MD Céline Tard
email: celinetard@gmail.com

Arnaud Delval

David Devos

Renaud Lopes

Pierre Lenfant

Kathy Dujardin

Claude Hossein-Foucher

Franck Semah

Alain Duhamel

Luc

Luc Defebvre

Florence Le Jeune

Caroline Moreau

Brain metabolic abnormalities during gait with freezing in Parkinson's disease

Keywords: PET, associative somatosensory cortex, premotor cortex, temporopolar area, executive-attention network

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INTRODUCTION

Freezing of gait (FoG) is defined as a brief, episodic absence or marked reduction of forward progression of the feet despite the intention to walk [START_REF] Nutt | Freezing of gait: moving forward on a mysterious clinical phenomenon[END_REF]. This phenomenon affects about three quarters of people with Parkinson's disease (PD) [START_REF] Macht | Predictors of freezing in Parkinson's disease: a survey of 6,620 patients[END_REF]. Dopaminergic medications do not alleviate FoG in 15% of patients and produce only partial relief in 44% of patients [START_REF] Perez-Lloret | Prevalence, Determinants, and Effect on Quality of Life of Freezing of Gait in Parkinson Disease[END_REF]. Even though severe dopaminergic striatal denervation has been described in models of freezing (even in other parkinsonian syndromes than in idiopathic PD [START_REF] Park | Functional brain imaging in pure akinesia with gait freezing: [18F] FDG PET and [18F] FP-CIT PET analyses[END_REF]), imaging studies of patients with PD have shown that the mechanism of FoG also involves non-dopaminergic pathways [START_REF] Bartels | Striatal dopa and glucose metabolism in PD patients with freezing of gait[END_REF]. A better understanding of the neural basis of FoG would probably boost the development of effective therapeutic approaches.

The variable, episodic nature of FoG makes it difficult to study this phenomenon in the laboratory [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF][START_REF] Snijders | Clinimetrics of freezing of gait[END_REF]. Furthermore, imaging studies of FoG are technically complex because the freezing occurs during gait (i.e. when the person in the vertical position, rather than in the horizontal position required for concomitant MRI acquisition) [START_REF] Bartels | Brain imaging in patients with freezing of gait[END_REF][START_REF] Maillet | Imaging gait disorders in parkinsonism: a review[END_REF][START_REF] Herman | Neuroimaging as a window into gait disturbances and freezing of gait in patients with Parkinson's disease[END_REF]Shine et al., 2013d). Recently, researchers have found new ways of studying FoG by using mental imagery of gait [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF][START_REF] Crémers | Hemispheric specialization during mental imagery of brisk walking[END_REF][START_REF] Maillet | Neural substrates of levodopa-responsive gait disorders and freezing in advanced Parkinson's disease: A kinesthetic imagery approach[END_REF]Peterson et al., 2014aPeterson et al., , 2014b) ) and virtual reality tasks (Shine et al., 2013a(Shine et al., , 2013b(Shine et al., , 2013c;;Shine et al., 2011b). Other functional MRI approaches are based on creating the equivalent of FoG for the upper limbs (Vercruysse et al., 2014a) or performing alternating foot movements in the supine position (Shine et al., 2013a(Shine et al., , 2013b(Shine et al., , 2013c;;Shine et al., 2011b) in order to individualize motor blocks that could be time-locked with changes in brain perfusion.

These experiments evidenced corticosubcortical decoupling during freezing, with (i) hypoactivation of the basal ganglia, thalamus and sensorimotor regions and (ii) hyperactivation of the frontoparietal cortical regions (Shine et al., 2013a;Vercruysse et al., 2014a). Namely, the Brodmann area 6 -including SMA [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF]Vercruysse et al., 2014a;[START_REF] Maillet | Neural substrates of levodopa-responsive gait disorders and freezing in advanced Parkinson's disease: A kinesthetic imagery approach[END_REF], the pre-SMA (Shine et al., 2013b) and the dorsolateral prefrontal cortex (Vercruysse et al., 2014a)-, is of interest because it is known to be directly involved in locomotor networks used to compensate for gait impairment in patients with PD [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF]Shine et al., 2013b;Vercruysse et al., 2014b;Peterson et al., 2014b), potentially via the hyper-direct pathway from the pre-SMA to the STN (Shine et al., 2013d), However, mental imagery and virtual reality do not fully reflect "real life" conditions because (i) the patient's ability to imagine him/herself performing gait may vary [START_REF] Cohen | Freezing of gait is associated with a mismatch between motor imagery and motor execution in narrow doorways, not with failure to judge doorway passability[END_REF][START_REF] Van Der Meulen | The influence of individual motor imagery ability on cerebral recruitment during gait imagery[END_REF] and (ii) the supine position required for MRI is not physiologically normal for gait because it fails to simulate movement of the centre of gravity during movement [START_REF] Massion | Movement, posture and equilibrium: interaction and coordination[END_REF][START_REF] Karim | Functional MR imaging of a simulated balance task[END_REF].

