduction in axial, limb and orolingual AIMs. Nevertheless, the effect of 8 mg/kg clozapine was close to reaching statistical significance (−41% versus control values, p = 0.059; Fig. 5A). This dose of clozapine markedly reduced also the locomotive AIMs (−80%, p < 0.05; Fig. 5B) and tended to negatively affect performance on the

Fig. 1. Amantadine (AMA; 20 and 40 mg/kg s.c.) effectively reduced (A) axial, limb and orolingual abnormal involuntary movements (AIMs) but not (B) locomotive AIMs in l-DOPA-treated dyskinetic rats (N = 9–10). AMA did not impair (C) accelerating rotarod performance. Data are expressed as percent of a respective score in the same animals treated with l-DOPA + vehicle on the preceding or consecutive day ± S.E.M. Control bar represents a group treated with l-DOPA and vehicle for 2 consecutive days. * p < 0.05 (Wilcoxon signed rank test).

rotarod (−50% versus control values in time spent on the rod; p = 0.092; Fig. 5C). 3.3. Compounds targeting noradrenergic transmission All the doses of propranolol tested (5, 10 and 20 mg/kg) caused a significant reduction in the rats’ axial, limb and orolingual AIM scores, the two higher doses (10 and 20 mg/kg)

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Fig. 2. Riluzole (RILU; 2 and 4 mg/kg i.p.) decreased (A) axial, limb and orolingual abnormal involuntary movements (AIMs) in l-DOPA-treated dyskinetic rats (N = 9–10). In contrast, neither (B) locomotive AIMs nor (C) accelerating rotarod performance were reduced by any dose RILU. Data are expressed as % of a respective score in the same animals treated with l-DOPA + vehicle on the preceding or consecutive day ± S.E.M. Control bar represents a group treated with l-DOPA and vehicle for 2 consecutive days. * p < 0.05 (Wilcoxon signed rank test).

Fig. 3. Fluoxetine (FLUOX; 2.5 and 5 mg/kg i.p.) did not significantly reduce (A) axial, limb and orolingual abnormal involuntary movements (AIMs) in lDOPA-treated dyskinetic rats (N = 9–10). Neither dose of FLUOX significantly affected (B) locomotive AIMs and (C) accelerating rotarod performance. Data are expressed as % of a respective score in the same animals treated with l-DOPA + vehicle on the preceding or consecutive day ± S.E.M. Control bar represents a group treated with l-DOPA and vehicle for 2 consecutive days.

producing about the same effect (−62 and −64%; Fig. 6A). At the dose of 20 mg/kg, propranolol significantly reduced also the rats’ locomotive AIMs scores (−57%; Fig. 6B), and showed a slight trend to reduce the time spent on the rod (p = 0.097, −40%; Fig. 6C). Clonidine dose-dependently attenuated the rats’ axial, limb and orolingual AIM scores, producing a reduction by 25, 47 and

82% at the doses of 0.01, 0.1 and 1 mg/kg, respectively (p < 0.05 for each dose Fig. 7A). However, only the lowest dose had a specific effect on dyskinesia as opposed to general mobility. Indeed, the two higher doses of clonidine suppressed also the rats’ rotational behaviour (locomotive AIMs; Fig. 7B) and the time spent on the rod (reduction by 64% by 0.01 mg/kg clonidine; the highest dose was not evaluated in this test; Fig. 7C).

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Fig. 4. Buspirone (BUSP; 1, 2, and 4 mg/kg i.p.) alleviated (A) axial, limb and orolingual abnormal involuntary movements (AIMs) but not (B) locomotive AIMs in l-DOPA-treated dyskinetic rats (N = 9–10). However, the intermediate and the highest dose of BUSP impaired (C) accelerating rotarod performance. Data are expressed as % of a respective score in the same animals treated with l-DOPA + vehicle on the preceding or consecutive day ± S.E.M. Control bar represents a group treated with l-DOPA and vehicle for 2 consecutive days. * p < 0.05 (A, Wilcoxon signed rank test; C, paired Student’s t-test).

