Cristina Alcacer

Emanuela Santini

Emmanuel Valjent

Florence Gaven

Jean-Antoine Girault

Dr Denis Hervé
email: denis.herve@inserm.fr

Extracellular Signal-Regulated Kinase-Dependent Signaling in L-3,4-Dihydroxyphenylalanine-Induced Dyskinesia

INTRODUCTION

Parkinson's disease (PD) results from neurodegeneration of nigrostriatal dopamine (DA) neurons. Despite innovative therapies, the DA precursor L-DOPA remains the drug of reference. However, after an initial period of full efficacy, this treatment is complicated by L-DOPA-induced dyskinesia (LID) with an incidence of 30-50% after 5 years and 60% after 10 years [START_REF] Rascol | Medical treatment of levodopa-induced dyskinesias[END_REF][START_REF] Van Gerpen | Levodopa-associated dyskinesia risk among Parkinson disease patients in Olmsted County, Minnesota, 1976-1990[END_REF]. Better understanding of the neuronal mechanisms underlying the development of LID is therefore important to identify therapeutic strategies [START_REF] Cenci | Dopamine dysregulation of movement control in L-DOPA-induced dyskinesia[END_REF][START_REF] Jenner | Molecular mechanisms of L-DOPA-induced dyskinesia[END_REF]. DA D1 receptor (D1R) agonists are more efficient than D2R agonists to induce dyskinesia in animal models and PD patients [START_REF] Calon | Chronic D1 and D2 dopaminomimetic treatment of MPTP-denervated monkeys: effects on basal ganglia GABA(A)/benzodiazepine receptor complex and GABA content[END_REF][START_REF] Rascol | Induction by dopamine D1 receptor agonist ABT-431 of dyskinesia similar to levodopa in patients with Parkinson disease[END_REF][START_REF] Rascol | Development of dyskinesias in a 5-year trial of ropinirole and L-dopa[END_REF][START_REF] Carta | Behavioral and biochemical correlates of the dyskinetic potential of dopaminergic agonists in the 6-OHDA lesioned rat[END_REF]. D1Rs are necessary for LID development [START_REF] Westin | Spatiotemporal pattern of striatal ERK1/2 phosphorylation in a rat model of L-DOPA-induced dyskinesia and the role of dopamine D1 receptors[END_REF][START_REF] Darmopil | Genetic inactivation of dopamine D1 but not D2 receptors inhibits L-DOPA-induced dyskinesia and histone activation[END_REF]. D1R signaling through cAMP-dependent protein kinase (PKA) and 32-kDa DA-and cAMP-regulated phosphoprotein (DARPP-32) is activated by L-DOPA in 6-OHDA-lesioned rodents [START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF][START_REF] Lebel | Striatal inhibition of PKA prevents levodopainduced behavioural and molecular changes in the hemiparkinsonian rat[END_REF] or MPTP-treated monkeys [START_REF] Santini | Distinct changes in cAMP and extracellular signal-regulated protein kinase signalling in L-DOPA-induced dyskinesia[END_REF]. Strong D1R-dependent activation of extracellular signal-regulated kinase (ERK) [START_REF] Gerfen | D1 dopamine receptor supersensitivity in the dopamine-depleted striatum results from a switch in the regulation of ERK1/2/MAP kinase[END_REF][START_REF] Pavon | ERK phosphorylation and FosB expression are associated with L-DOPA-induced dyskinesia in hemiparkinsonian mice[END_REF][START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF][START_REF] Westin | Spatiotemporal pattern of striatal ERK1/2 phosphorylation in a rat model of L-DOPA-induced dyskinesia and the role of dopamine D1 receptors[END_REF][START_REF] Rylander | Pharmacological modulation of glutamate transmission in a rat model of L-DOPA-induced dyskinesia: effects on motor behavior and striatal nuclear signaling[END_REF]Santini et al., 2009b) and mTOR signaling (Santini et al., 2009a) is also observed in striatonigral medium-sized spiny neurons (MSNs). Pharmacological or genetic blockade of these pathways supports their necessary role in LID [START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF]Santini et al., 2009a;[START_REF] Bateup | Distinct subclasses of medium spiny neurons differentially regulate striatal motor behaviors[END_REF][START_REF] Fasano | Inhibition of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) signaling in the striatum reverts motor symptoms associated with L-dopainduced dyskinesia[END_REF][START_REF] Lebel | Striatal inhibition of PKA prevents levodopainduced behavioural and molecular changes in the hemiparkinsonian rat[END_REF]. However, the cause of their activation in DA-deprived striatum and their relative contribution in LID development are unknown. Levels of D1R [START_REF] Shinotoh | Dopamine D1 receptors in Parkinson's disease and striatonigral degeneration: a positron emission tomography study[END_REF][START_REF] Turjanski | In vivo studies on striatal dopamine D1 and D2 site binding in L-dopa-treated Parkinson's disease patients with and without dyskinesias[END_REF][START_REF] Hurley | Dopamine D(1) receptor expression in human basal ganglia and changes in Parkinson's disease[END_REF] and major mediators of D1R-signaling, including PKA and DARPP-32 [START_REF] Girault | Striatal phosphoproteins in Parkinson disease and progressive supranuclear palsy[END_REF][START_REF] Nishino | Transmembrane signalling systems in the brain of patients with Parkinson's disease[END_REF] are unchanged in PD and its animal models. In MSNs D1Rs and adenosine A2A receptors (A2AR) are coupled to adenylyl-cyclase through a heterotrimeric Gprotein comprising Gαolf, Gβ2, and Gγ7 subunits [START_REF] Drinnan | G(olf) in the basal ganglia[END_REF][START_REF] Herve | G(olf) and Gs in rat basal ganglia: possible involvement of G(olf) in the coupling of dopamine D1 receptor with adenylyl cyclase[END_REF][START_REF] Kull | Adenosine A(2A) receptors are colocalized with and activate g(olf) in rat striatum[END_REF][START_REF] Zhuang | G(olf)alpha mediates dopamine D1 receptor signaling[END_REF][START_REF] Corvol | Galpha(olf) is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum[END_REF][START_REF] Schwindinger | Loss of G protein gamma 7 alters behavior and reduces striatal alpha(olf) level and cAMP production[END_REF][START_REF] Schwindinger | Adenosine A2A receptor signaling and golf assembly show a specific requirement for the gamma7 subtype in the striatum[END_REF]. Levels of Gαolf increase in DA-denervated striatum of rats and PD patients [START_REF] Herve | G(olf) and Gs in rat basal ganglia: possible involvement of G(olf) in the coupling of dopamine D1 receptor with adenylyl cyclase[END_REF][START_REF] Marcotte | Striatal G-proteins: effects of unilateral 6hydroxydopamine lesions[END_REF][START_REF] Penit-Soria | Levels of stimulatory G protein are increased in the rat striatum after neonatal lesion of dopamine neurons[END_REF][START_REF] Corvol | Persistent increase in olfactory type G-protein alpha subunit levels may underlie D1 receptor functional hypersensitivity in Parkinson disease[END_REF][START_REF] Rangel-Barajas | L-DOPAinduced dyskinesia in hemiparkinsonian rats is associated with up-regulation of adenylyl cyclase type V/VI and increased GABA release in the substantia nigra reticulata[END_REF]. Since Gαolf levels are a limiting factor for receptor signaling [START_REF] Corvol | Galpha(olf) is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum[END_REF][START_REF] Corvol | Quantitative changes in Galphaolf protein levels, but not D1 receptor, alter specifically acute responses to psychostimulants[END_REF], Gαolf up-regulation is a plausible but untested mechanism for the D1R hypersensitivity in LID-developing animals.

The objective of this study was to examine the association of Gαolf with LID and evaluate the respective contribution of cAMP-and ERK-mediated pathways in LID. We used a mouse model of hemiparkinsonism treated with L-DOPA [START_REF] Lundblad | A model of L-DOPA-induced dyskinesia in 6-hydroxydopamine lesioned mice: relation to motor and cellular parameters of nigrostriatal function[END_REF] and heterozygous mutant mice lacking one allele of the Gαolf gene (Gnal) [START_REF] Belluscio | Mice deficient in G(olf) are anosmic[END_REF][START_REF] Corvol | Quantitative changes in Galphaolf protein levels, but not D1 receptor, alter specifically acute responses to psychostimulants[END_REF]. This approach allowed us to parse the role of these various signaling components in the apparition of LID.

MATERIAL AND METHODS

Animals

We used 8-week-old C57Bl/6J male mice. Mice heterozygous for a null mutation of the gene encoding Gαolf (Gnal) kindly provided by L. Belluscio [START_REF] Belluscio | Mice deficient in G(olf) are anosmic[END_REF] were backcrossed for eight generations with C57Bl/6J wild type mice and mated with C57Bl/6J mice for producing heterozygous Gnal mutant mice and wild type littermates. Animals less than 30-week old were used for the experiments. The mice were maintained in a 12 h lightdark cycle, in stable conditions of temperature (22°C), with free access to food and water. All the experiments were in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals (decree 87-848) and were conducted under the approval of the "Direction Départementale de la Protection des Populations de Paris" (authorization number C-75-828, license B75-05-22).

6-OHDA lesions and post-operative care

Mice were anesthetized with a mixture of xylazine (10 mg/mL) and pentobarbital (25 mg/mL) (Centravet Plancoet, La Milière, France), and mounted in a digitalized stereotactic frame (Stoelting Europe, Dublin, Ireland) equipped with a mouse adaptor. 6-OHDA-HCl (3.0 mg/mL, Sigma-Aldrich, Lyon, France) was dissolved in a solution containing 0.2 g/L ascorbic acid and 9 g/L NaCl. Mice received two unilateral injections (2 x 2 µL) of 6-OHDA into the right striatum at the following coordinates according to the mouse brain atlas of [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF]: AP= +1.2 mm, L= +2.1 mm, DV= -3.2 mm; and AP= +0.6 mm, L= +2.4 mm, DV= -3.2 mm. Each injection was performed with a 36-gauge, 50mm long stainless steel cannula connected to a syringe pump (MTI Corporation, UC Berkeley, USA) by a polyethylene catheter, at a slow rate of 0.25 µL/min to minimize tissue damage. After the injection, the cannula was left in place for an additional 4 min before being slowly retracted. Sham mice were injected with vehicle only (ascorbic acid in saline).