Otherwise, network-based hypotheses have been tested by using (i) diffusion tensor imaging to study anatomic disconnection (and particularly disconnection from the pedunculopontine nucleus) [START_REF] Schweder | Connectivity of the pedunculopontine nucleus in parkinsonian freezing of gait[END_REF]Tessitore et al., 2012b;[START_REF] Fling | Asymmetric pedunculopontine network connectivity in parkinsonian patients with freezing of gait[END_REF][START_REF] Peterson | Dual-task interference and brain structural connectivity in people with Parkinson's disease who freeze[END_REF][START_REF] Youn | Alternations of mean diffusivity of pedunculopontine nucleus pathway in Parkinson's disease patients with freezing of gait[END_REF] and (ii) the blood-oxygen-level-dependent signal to assess functional reorganization of the default mode and locomotor networks [START_REF] Pappatà | Mild cognitive impairment in drug-naive patients with PD is associated with cerebral hypometabolism[END_REF]Tessitore et al., 2012b;[START_REF] Fling | Functional Reorganization of the Locomotor Network in Parkinson Patients with Freezing of Gait[END_REF] for reviews, see [START_REF] Bartels | Brain imaging in patients with freezing of gait[END_REF][START_REF] Maillet | Imaging gait disorders in parkinsonism: a review[END_REF][START_REF] Herman | Neuroimaging as a window into gait disturbances and freezing of gait in patients with Parkinson's disease[END_REF]Vercruysse et al., 2014a). Lastly, brain atrophy has been measured in patients with FoG, with divergent results (Tessitore et al., 2012a;[START_REF] Sunwoo | Thalamic volume and related visual recognition are associated with freezing of gait in non-demented patients with Parkinson's disease[END_REF][START_REF] Rubino | Does a volume reduction of the parietal lobe contribute to freezing of gait in Parkinson's disease?[END_REF]; this may be related to the fact that FoG is frequently associated with cognitive impairment [START_REF] Herman | Gray matter atrophy and freezing of gait in Parkinson's disease: Is the evidence black-on-white?[END_REF]. Furthermore, cognitive dysfunction can modify brain metabolism [START_REF] Bohnen | Cerebral glucose metabolic features of Parkinson disease and incident dementia: longitudinal study[END_REF][START_REF] Pappatà | Mild cognitive impairment in drug-naive patients with PD is associated with cerebral hypometabolism[END_REF]. Thus, it is important to recruit non-demented, cognitively matched patients to better understand the pathophysiology of FoG itself and avoid potential confounding aspects as attentional or executive impairment, that are often linked with FoG [START_REF] Yogev-Seligmann | The role of executive function and attention in gait[END_REF][START_REF] Vandenbossche | Freezing of Gait in Parkinson Disease Is Associated With Impaired Conflict Resolution[END_REF].

In the present study, we adopted a strategy based on current hypotheses about FoG (Nutt et al., 2011;[START_REF] Nieuwboer | Characterizing freezing of gait in Parkinson's disease: Models of an episodic phenomenon[END_REF]. In fact, FoG appears to be just one of several abnormalities that occur during continuous gait [START_REF] Hausdorff | Impaired regulation of stride variability in Parkinson's disease subjects with freezing of gait[END_REF][START_REF] Chee | Gait freezing in Parkinson's disease and the stride length sequence effect interaction[END_REF][START_REF] Vercruysse | Freezing in Parkinson's disease: A spatiotemporal motor disorder beyond gait[END_REF], including start hesitation [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF], trembling in place [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF], sequence effects [START_REF] Chee | Gait freezing in Parkinson's disease and the stride length sequence effect interaction[END_REF] and elevated step variability [START_REF] Hausdorff | Impaired regulation of stride variability in Parkinson's disease subjects with freezing of gait[END_REF]. Hence, the gait impairments in patients with FoG are probably more complex than the FoG phenomenon itself since they encompass FoG episodes. Some mental imaging protocols have studied gait disorders in general in freezers [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF][START_REF] Crémers | Hemispheric specialization during mental imagery of brisk walking[END_REF][START_REF] Maillet | Neural substrates of levodopa-responsive gait disorders and freezing in advanced Parkinson's disease: A kinesthetic imagery approach[END_REF]. In order to dissociate gait abnormalities and FoG phenomenon, restrictive FoG-time-locked imaging studies have also been performed (Shine et al., 2013a(Shine et al., , 2013b)), but might therefore fail to observe fundamental metabolic impairments in freezers during gait [START_REF] Nutt | Freezing of gait: moving forward on a mysterious clinical phenomenon[END_REF][START_REF] Nieuwboer | Characterizing freezing of gait in Parkinson's disease: Models of an episodic phenomenon[END_REF]Shine et al., 2013d).