Fig. 5. Clozapine (CLOZ; 4 and 8 mg/kg i.p.) failed to significantly decrease (A) axial, limb and orolingual abnormal involuntary movements (AIMs) in lDOPA-treated dyskinetic rats (N = 9–10). However, the higher dose of clozapine produced a marked decrease in (B) locomotive AIMs. CLOZ did not significantly impair (C) accelerating rotarod performance. Data are expressed as % of a respective score in the same animals treated with l-DOPA + vehicle on the preceding or consecutive day ± S.E.M. Control bar represents a group treated with l-DOPA and vehicle for 2 consecutive days. * p < 0.05 (Wilcoxon signed rank test).

Yohimbine was tested at the doses of 2 and 10 mg/kg. Both doses produced a large and significant reduction in the axial, limb and orolingual AIM scores (by 44 and 91%, Fig. 8A), but also worsened the rats’ rotarod performance (approximately 40% reduction by 2 mg/kg yohimbine, p < 0.05 versus vehicle; Fig. 8C). The higher dose of yohimbine (10 mg/kg) was not tested on the rotarod because it had a motor depressant effect that was evident already upon visual inspection of the rats. This

dose produced a large reduction in locomotive AIMs scores (approximately 60%; p < 0.05, Fig. 8B). 3.4. Compound targeting cholinergic transmission Mecamylamine, a non-selective nicotinic antagonist, had no effect on either dyskinesia or rotarod performance in the lDOPA-treated dyskinetic rats (Fig. 9A–C).

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Fig. 6. Propranolol (PROP; 5, 10 and 20 mg/kg i.p.) reduced (A) axial, limb and orolingual abnormal involuntary movements (AIMs) in l-DOPA-treated dyskinetic rats (N = 9–10). Only the highest dose of PROP (20 mg/kg) impaired (B) locomotive AIMs. None of the studied doses of PROP was found to significantly impair (C) accelerating rotarod performance. Data are expressed as % of a respective score in the same animals treated with l-DOPA + vehicle on the preceding or consecutive day ± S.E.M. Control bar represents a group treated with l-DOPA and vehicle for 2 consecutive days. * p < 0.05 (Wilcoxon signed rank test).

Fig. 7. Clonidine (CLON; 0.01, 0.1 and 1 mg/kg i.p.) dramatically reduced (A) axial, limb and orolingual abnormal involuntary movements (AIMs) in l-DOPAtreated dyskinetic rats (N = 9–10). The two higher doses of CLON (0.1 and 1 mg/kg) caused significant reductions in (B) locomotive AIMs. Already the intermediate dose of CLON (0.1 mg/kg) disrupted (C) accelerating rotarod performance. Data are expressed as % of a respective score in the same animals treated with l-DOPA + vehicle on the preceding or consecutive day ± S.E.M. Control bar represents a group treated with l-DOPA and vehicle for 2 consecutive days. * p < 0.05 (A and B, Wilcoxon signed rank test; C, paired Student’s t-test).

4. Discussion Most pharmacological studies of LID have been performed in nonhuman primates intoxicated with MPTP (for review, see refs. [51,58]). When treated with l-DOPA, MPTP-lesioned monkeys can develop choreiform dyskinesias that are remarkably similar to those seen in PD patients. However, there are significant

differences in the phenomenological and pharmacological characteristics of LID between nonhuman primate species [39,68]. Moreover, the economical and logistical constraints associated with the use of nonhuman primate models preclude their widespread application in high-throughput drug screening programs. More cost-effective models of LID can be obtained in

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Fig. 8. Yohimbine (YOH; 2 and 10 mg/kg i.p.) markedly reduced (A) axial, limb and orolingual abnormal involuntary movements (AIMs) in l-DOPA-treated dyskinetic rats (N = 9–10). The higher dose of YOH (10 mg/kg) decreased (B) locomotive AIMs. Already the low dose of 2 mg/kg, YOH disrupted (C) accelerating rotarod performance. Data are expressed as % of a respective score in the same animals treated with l-DOPA + vehicle on the preceding or consecutive day ± S.E.M. Control bar represents a group treated with l-DOPA and vehicle for 2 consecutive days. * p < 0.05 (A and B, Wilcoxon signed rank test; C, paired Student’s t-test).