Post-operative care was needed to minimize the mortality rate. Mice were let on a warm plate during about 24 h after surgery in order to avoid hypothermia. To reduce suffering, mice received subcutaneous (s.c.) injections of a non-steroidal anti-inflammatory drug (flunixin meglumine, 4 mg/kg, Sigma-Aldrich), just after the surgery and twice daily during 2 days post-surgery. All the lesioned mice were examined daily and the weakest animals received injections of sucrose 5% (10 mL/kg, s.c.) and intraperitoneal (i.p.) saline (10 mL/kg) to avoid dehydration. Concentrated milk was provided to all the lesioned animals during 3 days after the operation. Mice were allowed to recover for 4 weeks before behavioral evaluation and drug treatment. Lesions were assessed at the end of experiments by determining the striatal levels of tyrosine hydroxylase (TH) using immunohistochemistry or immunoblotting (see below). Only animals with a TH-depleted area above 80% of the striatum area and/or TH level reduction by more than 80% in the lesioned striatal area as compared to the control side were included in the analyses (i.e. about 85% of surviving lesioned mice).

L-DOPA and benserazide treatments

L-DOPA and the peripheral DOPA decarboxylase inhibitor benserazide hydrochloride (Sigma-Aldrich) were dissolved together in physiological saline solution (NaCl, 9 g/L). L-DOPA and benserazide hydrochloride were injected i.p. at a dose of 20 and 12 mg/kg, respectively in a volume of 10 mL/kg of body weight. For acute treatments mice were euthanasied 30 min after the first injection of L-DOPA and benserazide. The chronic treatment consisted in a daily single injection of L-DOPA and benserazide during 10 days. Mice were sacrificed on day 11, 30 min after the last injection.

Behavioral tests

Cylinder test. On day 1 of chronic treatment, the anti-parkinsonian effects of L-DOPA on sensorimotor function were evaluated in the cylinder test [START_REF] Lundblad | Pharmacological validation of behavioural measures of akinesia and dyskinesia in a rat model of Parkinson's disease[END_REF]. Each lesioned mouse was tested before the beginning of L-DOPA therapy and 1 h after the first injection of L-DOPA plus benserazide. Mice were placed one by one in glass cylinders (10 cm diameter and 15 cm height) and videorecorded for 5 min without previous habituation to the glass cylinder. Once introduced into the cylinder, the mice showed an exploratory behavior reflected by rearing and forepaw contacts on the wall. The number of contacts with the right or left forepaw was counted in blind conditions regarding mouse genotype. To eliminate accidental touches without physiological meaning, we counted only contacts in which the animal supported its body weight on the paw with extended digits. The use of the impaired (left) forepaw was expressed as a percentage of the total number of contacts on the wall.

Abnormal Involuntary Movements (AIMs).

6-OHDA-lesioned mice were chronically treated with L-DOPA for 10 days. AIMs were assessed on day 5 (two experiments) and day 10 (all experiments) in blind conditions regarding mouse genotype, using a previously validated scale for scoring LID in mouse [START_REF] Lundblad | A model of L-DOPA-induced dyskinesia in 6-hydroxydopamine lesioned mice: relation to motor and cellular parameters of nigrostriatal function[END_REF][START_REF] Lundblad | Pharmacological validation of a mouse model of l-DOPA-induced dyskinesia[END_REF]. Immediately after L-DOPA administration, mice were placed in separate cages and abnormal movements were assessed for 1 min (monitoring period) every 20 min, over a period of 140 min. Abnormal movements, clearly distinct from natural stereotyped behaviors (i.e., grooming, sniffing, rearing, and gnawing), were classified into four different subtypes: locomotive (tight contralateral turns), axial (contralateral dystonic posture of the neck and upper body), limb (jerky and fluttering movements of the limb contralateral to the side of the lesion), and orofacial (vacuous jaw movements and tongue protrusions) AIMs. Each subtype was scored on a severity scale from 0 to 4: 0, absent; 1, occasional; 2, frequent; 3, continuous; 4, continuous and not interruptible by external stimuli. The total AIMs score corresponded to the sum of individual scores for each AIM subtype. A composite score was obtained by the addition of scores for axial, limb and orofacial AIMs (ALO score). The ALO score is considered to more closely reflect the human dyskinetic behavior than the locomotive AIMs score (LOC score) [START_REF] Lundblad | Pharmacological validation of behavioural measures of akinesia and dyskinesia in a rat model of Parkinson's disease[END_REF]. To identify the extreme behaviors highly and weakly dyskinetic mice were separated by a quartile analysis based on total AIMs scores.

Tissue preparation and immunofluorescence

Thirty minutes after the last injection of L-DOPA, mice were rapidly anesthetized with pentobarbital (500 mg/kg, i.p., Sanofi-Aventis) and perfused transcardially with 40 g/L paraformaldehyde in phosphate buffered saline (PBS, 0.1 M sodium phosphate, 0.14 M NaCl), pH 7.4. Brains were postfixed overnight in the same solution at 4°C. The following day, 30-µm-thick sections were cut with a Vibratome (Leica, Nussloch, Germany) and stored at -20°C in a buffered solution containing 30% volume/volume (v/v) ethylene glycol, 30% (v/v) glycerol, and 0.1 M sodium phosphate pH 7.4, until they were processed for immunofluorescence. Brain regions were identified using a mouse brain atlas [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF], and sections at +0.86 mm from the bregma (dorsal striatum) were selected and processed as follows.

Day 1. Free floating sections were rinsed in Tris-buffered saline (TBS; 0.10 M Tris, 0.14 M NaCl), pH 7.4, incubated for 5 min in TBS containing 3% H 2 O 2 (v/v) and 10% methanol (v/v), and rinsed three times 10 min in TBS. After 20-min incubation in 0.2% Triton X-100 in TBS (v/v), sections were rinsed three times in TBS, blocked with BSA 30 g/L in TBS, and incubated overnight (or longer as indicated) at 4°C with the primary antibodies. For detection of phosphorylated proteins, 0.1 M NaF was included in all buffers and incubation solutions. Antibodies were a mouse monoclonal antibody for tyrosine hydroxylase (TH) (1:500 with an incubation ≥ 2 days; Sigma-Aldrich), a rabbit polyclonal antibody for Gαolf protein (1:500) [START_REF] Herve | Galpha(olf) levels are regulated by receptor usage and control dopamine and adenosine action in the striatum[END_REF], rabbit polyclonal antibodies for phospho-Thr202/Tyr204-ERK1/2 (phospho-ERK, 1:400; Cell Signaling Technology), phospho-Ser10-acetyl-Lys14histone H3 (pAcH3, 1:500, Upstate Ltd, UK), and phospho-(Ser/Thr) PKA substrate (phospho-PKA substrate, 1:500; Cell Signaling Technology).

Day 2. Following incubation with primary antibodies, sections were rinsed three times for 10 min in TBS and incubated for 45 min with goat Cy3-coupled (1:400; Jackson Laboratory, Bar Harbor, ME, USA) or goat A488-coupled (1:400; Invitrogen) secondary antibodies. Finally, sections were rinsed for 10 min twice in TBS and twice in Tris buffer (0.25 M Tris, pH 7.4) before mounting in Vectashield (Vector Laboratories). For immunofluorescence detection with Odyssey-LI-COR infrared fluorescent detection system (see below), suitable secondary antibodies were used: IRDye 700DX conjugated anti-rabbit IgG and IRDye 800CW conjugated anti-mouse IgG (Rockland Immunochemical Inc., Gilbertsville, PA, USA), diluted in TBS (1/1000). After rinsing as above, sections were mounted on glass slides and dried 2 h. For determining the TH-depleted area in the lesioned mice, TH immunoreactivity was revealed by immunohistochemistry using biotinylaled antibodies, avidine-biotine-peroxidase complex (Vector Laboratories) and 3,3'diaminobenzidine (DAB) staining [START_REF] Valjent | Addictive and non-addictive drugs induce distinct and specific patterns of ERK activation in mouse brain[END_REF].

Confocal microscopy analysis of brain sections

Confocal microscopy and image analysis were performed at the Institut du Fer à Moulin imaging facility. Images from each region of interest were obtained with a sequential laserscanning confocal microscope (SP5; Leica) using the same adjustments for all the sections from one experiment. The number of phospho-ERK-positive cells was counted manually in 375 x 375 µm confocal images taken in the lesioned and unlesioned dorsolateral striatum in blind conditions concerning the mouse genotype. MacBiophotonics Image J was used to count automatically the number of nuclei positive for pAcH3 and phospho-PKA substrate. TH and Gαolf immunofluorescence intensity was quantified in the striatum with MacBiophotonics Image J and the data represented mean gray levels above background value in 375 x 375 µm confocal images. The background level was evaluated by measuring the mean gray values in large fiber bundles passing through the striatum. All measurements were carried out in blind conditions regarding the mouse treatment and genotype.

LI-COR analysis of brain sections

In some experiments, TH, Gαolf and pAcH3 immunofluorescence intensity was measured using the Odyssey-LI-COR infrared fluorescent detection system (LI-COR, Lincoln, NE, USA). This system commonly used to quantify immunoblots can also be used to quantify immunofluorescence in regions of interest of tissue sections. All sections were scanned together at 21-µm resolution. The regions of interest were drawn as a circle of 1 mm of diameter in the dorsolateral striatum on the lesioned and unlesioned sides. The mean fluorescence intensity was measured in the regions of interest and the background determined in the cerebral cortex, a non-immunoreactive region. The validity of this approach to evaluate Gαolf levels was tested by determining the relationship between the fluorescence intensity and the amount of striatal tissue. Samples containing increasing amounts of Gαolf were prepared by mixing extracts (10 mg/mL protein in 2%, weight/vol SDS) of striatum and cortex that expresses and does not express Gαolf, respectively. Two µL of these samples were spotted on nitrocellulose membrane (Hybond ECL, GE Healthcare Europe, Velizy-Villacoublay, France). After fixation in a Ponceau S solution (Sigma-Aldrich), the membrane was blocked in BSA (30 g/L in TBS) and incubated in anti-Gαolf primary and IRDyeconjugated anti-rabbit secondary antibodies as described above. The mean fluorescence intensity of each dot was measured using the Odyssey-LI-COR detection system and the background determined as the fluorescence intensity of the dots containing only cortex extract. In parallel, free floating brain sections were incubated in the same solutions as the nitrocellulose membrane and mean fluorescence intensity was measured in various striatal areas as described above.

