Calciumpermeable AMPA receptors are ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2010 | 114 | 499–511

doi: 10.1111/j.1471-4159.2010.06776.x

,

*Faculty of Life Sciences, University of Manchester, Manchester, UK Greater Manchester Neurosciences Centre, Salford, UK àBasal Ganglia Pathophysiology Laboratory, Lund University, Lund, Sweden

Abstract Overactivity of striatal a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors is implicated in the pathophysiology of L-DOPA-induced dyskinesia (LID) in Parkinson’s disease (PD). In this study, we evaluated the behavioural and molecular effects of acute and chronic blockade of Ca2+-permeable AMPA receptors in animal models of PD and LID. The acute effects of the Ca2+-permeable AMPA receptor antagonist 1-trimethylammonio-5-(1adamantane-methylammoniopentane) dibromide hydrobromide (IEM 1460) on abnormal involuntary movements (AIMs) in the 6-hydroxydopamine (6-OHDA)-lesioned rat and LID in the MPTP-lesioned non-human primate were assessed. Subsequently, the effects of chronic treatment of 6-OHDAlesioned rats with vehicle, L-DOPA/benserazide (6/15 mg/kg, i.p.) + vehicle or L-DOPA + IEM 1460 (3 mg/kg, i.p.) on behavioural and molecular correlates of priming for LID were evaluated. In the 6-OHDA-lesioned rat and MPTP-lesioned

non-human primate, acute treatment with IEM 1460 (1–3 mg/ kg) dose-dependently reduced LID without adverse effects on motor performance. Chronic co-treatment for 21 days with IEM 1460 reduced the induction of AIMs by L-DOPA in the 6-OHDA-lesioned rat without affecting peak rotarod performance, and attenuated AIMs score by 75% following L-DOPA challenge (p < 0.05). Chronic IEM 1460 treatment reversed L-DOPA-induced up-regulation of pre-proenkephalin-A, and normalised pre-proenkephalin-B mRNA expression in the lateral striatum, indicating an inhibition of both behavioural and molecular correlates of priming. These data suggest that Ca2+-permeable AMPA receptors are critically involved in both the induction and subsequent expression of LID, and represent a potential target for anti-dyskinetic therapies. Keywords: animal models, calcium, glutamate, L-DOPAinduced dyskinesia, Parkinson’s disease, a-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid. J. Neurochem. (2010) 114, 499–511.

Treatment with the dopamine precursor L-DOPA improves motor symptoms of Parkinson’s disease (PD) (Cotzias et al. 1967; Fahn et al. 2004), and is the mainstay of antiparkinsonian therapy. However, motor complications of LDOPA treatment, including L-DOPA-induced dyskinesia (LID), occur in around 40% of patients after 5 years of treatment (Fahn 2000; Ahlskog and Muenter 2001). LID is linked to pathological corticostriatal synaptic plasticity (Picconi et al. 2003), which is mediated by molecular changes, such as altered expression of striatal opioid peptides (Andersson et al. 1999; Cenci and Lundblad 2006). Such changes are long-lasting and implicated in the process of priming, whereby dyskinesia may be elicited by subsequent doses of dopaminergic agents (Blanchet et al. 2004). The mechanisms involved in the induction and expression of LID are complex and involve the interaction of several neuro-

transmitter systems (Brotchie 2005), including a critical role for enhanced corticostriatal glutamatergic transmission (Chase and Oh 2000). Received March 16, 2010; revised manuscript received April 13, 2010; accepted April 20, 2010. Address correspondence and reprint requests to Dr Christopher Kobylecki, Department of Neurology, Greater Manchester Neurosciences Centre, Salford Royal Hospital, Stott Lane, Salford, M6 8HD, UK. E-mail: [email protected] Abbreviations used: 6-OHDA, 6-hydroxydopamine; AIM, abnormal involuntary movement; ALO, axial, limb and orolingual; AMPA, aamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; CaMKII, calcium–calmodulin-dependent protein kinase; ERK, extracellular signalrelated kinase; GluR, glutamate receptors; IEM 1460, 1-trimethylammonio-5-(1-adamantane-methylammoniopentane) dibromide hydrobromide; LID, L-DOPA-induced dyskinesia; NHP, non-human primate; PD, Parkinson’s disease; PPE, pre-proenkephalin; SSC, saline-sodium citrate.

