Oleoylethanolamide reduces L-DOPA-induced dyskinesia via TRPV1 receptor in a mouse model of Parkinson´s disease

Neurobiology of Disease 62 (2014) 416–425

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Oleoylethanolamide reduces L-DOPA-induced dyskinesia via TRPV1 receptor in a mouse model of Parkinson´s disease Ramiro González-Aparicio, Rosario Moratalla ⁎ Instituto Cajal, Consejo Superior de Investigaciones Científicas, CSIC, Madrid, Spain CIBERNED, ISCIII, Madrid, Spain

a r t i c l e

i n f o

Article history: Received 10 August 2013 Revised 2 October 2013 Accepted 6 October 2013 Available online 17 October 2013 Keywords: OEA Endocannabinoids Cannabinoids Capsaicin PPARα receptors

a b s t r a c t The long-term use of levodopa (L-DOPA) in Parkinson's disease (PD) results in the development of abnormal involuntary movements called L-DOPA-induced dyskinesias. Increasing evidences suggest that the endocannabinoid system may play a role in the modulation of dyskinesias. In this work, we assessed the antidyskinetic effect of the endocannabinoid analog oleoylethanolamide (OEA), an agonist of PPARα and antagonist of TRPV1 receptors. We used a hemiparkinsonian model of PD in mice with 6-OHDA striatal lesion. The chronic L-DOPA treatment developed intense axial, forelimb and orolingual dyskinetic symptoms, as well as contralateral rotations. Treatment with OEA reduced all these symptoms without reducing motor activity or the therapeutic motor effects of L-DOPA. Moreover, the OEA-induced reduction in dyskinetic behavior correlated with a reduction in molecular correlates of dyskinesia. OEA reduced FosB striatal overexpression and phosphoacetylation of histone 3, both molecular markers of L-DOPA-induced dyskinesias. We found that OEA antidyskinetic properties were mediated by TRPV1 receptor, as pretreatment with capsaicin, a TRPV1 agonist, blocked OEA antidyskinetic actions, as well as the reduction in FosB- and pAcH3-overexpression induced by L-DOPA. This study supports the hypothesis that the endocannabinoid system plays an important role in the development and expression of dyskinesias and might be an effective target for the treatment of L-DOPA-induced dyskinesias. Importantly, there was no development of tolerance to OEA in any of the parameters we examined, which has important implications for the therapeutic potential of drugs targeting the endocannabinoid system. © 2013 Elsevier Inc. All rights reserved.

Introduction Parkinson's disease (PD) is a progressive, chronic and age-associated neurodegenerative disorder characterized by a dramatic depletion of dopamine (DA) in the striatum due to loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Levodopa (L-DOPA) is the most commonly used treatment for PD. Initially, it improves all PD symptoms, but its therapeutic efficacy wanes with time and side effects appear in chronically treated patients, including motor fluctuations, “on-off” periods, and abnormal involuntary movements (AIMs) known as L-DOPA-induced dyskinesias (Jenner, 2004; Marsden, 1994). The development of dyskinesias is attributed to a sequence of striatal events that occur following repeated L-DOPA administration, including pulsatile stimulation of DA receptors and the complete denervation of areas within the striatum. These result in sensitization of responses to DA, and shortand long-term maladaptative synaptic plasticity (Cenci and Lundblad, 2006; Pavón et al., 2006). Several studies have associated dyskinesias

⁎ Corresponding author at: Instituto Cajal, Avda. Doctor Arce 37, 28002 Madrid, Spain. Fax: +34 91 585 4754. E-mail address: [email protected] (R. Moratalla). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nbd.2013.10.008

with increased expression of diverse molecular markers, including overexpression of FosB and dynorphin, phosphorylation of ERK1/2 and phospho-acetylation of histone 3 (Pavón et al., 2006; Santini et al., 2007). Interestingly, only the D1 dopamine receptor, not D2, seems to be critical in these underlying molecular changes (Darmopil et al., 2009; Murer and Moratalla, 2011; Santini et al., 2009). Increasing evidence suggests that non-dopaminergic mechanisms also contribute to dyskinesias (Brotchie, 2005), including several lines of evidence indicating a role for the endocannabinoid system. The endocannabinoid system is particularly abundant in the basal ganglia circuit (Bisogno et al., 1999; Devane et al., 1992; Egertova et al., 2003; Mailleux and Vanderhaeghen, 1992; Martín et al., 2008; Suárez et al., 2011), and cannabinoid modulation of GABA and glutamate synaptic transmission has important influences on all dopamine-mediated motor mechanisms (Di Marzo et al., 1998; Julian et al., 2003; Martín et al., 2008). The receptors primarily involved in cannabinoid-mediated effects are CB1 and CB2, but the endocannabinoid system also includes nuclear peroxisome proliferator-activated receptor α (PPARα) and transient receptor potential vanilloid subtype 1 (TRPV1) (Devane et al., 1988; Munro et al., 1993; Smart et al., 2000; Sun and Bennett, 2007). Based on well documented changes in CB1 expression and endocannabinoid levels in response to L-DOPA administration (Ferrer et al., 2003; Zeng et al., 1999), cannabinoids have been investigated in many