Immunoblotting

Mice were sacrificed by decapitation 30 min after L-DOPA plus benserazide injection. The heads of the animals were immediately immersed in liquid nitrogen for 6 s. The brains were removed and the striata dissected out within 20 s on an ice-cold surface, sonicated in 750 µL of 10 g/L sodium dodecyl sulfate (SDS), and placed at 100°C for 10 min. The effectiveness of this extraction procedure in preventing protein phosphorylation and dephosphorylation, hence ensuring that the level of phosphoproteins measured ex vivo reflects the in vivo situation, has been previously tested [START_REF] Svenningsson | Regulation of the phosphorylation of the dopamine-and cAMP-regulated phosphoprotein of 32 kDa in vivo by dopamine D1, dopamine D2, and adenosine A2A receptors[END_REF]. Aliquots (5 µL) of the homogenate were used for protein determination using a bicinchoninic acid assay kit (Pierce Europe, Oud Beijerland, the Netherlands). Equal amounts of protein (20 µg) were separated by 10% (w/v) polyacrylamide gel electrophoresis in the presence of SDS and transferred overnight to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Uppsala, Sweden) [START_REF] Towbin | Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications[END_REF]. The membranes were immunoblotted using phospho-Ser845 (Cell Signaling Technology, Beverly, MA) and phosphorylation state-independent (Upstate Biotechnology) glutamate AMPA receptor subunit 1 (GluA1) antibodies, phospho-Thr34 rabbit polyclonal and phosphorylation state-independent mouse monoclonal DARPP-32 antibodies (both gifts of P. Greengard, the Rockefeller University, New York), and TH monoclonal mouse antibodies (Chemicon, Temecula, CA). Secondary antibodies (1/5000) were IRDye 700DX conjugated anti-rabbit IgG and IRDye 800CW conjugated anti-mouse IgG (Rockland Immunochemical Inc., Gilbertsville, PA, USA). Their binding was quantified using an Odyssey-LI-COR infrared fluorescent detection system (LI-COR, Lincoln, NE, USA). The levels of phosphoproteins were normalized for the amount of the corresponding total protein detected in the sample.

Statistics

Correlations between variables were estimated using multiple regression analysis. Multiple comparisons were analyzed using two-way ANOVA followed by Bonferroni post hoc tests for specific comparisons. For 2-group comparisons two-tailed paired or unpaired Student ttest was used.

RESULTS

Gαolf is up-regulated in the 6-OHDA-lesioned dorsolateral mouse striatum

Increased Gαolf protein levels have been reported in the dorsal striatum of 6-OHDA-lesioned rats [START_REF] Herve | G(olf) and Gs in rat basal ganglia: possible involvement of G(olf) in the coupling of dopamine D1 receptor with adenylyl cyclase[END_REF][START_REF] Marcotte | Striatal G-proteins: effects of unilateral 6hydroxydopamine lesions[END_REF][START_REF] Penit-Soria | Levels of stimulatory G protein are increased in the rat striatum after neonatal lesion of dopamine neurons[END_REF][START_REF] Corvol | Persistent increase in olfactory type G-protein alpha subunit levels may underlie D1 receptor functional hypersensitivity in Parkinson disease[END_REF][START_REF] Rangel-Barajas | L-DOPAinduced dyskinesia in hemiparkinsonian rats is associated with up-regulation of adenylyl cyclase type V/VI and increased GABA release in the substantia nigra reticulata[END_REF] and in the putamen of PD patients [START_REF] Corvol | Persistent increase in olfactory type G-protein alpha subunit levels may underlie D1 receptor functional hypersensitivity in Parkinson disease[END_REF]. In contrast, Gαolf levels after DA depletion had never been explored in the hemiparkinsonian mouse model. In a first experiment we investigated the levels of Gαolf in the dorsal striatum of 6-OHDA-lesioned mice. Twelve mice (55% of the operated mice) survived and were well lesioned [see Methods for criteria]. We evaluated the lesion-induced changes in Gαolf and other markers in striatal sections using two different methods of immunofluorescence measurement with either the Odyssey-LI-COR system (Fig. 1A-D) or confocal microscopy (Fig. 1E,F).

The striatal 6-OHDA injections induced a dramatic decrease in TH immunofluorescence detected with Odyssey-LI-COR (Fig. 1A). TH immunoreactivity consistently disappeared in the most dorsolateral part of the striatum, although the lesion extension in the medioventral part varied from one animal to another. To detect the precise extension of D1R hypersensitivity within the striatum and to measure the levels of Gαolf specifically in this region, we used the L-DOPA-induced increase in phospho-Ser10, acetyl-Lys14 histone H3 (pAcH3), a prominent response directly linked to D1R hypersensitivity (Santini et al., 2009b;[START_REF] Cenci | Maladaptive striatal plasticity in L-DOPA-induced dyskinesia[END_REF]. Lesioned mice were treated with L-DOPA (20 mg/kg) and benserazide (12 mg/kg) 30 min before the sacrifice, a treatment which does not alter Gαolf levels (Corvol et al., 2004 and Alcacer and Hervé, unpublished observations), and the distribution of pAcH3-positive cells was determined in the striatum (Fig. 1B). An intense pAcH3 signal was detected in response to L-DOPA treatment in the dorsolateral part of the TH-depleted striatum (Fig. 1B). Interestingly the area of strong pAcH3-immunoreactivity was consistently more restricted than the TH-depleted zone (Fig. 1A,B).

In order to validate the use of Odyssey-LI-COR for evaluating Gαolf levels in tissue, we compared immunofluorescence signals measured in striatal sections with those obtained with various concentrations of striatal homogenates in dot blot (Fig. 1C). In dot blot, fluorescence intensity was linearly related with the concentration of striatal tissue (and thus of Gαolf) on a wide scale. Using this approach, Gαolf immunofluorescence was found to increase in the dorsolateral part of the striatum where TH immunofluorescence was absent and pAcH3 present, as compared to the contralateral side (Fig. 1D). Very similar results were obtained using confocal microscopy (Fig. 1E andF). Since quantification of images from confocal microscope provided results similar to those with Odyssey-LI-COR, we used confocal imaging in the rest of the study. Thus, our results revealed a significant increase in Gαolf immunoreactivity within the DA-deficient region of the striatum of 6-OHDA-lesioned mice, coincident with the area of increased D1R signaling.

Striatal Gαolf increase correlates with dyskinetic behavior

Since Gαolf immunoreactivity was higher in the mouse dorsolateral striatum after the lesion, we examined whether this increase was maintained after chronic treatment with L-DOPA and whether it was associated with the development of LID. To address this question 6-OHDAlesioned mice received a 10-day treatment with L-DOPA (20 mg/kg per day) and benserazide (12 mg/kg) referred to as "L-DOPA" below. In a series of six experiments, 65 mice (68 % of the operated mice) survived the operation and displayed a decrease in striatal TH levels greater than 80%. One group received a single injection of L-DOPA (n = 21), another group was chronically treated with L-DOPA during 10 days (n = 44). An additional control group of 6 sham-lesioned mice also received L-DOPA chronically. Thirty minutes after the last injection of L-DOPA, all the animals were sacrificed to compare the Gαolf levels in their 6-OHDA-injected and intact striata by confocal immunofluorescence analysis. For comparison between different animals and experiments we used the immunofluorescence on the lesioned side/ immunofluorescence on the unlesioned side ratio (Fig. 2A). As expected, in the shamoperated mice, the Gαolf immunofluorescence intensities were similar in the vehicle-injected and intact striata (Fig. 2A). In contrast, striatal Gαolf immunofluorescence was significantly higher in the 6-OHDA-lesioned striatum than in the contralateral striatum in mice that had received one or repeated injections of L-DOPA (Fig. 2A).

Since among chronically L-DOPA-treated mice some animals developed intense LID but not others, we examined whether the levels of Gαolf differed between these two groups. To perform this analysis, we separated the highly dyskinetic mice (i.e. those having total AIMs scores in the quartile of highest values, higher than 33.5, n = 11) and the weakly dyskinetic mice (total AIMs score in the quartile of lowest values, lower than 12.5, n = 11), among a total number of 44 animals in 3 distinct experiments. The mice in both groups displayed more than 80% reduction in TH in the dorsolateral striatum (data not shown). In lesioned mice, we did not detect any significant correlation between TH depletion and AIM score (data not shown). In the quartile of highly dyskinetic mice, Gαolf immunofluorescence was higher in the lesioned striatum than in the contralateral unlesioned striatum (Fig. 2B). In contrast, in the quartile of weakly dyskinetic mice Gαolf immunofluorescence was similar on the two sides (Fig. 2B). We then examined whether there was a correlation between the levels of Gαolf and the intensity of AIMs, including all the lesioned animals that had received a chronic L-DOPA treatment. Although the experimental points appeared quite disperse, statistical analysis revealed a significant positive correlation between Gαolf immunofluorescence and the total AIMs score (Fig. 2C), the locomotor component of LID (LOC, Fig. 2D), and the composite score of axial-limb-orofacial (ALO) AIMs (Fig. 2E), which have been suggested to be more similar to the human LID than the locomotive AIMs [START_REF] Lundblad | Pharmacological validation of behavioural measures of akinesia and dyskinesia in a rat model of Parkinson's disease[END_REF]. From these experiments, we concluded that the increase in Gαolf in the dorsolateral striatum of hemiparkinsonian mice was somehow associated with the severity of dyskinetic behavior. This result suggested a possible implication of Gαolf striatal up-regulation in the development of LID in the mouse model of hemiparkinsonism.

Effects of 6-OHDA lesions on Gαolf in Gnal +/-mice

Given the positive correlation between Gαolf increases and dyskinesia, we used a genetic model in which Gαolf levels are decreased to examine the consequences of this reduction on LID and signaling responses. Homozygous Gαolf knockout mice (Gnal -/-) have a severe phenotype due to the combination of olfactory and striatal deficits [START_REF] Belluscio | Mice deficient in G(olf) are anosmic[END_REF][START_REF] Zhuang | G(olf)alpha mediates dopamine D1 receptor signaling[END_REF][START_REF] Corvol | Galpha(olf) is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum[END_REF][START_REF] Herve | Galpha(olf) levels are regulated by receptor usage and control dopamine and adenosine action in the striatum[END_REF]. These mice usually die in the early post-natal period and could not be used in our study. In contrast, Gnal +/-mice which develop and breed normally, provide a very interesting model since they display a decrease of about 50% in Gαolf protein levels (Fig. 3A) and a 30-50% deficit in D1R-or A2AR-activated cAMP production in vitro when compared to wild type littermates [START_REF] Corvol | Galpha(olf) is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum[END_REF][START_REF] Corvol | Persistent increase in olfactory type G-protein alpha subunit levels may underlie D1 receptor functional hypersensitivity in Parkinson disease[END_REF]. Since striatal Gαolf immunofluorescence was increased by about 50% in the lesioned striatum of dyskinetic mice whereas it was similar to the unlesioned tissue in weakly dyskinetic mice (see Fig. 2B), Gnal +/-mice in which Gαolf was reduced but not absent were a good model for testing the specific implication of Gαolf up-regulation.