2010 The Authors Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 499–511

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a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluR) are tetrameric structures composed of combinations of subunits named GluR1–4 (Keinanen et al. 1990). Expression of mRNA and protein for all subunits has been identified in striatal neurones, indicating the contribution of AMPA receptors to corticostriatal transmission (Stefani et al. 1998). RNA editing of glutamine to arginine at codon 607 of the GluR2 subunit, which is essentially complete within the CNS, renders GluR2containing AMPA receptors impermeable to Ca2+ ions (Burnashev et al. 1992; Seeburg et al. 1998). Other posttranscriptional modifications, such as alternative splicing, alter AMPA receptor function (Sommer et al. 1990), while serine phosphorylation of GluR1 subunits enhances channel opening and conductance (Banke et al. 2000). While the role of NMDA glutamate receptors in striatal synaptic plasticity has been well characterised (Calabresi et al. 1992), the contribution of AMPA receptors is less clear. However, studies in the hippocampus have demonstrated early recruitment of GluR2-lacking, Ca2+-permeable AMPA receptors to the synaptic membrane in the initiation phase of long-term potentiation (Plant et al. 2006). The non-competitive AMPA receptor antagonist talampanel reduces LID in the MPTP-lesioned non-human primate (NHP) model of PD (Konitsiotis et al. 2000), suggesting a role for overactive AMPA receptor transmission in LID. This hypothesis is supported by findings of increased AMPA receptor binding in the lateral striatum of dyskinetic MPTPlesioned NHPs (Calon et al. 2002b) and parkinsonian patients (Calon et al. 2003). Enhanced phosphorylation and trafficking of AMPA receptor subunits to striatal synapses is also described in animal models of LID (Ba et al. 2006; Santini et al. 2007; Silverdale et al. 2010). Given the key role of Ca2+-dependent signalling pathways in the pathophysiology of LID (Konradi et al. 2004), specific blockade of Ca2+-permeable AMPA receptors represents a potential target both of anti-dyskinetic therapies and interventions directed at prevention of dyskinesia. However, the individual roles of Ca2+-permeable AMPA receptors in the induction and expression of dyskinesia are not clear; in particular, they have yet to be examined in the MPTPlesioned NHP model of LID. Here, we clarify the role of transmission by Ca2+permeable AMPA receptors in the induction and subsequent expression of LID. We first performed a dose-finding study of the selective Ca2+-permeable AMPA receptor antagonist, 1-trimethylammonio-5-(1-adamantane-methylammoniopentane) dibromide hydrobromide (IEM 1460), in the 6-hydroxydopamine (6-OHDA)-lesioned rat model of LID, and subsequently confirmed our findings in the dyskinetic MPTP-lesioned NHP. We then assessed behavioural and molecular correlates of dyskinesia induction and priming in 6-OHDA-lesioned rats receiving chronic treatment with L-DOPA and IEM 1460.

Methods All animal studies were carried out in accordance with the U.K. Animals (Scientific Procedures) Act 1986. Rodent studies Unilateral 6-OHDA lesion of the medial forebrain bundle Male Sprague-Dawley rats (280–310 g; Charles River, Margate, UK) were maintained at constant temperature and humidity on a 12 h light/dark cycle (light on from 8 AM to 8 PM), and were allowed free access to food and water. 6-OHDA lesions were performed as previously described (Henry et al. 1999a). Thirty minutes prior to surgery, rats were injected with pargyline (5 mg/ kg, i.p.; Sigma-Aldrich, Gillingham, UK) and desipramine (25 mg/ kg, i.p.; Sigma-Aldrich) in a volume of 1 mL/kg sterile saline. 6-OHDA hydrobromide (Sigma-Aldrich) was dissolved in ice-cold sterile water (5 mg/mL) and 2.5 lL was injected manually into the right medial forebrain bundle over 5 min under isoflurane anaesthesia. The needle was left in place for a further 5 min to avoid reflux along the track. Stereotaxic coordinates, in mm relative to bregma, were: A = )2.8, L = )2.0, V = )8.6 (Paxinos and Watson 1998). Drug treatments and behavioural analysis Cylinder test Three weeks post-lesion, animals were screened for inclusion in experimental groups using the cylinder test, as previously described (Kirik et al. 2000). Only animals with a cylinder test performance of < 30% were included in the study. It has previously been shown that this degree of impairment corresponds to > 90% depletion of striatal dopaminergic terminals (Lundblad et al. 2004; Mela et al. 2007). Abnormal involuntary movements (AIMs) assessment Abnormal involuntary movements were assessed as previously described (Lundblad et al. 2002; Winkler et al. 2002), with some modifications. Immediately after drug administration, rats were placed into transparent Perspex observation boxes and allowed to acclimatise. AIMs were scored for 1 min every 30 min after drug administration, for 3 h. Locomotor, axial, orolingual and limb AIMs, as defined previously were scored according to previously published protocols: 0 = absent; 1 = occasional, present < 50% of time; 2 = frequent, present > 50% of time; 3 = continuous, interrupted by sensory stimulus (e.g, hand movement); 4 = continuous, not interrupted by sensory stimulus (Lundblad et al. 2002; Winkler et al. 2002). The sum of axial, limb and orolingual (ALO) AIMs scores was calculated for each animal. Sum locomotor scores were calculated and analysed separately, since rotational behaviour is thought to derive from a different neurobiological substrate to ALO AIMs (Lundblad et al. 2002). Rotarod assessment Animals received training on the rotarod (Med Associates, Georgia, VT, USA) at 3 weeks post-6-OHDA lesion, using a similar protocol to that previously described (Rozas and Labandeira Garcia 1997; Rozas et al. 1997). Over four consecutive days, a series of fixed and accelerating speeds were used to accustom the animals to the


Calcium-permeable AMPA receptors and dyskinesia | 501

rotarod. During drug experiments, rotarod performance at 60 min post-drug administration was measured, as this has been shown to be the point of peak motor response to L-DOPA in 6-OHDAlesioned rats (Dekundy et al. 2007). Drug treatment L-DOPA methyl ester hydrochloride (Sigma-Aldrich) and benserazide hydrochloride (Sigma-Aldrich) were dissolved in sterile 0.9% saline and administered at a volume of 1 mL/kg. IEM 1460 (Tocris Bioscience, Bristol, UK) was dissolved in sterile water and administered at a volume of 1 mL/kg, 15 min before administration of L-DOPA/benserazide. Experimental design Experiment 1. Behavioural effects of acute blockade of Ca2+permeable AMPA receptors on established L-DOPA-induced AIMs Rats with 6-OHDA lesions received daily treatment for 21 days with L-DOPA/benserazide (6/15 mg/kg, i.p.), commencing 5 weeks postlesion. After this, rats received twice-weekly treatment with LDOPA/benserazide (6/15 mg/kg) to maintain stable levels of AIMs (Lee et al. 2000a). Animals showing ALO AIM scores of ‡ 10 per monitoring period were selected for behavioural study. Animals were treated with IEM 1460 (1 and 3 mg/kg, i.p.) or vehicle, and LDOPA/benserazide (6/15 mg/kg, i.p.). The acute effects of drug treatments on AIMs and rotarod performance were assessed during separate sessions, with a minimum of 3 days between each behavioural assessment. Experiment 2. Behavioural and molecular effects of chronic blockade of Ca2+-permeable AMPA receptors on dyskinesia priming Unilaterally 6-OHDA-lesioned rats were allocated to three treatment groups according to baseline cylinder test and rotarod performance, to ensure that these measures were matched between groups. The groups received the following treatment for 21 days: (i) vehicle (n = 7), (ii) L-DOPA/benserazide (6/15 mg/kg, i.p.) + vehicle (n = 6) and (iii) L-DOPA/benserazide (6/15 mg/kg, i.p.) + IEM 1460 (3 mg/kg, i.p.) (n = 7). The following behavioural assessments were performed: AIMs (on days 1, 7, 14, 21) and rotarod performance (on days 2, 8, 15, 20). On day 22, all animals received L-DOPA/benserazide (6/15 mg/kg, i.p.) and AIMs were scored to assess the effect of chronic treatments on priming for dyskinesia. Animals were killed on day 24 by exposure to rising concentrations of CO2 followed by cervical dislocation; the brains were rapidly removed, snap-frozen in isopentane at )35C and stored at )80C.