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studies as potential inhibitors of L-DOPA-associated motor disturbances. In animal models of Parkinsonism, both cannabinoid agonists (Fox et al., 2002; Martínez et al., 2012; Morgese et al., 2007; Walsh et al., 2010) and antagonists (Cao et al., 2007; Kelsey et al., 2009; Segovia et al., 2003; van der Stelt et al., 2005) have demonstrated antidyskinetic properties. In contrast, in clinical trials, only cannabinoid agonists seem to exert antidyskinetic effects (Carroll et al., 2004; Mesnage et al., 2004; Sieradzan et al., 2001). These apparently paradoxical effects may be explained in part by the fact that endocannabinoids modulate TRPV1 receptor activity (Smart et al., 2000; van der Stelt and Di Marzo, 2004; Zygmunt et al., 1999) as well as CB1 activity. CB1 and TRPV1 receptors are co-expressed in several brain areas (Cristino et al., 2006) and appear to play opposite roles in diverse actions, including L-DOPAinduced dyskinesias (Morgese et al., 2007). Oleoylethanolamide (OEA) (Fu et al., 2003; Lo Verme et al., 2005; Rodríguez de Fonseca et al., 2001) is a structural analog of the endocannabinoid anandamide and is co-synthesized with palmitoylethanolamide (PEA) and other acylethanolamides on demand. Generated from lipid membrane precursors, both PEA and OEA are primarily inactivated by hydrolysis by fatty acid amide hydrolase (FAAH; Di Marzo, 2008; Labar and Michaux, 2007; Mackie, 2006). In addition, OEA, PEA and other acylethanolamides can potentiate anandamide response through “entourage effects”, mainly via TRPV1 receptor (García et al., 2009; Ho et al., 2008; Smart et al., 2002). Interestingly, OEA does not bind to CB1, but has been identified as an endogenous ligand for PPARα (Fu et al., 2003; Lo Verme et al., 2005) and as an antagonist for TRPV1 (Almasi et al., 2008; Thabuis et al., 2008; Wang et al., 2005). Both PPARα and TRPV1 receptors are present in basal ganglia structures, including the striatum, substantia nigra and globus pallidus (Fernández-Ruiz, 2009; Galán-Rodríguez et al., 2009). In the work reported here, we assessed the effect of systemic administration of OEA in L-DOPA-induced dyskinesias and investigated the underlying molecular mechanisms. Materials and methods Animals Adult male C57⁄BL6 mice (Cajal Institute, Consejo Superior de Investigaciones Científicas, Madrid, Spain) were housed in standard Plexiglas cages with a maximum of six animals per cage and ad-libitum access to food and water. The environmental conditions were strictly controlled with a 12 h light⁄dark cycle, a temperature of 22 °C and 44% humidity. All animal maintenance and experimental procedures were in accordance with the guidelines laid out in the European Union Council Directive (86⁄609⁄EEC). All efforts were made to minimize the number of animals used in this study. The experimental protocols involving animals were approved by the Consejo Superior de Investigaciones Científicas ethics committee. Striatal unilateral 6-OHDA lesion Mice were anesthetized with an intraperitoneal (i.p.) injection of 200 mg⁄kg of 2,2,2-tribromoethanol (Sigma-Aldrich) and placed in a stereotaxic frame with a mouse adapter (David Kopf Instruments, Tujunga, CA, USA). An antibiotic cream was used to protect the eyes from drying during surgery. At 30 min before lesion, the mice received an i.p. injection of 20 mg⁄kg of the noradrenaline reuptake inhibitor desipramine hydrochloride (Sigma-Aldrich) to protect the noradrenergic neurons from 6-OHDA neurotoxicity (Breese and Traylor, 1971). Using a Hamilton syringe (Hamilton, Bonaduz, Switzerland), 4μL of 6-OHDA-HBr solution (5 μg/μL) in 0.02% ascorbic acid (SigmaAldrich) were injected in the left striatum in two deposits at the following stereotaxic coordinates (mm from bregma): AP, +0.65; L, −2.0; V1, −4 and V2, −3.5 (Paxinos and Franklin, 2004), targeting the dorsolateral striatum. After the injection, the skin was sutured and the animals were removed from the