Wild type (Gnal +/+) and heterozygous (Gnal +/-) mice were lesioned with 6-OHDA and, after a 4-week recovery, treated with L-DOPA during 10 days (17 well lesioned mice in each group survived the whole experimental procedure, corresponding to about 60% of operated mice). They were killed 30 min after the last L-DOPA injection and proteins of interest were analyzed by immunofluorescence or immunoblotting. Immunoblotting showed that the two groups of mice displayed the same degree of TH reduction in their lesioned striatum indicating that 6-OHDA had a similar ability to destroy DA innervation in the two genotypes (Fig. 3B). Immunohistochemistry indicated that the TH-depleted area was similar in wild type and Gnal +/-mice (87 ± 2 % and 92 ± 2% of the total striatum area, respectively, Student t-test, t = 1.48, DF = 18, not significant). To compare the changes in Gαolf immunofluorescence in mutant and wild type mice, three sections from each animal were labeled and quantified using confocal imaging in 3 independent experiments and the results were averaged (Fig. 3C). As expected, the levels of Gαolf detected with this method were lower in unlesioned striatum of mutant mice compared to wild type (Fig. 3C). Interestingly, Gαolf immunofluorescence levels in Gnal +/-mice were increased in the lesioned striatum when compared to the contralateral intact striatum (Fig. 3C). The relative increase on the lesioned side compared to the contralateral side was similar in the two genotypes (Gnal +/+ 132 ± 5 %, Gnal +/-, 145 ± 6 %, mean ± SEM, Student t-test t = 1.6, DF = 18, not significant). However, the levels of Gαolf immunofluorescence in the lesioned striatum of mutant mice remained below those in the intact striatum of wild type mice (-16%, Fig. 3C). Similar results were observed using the Odyssey-LI-COR system (data not shown). Altogether these results showed that the mechanisms of Gαolf increase following 6-OHDA lesion were unaltered in heterozygous mutant mice, but that the gene dosage effect maintained the levels of the protein below those in unlesioned wild type mice.

Behavioral responses of hemiparkinsonian Gnal +/-mice are similar to wild type

During L-DOPA treatment, the 6-OHDA-lesioned Gnal +/+ and Gnal +/-mice were evaluated in behavioral tests. Mice were first subjected to the cylinder test before (baseline) and after the first administration of L-DOPA. Before L-DOPA, Gnal +/+ and Gnal +/-mice displayed a marked reduction in the usage of the forelimb contralateral to the lesion (Fig. 4A), reflecting the hypokinetic effects of 6-OHDA-lesion. A similar recovery of the forelimb usage was observed in both genotypes 1 h after L-DOPA treatment. Thus, the anti-parkinsonian effect of L-DOPA appeared to be preserved in Gnal +/-mice.

At the end of the 10-day L-DOPA treatment, the animals were scored for the various types of AIMs. No significant difference was observed between Gnal +/-and Gnal +/+ mice in axial, limb and orofacial AIMs induced by chronic L-DOPA treatment, and the ALO composite score was not significantly changed in the mutant mice (Fig. 4B). Similarly, no significant difference in locomotive score was observed between the two genotypes (Fig. 4C). The total AIMs score and the time course of L-DOPA effects were not significantly affected by Gnal haplodeficiency (Fig. 4D). We also compared LID in Gnal +/-and wild type mice after only 5 days of L-DOPA treatment (Fig. 4E) and we detected no significant difference between the two genotypes, suggesting the absence of alteration in the LID development in Gnal +/-mice. Thus our results clearly showed that the partial deficit in Gαolf in heterozygous mice did not change the incidence and intensity of LID following a 10-day treatment.

L-DOPA-induced cAMP-dependent phosphorylation is dramatically reduced in the lesioned striatum of Gnal +/-mice

We have previously reported a dissociated alteration of signaling pathways in Gnal +/-mice in response to psychostimulants in vivo, with markedly decreased cAMP-dependent phosphorylation and normal ERK signaling [START_REF] Corvol | Quantitative changes in Galphaolf protein levels, but not D1 receptor, alter specifically acute responses to psychostimulants[END_REF]. It was therefore particularly interesting to examine the signaling responses to L-DOPA in Gnal +/-mice. Since Gαolf is directly involved in the stimulation of cAMP/PKA signaling [START_REF] Corvol | Quantitative changes in Galphaolf protein levels, but not D1 receptor, alter specifically acute responses to psychostimulants[END_REF], we compared the phosphorylation of several PKA substrates in chronically L-DOPA-treated Gnal +/-and wild type mice. In Gnal +/+ (n = 9) and Gnal +/-(n = 11) mice, we evaluated the number of neurons immunolabeled with an antibody recognizing the phosphorylated form of the PKA substrate consensus sequence, which has been validated for assessing the activity of cAMP/PKA pathway in neurons by immunofluorescence [START_REF] Sindreu | Ca2+ -stimulated adenylyl cyclases regulate ERK-dependent activation of MSK1 during fear conditioning[END_REF]. A dramatic increase in the number of immunopositive neurons was observed after L-DOPA in the lesioned striatum of wild type mice showing a strong responsiveness of cAMP/PKA signaling (Fig. 5A andB). This increase was significantly lower in the lesioned striatum of Gnal +/indicating a reduction of cAMP/PKA signaling in these mice. The persistence of some level of activation may correspond to a persistent partial PKA activation or to the reaction of the antibody with proteins phosphorylated by other kinases. Therefore, in a different set of lesioned animals, we examined by immunoblotting the phosphorylation of two well characterized PKA substrates, AMPA receptor subunit GluA1 at Ser845 [START_REF] Snyder | Regulation of phosphorylation of the GluR1 AMPA receptor in the neostriatum by dopamine and psychostimulants in vivo[END_REF] and DARPP-32 at Thr34 [START_REF] Hemmings | DARPP-32, a dopamine-and adenosine 3':5'-monophosphate-regulated neuronal phosphoprotein. I. Amino acid sequence around the phosphorylated threonine[END_REF]. Phosphorylation of these sites has been shown to be correlated with the occurrence of LID [START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF]. In the lesioned striatum of Gnal +/+ mice, L-DOPA treatment induced an increased phosphorylation of GluA1 and DARPP-32 when compared with the unlesioned striatum (Fig. 5C-E), in agreement with previous results [START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF]. In contrast, these responses were absent in Gnal +/-mice for Ser845-GluA1 (Fig. 5C,D) and markedly diminished for Thr34-DARPP-32 (Fig. 5C,E). A slight increase in baseline DARPP-32 phosphorylation was observed in Gnal +/-mice but it was not significant and of uncertain meaning given the variability in baseline DARPP-32 phosphorylation between animals. These results show that following a chronic treatment with L-DOPA, in vivo activation of the cAMP/PKA phosphorylation pathway is severely altered in Gnal +/-mice, in spite of the relative increase in Gαolf protein induced by the lesion as compared to the unlesioned striatum. This suggests a threshold effect of the amount of Gαolf for cAMP/PKA pathway activation.

Hypersensitivity of L-DOPA-induced activation of the ERK pathway is preserved in the lesioned striatum of Gnal +/-mice

The second signaling pathway that has been reported to be strongly activated by L-DOPA in the lesioned striatum is the ERK cascade [START_REF] Gerfen | D1 dopamine receptor supersensitivity in the dopamine-depleted striatum results from a switch in the regulation of ERK1/2/MAP kinase[END_REF][START_REF] Pavon | ERK phosphorylation and FosB expression are associated with L-DOPA-induced dyskinesia in hemiparkinsonian mice[END_REF][START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF][START_REF] Westin | Spatiotemporal pattern of striatal ERK1/2 phosphorylation in a rat model of L-DOPA-induced dyskinesia and the role of dopamine D1 receptors[END_REF]. The activation of this pathway is D1R-dependent [START_REF] Gerfen | D1 dopamine receptor supersensitivity in the dopamine-depleted striatum results from a switch in the regulation of ERK1/2/MAP kinase[END_REF][START_REF] Westin | Spatiotemporal pattern of striatal ERK1/2 phosphorylation in a rat model of L-DOPA-induced dyskinesia and the role of dopamine D1 receptors[END_REF] and takes place in striatonigral MSNs which express this receptor (Santini et al., 2009b), although prolonged L-DOPA treatment was found to activate ERK pathway in the small population of cholinergic interneurons [START_REF] Ding | Enhanced striatal cholinergic neuronal activity mediates L-DOPA-induced dyskinesia in parkinsonian mice[END_REF]. Thirty minutes after injection of L-DOPA, following a 10-day treatment, we observed a dramatic increase in the number of neurons immunofluorescent for the di-phosphorylated active form of ERK1/2 (pERK) in the lesioned striatum as compared to the unlesioned side in Gnal +/+ mice (Fig. 6A). A similar response was observed in Gnal +/-mutant mice (Fig. 6A). We also analyzed the phosphorylation of ERK2 by immunoblotting and found no difference between the two genotypes (pERK2/total ERK2: +/+, unlesioned, 100 ± 4, 6-OHDA, 142 ± 11; +/-, unlesioned, 102 ± 3, 6-OHDA 141 ± 6, means ± SEM. Two-way ANOVA, lesion effect F (1,57) = 42.1 p < 0.001, genotype effect F (1,57) = 0.01, not significant, interaction F (1,57) = 0.03, not significant). We then examined pAcH3 immunoreactivity that is known to be dependent on ERK activation and is strongly increased by L-DOPA in 6-OHDA-lesioned striatonigral MSNs [START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF]Santini et al., 2009b). A dramatic increase in the number of pAcH3-positive neurons was observed in the lesioned striatum of wild type mice (Fig. 6B). A virtually identical response was observed in Gnal +/-mice (Fig. 6B). Thus, there was a marked contrast between the identical pERK responses to L-DOPA in Gnal +/+ and +/-mice, and the clear reduction in the PKA pathway response in Gnal +/-mice.