In situ hybridisation histochemistry Tissue preparation Coronal 20 lm sections of rostral striatum, 1.7 mm anterior to bregma (Paxinos and Watson 1998) were cut using a cryostat (Bright, Huntingdon, UK) and thaw-mounted onto gelatin-subbed slides. Cut sections were desiccated and stored at )80C. Sections were fixed in 4% paraformaldehyde (BDH, Lutterworth, UK) as previously described (Henry et al. 1999a). Labelling of oligonucleotide probes Synthetic oligonucleotide probes (35–40 mer) were synthesised (Invitrogen, Paisley, UK) according to previously published sequen-

ces (Henry et al. 1999a). The pre-proenkephalin A (PPE-A) probe was complementary to bases 343–384 of rat PPE-A gene (GenBank accession no. M28263), sequence 5¢-CTTCATGAAGCCTCCATA CCGTTTGATGAACCCCCTATACTT-3¢. The pre-proenkephalin B (PPE-B) probe was complementary to bases 754–798 of rat PPE-B gene (GenBank accession no. M10088), sequence 5¢-GCTCCTCTT GGGGTATTTGCGCAAAAAGCCGCCATAGCGTTTGGC-3¢. Oligonucleotide probes (4 pmol) were 3¢-end-labelled at an incubation temperature of 37C for 60 min with the following reactants: 16.8 lL diethylpyrocarbonate-treated H2O; 8.0 lL terminal transferase buffer (Promega, Southampton, UK); 3.2 lL [35S]dATP (40 lCi; Perkin-Elmer, Beaconsfield, UK); and 2.0 lL recombinant terminal transferase (60 u; Promega). The labelled probe was purified using illustra MicroSpin G-15 columns (GE Healthcare Life Sciences, Amersham, UK) and dissolved in 200 lL of 1 M dithiothreitol (Sigma-Aldrich). The efficiency of radioactive labelling was calculated by counting 1 lL of the eluted probe in a liquid scintillation counter (Tricarb 1500; Perkin-Elmer, UK). In situ hybridisation Labelled probe was added to hybridisation buffer to achieve activity counts of 3 · 106 per mL. Hybridisation buffer consisted of 50% v/v formamide; 20% 20· saline-sodium citrate buffer (SSC); 10% 50· Denhardt’s solution; 2% salmon testes DNA (10 mg/mL); 2% 1 M dithiothreitol; 1% polyadenylic acid and 5 g dextran sulphate (all Sigma-Aldrich). Slides were placed into air-tight hybridisation boxes containing paper towels soaked in 50% formamide. Fifty lL of labelled hybridisation solution was added to each section, and slides were covered with Parafilm coverslips (Pechiney, Chicago, IL, USA) and incubated in a hybridisation oven (Stuart Scientific, Stone, UK) at 42C for 18 h. Following hybridisation, parafilm was lifted off and slides were washed for 30 min in 1· SSC at 25C, 30 min in 1· SSC at 55C and 10 min in 0.1· SSC at 55C, before being dehydrated in 70% and 95% ethanol for 2 min each. Slides were allowed to dry before being placed in cassettes apposed to autoradiographic film (Kodak Biomax MR; Sigma-Aldrich) for 1–2 weeks at 4C. Autoradiographic [14C] microscale standards (31–833 nCi/g; GE Healthcare Life Sciences) were placed in each cassette. Films were developed using an automated developer. Image analysis Densitometric analysis of autoradiographic films was performed using Image ProPlus v5.1 (Media Cybernetics, Wokingham, UK). Images were grabbed using a digital camera and corrected for background signal. Optical density was measured in the dorsolateral and ventrolateral striatal quadrants, and mean values for each quadrant were calculated for each animal. Optical density values from microscale standards were used to plot a standard curve, and tissue equivalent values of radioactivity were derived using linear regression in Prism v5.0 (GraphPad Software, La Jolla, CA, USA). High-performance liquid chromatography To assess the extent of the 6-OHDA lesion, HPLC for dopamine and its metabolites was performed as previously described on tissue scraped from rostral striatal coronal sections (Nash and Brotchie 2000).