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stereotaxic instrument and placed on a heating pad for 30 min. During the first days after surgery the animals received injections of saline solution to prevent dehydration, as well as supplementary food. Three weeks after the lesion, success of the surgeries was verified by cylinder test (Kirik et al., 2000). Briefly, the animal is allowed to move freely in a transparent cylinder during 3 min, and the number of left and right forepaw contacts is counted. Hemiparkinsonian mice with a lesion in the left dorsal striatum present a significant impairment in the contralateral (right) forepaw use, indicative of forepaw use asymmetry. L-DOPA treatment Mice recovered for 3–4 weeks after the lesion, and then were subjected to treatment with L-DOPA for 18 days. During this treatment period, 6-OHDA-operated animals received a daily i.p. injection of 10 mg/kg benserazide hydrochloride (Sigma-Aldrich), a peripheral blocker of L-DOPA decarboxylase, and followed 20 min later by an i.p. injection of 20 mg/kg of L-DOPA methyl ester (Sigma-Aldrich, Madrid, Spain). OEA (Tocris, Bristol, UK) at 0.5, 5 or 20 mg/kg, or vehicle (10% DMSO in saline solution) was administrated i.p. 15 min before L-DOPA treatment. In addition, GW 6471 (Tocris, Bristol, UK) (2.5 mg/kg) or capsaicin (Tocris, Bristol, UK) (1 mg/kg) were injected i.p. 15 min before OEA. The doses were chosen according to previous reports (Demirbilek et al., 2004; Lee et al., 2011; Reyes-Cabello et al., 2012). Behavioral measurements Abnormal involuntary movements (AIMs) The AIMs were studied at 40 min following L-DOPA administration. According to their topographical distribution, the AIMs were divided into three categories: axial (lateral deviation of the trunk, neck, and head toward the contralateral side, leading to a loss of the orthostatic equilibrium), forelimb (jerky movements of the forelimb contralateral to the lesion with choreic or ballistic nature) and orolingual (discrete vertical (open and close) jaw movements toward the contralateral side, sometimes including tongue protrusion). Each animal was evaluated on both amplitude and frequency (Cenci and Lundblad, 2007). Briefly, the amplitude of each category of AIM was rated on a scale from 1 to 4 according to the maximum amplitude observed in the evaluation period. And the frequency scale (rated from 0 to 4) scores as previously shown (Suárez et al., in press). For the evaluations, each mouse was introduced into a glass cylinder (4 cm diameter) and observed for a 2 min period for orofacial dyskinesias and another 2 min for the rest of the AIMs. The day before the sacrifice, the AIMs were recorded for 1min every 20min to see the general time course. The data are expressed as frequency x amplitude for each AIM category, or its sum for the total global score. Contralateral turns and locomotor activity Rotation tests were carried out in a rotometer system and only completed turns (360°) were counted. The mice were placed in rotometer bowls and, after 5 min of habituation, rotational data was registered for 15 min, 5 min after L-DOPA treatment. The horizontal and vertical activities were recorded as indicated (Centonze et al., 2003; Granado et al., 2008c) using a multicage activity meter system (Columbus Instruments, Columbus, OH) with a set of 8 individual cages measuring 20 × 20 × 28 cm. Horizontal movement was detected by 2 arrays of 16 infrared beams, whereas a third array positioned 4 cm above the floor detected vertical movement. The software allowed a distinction to be made between repetitive interruptions of the same photobeam and interruptions of adjacent photobeams. This latter measure was used as an index of ambulatory activity. The mice were habituated to the cages in 15 min sessions for 3 days. Lesioned mice were recorded for 200 min following the infusion of L-DOPA. Unlesioned animals were recorded for 90 min immediately after the injection of OEA or vehicle, both in acute or chronic experiments.

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Rotarod test Motor coordination was measured in the rotarod test (Ugo Basile, Rome, Italy) at crescent speed. Each day, mice had a 1 min training session in the immobile rod. If the mouse fell from the rotarod during the training session, it was placed back. Then the performance of the mice was tested in 5 min sessions every 20 min. Thus, the speed of the rod was turned on up to 40 rpm for five minutes. The latency to fall off the rod was measured on consecutive days in lesioned mice following the L-DOPA administration, or on just one day for unlesioned OEA/ vehicle treated animals.

Statistical analysis Behavioral data and the group comparisons of dyskinesia intensity scores were analyzed by one-way or two-way ANOVA followed by a post hoc Student Newman–Keuls test. For analysis of inmunohistochemistry quantification data, we used the one-way ANOVA followed by Student Newman–Keuls test. Data are expressed as mean ± standard error of the mean. The minimum level of significance was p b 0.05. For all statistical studies, SigmaStat 2.03 program was used. Results

Tissue preparation

Effects of OEA on motor functions in non-lesioned mice

Following behavioral analysis, animals were euthanized 1 h after the last injection of L-DOPA with an overdose of pentobarbital (Laboratorios Normon, Madrid, Spain), injected intracardially with 0.5 mL of 1% heparin (Rovi, Madrid, Spain) and then perfused with 10 mL of saline and 100 mL of 4% paraformaldehyde in phosphate buffer (pH 7.4). The brains were post-fixed for 24 h and were then transferred to a solution of 0.1 mM phosphate buffer containing 0.02% sodium azide for storage at 4 °C. To obtain regular blocks, the brains were further immersed in 3% agarose and cut in coronal sections (30 μm thick) using a vibratome (Leica, Wetzlar, Germany).

Since cannabinoids are known to induce hypo-locomotor effects, we tested whether OEA modifies normal activity by observing its effect on spontaneous motor tests in naïve mice. We first performed a doseresponse curve, measuring motor activity for 90 min after treatment with OEA. Following this acute treatment, only the higher dose of OEA, 20 mg/kg, significantly reduced horizontal (Fig. 1A) and vertical (Fig. 1B) activity. This reduction was observed picked between 20– 30 min post-injection (Fig. 1C). Similarly, on the rotarod test, only the 20 mg/kg dose significantly reduced time on the rod, at 20 min after OEA administration (Fig. 1D), in line with the peak of hypo-locomotor effects. We used the 5 mg/kg dose of OEA for subsequent experiments because it had no effect on baseline motor activity. To verify that chronic treatment does not modify this behavior, we treated mice with 5 mg/kg OEA for two weeks and measured motor activity daily for 90 min. Chronic treatment did not significantly affect either horizontal or vertical activity over time (Fig. 1E–F).