We used here a technique based on the measurement of glucose uptake (a proxy marker for brain metabolism) that has already been used to study (i) gait disorders in progressive supranuclear palsy [START_REF] Zwergal | Functional disturbance of the locomotor network in progressive supranuclear palsy[END_REF] and (ii) pure akinesia with gait freezing [START_REF] Park | Functional brain imaging in pure akinesia with gait freezing: [18F] FDG PET and [18F] FP-CIT PET analyses[END_REF]. Positron emission tomography (PET) of [ 18 F]-fluorodeoxyglucose ([ 18 F]-FDG, a glucose analogue that can cross the blood-brain barrier) is known to reflect the brain cells' metabolism over the 20 to 30 minutes following the injection of the radiotracer. The subsequent stabilization of radiotracer levels enables image acquisition after this timepoint.

Hence, if the subject performs actual gait in the 30 minutes following injection of [ 18 F]-FDG, uptake of the radiotracer will reflect the brain's metabolism during this motor activity, making it a real activation imaging [START_REF] La Fougère | Real versus imagined locomotion: a [18F]-FDG PET-fMRI comparison[END_REF]. In the present study, we explored the brain's metabolism during gait with FoG by comparing two matched populations of non-demented PD patients presenting (or not) off-drug FoG during a standardized, 30-minute series of FoG-triggering gait trajectories (referred to hereafter as the "FoG trajectories") [START_REF] Snijders | Clinimetrics of freezing of gait[END_REF]. We then compared brain activation at rest with brain activation immediately after performance of the FoG trajectories (i.e. reflecting metabolism during uptake of the radiotracer).

We hypothesized that compensatory locomotor networks, involving namely the SMA, could be recruited during this high-level gait task in the non-freezers patients, whereas freezers patients could display less activation of this area. Concerning the subcortical structures (basal ganglia and mesencephalic locomotor region -MLR-), we hypothesized that gait with a real load and intensive proprioceptive afferences could modify the data previously determined by fMRI [START_REF] Dietz | Locomotor activity in spinal man: significance of afferent input from joint and load receptors[END_REF]. Finally, we expected a potential deregulation of the compensatory networks in freezers patients.

METHODS

Subjects

We recruited a group of PD patients with FoG in the off-drug condition [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF] (the FoG group, n=11) and a matched group of PD patients without FoG (the non-FoG group, n=11). The study's objectives and procedures were approved by the local investigational review board. Each participant gave his/her written consent to participation in the study.