rodents. We have reported that 6-OHDA-lesioned rats treated with l-DOPA exhibit abnormal movements and postures reminiscent of LID. The rat AIMs mainly affect the side of the body contralateral to the lesion (i.e., the “parkinsonian” side of the body), and show many functional and phenomenological analogies to LID in patients. On a phenomenological level, rat AIMs are complex and involve many different muscle groups

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Fig. 9. Mecamylamine (MEC; 2, 4 mg/kg s.c.) exerted no effects on either (A) axial, limb and orolingual abnormal involuntary movements (AIMs); (B) locomotive AIMs; or (C) accelerating rotarod performance in l-DOPA-treated dyskinetic rats (N = 9–10). Data are expressed as % of a respective score in the same animals treated with l-DOPA + vehicle on the preceding or consecutive day ± S.E.M. Control bar represents a group treated with l-DOPA.

[26] (videoclips can be found at [24]). On a functional level, these movements are involuntary and disabling [59,63,65,103], as is LID in PD patients [45]. The severity of rat AIMs can be quantified using a rating scale well aligned with that used in many clinical studies [102]. A first pharmacological validation of the rat LID model was carried out by Lundblad et al. [63], who showed that AIMs are specifically induced by l-DOPA, as opposed to antiparkinsonian treatments that do not produce dyskinesia in primates. The same study [63] reported reductions

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in l-DOPA-induced AIMs by some compounds with demonstrated antidyskinetic efficacy in nonhuman primate models of LID. In the present work, we have carried out a more extensive and detailed pharmacological characterization of the rat LID model applying a behavioural testing routine whereby the AIM rating scale was used to assess antidyskinetic drug effects, while the rotarod test (measuring general motor dexterity and locomotor coordination in rodents [24]) was used to detect deleterious effects of the same treatments on the animals’ motor performance. Using this approach, we have evaluated the potential antidyskinetic efficacy of eight clinically available drugs, which have been shown to reduce the severity of LID in MPTPintoxicated monkeys and/or in PD patients (Table 1). Different doses of each compound were thus evaluated sequentially on the l-DOPA-induced AIMs scale and on the rotarod test. Performance on each of these tests was assessed using a randomized cross-over design after combined administration of a given compound plus l-DOPA versus l-DOPA and the compound’s vehicle. Although we have previously reported antidyskinetic effects of amantadine, clozapine and yohimbine in the rat AIMs model [63], this is the first report showing dose-responses for these compounds. Furthermore, we report about the effects of an additional 5 compounds (buspirone, clonidine, fluoxetine, propranolol, and riluzole), which had never been previously tested in the rat AIMs model. The non-selective nicotinic antagonist, mecamylamine [2,28], also evaluated in this study, had not previously been tested in any animal model of LID (see Tables 1 and 2). The antidyskinetic action of the above compounds is mainly targeted on three neurotransmitter systems, i.e., glutamate, serotonin and noradrenaline. The nicotinic receptor antagonist (mecamylamine) was tested in order to rule out a contribution of this class of receptors to the antidyskinetic effect of amantadine (cf. Table 1). 4.1. Compounds targeting glutamatergic transmission An overactive glutamate transmission has been strongly implicated in the pathophysiology of both PD motor symptoms and LID. Experimental studies have revealed an association of LID with an enhanced glutamate release in the striatum [95] and with an increased expression [20,46] and phosphorylation [27] of ionotropic glutamate receptors. Accordingly, ionotropic glutamate receptor antagonists have been shown to alleviate LID both in MPTP-treated monkeys and in PD patients [13,44,54,85,101]. Amantadine, a compound with multiple mechanisms of action including NMDA receptor blockade [56], is the only antiparkinsonian drug that is broadly used for the palliative treatment of LID [14,30,62,75,84,91,99], although not all PD patients benefit from this treatment [29]. Interestingly, efficacy of memantine, a NMDA antagonist structurally similar to amantadine [86] has been assessed in small clinical trials in PD patients, but with conflicting results [47,60,73]. Riluzole is another anti-glutamate compound that has been approved for clinical use [104]. The main action of riluzole consists in blocking voltage-dependent sodium channels with preferential