Inmunohistochemistry Immunostaining was carried out in free-floating sections using a standard avidin–biotin immunocytochemical protocol (Darmopil et al., 2008; Granado et al., 2008b; Grande et al., 2004; Rivera et al., 2002) with the following rabbit antisera: tyrosine hydroxylase (TH; 1:1000; Chemicon, Temecula, California), FosB (1:7500, Santa Cruz Biotechnology, Santa Cruz, California), and antiphospho (Ser10)-acetyl (Lys14)-Hystone 3 (pAcH3; 1:500; Upstate, Cell Signaling Solutions, Lake Placid, New York). Briefly, after incubation with primary antibody (overnight), the sections were washed and incubated with biotinylated secondary anti-rabbit antibody (1:500) (Vector Laboratories) for 1 h at room temperature. After washing, the sections were incubated with streptavidin (Zymed, San Francisco, CA, USA) for 1 h and antibody staining was developed using DAB (Sigma-Aldrich). After developing the reaction, stained sections were mounted, dried, dehydrated, and coverslipped with Permount mounting medium (Fisher Chemicals, Fair Lawn, NJ, USA).

Cellular density: FosB- and pAcH3-ir neuron quantification The quantification of the completely denervated area was measured using Neurolucida software (Microbrightfield, Colchester, VT, USA). The borders of the areas of interest in lesioned striata (complete dorsal striatum and FosB-ir stained dorsal striatum) were outlined from a live image with a 5 × objective. The images were then exported to Neuroexplorer (Microbrightfield) to determine the total striatal area and the relative area of FosB-ir stained striatum. The data are expressed as the % of FosB-ir striatal area in relation to total striatal area. Regarding the quantification of FosB and pAcH3 immunoreactivity, it was carried out using an image analysis system (AIS, Imaging Research, Linton, United Kingdom) as previously shown (Ares-Santos et al., 2012; Espadas et al, 2012; Granado et al., 2008a, 2011). For all sides, immunostaining intensity and number of immunolabeled nuclei were determined using five serial rostrocaudal sections per animal and three counting frames (dorsal, dorsolateral and lateral) per section (0.091 mm2 each frame). Images were digitized with a Leica microscope under 40× lens. Before counting, images were thresholded at a standardized gray-scale level. The data are presented as number of stained nuclei per mm2 (mean ± standard error of the mean) in the lesioned striatum.

OEA reduces the development of L-DOPA-induced dyskinesias and contralateral turns in mice with unilateral 6-OHDA lesions 6-OHDA lesioned mice undergoing chronic L-DOPA administration showed increasingly severe AIMs over time (axial, limb and orolingual dyskinesias). All abnormal movements increased significantly in frequency and severity during the first week of L-DOPA treatment. After this, the intensity of the movements remained stable until the end of the experiment. Systemic administration of OEA (5 mg/kg) 15 min before L-DOPA significantly attenuated all dyskinetic symptoms both when assessed individually (axial, forelimb and orolingual; Fig. 2A–C), and in a global score (Fig. 2D). No AIMs were observed in control animals lesioned with 6-OHDA receiving saline instead of L-DOPA (Fig. 2D). Other doses of OEA (0.5, 20 mg/kg) did not modify the occurrence or intensity of any of the dyskinetic symptoms. The anti-dyskinetic effects of OEA followed a U-shaped curve in which medium doses were effective while lower or higher doses were not. The global time courses revealed that dyskinesias are present from 20 to 160min following L-DOPA administration. OEA 5mg/kg was effective in reducing AIMs within the first 100 min, including the peak-dose dyskinesias interval (Fig. 2E), and in reducing the intensity and the duration of dyskinetic symptoms. Treatment with L-DOPA also induced a high contralateral turning behavior beginning on the first treatment day and remaining until the end of the experiment. Only the 5 mg/kg dose of OEA was able to significantly reduce contralateral rotations (Fig. 2F). No ipsilateral turns was observed with this dose of OEA. OEA enhances the motor coordination effects of L-DOPA We next explored whether the anti-dyskinetic dose of OEA reduced the therapeutic effect of L-DOPA. We administered L-DOPA and performed the rotarod test at 40 min (when dyskinesias are highest) and 180 min (when dyskinesias have clearly disappeared but the therapeutic effects of L-DOPA still remain). In addition, we recorded horizontal and

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Fig. 1. Motor effects of OEA in naïve animals. Graphs show acute (A–D) and chronic (E, F) motor effects of OEA treatment in naive animals. Only the highest dose of OEA, 20 mg/kg, significantly reduces horizontal and vertical activity, and time on the rotarod test. Acute or chronic OEA treatment with 5 mg/kg does not alter motor activity or motor coordination. Data points represent beam breaks or time, mean ± standard error of the mean, *p b 0.05; **p b 0.001 vs vehicle, one- and two-way ANOVA, n = 8–10 per group.

vertical activity for 20 min at each of these time points (Fig. 3). Pretreatment with OEA significantly increased the time on the rod at 40 min, but had no effect at 180 min, compared with L-DOPA alone (Fig. 3A–B). Moreover, OEA did not modify horizontal (Fig. 3C–D) or vertical (Fig. 3E–F) locomotor activity-induced by L-DOPA, suggesting that OEA reduces dyskinetic symptoms induced by L-DOPA without modifying its motor effect, thereby improving its therapeutic efficacy.