Twenty-two outpatients with PD diagnosed according to Gibb's criteria (1988) were enrolled from the active case file of the Movement Disorders Department at Lille University Hospital (Lille, France). We first screened PD patients with off-drug FoG [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF][START_REF] Espay | On" state freezing of gait in Parkinson disease: a paradoxical levodopa-induced complication[END_REF] (forming the FoG group) on the basis of their answer to item 3 of the FoG questionnaire [START_REF] Giladi | Validation of the freezing of gait questionnaire in patients with Parkinson's disease[END_REF]. We next checked that potentially eligible patients displayed FoG episodes during specific FoG trajectories [START_REF] Snijders | Clinimetrics of freezing of gait[END_REF] in the off-drug state but not in the "supra-on-drug" state (i.e. after an acute intake of at least one and a half times the usual dose of levodopa) [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF][START_REF] Espay | On" state freezing of gait in Parkinson disease: a paradoxical levodopa-induced complication[END_REF]. We then included non-freezer PD patients matched for age, gender, cognitive efficiency (according to the Mattis Dementia Rating Scale [START_REF] Schmidt | The Mattis Dementia Rating Scale: normative data from 1,001 healthy volunteers[END_REF][START_REF] Green | Validity of the Mattis Dementia Rating Scale for detection of cognitive impairment in the elderly[END_REF]) and overall motor severity, in order to form the non-FoG group. The exclusion criteria included the inability to walk unaided in the off-drug condition, the use of deep brain stimulation, the presence of neurological disorders other than PD, dementia (as defined by the Movement Disorders Society criteria [START_REF] Emre | Clinical diagnostic criteria for dementia associated with Parkinson's disease[END_REF] and by a Mattis Dementia Rating Scale score of 130 or less out of 144 recorded in the 6 months preceding the PET acquisition [START_REF] Schmidt | The Mattis Dementia Rating Scale: normative data from 1,001 healthy volunteers[END_REF]) and major depression (according to the DSM IV criteria (American Psychiatric Association., 1994)). To ensure that all FoG episodes were dopasensitive, patients not taking dopaminergic medications as part of their usual therapeutic regimen were excluded from the study. Neuropsychological assessments were performed in on-drug state, including Mini Mental State Examination [START_REF] Folstein | Mini-mental state". A practical method for grading the cognitive state of patients for the clinician[END_REF], Mattis Dementia Rating Scale [START_REF] Green | Validity of the Mattis Dementia Rating Scale for detection of cognitive impairment in the elderly[END_REF], Hamilton Anxiety Scale (Hamilton, 1959), Montgomery and Asberg Depression Rating Scale [START_REF] Montgomery | A new depression scale designed to be sensitive to change[END_REF] and Lille Apathy Rating Scale [START_REF] Sockeel | The Lille apathy rating scale (LARS), a new instrument for detecting and quantifying apathy: validation in Parkinson's disease[END_REF] (Table 1).

Although clinical evaluations and PET scans were performed under off-drug conditions, the participants had been on their usual, stable medication regimen for at least 3 months prior to inclusion.

Experimental design

Clinical observations and nuclear medicine procedures were all performed under off-drug conditions (i.e. after the withdrawal of dopaminergic therapy for at least 12 hours) [START_REF] Langston | Core assessment program for intracerebral transplantations (CAPIT)[END_REF].

We evaluated the brain's metabolism of [ 18 F]-FDG first at rest and then immediately after continuous gait performed during radiotracer uptake. The time interval between these two acquisitions was between one and four weeks. In the resting condition, the participant lay still in the supine position throughout the 30-minute tracer uptake and stabilization period.

A 15-minute PET scan was then performed. In the gait condition, the participant performed FoG trajectories [START_REF] Snijders | Clinimetrics of freezing of gait[END_REF] for the 30 minutes immediately following the injection of [ 18 F]-FDG (i.e. during radiotracer uptake and stabilization). The subsequent 15-minute emission scan reflected brain metabolism during the FoG trajectories.

Actual gait was performed for 30 minutes before the PET acquisition because this corresponds to the time required for [ 18 F]-FDG uptake and stabilization in the brain [START_REF] Lucignani | Measurement of regional cerebral glucose utilization with fluorine-18-FDG and PET in heterogeneous tissues: theoretical considerations and practical procedure[END_REF][START_REF] Shimoji | Measurement of cerebral glucose metabolic rates in the anesthetized rat by dynamic scanning with 18F-FDG, the ATLAS small animal PET scanner, and arterial blood sampling[END_REF]. The patient performed the FoG trajectories [START_REF] Snijders | Clinimetrics of freezing of gait[END_REF] throughout the radiotracer uptake period, so that environmental triggers elicited as many FoG episodes as possible.

The FoG trajectory was standardized, as previously described by [START_REF] Snijders | Clinimetrics of freezing of gait[END_REF]: all patients initiated gait and sought to pass through a narrow (80-cm-wide) passage a few metres later. This was followed by a full (360°) turn to the right, a full turn to the left, a turn and half (540°) to the right, a turn and half to the left, a full turn to the right as quickly as possible, a full turn to the left as quickly as possible, a turn and half to the right as quickly as possible, a turn and half to the left as quickly as possible and then a "go" while counting backwards in threes (starting from a number between 100 and 200), (Figure 1 and the video in the Supplemental data). The trajectories were performed continuously for the 30 minutes between radiotracer injection and PET acquisition.