Non-human primate studies MPTP administration Parkinsonism was induced in common marmosets (Callithrix jacchus) obtained from a purpose-bred colony as previously described (Fox et al. 2001). MPTP hydrochloride (1–2 mg/kg; s.c.; Sigma-Aldrich) was administered once daily for 5 days. Parkinsonian signs were stable after 10–14 weeks; after this, LDOPA/benserazide was administered for 3 weeks until stable and reproducible dyskinesia was elicited. Behavioural assessment and drug treatments Behavioural analysis Behavioural analysis was performed as previously described (Henry et al. 1999b; Fox et al. 2001). Animals were placed in observation cages immediately after drug administration. Motor activity was measured using an infrared-based activity monitoring system (Excalibur, Manchester, UK), which provides a measure of total motor activity in 5-min bins. Behaviour was video-recorded for a total of 4 h following drug administration. Parkinsonism and dyskinesia were scored over a 10-min period at 30-min intervals, using a behavioural rating scale described previously (Henry et al. 1999b; Fox et al. 2001). Dyskinesia is rated on a scale from 0 to 4, where 1 = mild, present < 30% of time; 2 = moderate, present > 30% of time; 3 = marked, present < 70% of time; 4 = severe, present > 70% of time. The composite parkinsonian disability score was calculated using a formula previously described, where disability = [18 ) (Range of movement · 2) + (Bradykinesia · 3) + (Posture · 9)] (Fox et al. 2001). The parkinsonian disability and dyskinesia scores for 0–1, 1–2 and 2–3 h post-drug administration were obtained by calculating the sum of the scores in each of these time periods. In addition, total ‘on-time’ was calculated from locomotor activity counts as previously described (Hill et al. 2003). Drug treatments L-DOPA/benserazide (12.5/3.1 mg/kg; p.o.) was administered as Madopar (Roche, Indianapolis, IN, USA) 62.5 mg dissolved in apple juice, in a volume of 2.5 mL/kg body weight. IEM 1460 (Tocris Biosciences, Bristol, UK) was dissolved in sterile water and given s.c. at a volume of 1 mL/kg body weight, at the same time as L-DOPA administration. Experimental design Behavioural effects of acute blockade of Ca2+-permeable AMPA receptors on established LID in MPTP-lesioned NHPs MPTP-lesioned marmosets previously primed with L-DOPA received titrated doses of L-DOPA (12–20 mg/kg, p.o.) providing optimum relief of parkinsonian disability and inducing a similar level of dyskinesia. During the study, marmosets received twiceweekly L-DOPA (12.5 mg/kg, p.o.) to maintain a stable level of dyskinesia. Animals received acute doses of IEM 1460 (0.1, 0.3, 1.0 and 3.0 mg/kg) in combination with L-DOPA, and the effects on parkinsonian disability and dyskinesia were assessed. Statistical analysis Statistical analysis was performed using Prism v5.0 (GraphPad Software); a significance level of p < 0.05 was used for all analyses.

Friedman’s non-parametric ANOVA followed by Dunn’s test for multiple comparisons was used to analyse non-parametric behavioural data, such as rat AIMs scores and NHP parkinsonism and dyskinesia scores. Rotarod performance and motor activity counts were analysed using one-way repeated measures ANOVA followed by Dunnett’s test. Analysis of AIMs data from rat experimental group 2 was done using two-way ANOVA to determine the effect of time and treatment group over several testing sessions. For each individual testing session, non-parametric statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. Analysis of behavioural data from the final AIMs rating session on day 22 was carried out using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. Rotarod data from experimental group 2 were analysed using two-way repeated measures ANOVA followed by Bonferroni’s test for multiple comparisons. Analysis of in situ hybridisation and HPLC data was performed using two-way ANOVA (using ‘lesion’ and ‘treatment group’ as factors) followed by Bonferroni’s test for multiple comparisons.

Results Acute blockade of Ca2+-permeable AMPA receptors reduces established dyskinesia 6-OHDA-lesioned rat studies Following administration of IEM 1460 (1, 3 mg/kg) or vehicle with L-DOPA/benserazide to 6-OHDA-lesioned rats, there was a significant effect of treatment group on total ALO AIMs (Friedman statistic, Fr = 16.00; p < 0.0001). Total ALO AIMs were reduced by 62% by IEM 1460 3 mg/kg compared with vehicle (Dunn’s multiple comparison test, p < 0.001), but not by IEM 1460 1 mg/kg (p > 0.05; Fig. 1a). Following administration of IEM 1460 (1, 3 mg/kg) or vehicle with L-DOPA/benserazide to 6-OHDA-lesioned rats, there was a significant effect of treatment group on total locomotor score (Fr = 10.41; p = 0.0024), which was reduced by 50% by IEM 1460 3 mg/kg (p < 0.05), compared with vehicle (Fig. 1b). There was no significant effect of treatment group on peak rotarod performance (F2,7 = 0.504; p = 0.615; Fig. 1c). MPTP-lesioned marmoset studies Following administration of IEM 1460 (0.1, 0.3, 1.0, 3.0 mg/ kg) or vehicle with L-DOPA (Fig. 2a), there was no significant effect of treatment group on parkinsonian disability score at 0–1 h (Fr = 6.705; p = 0.152; Fig. 3a), 1–2 h (Fr = 2.413; p = 0.660; Fig. 3b) or 2–3 h (Fr = 1.570; p = 0.814; Fig. 3c) post-drug administration. There was a significant effect of treatment group on locomotor ‘on-time’ (F4,24 = 6.521; p = 0.001), which was increased by 35% by IEM 1460 0.3 mg/kg (p < 0.01), compared with vehicle (Fig. 2c). Following administration of IEM 1460 (0.1, 0.3, 1.0, 3.0 mg/kg) or vehicle with L-DOPA (Fig. 2b), there was a



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Fig. 1 Behavioural effects of IEM 1460 (1, 3 mg/kg, i.p.) or vehicle administered with L-DOPA/benserazide (6/15 mg/kg, i.p.) in the unilateral 6-hydroxydopamine-lesioned rat model of Parkinson’s disease. (a) Total L-DOPA-induced axial, limb and orolingual abnormal involuntary movements. (b) Total L-DOPA-induced locomotor scores. Points represent individual scores and bars median values (n = 8). *p < 0.05, ***p < 0.001 vs. vehicle (Friedman ANOVA followed by Dunn’s multiple comparison test). (c) Peak rotarod performance. Bars represent mean ± SEM (n = 8).