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Fig. 2. Effect of OEA on L-DOPA-induced dyskinesia and contralateral rotation. 5 mg/kg OEA significantly decreases all subtypes of dyskinesia, either separately (axial (A), forelimb (B), and orolingual (C)), or expressed as total global score (D). (E) Time course of the total dyskinesia score on day 18 of treatment. (F) Effect of OEA on the right turns induced by L-DOPA. Only the dose of 5 mg/kg significantly decreases dyskinetic symptoms and the number of right turns. Data represent the mean ± standard error of the mean, *p b 0.05; **p b 0.001 vs vehicle, two-way ANOVA, n = 7–13 per group.

duration of the symptoms (Fig. 4E). Similarly, GW 6471 did not significantly modify L-DOPA-induced contralateral turns or the reduction induced by pre-administration of OEA 5 mg/kg (Fig. 4F), suggesting that PPARα is not involved in the antidyskinetic effect of OEA. The anti-dyskinetic effect of OEA is mediated by TRPV1 receptors

The anti-dyskinetic effect of OEA is not mediated by PPARα receptors To see if the effect of OEA was mediated by PPARα receptors, we injected the PPARα antagonist GW 6471 (2.5 mg/kg, i.p.) 15 min before OEA administration and measured dyskinetic behavior (Fig. 4). GW 6471 did not significantly modify the presence of L-DOPA-induced dyskinesias, or the anti-dyskinetic effect of OEA on axial, forelimb and orolingual symptoms (Fig. 4A–C), on the total score (Fig. 4D) or on the

To test whether the vanilloid TRPV1 receptor has any role in the antidyskinetic effect of OEA, capsaicin (1 mg/kg, i.p.), a TRPV1 agonist, was injected 15 min before OEA. Dyskinesias and contralateral turns were measured on alternate days after L-DOPA administration (Fig. 5). Capsaicin significantly blocked the anti-dyskinetic effects of OEA as observed for each of the dyskinetic symptoms individually as well as in the total dyskinetic score (Fig. 5A–D). This blockade was maintained throughout the course of the L-DOPA effect (Fig. 5E). Capsaicin had no

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Fig. 3. Effects of OEA in the therapeutic action of L-DOPA. OEA improves time on the rod in lesioned mice at 40 min post-injection of L-DOPA, when dyskinesia peaks (A). However, it does not modify time on the rod at 180 min post-L-DOPA, by which time dyskinesia has disappeared (B). In addition, OEA does not modify either horizontal (C, D) or vertical (E, F) activity recorded 40 and 180 min following L-DOPA injection. Data points represent beam breaks or time, mean ± standard error of the mean, *p b 0.05 vs vehicle, two-way ANOVA, n = 7–8 per group.

effect on the level of the individual or global dyskinesia scores induced by L-DOPA alone or on the time-course (Fig. 5A–E). These results indicate that the stimulation of TRPV1 receptors with capsaicin specifically abolished the anti-dyskinetic effects of OEA. Similar effects were observed for contralateral rotations: L-DOPA-induced contralateral turns were significantly reduced by OEA (5 mg/kg) and pretreatment with capsaicin abolished this reduction (Fig. 5F), suggesting that TRPV1 receptors are involved in this effect of OEA.

OEA, GW 6471 and capsaicin have no effect on lesion size In this model, the intensity of dyskinesias correlates with the extent of the lesion. To rule out an effect of OEA, GW 6471 or capsaicin on lesion size, we analyzed the percentage of striatal areas that were completely denervated in all groups and found no significant differences in lesion

Fig. 4. The decrease in dyskinesia and contralateral rotation induced by OEA is not mediated by PPARα. Pretreatment with GW 6471 (2.5 mg/kg) does not abolish the antidyskinetic effect of OEA, either individual dyskinetic symptoms (axial (A), forelimb (B), and orolingual (C)), or global score (D). (E) Time course of the global dyskinetic score on day 18 of treatment. (F) Pretreatment with GW 6471 does not abolish the reduction of contralateral rotation caused by OEA. Data represent the mean ± standard error of the mean, *p b 0.05; **p b 0.001 vs vehicle, two-way ANOVA, n = 7–13 per group.

size (Fig. 6). In addition, none of the treatments modified the pattern of FosB expression, confirming that the dyskinetic behavior observed in the different groups was due to the pharmacological treatments and not to differences in the extent of the lesions.

OEA reduces intensity of L-DOPA-induced FosB expression via TRPV1 receptors Although there was no effect of OEA on the pattern of FosB expression induced by L-DOPA, treatment with 5mg/kg OEA reduced the intensity of L-DOPA-induced FosB expression in the denervated striatum (Fig. 7A). Both the number and intensity of FosB-positive nuclei in the striatum were reduced. This reduction in level of FosB expression correlates well with the reduced intensity of dyskinesias observed with OEA pre-

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Fig. 5. The decrease in dyskinesia and contralateral rotation induced by OEA is mediated by TRPV1. Pretreatment with capsaicin (1 mg/kg) abolishes the antidyskinetic effect of OEA, either individual dyskinetic symptoms (axial (A), forelimb (B), and orolingual (C)), or the global dyskinetic score (D). (E) Time course of global dyskinetic score on day 18 of treatment. (F) Pretreatment with capsaicin does not abolish the reduction of contralateral rotation caused by OEA. Data represent the mean ± standard error of the mean, *p b 0.05; **p b 0.001 vs vehicle, two-way ANOVA, n = 7–13 per group.

treatment (Fig. 6D), which had no effect on the pattern of striatal FosB expression (Fig. 6B). Pretreatment with capsaicin significantly blocked the reduction of striatal FosB-ir neurons induced by OEA, whereas GW 6471 did not. Neither capsaicin nor GW 6471 alone altered the pattern or intensity of FosB-ir induced by L-DOPA (Fig. 7B). These results indicate that OEA partially reduces L-DOPA-induced FosB expression in the striatum in a TRPV1 dependent way (Fig. 7C).