Table 2 Summary of the effects of the drugs investigated in the 6-OHDA-lesioned rat model of l-DOPA-induced dyskinesia Drug Amantadine

Dose [mg/kg] 20 40

ALO AIMs

Lo AIMs

Time on the rod

↓ ↓↓

↔ ↔

N/A ↔

Buspirone

1 2 4

↓ ↓↓ ↓↓↓

↔ ↔ ↔

↔ ↓ ↓

Clonidine

0.01 0.1 1

↓ ↓↓ ↓↓↓

↔ ↓↓↓ ↓↓↓

↔ ↓↓↓ N/A

Clozapine

4 8

↔ ↔*

↔ ↓↓↓

↔ ↔

Fluoxetine

2.5 5

↔ ↔*

↔ ↔

↔ ↔

Mecamylamine

2 4

↔ ↔

↔ ↔

↔ ↔

↓ ↓↓↓ ↓↓↓

↔ ↔ ↓↓

↔ ↔ ↔

↓ ↓

↔ ↔

↔ ↔

↓↓ ↓↓↓

↔ ↓↓↓

↓↓ N/A

Propranolol

Riluzole Yohimbine

5 10 20 2 4 2 10

Effects of drugs on axial, limb and orolingual (ALO) abnormal involuntary movements (AIMs), locomotive (Lo) AIMs, and time spent on an accelerating rotarod were evaluated after administration of l-DOPA plus the drug of interest vs. l-DOPA and the drug’s vehicle. Symbol description: (↓), moderate reduction (≤40%) in the given motor parameter; (↓↓), strong reduction (41–60%) in the given motor parameter; (↓↓↓), very strong reduction (>60%) in the given motor parameter; (↔), no statistically significant change; N/A, data not available. * Strong trend towards reduction close to statistical significance (p ≤ 0.06).

influence on glutamatergic nervous terminals, although it can also exert non-competitive antagonism at NMDA receptors [31]. Riluzole has been shown to alleviate LID without worsening parkinsonism in an open-label study in advanced PD patients [74], although it has been found ineffective against apomorphine-induced dyskinesia in a pilot study [17]. In the present study, both amantadine and riluzole were able to dose-dependently reduce the axial, limb and orolingual AIM scores in the rat model of LID. Significant antidyskinetic effects were seen with doses of the drugs that did not worsen the animals’ overall motor performance on the rotarod. The largest reduction in AIM scores (approximately 50%) was produced by amantadine at the dose of 40 mg/kg. The extent of this reduction is quite in agreement with the best results reported in dyskinetic PD patients [62,75]. In patients, this extent of LID attenuation by amantadine is not associated with a worsening of the antiparkinsonian action of l-DOPA [62,75], which is also in agreement with our findings in the rat model. It should be mentioned that the unique antidyskinetic profile of amantadine may not be solely related to the NMDA receptor blockade. At the doses used in the present study, this compound may also target nicotinic [19,70] and sigma [55,87] receptors as well as the aromatic l-amino acid decarboxylase [38]. In this study,