Fig. 2 Effects of IEM 1460 (0.1, 0.3, 1.0, 3.0 mg/kg, s.c.) or vehicle on the time course of L-DOPA-induced motor behaviour in the MPTPlesioned common marmoset model of Parkinson’s disease. (a) Time course of parkinsonian disability. Points represent median values at each time point (n = 7). Parkinsonian disability is expressed on the y axis as follows: 0 = none, 9 = mild, 18 = moderate, 27 = marked, 36 = severe, according to the behavioural rating system described. (b) Time course of L-DOPA-induced dyskinesia. Points represent median values at each time point. Dyskinesia score is expressed on the y axis as follows: 0 = none, 1 = mild, 2 = moderate, 3 = marked, 4 = severe, according to the behavioural rating system described. (c) Locomotor on-time following drug administration. Bars represent mean ± SEM (n = 7). **p < 0.01 vs. vehicle (one-way ANOVA followed by Dunnett’s test).




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Fig. 3 Effects of IEM 1460 (0.1, 0.3, 1.0, 3.0 mg/kg, s.c.) or vehicle on L-DOPA-induced motor behaviour in the MPTP-lesioned common marmoset model of Parkinson’s disease. (a) Parkinsonian disability at (a) 0–1 h, (b) 1–2 h and (c) 2–3 h post-drug administration. Points represent the sum of behavioural scores for individual animals in this time period (n = 7) and bars median values. Parkinsonian disability is expressed on the y axis as follows: 0 = none, 18 = mild, 36 = moderate, 54 = marked, 72 = severe, according to the behavioural rating

system described. Dyskinesia at (d) 0–1 hours, (e) 1–2 hours and (f) 2–3 hours post-drug administration. Points represent the sum of behavioural scores for individual animals in this time period (n = 7) and bars median values. Dyskinesia score is expressed on the y axis as follows: 0 = none, 2 = mild, 4 = moderate, 6 = marked, 8 = severe, according to the behavioural rating system described. *p < 0.05, **p < 0.01 vs. vehicle (Friedman ANOVA followed by Dunn’s multiple comparison test).

significant effect of treatment group on dyskinesia score at 0–1 h (Fr = 16.45; p = 0.0025), 1–2 h (Fr = 16.51; p = 0.0024) and 2–3 h (Fr = 16.79; p = 0.0021) post-drug

administration. During the 0–1 h time period, dyskinesia was reduced by 40% by IEM 1460 1.0 mg/kg compared with vehicle (p < 0.05; Fig. 3d). During the 1–2 h time period,



treatment group on day 1 (Kruskal–Wallis statistic, H = 13.87; p = 0.001), day 7 (H = 17.66; p = 0.0001), day 14 (H = 17.66; p = 0.0001) and day 21 (H = 17.70; p = 0.0001). ALO AIM scores were higher in the L-DOPA + vehicle group on day 1 (p < 0.01) and days 7, 14 and 21 (all p < 0.001) compared with vehicle. There were no significant differences between AIM scores in the vehicle and L-DOPA + IEM 1460 groups, or L-DOPA + vehicle and L-DOPA + IEM 1460 groups, at any testing session (p > 0.05). During the chronic treatment period, there were significant effects of time (F4,68 = 4.549; p < 0.0026), treatment group (F2,68 = 8.450; p = 0.0028) and time · treatment interaction (F8,68 = 5.851; p < 0.0001) on rotarod performance (Fig. 4b). Post hoc analysis revealed rotarod performance to be higher in the L-DOPA + vehicle group compared with vehicle on day 2 (p < 0.001), day 8 (p < 0.05) and day 15 (p < 0.05). Rotarod performance was higher in the L-DOPA + IEM 1460 group compared with vehicle on day 2, day 8 (both p < 0.001) and day 15 (p < 0.01). There were no differences between L-DOPA + vehicle and L-DOPA + IEM 1460 groups at any time point (p > 0.05). Following L-DOPA challenge on day 22, there was a significant effect of treatment group on total ALO AIM

dyskinesia was reduced by IEM 1460 1.0 mg/kg (43%; p < 0.05) and 3.0 mg/kg (52%; p < 0.01; Fig. 3e) compared with vehicle. During the 2–3 h time period, dyskinesia was reduced by 78% by IEM 1460 3.0 mg/kg compared with vehicle (p < 0.01; Fig. 3f). Chronic blockade of Ca2+-permeable AMPA receptors in the 6-OHDA-lesioned rat attenuates dyskinesia priming There was a significant effect of lesion (F1,34 = 48.29; p < 0.0001), but no effect of treatment group (F2,34 = 1.454; p = 0.248) or lesion · treatment interaction (F2,34 = 1.311; p = 0.283) on striatal dopamine levels as measured by HPLC. Striatal dopamine was reduced in the lesioned compared with unlesioned side in the vehicle (94%, p < 0.01), L-DOPA + vehicle (93%, p < 0.05) and L-DOPA + IEM 1460 groups (97%, p < 0.001). There were no significant differences between the lesioned striata of the three treatment groups (p > 0.05). During the chronic 21-day treatment period, there were significant effects of time (F3,51 = 29.59; p < 0.0001), treatment group (F2,51 = 144.3; p < 0.0001) and time · treatment interaction (F6,51 = 15.65; p < 0.0001) on ALO AIM scores (Fig. 4a). Non-parametric analysis of ALO AIM scores at each testing session revealed a significant effect of Vehicle