FoG evaluation

We used a stopwatch to measure the cumulative duration of FoG episodes during the 30minute FoG trajectories preceding the PET acquisition [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF]. Each observer tagged the onset of a FoG episode by pressing the stopwatch's button and holding it down until the end of the episode. FoG was considered to be (i) paroxysmal, very small shuffling steps with minimal forward movement (contrasting with the patients' previous steps), (ii) leg trembling in the absence of effective forward motion or (iii) complete akinesia (i.e. no observable motion of the legs) [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF]. The end of each episode of FoG was defined as the time when the patient took an effective step with a relatively normal step length and swing phase [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF]. Data on all subtypes of FoG (start hesitation, trembling in place and FoG when turning, when approaching a narrow gap or when preparing to stop) were pooled. Hence, the percent time with FoG was defined as the ratio of the cumulative duration of FoG episodes to the total test duration (30 minutes). We choose to record the percent time with FoG in order to obtain an objective measure of freezing [START_REF] Shine | Assessing the utility of Freezing of Gait Questionnaires in Parkinson's Disease[END_REF]; this criterion is more reliable and more accurate than the number of FoG episodes or the mean duration of a FoG episode [START_REF] Morris | A comparison of clinical and objective measures of freezing of gait in Parkinson's disease[END_REF].

PET data

All participants underwent PET scans at the same centre (the Nuclear Medicine Department at Lille University Hospital, Lille, France). The same acquisition and image reconstruction procedures were used for the resting and gait conditions in all patients.

PET data acquisition

Data were acquired on an Advance SL PET/CT system (GE Medical Systems, General Electric Company, Chalfont St. Giles, UK) with a 5 mm full-width at half-maximum and a 30 cm transaxial field of view. Participants were instructed to fast before the scans, and the patient's blood glucose level was always checked prior to intravenous injection of between 185 and 198 MBq of [ 18 F]-FDG. Thirty minutes later (i.e. during the radiotracer's stability window from 20 minutes to 90 minutes after injection, reflecting the uptake during the first 20 minutes [START_REF] Sokoloff | The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat[END_REF]), a low-dose CT scan of the brain was acquired for attenuation correction of the PET data. Emission images were subsequently acquired in three-dimensional mode. The images were reconstructed iteratively using an orderedsubset expectation-maximization algorithm (with two iterations and 21 subsets) in a 256x256 matrix.

PET data processing

The University, Montreal, Canada) using an affine transformation (with 12 parameters for rigid transformations) [START_REF] Friston | Commentary and opinion: II. Statistical parametric mapping: ontology and current issues[END_REF]. To increase the signal-to-noise ratio, the images were smoothed by convolution with an isotropic Gaussian kernel (12 mm full-width at halfmaximum). An overall normalization was applied by including each subject's mean global activity as a covariate of no interest. Thus, our patient-by-patient analysis focused on individual differences in regional brain activity as a proportion of overall brain activity.

PET data analysis

First, the Talairach applet [START_REF] Lancaster | Automated labeling of the human brain: a preliminary report on the development and evaluation of a forward-transform method[END_REF][START_REF] Lancaster | Automated Talairach atlas labels for functional brain mapping[END_REF] (Research Imaging Institute of the University of Texas Health Science Center San Antonio (UTHSCSA) was used to determine the location of the nearest grey matter for each significant peak (from the wholebrain analysis). Since the applet uses Talairach space, the coordinates were first translated from MNI space with the MNI-Talairach Coordinate Converter (www.bioimagesuite.org), as presented in Tables. Next, in order to specifically explore areas of the cortex, brainstem and basal ganglia, we defined a number regions of interest (ROIs): the primary motor cortex, dorsal premotor area, dorsolateral prefrontal cortex, medial prefrontal cortex, posterior parietal cortex, subthalamic nucleus, thalamus, putamen, globus pallidus, caudate nucleus, ventral striatum and MLR, as described by Shine et al. (2013a). The coordinates of each ROI were registered on each PET scan (normalized in MNI space).

Statistical analysis

When considering clinical data, intergroup differences in continuous variables were evaluated with an unpaired Student's t test (for variables with a normal distribution according to the Shapiro-Wilk test) or a Wilcoxon test (for non-normal distributions). A chi-squared test was used for categorical variables. The threshold for statistical significance was set to p<0.05. All statistical analyses of clinical data were performed with IBM SPSS for Windows software (version 16.0, IBM, Armonk, NY, USA).