Since chronic treatment by L-DOPA may change the response of ERK pathway [START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF][START_REF] Cenci | Maladaptive striatal plasticity in L-DOPA-induced dyskinesia[END_REF][START_REF] Ding | Enhanced striatal cholinergic neuronal activity mediates L-DOPA-induced dyskinesia in parkinsonian mice[END_REF], we wondered whether this treatment could blunt differences between Gnal +/+ and +/-mice. We therefore examined in a different batch of mice the responses to a single acute injection of L-DOPA. A very strong increase in pERK-positive (Fig. 6C) and pAcH3-positive (Fig. 6D) cells was observed in the lesioned striatum and this effect was identical in wild type and heterozygous Gnal mutant mice. These results clearly show that the decrease in Gαolf did not alter ERK activation in response to L-DOPA.

DISCUSSION

The present study addressed the involvement of Gαolf up-regulation in LID in a mouse model of hemiparkinsonism. The use of mice partially deficient in Gαolf (Gnal +/-) allowed us to determine its contribution to LID and to intracellular signaling cascades presumably involved in LID development or expression.

Gαolf up-regulation in dyskinetic mice

We replicated in mice the up-regulation of Gαolf protein observed in the dorsal striatum of hemiparkinsonian rats and PD patients [START_REF] Herve | G(olf) and Gs in rat basal ganglia: possible involvement of G(olf) in the coupling of dopamine D1 receptor with adenylyl cyclase[END_REF][START_REF] Marcotte | Striatal G-proteins: effects of unilateral 6hydroxydopamine lesions[END_REF][START_REF] Penit-Soria | Levels of stimulatory G protein are increased in the rat striatum after neonatal lesion of dopamine neurons[END_REF][START_REF] Corvol | Persistent increase in olfactory type G-protein alpha subunit levels may underlie D1 receptor functional hypersensitivity in Parkinson disease[END_REF][START_REF] Rangel-Barajas | L-DOPAinduced dyskinesia in hemiparkinsonian rats is associated with up-regulation of adenylyl cyclase type V/VI and increased GABA release in the substantia nigra reticulata[END_REF]. Previous data suggested that this up-regulation was post-transcriptional [START_REF] Herve | G(olf) and Gs in rat basal ganglia: possible involvement of G(olf) in the coupling of dopamine D1 receptor with adenylyl cyclase[END_REF] and resulted from a lack of usage of the receptor [START_REF] Herve | Galpha(olf) levels are regulated by receptor usage and control dopamine and adenosine action in the striatum[END_REF]. Although its precise mechanisms are still unknown, a parallel can be made with the highly related Gαs whose degradation increases following its activation in cultured cells [START_REF] Levis | Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity[END_REF]. In support of this hypothesis treatment of lesioned rats with L-DOPA or a D1R agonist normalized Gαolf levels [START_REF] Corvol | Persistent increase in olfactory type G-protein alpha subunit levels may underlie D1 receptor functional hypersensitivity in Parkinson disease[END_REF][START_REF] Rangel-Barajas | L-DOPAinduced dyskinesia in hemiparkinsonian rats is associated with up-regulation of adenylyl cyclase type V/VI and increased GABA release in the substantia nigra reticulata[END_REF]. However, Gαolf increase was observed in the putamen of PD patients who had received a prolonged L-DOPA treatment [START_REF] Corvol | Persistent increase in olfactory type G-protein alpha subunit levels may underlie D1 receptor functional hypersensitivity in Parkinson disease[END_REF]. The present study shows a correlation between LID and maintenance of increased Gαolf levels. When the mice were stratified on AIMs intensity, animals with no or minimal dyskinesia had normal levels of Gαolf, whereas those with strong dyskinesia had the highest levels. If the degree of D1R usage was the only factor regulating Gαolf levels, their normalization should have been observed in all groups. Therefore our results, as those in patients [START_REF] Corvol | Persistent increase in olfactory type G-protein alpha subunit levels may underlie D1 receptor functional hypersensitivity in Parkinson disease[END_REF], suggest the existence of a dysregulation associated with the occurrence of dyskinesia and responsible for the "abnormal" persistence of high levels of Gαolf. It should be noted that no persistent increase in Gαolf was reported in dyskinetic L-DOPA-treated rats [START_REF] Rangel-Barajas | L-DOPAinduced dyskinesia in hemiparkinsonian rats is associated with up-regulation of adenylyl cyclase type V/VI and increased GABA release in the substantia nigra reticulata[END_REF]. We do not know whether this discrepancy results from differences between species or experimental protocols. Yet, the persistence of Gαolf increase in treated PD patients (see above) suggests that results in mice are relevant for the human disease.

Gαolf up-regulation contributes to the L-DOPA activation of the D1R/cAMP/PKA pathway in the lesioned striatum

The molecular mechanisms leading to the hypersensitivity of D1R-stimulated adenylylcyclase activity and signaling following DA lesion are unclear since the overall D1R concentration and its abundance at the plasma membrane are not increased [START_REF] Savasta | Different plasticity changes in D1 and D2 receptors in rat striatal subregions following impairment of dopaminergic transmission[END_REF][START_REF] Herve | G(olf) and Gs in rat basal ganglia: possible involvement of G(olf) in the coupling of dopamine D1 receptor with adenylyl cyclase[END_REF][START_REF] Shinotoh | Dopamine D1 receptors in Parkinson's disease and striatonigral degeneration: a positron emission tomography study[END_REF][START_REF] Berthet | Pharmacological analysis demonstrates dramatic alteration of D1 dopamine receptor neuronal distribution in the rat analog of L-DOPA-induced dyskinesia[END_REF]. Our results strongly support the role of Gαolf up-regulation in D1R-hypersignaling, since a marked decrease in the PKA stimulation was observed in Gnal +/-mutant mice. L-DOPA-induced phosphorylation was partially (general PKA-phosphosubstrates) or massively (phosphorylation of GluA1 or DARPP-32) decreased. Since in mutant mice the levels of Gαolf were below those in unlesioned wild type striatum, we can conclude that the increase in Gαolf is critical for D1R-dependent cAMP/PKA hypersensitivity. These results are in agreement with a previous study showing that the levels of Gαolf but not those of D1R are critical for the efficacy of the D1R/cAMP/PKA pathway [START_REF] Corvol | Quantitative changes in Galphaolf protein levels, but not D1 receptor, alter specifically acute responses to psychostimulants[END_REF]. They also account for the increased G-protein activation by D1R reported in MPTP-lesioned monkeys [START_REF] Aubert | Increased D1 dopamine receptor signaling in levodopainduced dyskinesia[END_REF]. Additional factors may contribute to the cAMP/PKA hypersensitivity in DA-lesioned striatum, including increased levels of adenylyl-cyclase5/6 (Rangel- [START_REF] Rangel-Barajas | L-DOPAinduced dyskinesia in hemiparkinsonian rats is associated with up-regulation of adenylyl cyclase type V/VI and increased GABA release in the substantia nigra reticulata[END_REF], or changes in phosphatases or phosphodiesterases levels and/or activities [START_REF] Meurers | Dopamine depletion induces distinct compensatory gene expression changes in DARPP-32 signal transduction cascades of striatonigral and striatopallidal neurons[END_REF].

Gαolf/cAMP/PKA hypersensitivity does not account for the strong activation of ERK by L-DOPA in dyskinetic animals

In animal models of PD, a strong ERK activation in response to L-DOPA is observed, correlated with the occurrence of LID [START_REF] Gerfen | D1 dopamine receptor supersensitivity in the dopamine-depleted striatum results from a switch in the regulation of ERK1/2/MAP kinase[END_REF][START_REF] Pavon | ERK phosphorylation and FosB expression are associated with L-DOPA-induced dyskinesia in hemiparkinsonian mice[END_REF][START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF][START_REF] Westin | Spatiotemporal pattern of striatal ERK1/2 phosphorylation in a rat model of L-DOPA-induced dyskinesia and the role of dopamine D1 receptors[END_REF][START_REF] Nicholas | Striatal histone modifications in models of levodopa-induced dyskinesia[END_REF]Santini et al., 2009b;[START_REF] Fasano | Inhibition of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) signaling in the striatum reverts motor symptoms associated with L-dopainduced dyskinesia[END_REF][START_REF] Santini | Distinct changes in cAMP and extracellular signal-regulated protein kinase signalling in L-DOPA-induced dyskinesia[END_REF][START_REF] Francardo | Impact of the lesion procedure on the profiles of motor impairment and molecular responsiveness to L-DOPA in the 6-hydroxydopamine mouse model of Parkinson's disease[END_REF]. ERK activation occurs in D1R-expressing striatonigral neurons [START_REF] Gerfen | D1 dopamine receptor supersensitivity in the dopamine-depleted striatum results from a switch in the regulation of ERK1/2/MAP kinase[END_REF]Santini et al., 2009b), and following prolonged treatment in cholinergic interneurons [START_REF] Ding | Enhanced striatal cholinergic neuronal activity mediates L-DOPA-induced dyskinesia in parkinsonian mice[END_REF]. The ERK pathway may be important at several levels including regulation of transcription [START_REF] Cenci | L-DOPA-induced dyskinesia in the rat is associated with striatal overexpression of prodynorphin-and glutamic acid decarboxylase mRNA[END_REF][START_REF] Aubert | Enhanced preproenkephalin-B-derived opioid transmission in striatum and subthalamic nucleus converges upon globus pallidus internalis in L-3,4dihydroxyphenylalanine-induced dyskinesia[END_REF] and translation, through the mTORC1 complex (Santini et al., 2009a). The precise molecular mechanism of ERK-signaling hypersensitivity is not known. In the present study, despite a dramatic reduction in cAMP/PKA response, the L-DOPA-induced ERK activation was not affected by Gnal haplodeficiency. This result strongly suggests that the ERK hyperresponsiveness following DA degeneration does not result from the hypersensitivity of the cAMP/PKA pathway and that the two responses can be dissociated. These results are in line with our previous data on responses to amphetamine in Gnal +/-mice, showing no alteration in the ERK activation despite dramatic reduction in PKA signaling activity [START_REF] Corvol | Quantitative changes in Galphaolf protein levels, but not D1 receptor, alter specifically acute responses to psychostimulants[END_REF]. A cAMP-independent synergy of D1R and NMDAR for the ERK activation exists in neurons in culture [START_REF] Pascoli | Cyclic adenosine monophosphate-independent tyrosine phosphorylation of NR2B mediates cocaine-induced extracellular signal-regulated kinase activation[END_REF]. In vivo an amplification of this synergy by cAMP/PKA/DARPP-32 appears critical, possibly due to the high concentrations of regulatory proteins like DARPP-32 and striatal-enriched tyrosine phosphatase (STEP) [START_REF] Valjent | Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum[END_REF]. The cause of the D1R-activated ERK pathway hypersensitivity remains to be identified in DA-lesioned mice, but could involve cAMP-independent mechanisms [START_REF] Gerfen | Differences between dorsal and ventral striatum in Drd1a dopamine receptor coupling of dopamine-and cAMP-regulated phosphoprotein-32 to activation of extracellular signal-regulated kinase[END_REF], including crosstalk between D1R and mGluR5 receptor [START_REF] Rylander | Pharmacological modulation of glutamate transmission in a rat model of L-DOPA-induced dyskinesia: effects on motor behavior and striatal nuclear signaling[END_REF] or dysregulation of protein phosphatases [START_REF] Meurers | Dopamine depletion induces distinct compensatory gene expression changes in DARPP-32 signal transduction cascades of striatonigral and striatopallidal neurons[END_REF]. It should be underlined however that a basal tonus of cAMP/PKA/DARPP-32 pathway appears required for ERK activation. For instance, the dramatic impairment of the cAMP pathway in Gnal -/-homozygous mutants prevented in vivo ERK activation by amphetamine [START_REF] Corvol | Quantitative changes in Galphaolf protein levels, but not D1 receptor, alter specifically acute responses to psychostimulants[END_REF]. In addition, in vivo complete inhibition of PKA [START_REF] Lebel | Striatal inhibition of PKA prevents levodopainduced behavioural and molecular changes in the hemiparkinsonian rat[END_REF] or genetic deletion of an amplifier of PKA action, DARPP-32 [START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF], decreased the L-DOPA-induced ERK activation of in lesioned mice. Accordingly the existence of a basal level of phospho-DARPP-32 in Gnal +/-mice may play a permissive role for ERK activation.