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a nicotinic receptor antagonist (mecamylamine) did not have any effect on the rat AIM scores, which suggests lack of a contribution of nicotinic receptor blockade to the antidyskinetic effect of amantadine. To date, there is no evidence that sigma receptors are implicated in LID (although a positron emission tomography study in PD patients has shown an association between LID severity and increased radioligand binding to sigma-receptors in the cerebellum [77]). Thus, it seems most likely that the beneficial actions of amantadine may result from its effects on both NMDA receptors and aromatic l-amino acid decarboxylase. The relative efficacy of riluzole in reducing the rat AIM scores was somewhat inferior to that of amantadine. Further dose increments of riluzole did not achieve a better antidyskinetic effect and caused muscle relaxation (data not shown). 4.2. Compounds targeting serotonergic transmission Serotonin is an important modulator of synaptic transmission and neurotransmitter release, and has widespread effects on monoamine pathways and glutamate systems in the brain (for review, see refs. [6,15,72,98]). Accumulating evidence implicates serotonergic mechanisms in the pathophysiology of LID. Several compounds that produce an elevation in brain extracellular serotonin levels have shown antidyskinetic efficacy in nonhuman primate models of LID and PD patients [36,41,50]. The mechanisms by which these compounds reduce LID are not fully understood but are believed to rely, at least in part, on the stimulation of 5-HT1A receptors [50]. Accordingly, the 5-HT1A agonist sarizotan has been found to alleviate l-DOPAinduced motor complications both in animal models of PD and in patients [5,11,83], and buspirone, an anxiolytic compound with prominent 5-HT1A-agonist properties, has been found to significantly lessen the severity of LID in small clinical trials in PD patients [16,53]. By activating presynaptic 5-HT1A receptors on serotonergic and glutamatergic terminals, an elevation of extracellular serotonin levels can actually reduce synaptic release of both serotonin and glutamate, respectively (for review, see refs. [6,72]). Moreover, serotonin axon terminals are likely to provide the main source of striatal l-DOPA conversion in severely DAdenervated rodents (reviewed in [25]), and selective 5-HT1A agonists have been shown to attenuate increases in striatal levels of extracellular DA after peripheral l-DOPA administration [52]. Another serotonin-dependent approach to the treatment of LID consists in blocking postsynaptic 5-HT2-type receptors [7,81], which have a wide distribution in the cortico-basal ganglia circuits, and can boost DA-dependent cellular responses in striatal neurons [100]. The atypical antipsychotic and multireceptor antagonist, clozapine, has low nanomolar affinity for serotonin 5-HT2 and 5-HT6 receptors [71]. This drug can significantly alleviate the severity of LID both in PD patients and in nonhuman primate models of PD [9,34,42]. Although clozapine can potentially worsen parkinsonism, low doses of this compound have been shown to reduce dyskinesia without compromising the anti-akinetic action of l-DOPA [42]. This effect has been attributed to the antagonistic properties of clozapine at 5-HT rather than DA receptors [34,42].