For functional imaging data, we first performed voxel-wise whole-brain analyses by using the flexible factorial design in SPM5. Clusters of at least 30 contiguous voxels with a threshold two-tailed p value <0.005 were considered to be statistically significant. We included each of the following variables in turn: subject, group (FoG vs. non-FoG) and condition (resting vs. gait). We tested for a main effect of condition (resting vs. gait), a main effect of group (FoG vs. non-FoG) and a group x condition interaction. Both increases and decreases in glucose metabolism were analyzed. Post-hoc analyses were based on the "compare-populations one scan/subject" routine. For each voxel, a simple, fixed-effect T test was used to compare the two groups or pairs of conditions. For analyses of ROIs, spherical volumes with a 5 mm radius around the peak coordinates of each ROI were defined and a t test was used to compare the two groups (p voxel uncorrected =0.005).

Lastly, we explored correlations between brain metabolism during gait and the percent time with FoG in the FoG group with a simple regression analysis.

RESULTS

Characteristics of the study population

The FoG trajectories performed before the "gait" image acquisition were effective in eliciting FoG episodes. All patients in the FoG group presented FoG episodes, and the mean (± SD) total duration of FoG per patient was 12 (±7) minutes (i.e. 39% (± 23%) of the 30-minute FoG trajectory; range: 13-77%). No FoG episodes were recorded in the non-FoG group. Gait was more impaired in the FoG group, as shown in Table 1. Due to the FoG itself and the gait impairment between FoG episodes, the number of FoG trajectories completed was obviously lower and more variable in the FoG group. The time needed to complete one FoG trajectory was comprised between 2 and 13 min in the FoG group, compared with 1.5 to 1.8 min in the non-FoG group. Even though the FoG trajectory (which was the same for all patients) comprised several triggers, most of the FoG episodes in the FoG group were triggered by turning. previously [START_REF] Giladi | Construction of freezing of gait questionnaire for patients with Parkinsonism[END_REF][START_REF] Giladi | Freezing of gait in PD: prospective assessment in the DATATOP cohort[END_REF] (Table 2). However, FoG scores (concerning off drug FoG) were not correlated with postural stability nor gait item in on drug (UPDRS II), but with gait item in off drug (UPDRS III). 3).

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The results of the ROI analysis are presented in Table 4. In the basal ganglia, activation after gait (compared with rest) was only significant for the right thalamus and the subthalamic nuclei. There were no differences for the putamen or the caudate nucleus.

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The FoG group

In a whole-brain analysis, the patients in the FoG group showed post-gait hypermetabolism (vs. rest) in the secondary visual cortex (BA18), premotor cortex (BA6), dorsolateral and anterior prefrontal cortices (BA9 and BA10), primary somatosensory cortex (BA3), cerebellum (the culmen, tonsil, and semi-lunar lobule), basal ganglia, temporoparietal junction (BA40, BA41 and the insula (BA13)) and cingulate cortex (BA30) (p<0.005). All the clusters withstood correction for the false discovery rate (<0.005 for the first eight clusters and <0.05 for the following clusters) (Table 3).

The results of the ROI analysis are presented in Table 4. There was overall activation after gait (vs. rest) in the basal ganglia (including the thalamus, subthalamic nucleus and putamen). In contrast, the globus pallidus and the MLR displayed significant hypometabolism after gait. The FoG group did not activate the medial prefrontal region after gait; in fact, there was a non-significant trend towards deactivation.

Comparison of brain metabolism after gait

in FoG and non-FoG patients (Figure 3)

When comparing the two groups, the non-FoG group showed greater activation of the temporopolar area (BA38), orbitofrontal area (BA11) and associative premotor cortex (BA8) (p<0.005). Conversely, the FoG group showed greater activation around the intraparietal sulcus in the paracentral lobule (BA5) (p<0.005).

In the ROI analysis, the FoG group showed hypermetabolism (relative to the non-FoG group) in the globus pallidus and left posterior parietal cortex and hypometabolism in the left dorsolateral prefrontal cortex.

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The results were generally similar when disease duration and gender were added as covariates (Table 5), with greater metabolic activity after gait in the premotor area, frontal eye fields (FEFs) and somatosensory association cortex in the non-FoG group and in the claustrum, cerebellum and primary visual cortex in the FoG group. Lastly, we observed activation of the temporopolar area and pars orbitalis in the FoG group when disease duration and gender were added as covariates. However, none of the clusters withstood correction for the false discovery rate. Only the 875-voxel cluster located in the right supplemental motor area (BA 6, around (37; 1; 57)) tended to withstand correction for the false discovery rate (0.254) and the family wise error (0.121).