OEA reduces L-DOPA-induced phosphoacetylation of histone 3 via TRPV1 receptors Chronic L-DOPA treatment of hemiparkinsonian mice increases phosphorylation and acetylation of histone 3 (pAcH3) in the lesioned striatum, with a pattern of expression very similar to that described

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Fig. 6. OEA, GW 6471 and capsaicin have no effect on lesion size. (A) Striatal pattern of LDOPA-induced molecular changes in the lesioned striatum of mice. (B) Pretreatment with OEA, capsaicin, or GW 6471 does not modify L-DOPA-induced FosB expression pattern. (C) Histogram showing the extent of the striatal lesions. No differences are found in lesion size. (D) Correlation between the number of striatal FosB-ir cells and the total dyskinesia score. Scale bar = 500 μm, V = vehicle, Cap = capsaicin, GW = GW 6471, n = 7–13 per group.

for FosB (Fig. 6A). In agreement with its effects on FosB expression and dyskinetic behavior, OEA at 5 mg/kg reduced the induction of pAcH3 following chronic L-DOPA treatment in Parkinsonian animals. The other doses of OEA had no effect (Fig. 8A). This effect of OEA was blocked by pretreatment with capsaicin, but not GW 6471. Neither capsaicin nor GW 6471 alone modified the pattern of pAcH3 expression induced by L-DOPA (Fig. 8B). These results suggest that OEA's partial blockade of L-DOPA-induced pAcH3 expression is TRPV1 dependent. Representative photomicrographs of pAcH3 in the lesioned striatum are shown in Fig. 8C.

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Fig. 7. OEA reduces L-DOPA-induced FosB expression via TRPV1 receptors. (A) Striatal quantification of FosB-positive cells after treatment with L-DOPA and OEA. Only the OEA 5 mg/kg dose significantly reduces FosB-ir expression induced by L-DOPA. (B) Striatal quantification of FosB-positive cells after pretreatment with capsaicin or GW 6471 and the effective dose of OEA 5 mg/kg. Pretreatment with capsaicin, but not with GW 6471, abolishes the OEA-mediated reduction of FosB-positive cells. (C) High power photomicrographs of FosB expression in lesioned striatum induced by the different treatments. Histograms represent number of FosB-positive nuclei, mean ± standard error of the mean, **p b 0.01 vs vehicle, one-way ANOVA. Scale bar = 50 μm, V = vehicle, Cap = capsaicin, GW = GW 6471, n = 7–13 per group.

Discussion We used a hemiparkinsonian mouse model of dyskinesia based on unilateral 6-OHDA striatal lesion (González-Aparicio et al., 2010; Sauer and Oertel, 1994), followed by chronic L-DOPA administration of a daily dose of 20 mg/kg. In this model animals progressively develop dyskinetic symptoms, including axial, forelimb and orolingual dyskinesias, as well as sensitization manifested by intense contralateral turn behavior (Bido et al., 2011; Darmopil et al., 2009; Pavón et al., 2006; Suárez et al., in press). In this study we show that: (1) OEA reduces both L-DOPAinduced dyskinesias and contralateral rotations; (2) The effective OEA dose (5 mg/kg) does not interfere with the therapeutic motor effects of L-DOPA; (3) OEA reduces L-DOPA-induced FosB and pAcH3 overexpression in lesioned striatum; and (4) Antidyskinetic actions of OEA are mediated by TRPV1, not PPARα receptors. Our results show significant antidyskinetic properties of OEA. The anti-dyskinetic effect of the 5 mg/kg dose of OEA was observed 40 min after L-DOPA administration, when dyskinesias were peaking, and persisted throughout the duration of dyskinesias over the first 100 min after L-DOPA-administration. This is in contrast with other cannabinoids that were primarily effective either within the first 10–20 min after L-DOPA (Fox et al., 2002), or at longer times after dosage (e.g. 100 min post-L-DOPA, Morgese et al., 2007, 2009) but not at the peak of dyskinetic symptoms. Moreover, this effect of OEA was maintained over the entire 18-day chronic administration protocol without development of tolerance. This is particularly interesting, since tolerance is a common cannabinoid characteristic that could complicate or negate therapeutic use. In previous studies with other cannabinoids, the drugs were not administered for more than three days; thus, the development

Fig. 8. OEA reduces L-DOPA-induced phosphoacetylation of histone 3 via TRPV1 receptors. (A) Striatal quantification of pAcH3-positive cells after treatment with L-DOPA and OEA. Only the OEA 5 mg/kg dose significantly reduces pAcH3-ir expression induced by LDOPA. (B) Striatal quantification of pAcH3-positive cells after pretreatment with capsaicin or GW 6471 and the effective dose of OEA 5 mg/kg. Pretreatment with capsaicin, but not with GW 6471, abolishes the OEA-mediated reduction of pAcH3-positive cells. (C) High power photomicrographs of pAcH3 expression in lesioned striatum induced by the different treatments. Histograms represent number of pAcH3-positive nuclei, mean ± standard error of the mean, *p b 0.05 vs vehicle, one-way ANOVA. Scale bar = 50 μm, V = vehicle, Cap = capsaicin, GW = GW 6471, n = 7–13 per group.