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Fig. 4 Behavioural pharmacology in unilateral 6-hydroxydopaminelesioned rats chronically treated with vehicle, L-DOPA/benserazide (6/ 15 mg/kg, i.p.) + vehicle, or L-DOPA/benserazide + IEM 1460 (3 mg/ kg, i.p.) for 21 days. (a) Development of axial, limb and orolingual abnormal involuntary movements (AIMs). Data are expressed as mean ± SEM (n = 6–7 per group). **p < 0.01, ***p < 0.001 vs. vehicle (Kruskal–Wallis test followed by Dunn’s multiple comparison test). (b)

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Peak rotarod performance. Data are expressed as mean ± SEM (n = 6–7 per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle (two-way ANOVA followed by Bonferroni’s test for multiple comparisons). (c) Dyskinesia priming and (d) locomotor behaviour following LDOPA challenge. Points represent individual AIM scores and bars median values. **p < 0.01 vs. vehicle; #p < 0.05 vs. L-DOPA + vehicle (Kruskal–Wallis test followed by Dunn’s multiple comparisons test).



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score (H = 12.73; p = 0.0017; Fig. 4c). ALO AIM score was increased in the L-DOPA + vehicle group (p < 0.01), but not the L-DOPA + IEM 1460 group (p > 0.05), compared with vehicle. ALO AIM score was reduced by 75% in the L-DOPA + IEM 1460 group compared with L-DOPA + vehicle (p < 0.05). There was a significant effect on total locomotor score (H = 11.45; p = 0.0033; Fig. 4d), which was increased by L-DOPA + vehicle (p < 0.01) compared with vehicle, but not compared with L-DOPA + IEM 1460 (p > 0.05). Chronic blockade of Ca2+-permeable AMPA receptors reverses molecular correlates of dyskinesia priming Pre-proenkephalin A In the dorsolateral striatum, there was a significant effect of lesion (F1,34 = 73.64; p < 0.0001) and treatment group (F2,34 = 8.16; p = 0.0013) on PPE-A mRNA expression (Fig. 5), which was increased in the lesioned compared to unlesioned side in the vehicle (p < 0.001), L-DOPA + vehicle (p < 0.001) and L-DOPA + IEM 1460 groups (p < 0.001). PPE-A mRNA was increased in the lesioned striatum of the L-DOPA + vehicle group compared with vehicle (p < 0.001) and L-DOPA + IEM 1460 groups (p < 0.01). There was no significant difference between vehicle and L-DOPA + IEM 1460 groups (p > 0.05) (Fig. 6a). In the ventrolateral striatum, there was a significant effect of lesion (F1,34 = 49.52; p < 0.0001), treatment group (F2,34 = 14.45; p < 0.0001) and lesion · treatment interaction (F2,34 = 5.05; p = 0.012) on PPE-A mRNA expression (Fig. 5), which was increased in the lesioned compared with unlesioned side in the vehicle (p < 0.05), L-DOPA + vehicle (p < 0.001) and L-DOPA + IEM 1460 groups (p < 0.05). PPE-B mRNA was increased in the lesioned striatum of the L-DOPA + vehicle group compared with vehicle (p < 0.001) and L-DOPA + IEM 1460 groups (p < 0.001). There was no

L

Fig. 5 Pseudocolour transformations of representative autoradiographic images of in situ hybridisation in 6-hydroxydopaminelesioned rats treated with vehicle, L-DOPA/ benserazide (6/15 mg/kg, i.p.) + vehicle, or L-DOPA/benserazide + IEM 1460 (3 mg/ kg, i.p.). Images denote pre-proenkephalin A (PPE-A) and pre-proenkephalin B (PPEB) mRNA expression in the unlesioned (U) and lesioned (L) rostral striatum.

significant difference between vehicle and L-DOPA + IEM 1460 groups (p > 0.05) (Fig. 6b). Pre-proenkephalin B In the dorsolateral striatum, there was a significant effect of treatment group (F2,32 = 16.42; p < 0.0001) and lesion · treatment interaction (F2,32 = 11.54; p = 0.0002), but not lesion (F1,32 = 0.798; p = 0.388) on PPE-B mRNA expression (Fig. 5), which was decreased in the lesioned compared with unlesioned side in the vehicle group (p < 0.05), increased in the L-DOPA + vehicle group (p < 0.01), and unchanged in the L-DOPA + IEM 1460 group (p > 0.05). PPE-B mRNA was increased in the lesioned striatum of the L-DOPA + vehicle group compared with vehicle (p < 0.001) and L-DOPA + IEM 1460 groups (p < 0.001), and was increased in the L-DOPA + IEM 1460 group compared with vehicle (p < 0.01) (Fig. 6c). In the ventrolateral striatum, there was a significant effect of treatment group (F2,32 = 17.25; p < 0.0001) and lesion · treatment interaction (F2,32 = 13.86; p < 0.0001), but not lesion (F1,32 = 1.53; p = 0.225) on PPE-B mRNA expression (Fig. 5), which was decreased in the lesioned compared with unlesioned striatum in the vehicle group (p < 0.05), increased in the L-DOPA + vehicle group (p < 0.001), and unchanged in the L-DOPA + IEM 1460 group (p > 0.05). PPE-B mRNA was increased in the lesioned striatum of the L-DOPA + vehicle group compared with vehicle (p < 0.001) and L-DOPA + IEM 1460 groups (p < 0.001), and was increased in the L-DOPA + IEM 1460 group compared with vehicle (p < 0.01) (Fig. 6d).