Hypersensitivity of the D1R/Gαolf/cAMP pathway is not responsible for dyskinetic behavior but have a permissive role

A variety of dysregulations take place in DA-denervated striatum and are likely to concur to the occurrence of LID [START_REF] Cenci | Dopamine dysregulation of movement control in L-DOPA-induced dyskinesia[END_REF][START_REF] Jenner | Molecular mechanisms of L-DOPA-induced dyskinesia[END_REF][START_REF] Santini | Parkinson's disease: levodopa-induced dyskinesia and signal transduction[END_REF][START_REF] Calabresi | Levodopa-induced dyskinesias in patients with Parkinson's disease: filling the bench-to-bedside gap[END_REF][START_REF] Feyder | L-DOPA-Induced Dyskinesia and Abnormal Signaling in Striatal Medium Spiny Neurons: Focus on Dopamine D1 Receptor-Mediated Transmission[END_REF][START_REF] Gerfen | Modulation of striatal projection systems by dopamine[END_REF][START_REF] Murer | Striatal Signaling in L-DOPA-Induced Dyskinesia: Common Mechanisms with Drug Abuse and Long Term Memory Involving D1 Dopamine Receptor Stimulation[END_REF]. From a therapeutic stand-point it is important to dissect the mechanisms of induction and expression of LID, which can be targets for potential preventive or symptomatic treatments. Interfering with D1R [START_REF] Westin | Spatiotemporal pattern of striatal ERK1/2 phosphorylation in a rat model of L-DOPA-induced dyskinesia and the role of dopamine D1 receptors[END_REF][START_REF] Darmopil | Genetic inactivation of dopamine D1 but not D2 receptors inhibits L-DOPA-induced dyskinesia and histone activation[END_REF], cAMP/PKA [START_REF] Lebel | Striatal inhibition of PKA prevents levodopainduced behavioural and molecular changes in the hemiparkinsonian rat[END_REF], DARPP-32 [START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF][START_REF] Bateup | Distinct subclasses of medium spiny neurons differentially regulate striatal motor behaviors[END_REF], Ras-GRF1/ERK [START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF][START_REF] Fasano | Inhibition of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) signaling in the striatum reverts motor symptoms associated with L-dopainduced dyskinesia[END_REF], and mTOR (Santini et al., 2009a) decreases or prevents LID. Although these results suggest the involvement of all these signaling modules in the generation of LID, they do not prove that LID results from their up-regulation. They only show that their activity is necessary. Our results clarify this situation by showing that LID was still observed in Gnal +/-mice in spite of a marked, but not complete, decrease in D1R/cAMP/PKA signaling. In addition, in Gnal+/-mice the A2A receptor/cAMP/PKA signaling is known to be blunted [START_REF] Corvol | Galpha(olf) is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum[END_REF][START_REF] Herve | Galpha(olf) levels are regulated by receptor usage and control dopamine and adenosine action in the striatum[END_REF]. A decrease in A2A signaling would be expected to reduce LID development [START_REF] Xiao | Forebrain adenosine A2A receptors contribute to L-3,4dihydroxyphenylalanine-induced dyskinesia in hemiparkinsonian mice[END_REF], but LID was not decreased in Gnal+/-mice. Therefore we can rule out that increase of cAMP/PKA response is the main culprit for the occurrence of LID. As discussed above for ERK activation however, cAMP/PKA signaling appears necessary for LID. Indeed, the marked reduction of LID induced by pharmacological inhibition of PKA [START_REF] Lebel | Striatal inhibition of PKA prevents levodopainduced behavioural and molecular changes in the hemiparkinsonian rat[END_REF] or by general or targeted DARPP-32 knockout [START_REF] Santini | Critical involvement of cAMP/DARPP-32 and extracellular signalregulated protein kinase signaling in L-DOPA-induced dyskinesia[END_REF][START_REF] Bateup | Distinct subclasses of medium spiny neurons differentially regulate striatal motor behaviors[END_REF] shows the importance of this pathway. On the other hand, our results are compatible with a critical role of ERK in LID generation since its activation was similar in Gnal +/-and wild type mice.

In conclusion, this study allows better understanding the importance of various aspects of D1R-signaling hypersensitivity in the generation of LID and of their relationships. Our results suggest several important conclusions: i, Gαolf up-regulation is a conserved response to DAlesion and its persistence during L-DOPA treatment correlates with LID; ii, this up-regulation contributes to the hypersensitivity of the D1R/cAMP/PKA pathway, which was markedly decreased following reduction in Gαolf gene dosage; iii, hypersensitivity of cAMP/PKA signaling does not explain ERK strong activation, which was unaltered in Gnal mutant mice; iv, although the cAMP/PKA pathway is necessary for LID occurrence, its hypersensitivity is unlikely to be its main cause. These results are compatible with a model in which a minimal level of cAMP/PKA pathway is necessary for LID generation, whereas increased ERK signaling may play a direct causal role. Identification of the mechanisms leading to hypersensitivity of the ERK pathway following lesion of DA neurons is therefore an important objective of future studies.

Figure Legends

Figure 3

Gαolf and tyrosine hydroxylase levels in 6-OHDA-lesioned Gnal+/-and control mice (A) Quantification of Gαolf levels in mice heterozygous for a null mutation of Gnal gene (+/-) and their wild type littermates (+/+) by immunoblotting. Data are expressed as percentage of the mean in wild type mice and are means ± SEM (n = 4-6). Unpaired two-tailed Student's ttest: Gαolf t = 10.93, *** p < 0.0001. (B) Quantification of TH levels by immunoblotting. Data are expressed as percentage of the means of unlesioned (UL) side of Gnal +/+ mice and are means ± SEM (n = 7-8). Two-way ANOVA: effect of lesion: F (1,24) = 357, p < 0.0001; effect of genotype: F (1,24) = 0.01, not significant (NS); interaction: F (1,24) = 0.90, NS. Post hoc comparison (Bonferroni's test): *** p < 0.001, 6-OHDA vs. unlesioned (UL) for each genotype; (C) Quantification of Gαolf mean immunofluorescence (IF) intensity in 6-OHDAlesioned Gnal +/-and wild type control (+/+) mice chronically treated with L-DOPA. Gαolf IF was measured in the dorsolateral striatum by confocal microscopy in 3 independent experiments for each mouse and normalized to the mean values in wild type mice for each experiment. Two-way ANOVA analysis effect of the lesion: F (1,18) = 133, p < 0.0001; effect of the genotype: F (1,18) = 13.97, p < 0.01; interaction: F (1,18) = 1.81, NS. Post hoc comparison (Bonferroni's test): *** p < 0.001, 6-OHDA-lesioned vs. UL; ## p < 0.01 and ### p < 0.001, Gnal +/+ vs. Gnal +/-.

Figure 4

L-DOPA-induced dyskinesia is not altered in Gnal heterozygous mice. Heterozygous (+/-) Gnal mutant and wild type (+/+) mice were lesioned and treated with L-DOPA and benserazide during 10 days. (A) Forelimb use was determined using the cylinder test in the 6-OHDA lesioned mice before (-) and after administration of L-DOPA (+) on the first day of treatment. Data are means ± SEM (n = 7-8). Repeated-measure ANOVA (with the withinsubjects factor of treatment and the between-subjects factor of genotype): effect of treatment: F (1,26) = 56, p < 0.0001; effect of genotype: F (1,26) = 0.49, NS; interaction between genotype and treatment: F (n = 8-11). Repeatedmeasure two-way ANOVA (with the within-subjects factor of lesion and the between-subjects factor of genotype) effect of the lesion: F (1,17) = 54.5, p < 0.001, effect of the genotype: F (1,17) = 7.04, p < 0.02, and interaction: F (1,17) = 6.49, p < 0.05. Post-hoc comparison (Bonferroni's test): *** p < 0.001 and ** p < 0.01: 6-OHDA vs. UL. ## p < 0.01 Gnal +/-vs. Gnal +/+. (C) Immunoblot analysis using antibodies against phospho-Ser845-GluA1 (pSer845) and total GluA1, phospho-Thr34-DARPP-32 (pThr34) and total DARPP-32 in the unlesioned (UL) and 6-OHDA-lesioned striatum of Gnal +/+ and Gnal +/-mice, 30 min after the last injection of L-DOPA + benserazide. (D) Quantification of pSer845 normalized to total GluA1, expressed as percentage of unlesioned (UL) striatum of Gnal+/+ mice. Data are means ± SEM (n = 9-15). Two-way ANOVA, effect of the genotype; F (1,48) = 6.02, p < 0.02; effect of the lesion: F (1,48) = 10.8, p < 0.01; interaction: F (1,48) = 6.8, p < 0.05. Post hoc comparison (Bonferroni's test): *** p < 0.001, 6-OHDA vs. UL; ## p < 0.01 Gnal +/+ vs. Gnal +/-. (E) Same representation for pThr34 normalized to total DARPP-32. Data are means ± SEM (n = 5-9). Two-way ANOVA, effect of the genotype; F (1,22) = 1.13, NS; effect of the lesion: F (1,22) = 15.5, p < 0.001; interaction: F (1,22) = 10.1, p < 0.01. Post hoc comparison (Bonferroni's test): *** p < 0.001, 6-OHDA vs. UL; # p < 0.05 Gnal +/+ vs. Gnal +/-.