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Clinical and metabolic correlations (Table 6)

In the FoG group, the percent time with FoG was positively correlated with the activity of the cerebellum, paracentral lobule (BA5) and the right FEF (BA8). Conversely, the percent time with FoG was negatively correlated mainly with the activity in the orbitofrontal area, premotor cortex, the left SMA and temporal lobe. For the study population as a whole, we also found a positive correlation between the UPDRS III gait score and the activity of the cerebellum, FEFs (BA8) and basal ganglia after gait. In contrast, it was negatively correlated with the activity mainly in the orbitofrontal area, premotor cortex, SMAs and temporal lobe (Figure 4).

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DISCUSSION

This [ 18 F]-FDG-PET study is the first to evidence abnormal brain metabolic activation after actual gait in PD patients with FoG. The frontal and parietal cortical FDG uptake seen in non-FoG PD patients differed from that seen in patients with FoG; the latter displayed significant deregulation of the premotor area (notably the premotor cortex and SMA, which are involved in both the indirect cortical locomotor pathway [START_REF] La Fougère | Real versus imagined locomotion: a [18F]-FDG PET-fMRI comparison[END_REF] and attentional mechanisms) and the output of the basal ganglia (namely the globus pallidus-MLR complex). We will first discuss the frontoparietal network and its implication concerning locomotor adaptation according to the environment, then implication of other motor networks (basal ganglia and cerebellar loops) and their involvement in this type of gait.

As previously described for human locomotor control (Jahn et al., 2008a;[START_REF] La Fougère | Real versus imagined locomotion: a [18F]-FDG PET-fMRI comparison[END_REF], the classical components of the supraspinal locomotor network are indeed present in our PD patients. In fact, the brain areas involved in PD gait can be grouped together, as follows: (i) the basal ganglia (involved in motor program selection, [START_REF] Grillner | Neural bases of goaldirected locomotion in vertebrates--an overview[END_REF]),

(ii) the cerebellum (involved in rhythm generation, [START_REF] Grillner | Neurobiological bases of rhythmic motor acts in vertebrates[END_REF]) (iii) the sensory cortices (for external inputs in general, with a key role in proprioception for motor control in PD [START_REF] Almeida | An evaluation of sensorimotor integration during locomotion toward a target in Parkinson's disease[END_REF][START_REF] Jacobs | Abnormal proprioceptive-motor integration contributes to hypometric postural responses of subjects with Parkinson's disease[END_REF][START_REF] Schrader | Changes in processing of proprioceptive information in Parkinson's disease and multiple system atrophy[END_REF][START_REF] Konczak | Proprioception and motor control in Parkinson's disease[END_REF][START_REF] Tan | Proprioceptive deficits in Parkinson's disease patients with freezing of gait[END_REF]), (iv) the temporoparietal junction (for the multimodal sensory integration of external cues and updating of environmental information via a bottom-up mechanism, [START_REF] Corbetta | Control of goal-directed and stimulus-driven attention in the brain[END_REF][START_REF] Yang | An event-related FMRI study of exogenous orienting across vision and audition[END_REF]) and (v) the prefrontal cortex (via a top-down mechanism that regulates pertinent sensory inputs and adapts the motor gait program accordingly [START_REF] Corbetta | Control of goal-directed and stimulus-driven attention in the brain[END_REF][START_REF] Taylor | Subsecond changes in top down control exerted by human medial frontal cortex during conflict and action selection: a combined transcranial magnetic stimulation electroencephalography study[END_REF][START_REF] Cools | Top-down attentional control in Parkinson's disease: salient considerations[END_REF]).

Our results are generally consistent with published MRI perfusion data, i.e. in freezers patients an (i) hyperactivation within the paracentral lobule and basal ganglia and (ii) cerebellar dysfunction or (iii) corticosubcortical decoupling, (Shine et al., 2013a;Vercruysse et al., 2014b;[START_REF] Maillet | Neural substrates of levodopa-responsive gait disorders and freezing in advanced Parkinson's disease: A kinesthetic imagery approach[END_REF]. The differences with regard to the literature data are discussed below.