Discussion We report a series of behavioural pharmacological experiments designed to establish the role of Ca2+-permeable AMPA receptors in the induction and subsequent expression of LID. Initial experiments in the unilateral 6-OHDA-



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Fig. 6 Molecular correlates of dyskinesia priming in unlesioned (h) and lesioned ( ) rostral striatum of 6-hydroxydopamine-lesioned rats treated with vehicle, L-DOPA/benserazide (6/15 mg/kg, i.p.) + vehicle, or L-DOPA/benserazide + IEM 1460 (3 mg/kg, i.p.) for 21 days. Pre-proenkephalin A (PPE-A) mRNA expression in (a) dorsolateral and (b) ventrolateral rostral striatum. Bars represent mean ± SEM (n = 6–7

per group). (c) Pre-proenkephalin B (PPE-B) mRNA expression in (c) dorsolateral and (d) ventrolateral rostral striatum. Bars represent mean ± SEM (n = 6–7 per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs. unlesioned side; $$p < 0.01, $$$p < 0.001 vs. ipsilateral vehicle group; ##p < 0.01, ###p < 0.001 vs. ipsilateral L-DOPA + vehicle group (two-way ANOVA followed by Bonferroni’s test for multiple comparisons).

lesioned rat indicated an anti-dyskinetic effect of the Ca2+-permeable AMPA receptor antagonist, IEM 1460; these findings were confirmed by studies in the dyskinetic MPTP-lesioned marmoset. Chronic treatment with IEM 1460 in the 6-OHDA-lesioned rat attenuated behavioural and molecular correlates of priming for LID. These results indicate the critical role of Ca2+-permeable AMPA receptors in both priming and subsequent behavioural expression of LID.

are similar to those found to have behavioural effects in a rat model of kainate-induced epilepsy (Serdyuk and Gmiro 2007). Our method for dyskinesia priming in the 6-OHDAlesioned rat is similar to that previously reported (Cenci et al. 1998; Lundblad et al. 2002), with minor modifications. To investigate the priming process, all animals received L-DOPA alone 24 h following their last chronic treatment injection, as previously described (Mela et al. 2007). While no pharmacokinetic data have been published concerning the half-life of IEM 1460 in rats, it is likely that any acute effect of treatment would have abated at this point. To assess molecular correlates of priming, a further 48 h elapsed before animals were killed; this has been shown to be sufficient to obviate the acute effects of L-DOPA injection (Andersson et al. 1999; Mela et al. 2007).

Methodological considerations IEM 1460 is an adamantane-derivative drug acting at the polyamine site within the AMPA receptor ion channel (Stromgaard and Mellor 2004). It is a selective antagonist at GluR2-lacking AMPA receptors in vitro (Magazanik et al. 1997; Samoilova et al. 1999), acting via both open channel blockade and competitive antagonism (Schlesinger et al. 2005). While other adamantane derivatives have been shown to block other ligand-gated ion channels, including NMDA and nicotinic acetylcholine receptors (Antonov et al. 1995), IEM 1460 exhibits around 100 times greater efficacy at AMPA vs. NMDA receptors (Bolshakov et al. 2005). The nicotinic acetylcholine receptor antagonist mecamylamine has no effect on AIMs in the 6-OHDA-lesioned rat model of LID (Dekundy et al. 2007), making this potential mechanism of action unlikely. The doses of IEM 1460 used in this study

Symptomatic effects of blockade of Ca2+-permeable AMPA receptors In the 6-OHDA-lesioned rat, blockade of Ca2+-permeable AMPA receptors by IEM 1460 dose-dependently attenuated established AIMs. This effect was not associated with depression of motor behaviour as measured by the rotarod test. While rotational locomotor behaviour was reduced by up to 30%, the significance of this behaviour has been heavily debated (Lundblad et al. 2002; Marin et al. 2006),



and it may not accurately represent the therapeutic response to L-DOPA. Behavioural experiments suggest that clinically relevant motor depression is associated with reduction of both rotarod performance and locomotor behaviour (Dekundy et al. 2007). Having established a dose–response relationship for IEM 1460 in the 6-OHDA-lesioned rat model, we proceeded to confirm this finding in the dyskinetic MPTP-lesioned NHP model. Our findings confirm a dose-dependent anti-dyskinetic effect of IEM 1460, with the highest dose used showing the most prolonged action. The lack of any deleterious effect on the anti-parkinsonian response is again supportive of the findings in the rat model; indeed, at doses lacking an antidyskinetic effect IEM 1460 extended the ‘on-time’, a finding that warrants further investigation. We interpret these findings to show that Ca2+-permeable AMPA receptors are involved in the expression of dyskinesia once priming has occurred. While the anti-dyskinetic effect of non-competitive AMPA receptor antagonists has been established in the MPTP-lesioned NHP (Konitsiotis et al. 2000), this is the first published report of the efficacy of selective antagonists of Ca2+-permeable AMPA receptors in animal models of LID. Given the key role of pathologically enhanced corticostriatal plasticity in LID (Picconi et al. 2003), the striatum seems the most likely site of action. While most AMPA receptors assemble from pairing of GluR1–GluR2 dimers, and are therefore Ca2+-impermeable (Mansour et al. 2001), a small population of homomeric GluR1-containing receptors has also been identified (Brorson et al. 2004). Electrophysiological studies in striatal medium spiny neurones have identified a functional contribution of Ca2+-permeable AMPA receptors to corticostriatal transmission (Carter and Sabatini 2004). The enhanced phosphorylation of GluR1 seen in animal models of LID (Ba et al. 2006; Santini et al. 2007) therefore represents a potential substrate for enhanced functional activity of Ca2+permeable AMPA receptors (Roche et al. 1996; Lee et al. 2000b) Our behavioural findings suggest that increased functional activity of Ca2+-permeable AMPA receptors is likely to play a key role in dyskinesia expression across different animal model systems, and may well be relevant in patients with LID. Role of Ca2+-permeable AMPA receptors in priming for LID To establish the effect of Ca2+-permeable AMPA receptor blockade on dyskinesia priming, we selected for chronic study the dose of IEM 1460 that produced maximal antidyskinetic effects without impairment of rotarod performance (Dekundy et al. 2006; Mela et al. 2007). Over the 21-day chronic treatment period, L-DOPA + vehicle caused a marked increase in ALO AIMs over time compared with vehicle, while AIM scores in the L-DOPA + IEM 1460 group did not differ from the vehicle group. Chronic treatment with IEM 1460 did not adversely affect the motor response to