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Our results to some extent support the notion that pharmacological manipulation of serotonin transmission provides a valid approach to the treatment of LID. The rat axial, limb and orolingual AIMs were significantly reduced by the 5-HT1A agonist, buspirone, even at a dose that did not affect either locomotive AIMs or rotarod performance (i.e., 1 mg/kg). Fluoxetine (which blocks serotonin reuptake) and clozapine (which antagonizes 5-HT2A/C and 5-HT6 receptors) tended to attenuate the l-DOPA-induced axial, limb, and orolingual AIMs, although the effects did not reach significance (but see clozapine effect in ref. [63]). The higher dose of clozapine tended to produce a general reduction in the animals’ motor output as documented by a significant reduction in locomotive scores and a trend towards impaired rotarod performance. Based on the results obtained in the rat, it would seem that stimulation of 5-HT1A receptors is the most promising “serotonin-based” approach to the treatment of LID among the three outlined above. This conclusion finds support in the body of pharmacological studies so far performed in PD patients and nonhuman primate models of LID, which are concordant in pointing to 5-HT1A agonists as a promising approach for LID treatment, but have provided divergent results regarding the efficacy of either selective serotonin reuptake inhibitors or 5-HT2-type receptor antagonists (for a recent review, see ref. [39]). 4.3. Compounds targeting noradrenergic transmission Like serotonin, noradrenaline has broad neuromodulatory actions in the central nervous system (partially reviewed in refs. [15,76]). Manipulations of central noradrenergic pathways alter electrophysiological, neurochemical and behavioural indices of neurotransmission in the nigrostriatal DA system [67]. Accumulating evidence from both monkey models of LID and PD patients indicates that alpha2-adrenoceptor antagonists can alleviate LID [43,49,93], but see ref. [66]. The alpha2 adrenoceptor agonist, clonidine, has also shown antidyskinetic efficacy in MPTP-intoxicated monkeys [41], but this effect is likely to be indirect, and to rely on an overall reduced adrenoceptor stimulation. Indeed, clonidine decreases noradrenergic cell firing [1] and noradrenaline release [97] through stimulation of presynaptic alpha2-adrenoceptors. In addition to alpha2-adrenoceptors, beta-adrenoceptors may provide a viable target for antidyskinetic drug therapy. The beta-adrenoceptor blocker, propranolol, is a broadly used antihypertensive that is also endowed with anxiolytic, anticonvulsant, and anti-tremor properties [61,69]. This compound has been found to improve LID in PD patients [21], and MPTP-intoxicated monkeys [41]. Of the adrenoceptor agonists and antagonists tested in this study, propranolol had the most favourable profile, producing a conspicuous (−59%) reduction in axial, limb and orolingual AIM scores at a dose that did not disrupt rotarod performance (i.e., 10 mg/kg). Higher dose (i.e., 20 mg/kg) failed to offer any additional benefit and tended to impair motor performance. Clonidine produced a dose-dependent alleviation of rat LID, but only the lowest dose tested had a specific effect on dyskinesia (−25%) as opposed to general mobility. Yohimbine was very potent in reducing the rat AIMs but the higher dose tested

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(10 mg/kg) had overt motor depressant effects and caused a decrease in all the parameters under investigation. The lower dose of yohimbine (2 mg/kg) significantly alleviated axial, limb and orolingual AIMs without reducing the rats’ locomotive scores, but caused a decrease in the “time spent on the rod”. This decrease indicates that the drug had a negative impact on the animals’ general motor dexterity. It is here relevant to mention that cardiovascular side effects and tolerability issues have been a matter of concern in the clinical translation of alpha2 adrenoceptor as an antidyskinetic treatment (reviewed in ref. [39]). 4.4. General considerations The purpose of this study was to further verify the predictive validity of the rat LID model by comparing the efficacy of registered drugs to the effects reported in PD patients and nonhuman primate models of PD. This is the first study where potentially antidyskinetic compounds are evaluated using both the AIM rating scale and a sensitive test of general motor performance (rotarod). Taken together, the tested drugs modulated the rodent axial, limb and orolingual AIM scores in a manner that conformed well to the results obtained in dyskinetic primates. The recording of locomotive AIM scores and rotarod performance proved instrumental to defining the dose range for a specific antidyskinetic effect dissociated from a general reduction in motor output. Contrary to axial, limb and orolingual AIMs, locomotive AIMs seemed to provide a general measure of locomotor activity. Indeed, treatments that specifically alleviated the severity of trunk, limb and orofacial dyskinesia did not necessarily reduce the rats’ locomotive AIM scores. On the other hand, significant reductions in locomotive AIM scores were seen after treatments that visibly diminished the animals’ spontaneous motor activity. Some of these treatments tended to negatively affect the time spent on the rod. We have previously shown that the pattern of changes in locomotive AIMs produced by antiparkinsonian drug treatment follows very closely treatmentinduced changes in contralateral turning behaviour as assessed using automated rotometer counts [63]. The significance of contralateral turning behaviour in unilaterally 6-OHDA-lesioned rats is a matter of continuous debate (for review, see refs. [24,26,68]). It has recently been argued that testing 6-OHDAlesioned rats in rotometer bowls as opposed to open cages would increase the sensitivity of drug-induced rotational behaviour as a model of LID [89]. However, in our previous pharmacological characterization [63], antidyskinetic drugs that acutely reduced the rats’ axial, limb and orolingual AIM scores did not produce any significant reduction in the number of l-DOPA-induced contralateral turns as assessed in automated rotometer bowls. Taken together, our results support the notion that locomotive AIMs and contralateral rotation do not provide a rat equivalent of LID that is aligned with the measures of dyskinesia used both in the clinic and in nonhuman primate models. Nevertheless, locomotive AIMs and/or rotational counts may help to better understand the general pattern of effects produced by potential antidyskinetic treatment. In the present study, locomotive AIMs scores provided information that was complementary (not overlapping)