Figure 1 Unilateral 6 -

 16 Figure 1 Unilateral 6-OHDA lesion increases Gαolf levels in the dorsal striatum. Immunoreactivity was detected using Odyssey-LI-COR system (A-D) or confocal microscope (E, F). A, B, D: Serial coronal brain sections from a mouse that received a unilateral 6-OHDA injection into the right striatum. (A) Tyrosine hydroxylase immunofluorescence (TH IF). (B) Phosphoacetyl-histone H3 immunofluorescence (pAcH3 IF) showing the extent of signaling hypersensitivity in the dorsolateral part of the lesioned striatum. (C) Plot of Gαolf IF quantification with Odyssey-Li-Cor, as a function of the proportion of Gαolf protein in a dot blot assay. The samples were obtained by mixing various proportions of protein extracts from striatum (Str) expressing Gαolf and cortex (Cx) devoid of Gαolf. The immunofluorescence values were determined in parallel in two regions of the striatum, in two brain sections (i.e., non-homogenized striatal slices, right panel, Striatal regions). These values were within the linear range of the standard curve. (D) Gαolf immunofluorescence (Gαolf IF). Quantification of mean immunofluorescence intensity (IF) on the unlesioned (UL) and lesioned (6-OHDA) sides is shown in A, B, D in the dorsolateral part of the striatum (circled region of interest drawn in A). Data are means ± SEM (n = 11-13). Paired two-tailed Student's t-test: TH: t = 4.57; pAcH3: t = 12.8; Gαolf: t = 4.00. Scale bar, 1 mm. (E and F) Single confocal sections in the dorsolateral striatum showing TH (E) and Gαolf immunofluorescence (F). Data are means ± SEM (n = 8-12). Paired two-tailed Student's t-test: TH: t = 4.47, Gαolf: t = 3.19. Scale bar, 100 µm. ** p < 0.01, *** p < 0.001. a.u. arbitrary units.

Figure 2 Striatal

 2 Figure 2 Striatal Gαolf levels increase correlates with dyskinetic behavior. (A) Quantification of Gαolf immunofluorescence (IF) in the dorsal striatum of sham-operated mice treated with L-DOPA for 10 days (Sham, n = 6), 6-OHDA-lesioned mice treated with a single injection of L-DOPA (Acute, n = 21) or daily for 10 days (Chronic, n = 44). Data are expressed as percentage of the unlesioned (UL) side in each group and are means ± SEM. One-way ANOVA F (2,68) = 4.16, * p < 0.05. Post hoc comparison (Bonferroni's test): * p < 0.05 vs sham. (B) Comparison of Gαolf levels in the 25% 6-OHDA-lesioned chronically L-DOPAtreated mice in (A) that developed the weakest (total AIM score below 12.5, Low Dysk n = 11) and strongest (above 33.5, High Dysk, n = 11) dyskinesia. Data are means ± SEM. Unpaired two-tailed Student's t-test: t = 2.89, ** p < 0.01. (C-E) Correlation between Gαolf levels and (C) total AIMs score (r = 0.40, F (1,42) = 7.96, p < 0.01), (D) LOC (locomotor) AIMs (r = 0.41, F (1,42) = 8.56, p < 0.01), and (E) ALO (axial, limb, orofacial) AIMs (r = 0.34, F (1,42) = 5.38, p < 0.05).

  (1,26) = 0.09, NS. Post hoc comparison (Bonferroni's test): *** p < 0.001, before vs after L-DOPA. (B) Sum of axial, limb and orolingual (ALO) AIMs scored during 140-min period after L-DOPA. Comparison between +/+ (n = 18) and +/-(n = 23) mice. Data are means ± SEM. Unpaired two-tailed Student's t-test: t = 0.62, NS. (C) Sum of locomotive (LOC) AIMs scored during 140-min period after L-DOPA in the same animals. Data are means ± SEM. Unpaired two-tailed Student's t-test: t = 0.61, NS. (D) Time course of total AIMs (sum of axial, limb, orofacial and locomotive AIMs) scored every 20 min over a period of 140 min after the last L-DOPA administration. Repeated-measure two-way ANOVA (with the within-subjects factor of time and the between-subjects factor of genotype): effect of genotype F (1,234) = 0.18, NS, effect of time F (6,234) = 71, p < 0.0001; interaction: F (6,234) = 0.43, NS. (E) Total AIMs (sum of axial, limb, orofacial and locomotive AIMs) scored during 140min period after L-DOPA on days 5 of L-DOPA treatment. Data are means ± SEM. Student ttest, t = 0.181, DF = 25, NS.

Figure 5 L

 5 Figure 5 L-DOPA-induced PKA-dependent phosphorylation is markedly impaired in Gnal heterozygous mice. (A) Single confocal section showing phospho-PKA substrate immunofluorescence in the unlesioned (UL) and 6-OHDA-lesioned dorsal striatum of Gnal +/+ (upper panels) and Gnal +/-(lower panels) mice, 30 min after the last injection of L-DOPA + benserazide. Scale bar, 100 µm. (B) Number of phospho-PKA substrate (pPKA substrate) positive cells in the dorsolateral striatum of wild type and heterozygous mice. Data are means ± SEM of positive cells in 375 x 375 µm confocal images (n = 8-11). Repeatedmeasure two-way ANOVA (with the within-subjects factor of lesion and the between-subjects factor of genotype) effect of the lesion: F (1,17) = 54.5, p < 0.001, effect of the genotype: F (1,17) = 7.04, p < 0.02, and interaction: F (1,17) = 6.49, p < 0.05. Post-hoc comparison (Bonferroni's test): *** p < 0.001 and ** p < 0.01: 6-OHDA vs. UL. ## p < 0.01 Gnal +/-vs. Gnal +/+. (C) Immunoblot analysis using antibodies against phospho-Ser845-GluA1 (pSer845) and total

Figure 6

 6 Figure 6 Chronic or acute L-DOPA induces phosphorylation of ERK and histone H3 in the lesioned striatum of Gnal heterozygous mice. (A-B) Confocal sections through the dorsolateral striatum of unlesioned (UL) and 6-OHDA-lesioned dorsolateral striatum of Gnal +/+ (upper panels) and Gnal +/-(lower panels) mice, 30 min after the last injection of chronic L-DOPA and benserazide treatment showing phospho-ERK (pERK, A) and phosphoacetyl-histone H3 (pAcH3, B) immunofluorescence. Scale bar, 100 µm. Lower panels, number of pERK or pAcH3 positive cells in the dorsolateral striatum. Data are means ± SEM in 375 x 375 µm confocal images (n = 8-11). Repeated-measure two-way ANOVA (with the within-subjects factor of lesion and the between-subjects factor of genotype) (A) pERK : effect of the lesion F (1,17) = 67, p < 0.0001 effect of the genotype: F (1,17) = 0.89, NS; interaction F (1,17) = 1.03, NS; (B) pAcH3: effect of lesion: F (1,17) = 57, p < 0.0001; but no significant effect of genotype: F (1,17) = 0.02, NS; and no significant interaction between genotype and lesion F (1,17) = 0.01, NS. Post hoc comparison (Bonferroni's test): *** p < 0.001, 6-OHDA vs. UL. (C, D) Same as in A, B but in different groups of mice which received a single injection of L-DOPA (n = 4-8 per group). (C) pERK, effect of lesion F (1,20) = 893, p < 0.0001; effect of genotype: F (1,20) = 0.79, NS; interaction: F (1,20) = 0.79; NS. (D) pAcH3, effect of lesion: F (1,20) = 1231, p < 0.0001; effect of genotype: F (1,20) = 3.61, NS; interaction F (1,20) = 3.61, NS. Post hoc comparison (Bonferroni's test): *** p < 0.001, 6-OHDA vs. UL.

The work was supported by Inserm, Universite´ Pierre et Marie Curie, and grants from the Fondation pour la Recherche Me´dicale, the Fondation pour la Recherche sur le Cerveau, European Research Council, the Michael Stem Parkinson's Research Foundation, and Agence Nationale de la Recherche Grant ANR09-MNPS-014. The Girault and Hervé group is affiliated to the Ecole des Neurosciences de Paris-Ile-de-France. The authors are grateful to Gilberto Fisone for his help, support, and comments on this manuscript.Wethank Leonardo Belluscio for providing Gnal-/-mice, Sophie Longueville for genotyping, Natacha Roblot, Rachida Boukhari, and Yohann Bertelle for breading the mice, Charlotte Plestant for her help with image analysis, and the staff of the Institut du Fer a` Moulin Imaging facility.


Alexandre Barachant

Stéphane Bonnet

Marco Congedo

Christian Jutten

BCI Signal Classification using a Riemannian-based kernel

The use of spatial covariance matrix as feature is investigated for motor imagery EEG-based classification. A new kernel is derived by establishing a connection with the Riemannian geometry of symmetric positive definite matrices. Different kernels are tested, in combination with support vector machines, on a past BCI competition dataset. We demonstrate that this new approach outperforms significantly state of the art results without the need for spatial filtering.

Introduction

Brain-Computer Interfaces (BCIs) based on motor imagery have been well studied in the literature. For this type of BCI, the electrophysiological source of BCI control is based on spontaneous signals induced by voluntary changes in the dynamics of brain oscillations such as event related (de)synchronization (ERD/ERS) [START_REF] Pfurtscheller | Event-related EEG/MEG synchronization and desynchronization: basic principles[END_REF]. Motor Imagery (MI) is often used where movements are imagined by the subjects, which results in the activation of dedicated cortical areas (sensorimotor cortex) in given frequency bands. Thus this type of BCI is well suited for asynchronous BCI applications since the user can potentially perform actions without any stimulus from the exterior world.

The standard approach in MI-based EEG signal classification is to perform band-pass filtering, spatial filtering and linear classification, generally using Fisher's Linear Discriminant Analysis (LDA). The spatial filtering can be seen as a data-driven dimension reduction method that aims at promoting variance differences between two conditions. Sample covariance matrices are generally handled in the Euclidean space without considerations about the curvature of the space of Symmetric Positive Definite (SPD) matrices.