L-DOPA, as demonstrated by performance on the rotarod: therefore, any reduction in AIMs was not simply because of non-specific motor depression. The reduction in rotarod performance over time in both groups receiving L-DOPA is consistent with previous reports (Picconi et al. 2003). Following an L-DOPA challenge, animals which had received L-DOPA + IEM 1460 displayed significantly lower ALO AIM scores than those receiving L-DOPA + vehicle. Furthermore, AIM scores in the L-DOPA + IEM 1460 group were not significantly different to previously L-DOPA-naı¨ve animals in the vehicle group. There was, however, no difference in locomotor score between the L-DOPA-treated groups. These results suggest that chronic blockade of Ca2+permeable AMPA receptors is able specifically to interfere with the process of priming for LID, such that lower levels of dyskinesia are elicited by a subsequent L-DOPA challenge. Blockade of Ca2+-permeable AMPA receptors could interfere with the priming process via several potential mechanisms. Activation of extracellular signal-regulated kinase (ERK) 1/2 has been implicated in the development of LID in animal models (Santini et al. 2007; Westin et al. 2007), via phosphorylation of downstream proteins involved in gene transcription. Striatal ERK1/2 activation occurs in a Ca2+- and Ca2+–calmodulin-dependent protein kinase II (CaMKII)-dependent manner (Perkinton et al. 1999) which is blocked by AMPA receptor antagonists (Mao et al. 2004; Wang et al. 2004). Pharmacological blockade of ERK1/2 activation markedly reduces AIMs in the 6-OHDA-lesioned mouse model (Santini et al. 2007). Specific blockade of Ca2+-permeable AMPA receptors could therefore attenuate striatal ERK1/2 signalling and subsequent maladaptive plastic changes. It is also possible that Ca2+-permeable AMPA receptor blockade might itself modulate the abnormal phosphorylation of the GluR1 subunit implicated in LID. Both increased CaMKII-mediated phosphorylation of the S831 residue (Wessell et al. 2004; Ba et al. 2006) and protein kinase A-dependent phosphorylation of S845 (Ba et al. 2007; Santini et al. 2007) have been described in rodent models of LID. A reduction in CaMKII activation could therefore attenuate GluR1–S831 phosphorylation with resultant modulation of corticostriatal synaptic plasticity (Lee et al. 2000b, 2003). Anatomical studies of the basal ganglia indicate that PPEA, and its protein product enkephalin, is predominantly expressed in medium spiny neurones of the indirect pathway, while the dynorphin precursor PPE-B is mainly found in the direct pathway (Gerfen and Young 1988). Consistent with previous studies (Cenci et al. 1998; Duty et al. 1998; Ravenscroft et al. 2004), PPE-A expression was increased in the lateral striatum of 6-OHDA-lesioned animals, with a further increase following L-DOPA treatment which was reversed in animals receiving IEM 1460. Similarly, PPE-B



expression was markedly increased compared with vehicletreated animals following L-DOPA treatment alone. However, following co-treatment with L-DOPA and chronic IEM 1460, PPE-B expression was restored to pre-lesion (normal) levels. The molecular findings are therefore in agreement with the changes in behaviour following chronic blockade of Ca2+-permeable AMPA receptors. Taken together, these data support the hypothesis that chronic blockade of Ca2+-permeable AMPA receptors attenuates the process of dyskinesia priming. Striatal expression of both PPE-A and PPE-B has previously been found to correlate with severity of ALO AIMs in the 6-OHDAlesioned rat model of LID (Cenci et al. 1998). Increased lateral striatal PPE-A mRNA has been observed in parkinsonian patients with LID (Calon et al. 2002a), although it is not clear whether this is a pathogenic or compensatory mechanism. Consistent with our findings, chronic co-treatment with the competitive AMPA receptor antagonist LY293558 reduces L-DOPA-induced PPE-A expression in the 6-OHDA-lesioned rat (Perier et al. 2002). Numerous studies in animal models and dyskinetic parkinsonian patients have implicated up-regulation of PPE-B in dyskinesia priming (Cenci et al. 1998; Duty et al. 1998; Andersson et al. 1999; Henry et al. 2003), and reduction of behavioural expression of priming by chronic treatment with a metabotropic glutamate receptor antagonist was also associated with reduced PPE-B mRNA expression (Mela et al. 2007). Indeed, AMPA receptor antagonists exert a preferential effect on dyskinesia induced by dopamine D1 receptor agonists in the MPTP-lesioned NHP (Bibbiani et al. 2005), suggesting an effect on direct pathway responses implicated in priming.

Conclusions These findings indicate that Ca2+-permeable AMPA receptors are involved in the process of priming for, and the subsequent expression of, LID. Furthermore, the lack of adverse motor effects makes Ca2+-permeable AMPA receptors a potential target for anti-dyskinetic therapies.

Acknowledgements This study was funded by the Edmonds bequest for Parkinson’s disease research. The authors have no conflicts of interest. Many thanks to Steve McGuire and Ralph Davies for their technical assistance.

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