of tolerance cannot be ruled out (Martínez et al., 2012; Morgese et al., 2009). This absence of tolerance of OEA is critical, since only chronic L-DOPA treatment is effective for Parkinson's disease. The medium dose (5 mg/kg) of OEA also reduced the total number of contralateral turns. Contralateral turning is an index of behavioral sensitization, which has been shown to correlate with dyskinesia (Hodgson et al., 2009; Carta et al., 2007). Therefore, our results suggest that OEA may reduce dyskinesias by decreasing behavioral sensitization of contralateral rotation. L-DOPA-induced sensitization of contralateral turning depends primarily on the activation of sensitized D1R (Darmopil et al., 2009). This supersensitivity is caused by an increase in the post-synaptic scaffolding protein PSD95 in D1R-containing neurons, altering D1R trafficking (Berthet et al., 2009; Porras et al., 2012) and increasing downstream receptor signaling (Aubert et al., 2005), rather than a D1R overexpression. Our results are consistent with this: OEA reduced D1-dependent signaling, as demonstrated by a decrease in the molecular markers after L-DOPA treatment, concomitant with a reduction in behavioral sensitization. Although higher doses of OEA induce hypolocomotor effects (Rodríguez de Fonseca et al., 2001), the OEA-induced reduction in dyskinesias and contralateral rotations that we observed is not due to a general decrease in motor activity since the effective anti-dyskinetic dose (5 mg/kg) had no effect in any of the locomotor tests performed after acute or chronic administration. This is important since other groups have been unable to dissociate cannabinoid-mediated antidyskinetic effects from their locomotor suppressant effects (Lee et al., 2006; Segovia et al., 2003; Walsh et al., 2010). This dual effect is not unusual for cannabinoids: low and high cannabinoid doses have been shown previously to induce different and sometimes opposite

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effects (Guzmán et al., 2001; Sarne and Keren, 2004). Based on these dual dose-response curves, studies of cannabinoids require careful doseresponse of both anti-dyskinetic effects and effects on motor activity. Thus previous studies of anti-dyskinetic effects of a single cannabinoid dose may need to be re-evaluated. Consistent with the absence of locomotor effects, 5 mg/kg OEA improved performance on the rotarod test for hemiparkinsonian mice measured 40 min after L-DOPA delivery, indicating an improvement in motor coordination while dyskinesias are at peak intensity. At 180 min after L-DOPA, when dyskinesias have worn off, pretreatment with OEA had no effect on latency time on the rotarod. Similarly, pretreatment with OEA did not alter horizontal and vertical activities recorded at 180 min after L-DOPA. These results demonstrate that OEA does not alter L-DOPA's therapeutic effect and that the better performance of OEA-treated mice in the rotarod at 40 min is likely due to the decrease in dyskinetic symptoms. Several previous studies have shown that cannabinoids can reduce dyskinesias via CB1 receptors, as several CB1-specific agonists and antagonists significantly reduce dyskinesias in rats (Martínez et al., 2012; Morgese et al., 2007, 2009; Segovia et al., 2003; Walsh et al., 2010) and in MPTP-lesioned primates (Fox et al., 2002; van der Stelt et al., 2005). CB1 receptors are widely expressed in the basal ganglia motor circuit, including the striatum, globus pallidus and SNpr, the main output nuclei, so the activation of CB1 may correct the imbalance of GABAergic and glutamatergic signaling observed in the dyskinetic state (see Brotchie, 2005; Fernández-Ruiz, 2009). However, OEA does not bind to CB1 receptors (Piomelli et al., 1998; Rodríguez de Fonseca et al., 2001), so its antidyskinetic effects cannot be directly related to CB1 receptor stimulation. OEA has been identified as an endogenous agonist of nuclear receptor PPARα (Fu et al., 2003; Lo Verme et al., 2005) and also as an antagonist of the ionotropic TRPV1 receptor (Almasi et al., 2008; Thabuis et al., 2008; Wang et al., 2005). Our results indicate that the antidyskinetic effects of OEA are mediated by TRPV1 and not by PPARα. Although PPARα is present in the nigrostriatal circuit (Galán-Rodríguez et al., 2009; González-Aparicio et al., 2011) and has shown to mediate behavioral sensitization in other systems such as the opioid (Fernández-Espejo et al., 2009), pretreatment with GW 6471, a PPARα antagonist, did not modify the antidyskinetic or anti-rotational effects of OEA. In contrast pretreatment with the TRPV1 receptor agonist capsaicin abolished the antidyskinetic action of OEA. TRPV1 receptors are present in several basal ganglia structures such as the striatum, globus pallidus and SN. Thus, OEA may exert its antidyskinetic actions by modulating neurotransmission in the basal ganglia via TRPV1 (Marinelli et al., 2003). Moreover, Morgese et al. (2007) reported that increasing endocannabinoid levels by blocking FAAH reduced AIMs only if coadministered with TRPV1 antagonist capsazepine. Together, these findings suggest that blocking TRPV1 receptors is important to unmask the antidyskinetic effect of endocannabinoids. Further confirming the role of the TRPV1 receptor in mediating the effects of endocannabinoids, certain motor effects mediated by endocannabinoids are only reversed by the TRPV1 receptor antagonist capsazepine, and not by rimonabant, a CB1 receptor antagonist (de Lago et al., 2004; Lastres-Becker et al., 2002, 2003). These results support the importance of TRPV1 receptors in endocannabinoid-mediated motor effects, and explain our finding of an antidyskinetic effect of OEA based on its antagonism to TRPV1 receptors. The relationship between CB1 and TRPV1 may also be important for the anti-dyskinetic action of endocannabinoids. These receptors are coexpressed in the striatum, globus pallidus and SN. Several studies have shown that their activation has opposite effects in diverse actions, including changes in intracellular Ca+2 concentration (Szallasi and Di Marzo, 2000) and glutamate release (Marinelli et al., 2003). Other authors showed a functional cross-talk between CB1 and TRPV1 receptors when they are co-expressed in the same cell (Cristino et al., 2006; Hermann et al., 2003). This is interesting, because OEA could reinforce