This paper provides a simple way to take into account the Riemannian geometry for EEG signal classification. This approach has been successfully applied on radar signal processing and image processing [START_REF] Tuzel | Pedestrian detection via classification on Riemannian manifolds[END_REF]. A new kernel is derived by establishing a connection with the Riemannian geometry of SPD matrices. This kernel is tested in combination with support vector machines, although we could have applied the kernel trick to other classifiers like logistic regression. Encouraging results are presented that demonstrate the potential benefit of the approach. Another advantage of the presented method is that it can directly be applied without the need for spatial filtering [START_REF] Barachant | Riemannian geometry applied to BCI classification In LVA ICA-2010[END_REF]. In section 2, the kernel is introduced based on the Riemannian geometry. In section 3, results on BCI competition dataset are provided.

2 A new kernel for symmetric positive definite matrices

Introduction

EEG signals are often analysed on short-time segments where the signal is assumed to be wide-sense stationary. Let X ∈ R E×T be a trial of imaginary movement; E being the number of electrodes and T the epoch duration in number of samples. We further assume that the different EEG signals have been band-pass filtered by the acquisition system, usually in the µ and β frequency bands in our application. For each trial X p of known class y p ∈ {-1, 1}, one can estimate the covariance matrix of the EEG random signal by the E × E sample covariance matrix (SCM) : C p = 1/(T -1) X p X T p . However note that SCM is sensitive to outliers and either robust covariance estimation techniques or regularization can be applied to improve the estimation.

It is common practice in MI-based BCI to use spatial filtering for dimension reduction and variance enhancement between EEG trials coming from different classes [START_REF] Blankertz | Optimizing spatial filters for robust EEG single-trial analysis[END_REF]. The Common Spatial Patterns method (CSP) is for instance successfully applied as a mean to extract relevant features for the classification of EEG trials recorded during two motor imagery tasks. This technique aims at simultaneously diagonalizing the two intra-class covariance matrices obtained in the two conditions. This observation motivated us to investigate the direct use of spatial covariance matrix as input feature for EEG-based BCI signal classification.

SVM formulation

Support Vector Machine (SVM) is a popular linear classifier in BCI applications [START_REF] Jrad | sw-SVM: sensor weighting support vector machines for EEG-based brain-computer interfaces[END_REF]. Given a set of labelled feature vectors {(x p , y p )} P p=1 , this classification technique seeks to linearly separate data by finding an hyperplane (with normal vector w) that maximizes the margin, i.e. the distance between the hyperplane and the nearest points from each class, called support vectors. We refer to [START_REF] Schölkopf | Learning with Kernels: Support Vector Machines, Regularization, Optimization, and beyond[END_REF] and references herein for a detailed discussion on SVM. The decision function will be based on the sign of :

h(x) = b + P p=1 α p y p x p , x = b + w, x (1)
where ., . denotes the Euclidean scalar product between vectors. The term b is a bias and the {α p } P p=1 are the Lagrangian multipliers associated to the dual optimization problem [START_REF] Schölkopf | Learning with Kernels: Support Vector Machines, Regularization, Optimization, and beyond[END_REF]. Both quantities are estimated by quadratic programming. Most of the α p 's are null except for the support vectors. If data are not linearly separable in their native space, a mapping can be applied on the feature vector x to another (high-dimensional) transformed space. The transformation φ is generally non-linear and the decision function can be rewritten as :

h(x) = b + P p=1 α p y p φ(x p ), φ(x) H ( 2
)
where H is a reproducing kernel Hilbert space where the dot product is defined. The associated kernel k(., .) is defined by : k(x i ,

x j ) = φ(x i ), φ(x j ) H .
In most SVM applications, the φ-function is not explicitly expressed and solely the kernel is used. A common kernel is the Gaussian radial basis function :

k(x i , x j ) = exp -γ x i -x j 2 .
SVM is known to possess good generalization properties and to perform well in high-dimensional feature space. Finally the margin maximization cost function is often penalized with the introduction of slack variables whose effects are controlled by the hyper-parameter λ. This allows to enable soft margins where some feature vectors can be reasonably misclassified.

SVM applied on covariance matrix

In order to use sample covariance matrix C as input feature to SVM, a simple choice consists in vectorizing it : x = vec(C) and apply linear SVM classification on the resulting set [START_REF] Farquhar | A linear feature space for simultaneous learning of spatio-temporal filters in BCI[END_REF]. A more powerful approach consists in taking account the Riemannian geometry of the space of covariance matrices, i.e. the space of symmetric positive definite (SPD) matrices. Due to space constraints, we focus on the main points needed for the proposed algorithm.

The space of SPD E×E square matrices P (E) forms a differentiable manifold M of dimension E ⋆ = E (E+1)/2. At each point C (i.e. each covariance matrix), a scalar product can be defined in the associated tangent space T C M. The manifold is locally homomorphic to the Euclidean tangent space and distance computations in the manifold can be well approximated by distance computations in the tangent space. Let S 1 and S 2 be two tangent vectors (i.e. two symmetric matrices in our case), the scalar product in the tangent space at C can be defined by the relation :

S 1 , S 2 C = tr(S 1 C -1 S 2 C -1 ). (3)
Furthermore, we can define the logarithmic map to project locally all covariance matrices {C p } P p=1 , onto the tangent plane by :

S p = Log C (C p ) = C 1/2 logm C -1/2 C p C -1/2 C 1/2 (4)
where logm denotes the logarithm of a matrix. From this connection with Riemannian geometry, we define the mapping function as :

φ(C) = Log C ref (C) .
This choice yields after some manipulations to the kernel :

k(vec(C i ), vec(C j )) = φ(C i ), φ(C j ) C ref (5) = tr logm C -1/2 ref C i C -1/2 ref logm C -1/2 ref C j C -1/2 ref .
Here C ref is a free parameter that defines the point in SPD space where the tangent plane is computed. A common choice is to use the geometric mean of the P labelled covariance matrices {C p } P p=1 [START_REF] Moakher | A differential geometric approach to the geometric mean of symmetric positive-definite matrices[END_REF]. Alternatively, the choice C ref = I E yields to the simplified log-Euclidean kernel :

k(vec(C i ), vec(C j )) = tr [logm (C i ) logm (C j )] = logm (C i ) , logm (C j ) F ( 6
)
where subscript F stands for Frobenius. This kernel is investigated in [START_REF] Wang | Factor analysis based spatial correlation modeling for speaker verification In ISCSLP-2010[END_REF].

Experiments

In order to evaluate the performance of the proposed method, we have compared it to the classical signal processing chain in asynchronous MI-based BCI. The standard approach consists in band-pass filtering, spatial filtering (using CSP approach), log-variance feature extraction and Fisher's LDA classification [START_REF] Blankertz | Optimizing spatial filters for robust EEG single-trial analysis[END_REF]. This method is compared to linear SVM applied on vectorized covariance matrices and to kernel-based SVMs according to equation 5. Two tangent planes have been considered : one at identity I E and the other one at the geometric mean of all covariance matrices {C p } P p=1 .

Dataset

Dataset IIa of BCI competition IV are used for analysis. 22 electrodes are used (Fz, FC3, FC1, FCz, FC2, FC4, C5, C3, C1, Cz, C2, C4, C6, CP3, CP1, CPz, CP2, CP4, P1, Pz, P2, POz). The reference electrode is located on the left mastoid. A 8 -35 Hz band-pass filter has been applied on the original signals for all subjects. This dataset is composed of 9 subjects who performed 576 trials of right-hand (RH), left-hand (LH), tongue (TO) and both feet (BF) motor imagery (i.e. 144 trials per class). Since CSP is designed for binary classification, we have evaluated the average performance per subject, for all 6 possible pairs of mental tasks : BF/TO}. We have used 30-fold cross-validation to evaluate the generalization performance of the algorithms and the significance of the classification accuracy.

{LH

Results

The hyperparameter λ chosen equal to 10 in our experiments. As it can be observed from Table 1, kernel-based SVM approaches outperform both vectorized-SCM SVM method and CSP. A mean classification accuracy of 87% can be obtained whereas the CSP method is limited to 85%. Locating the tangent plane at the mean of all covariance matrices yields better results on average compared to the log-Euclidean choice of equation 6.

Comparing CSP and proposed kernel-based SVM on the individual sessions yields to Figure 1. It can be appreciated that except for few localized pairs of mental task, kernel-based SVM consistently outperforms CSP results. This remark is especially true for difficult binary classification cases (CSP performance below 80%) where the improvement brought by kernel-based SVM is significant.

We have also tested the significance of methods with respect to each other. Results are presented in Figure 2. This figure illustrates the p-values obtained for the 6 pairs of mental tasks. These values are obtained with a one-tailed dependent t-test (8 degrees of freedom) for paired samples, for test hypothesis {H 0 : µ 1 > µ 2 }. Both kernel-SVM proposed methods perform significantly better than the CSP (p < 0.05) for 4 out of 6 pairs of mental tasks. The performance of the two kernel-based SVMs differs significantly only for pair LH/BF. Nonetheless, the kernel using the tangent space at the geometric mean is always superior to the kernel using the tangent space at the identity. The slight increase of p-value in the RH/LH case could be futher reduced by optimizing the hyperparameter λ. Fig. 2: P-values for the 6 pairs of mental tasks in a subject-independent manner. Hypothesis H 0 : µ 1 > µ 2 , one-tailed dependent t-test (8 df) for paired samples.

Conclusion

This paper has proposed a new kernel for directly handling covariance matrices in classification methods. The approach is tested on a BCI competition dataset and outperforms significantly the conventional CSP method. This kernel could be employed in different applications where covariance matrices are the main ingredients of the feature extraction process. We have demonstrated in this work that the spatial filtering of electrodes, could be avoided without loss of performance. A future work will investigate the online use of this algorithm and the location update of the tangent space between BCI sessions and the minimum number of trials required to properly estimate the covariance matrices.

Fig. 1 :

 1 Fig. 1: Kernel SVM versus CSP classification accuracy comparison for each individual pair of mental tasks.

Table 1 :

 1 Average classification accuracy across the 9 subjects for 6 pairs of mental tasks.

		SVM kernel	SVM kernel SVM plain CSP
		Geometric Mean	Identity
	LH/RH	83.4	82.8	76.9	81.4
	LH/BF	89.9	89.5	82.2	86.9
	LH/TO	89.3	88.8	84	87.3
	RH/BF	88.8	88.2	81.1	86.3
	RH/TO	88.7	88	83.4	85.7
	BF/TO	82.1	81.4	74.9	80.8
	mean	87	86.4	80.4	84.7

/RH, LH/BF, LH/TO, RH/BF, RH/TO,