to that obtained using measures of “body” AIMs and rotarod performance. In our experience, treatments that specifically reduced dystonic and dyskinetic motor features without interfering with the rats’ normal activities (e.g., locomotion, exploration, grooming) produced a decrease in the axial, limb and orolingual AIM scores and did not affect the locomotive AIM scores nor the time spent on the rod. Treatments producing a marked and readily visible motor depression caused a significant reduction in all three parameters under investigation. There were also treatments that decreased the locomotive AIMs without affecting rotarod performance, and vice versa. We interpret these data as indicating that reductions in spontaneous locomotor activity and impaired performance in a semi-automated, learned task of general motor dexterity are not necessarily associated. In conclusion, the pharmacological data in this study support the validity of rat axial, limb and orolingual AIM scores as a model to evaluate potential antidyskinetic drugs for PD, and demonstrate the importance of including tests of general motor output in the drug screening process. The overall profile of effects produced by the tested compounds is quite in agreement with the results reported in nonhuman primate models of LID and/or in PD patients. Acknowledgements The part of this work performed in Sweden was supported by grants from the Swedish National Research Council, Thorsten and Elsa Segerfalks and Greta and Johan Kocks Foundations (to M.A.C.). References [1] Aghajanian GK, VanderMaelen CP. Alpha 2-adrenoceptor-mediated hyperpolarization of locus coeruleus neurons: intracellular studies in vivo. Science 1982;215:1394–6. [2] Anderson K, Fuxe K, Eneroth P, Agnati LF. Differential effects of mecamylamine on the nicotine induced changes in amine levels and turnover in hypothalamic dopamine and noradrenaline nerve terminal systems and in the secretion of adenohypophyseal hormones in the castrated female rat. Evidence for involvement of cholinergic nicotine-like receptors. Acta Physiol Scand 1984;120:489–98. [3] Andersson M, Hilbertson A, Cenci MA. Striatal fosB expression is causally linked with l-DOPA-induced abnormal involuntary movements and the associated upregulation of striatal prodynorphin mRNA in a rat model of Parkinson’s disease. Neurobiol Dis 1999;6:461–74. [4] Angrini M, Leslie JC, Shephard RA. Effects of propranolol, buspirone, pCPA, reserpine, and chlordiazepoxide on open-field behavior. Pharmacol Biochem Behav 1998;59:387–97. [5] Bara-Jimenez W, Bibbiani F, Morris MJ, Dimitrova T, Sherzai A, Mouradian MM, et al. Effects of serotonin 5-HT1A agonist in advanced Parkinson’s disease. Mov Disord 2005;20:932–6. [6] Barnes NM, Sharp T. A review of central 5-HT receptors and their function. Neuropharmacology 1999;38:1083–152. [7] Baron MS, Dalton WB. Quetiapine as treatment for dopaminergicinduced dyskinesias in Parkinson’s disease. Mov Disord 2003;18:1208–9. [8] Bennett Jr JP, Landow ER, Dietrich S, Schuh LA. Suppression of dyskinesias in advanced Parkinson’s disease: moderate daily clozapine doses provide long-term dyskinesia reduction. Mov Disord 1994;9:409–14. [9] Bennett Jr JP, Landow ER, Schuh LA. Suppression of dyskinesias in advanced Parkinson’s disease. II. Increasing daily clozapine doses suppress dyskinesias and improve parkinsonism symptoms. Neurology 1993;43:1551–5.

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