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the antidyskinetic actions of TRPV1 and CB1 receptors. For example, a blockade of TRPV1 may reinforce the CB1-mediated anti-dyskinetic effects of endocannabinoids that bind both CB1 and TRPV1 receptors such as anandamide (Morgese et al., 2007). OEA can enhance the effects of other CB1-acting endocannabinoids via “entourage” effects (Ben-Shabat et al., 1998; García et al., 2009; Ho et al., 2008; Smart et al., 2002), and enhancement in endocannabinoid tone protects against the development of seizures in viral-induced dyskinetic rats (Solbrig et al., 2005), so OEA may indirectly exert antidyskinetic effects via CB1 receptors. In 6-OHDA-treated rodents, the severity of L-DOPA-induced dyskinesias correlates with increased levels of FosB/ΔFosB (Andersson et al., 1999; Pavón et al., 2006) and pAcH3 (Darmopil et al., 2009) in striatonigral MSNs. Most studies with cannabinoids have only shown acute or subchronic (3 days) behavioral antidyskinetic results, without examining molecular events. We demonstrate that OEA 5 mg/kg reduced L-DOPA-induced striatal overexpression of FosB and pAcH3 at 60min post L-DOPA administration. Moreover, this reduction was mediated by the TRPV1 receptor, because it was abolished by pretreatment with capsaicin. This is the first demonstration of a correlation between sustained cannabinoid-mediated antidyskinetic effects and reduction of FosB and H3 molecular changes. These markers are linked to dopamine and glutamate receptor stimulation (Cenci and Konradi, 2010; Darmopil et al., 2009). It is possible that OEA reduces glutamate release, as the TRPV1 antagonist capsazepine does (Marinelli et al., 2003), reducing the molecular changes and dyskinesias. Reducing striatal glutamatergic inputs has an anti-dyskinetic effect (Bido et al., 2011; Dupre et al., 2011) and other cannabinoid agonists may decrease dyskinesia by modulating the glutamatergic input from cortico-striatal afferents (Gubellini et al., 2002). Another possibility is that OEA reduces striatal release of serotonin from brainstem neurons, at the same time reducing the release of the dopamine that accumulates as a false transmitter in these terminals following L-DOPA treatment (Carta et al., 2007). In conclusion, this study supports the hypothesis that the endocannabinoid system plays an important role in the development and expression of dyskinesias. The systemic administration of OEA reduces the development of dyskinesias and associated molecular changes in mice through a TRPV1-dependant pathway, opening new pharmacological strategies and suggesting a possible approach for the treatment of this neurological disorder. Acknowledgments This work was supported by grants from the Spanish Ministries de Economía y Competitividad and Sanidad y Política Social, ISCIII: BFU2010-20664, PNSD, RedRTA (RD06/0001/1011), CIBERNED ref. CB06/05/0055, and Comunidad de Madrid ref. S2011/BMD-2336 to RM. The authors would like to thank Mrs. Emilia Rubio and Mr. Marco de Mesa for their technical assistance, Mr. Mario Lewandowski for animal care and Dr. Noelia Granado, Mrs. Irene Ruiz de Diego, and Mr. Oscar Solís, for their excellent technical help. The authors report no biomedical financial interests or potential conflicts of interest. References Almasi, R., Szoke, E., Bölcskei, K., Varga, A., Riedl, Z., Sandor, Z., et al., 2008. Actions of 3methyl-N-oleoyldopamine, 4-methyl-N-oleoyldopamine and N-oleoylethanolamide on the rat TRPV1 receptor in vitro and in vivo. Life Sci. 82, 644–651. Andersson, M., Hilbertson, A., Cenci, M.A., 1999. 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. 6, 461–474. Ares-Santos, S., Granado, N., Oliva, I., O'Shea, E., Martin, E.D., Colado, M.I., et al., 2012. Dopamine D1 receptor deletion strongly reduces neurotoxic effects of methamphetamine. Neurobiol. Dis. 45, 810–820. Aubert, I., Guigoni, C., Håkansson, K., Li, Q., Dovero, S., Barthe, N., et al., 2005. Increased D1 dopamine receptor signaling in levodopa-induced dyskinesia. Ann. Neurol. 57, 17–26.

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