Balance inside the frontoparietal network after gait

First, the results of the whole-brain analysis revealed a premotor vs. parietal contrast, with greater premotor activation in the non-FoG PD group and greater parietal activation in the FoG group. The parietal cortex provides the input for the parietal-premotor and frontoparietal networks [START_REF] Battaglia-Mayer | Multiple levels of representation of reaching in the parieto-frontal network[END_REF]. In fact, the frontoparietal network may be one of the major functional substrates for modulated gait in PD because it is responsible for integrating external and internal modalities and comparing them with the goal (gait, in this case) [START_REF] Battaglia-Mayer | Multiple levels of representation of reaching in the parieto-frontal network[END_REF].

Role of attention

During gait, the motor program is continuously updated by incoming information. The frontotemporal networks ensure that goal-relevant information receives priority; for example, they will reorient attention if a visual trigger requiring adaptation of the motor program occurs. During modulated gait, the patient has to focus attention on obstacles on his/her path (e.g. when turning in a corridor or upon reaching some stairs) and then adapt his/her trajectory and cadence. However, the patient also has to filter out stimuli that are not behaviourally relevant (e.g. markings on the ground such as threshold strips, or unrelated vocal sounds). Lastly, the most important aspect is the assignment of an appropriate, patient-scaled behavioural response to each stimulus (rather than an exaggerated response in a confined space, for example).

The attentional system includes two networks: the dorsal one (including regions of the intraparietal sulcus, superior parietal cortex, FEF, premotor cortex (SMA), dorsolateral prefrontal cortex) tends to be active during focused, goal-directed attention to a particular target (here gait), whereas the ventral one (including the temporoparietal junction, inferior frontal gyrus, lateral and inferior frontal/prefrontal cortex and anterior insula) is associated with redirecting attention toward stimuli that are relevant to the immediate goal [START_REF] Corbetta | The reorienting system of the human brain: from environment to theory of mind[END_REF][START_REF] Asplund | A central role for the lateral prefrontal cortex in goal-directed and stimulus-driven attention[END_REF][START_REF] Frank | Stimulus-driven reorienting in the ventral frontoparietal attention network: the role of emotional content[END_REF], called stimulus-driven attention. The balance between dorsal and ventral subdivisions of the frontoparietal network requires determining the locus of attention, disengaging and reorienting attention as necessary [START_REF] Corbetta | Control of goal-directed and stimulus-driven attention in the brain[END_REF], especially by determining which stimuli are relevant or not according the task.

Normally, as task difficulty increases, activity suppression in the ventral network correlates positively with task performance, an effect thought to reflect the gating of irrelevant cues [START_REF] Frank | Stimulus-driven reorienting in the ventral frontoparietal attention network: the role of emotional content[END_REF]. It would be expected that during this high-asking attention task with several FoG trajectories, the stimuli integration level would decrease. Indeed, in the non-freezers patients, this task majority involves the dorsal network, including activation of the FEF, premotor cortex (SMA) and dorsolateral prefrontal cortex. In contrast, in the FoG group, there was a poor frontal activation in FoG, accordingly with less inhibition or gating from frontal to parietal structures [START_REF] Konishi | Contribution of working memory to transient activation in human inferior prefrontal cortex during performance of the Wisconsin Card Sorting Test[END_REF][START_REF] Downar | A multimodal cortical network for the detection of changes in the sensory environment[END_REF][START_REF] Downar | The effect of task relevance on the cortical response to changes in visual and auditory stimuli: an event-related fMRI study[END_REF] and then higher activation of the ventral network. Supporting the hypothesis of the impairment of interaction between dorsal and ventral ways in freezers patients, our study showed that the FoG group had less activation of the dorsal pathway than the non-FoG group after gait, mainly in SMA and FEF (Tables 345). Moreover, gait scores were positively correlated with the ventral way's activation (inferior frontal gyrus) (Table 6 and Figure 4).

The cingulate cortex was activated by FoG trajectories in both the FoG and non-FoG groups, whereas the temporopolar and orbitofrontal areas were activated in non-FoG patients (relative to FoG patients). Concerning the ventral frontal cortex (ventromedial -BA 10, 11 and 47-and ventrolateral -BA 44, 45 and 47-), its activation after gait was generally positively correlated with the gait score and negatively with the time of freezing.

Considering its role in decision making and in stimulus-outcome associations, it could be involved in facilitating changes of behaviour in case of unexpected outcomes [START_REF] Murray | What we know and do not know about the functions of the orbitofrontal cortex after 20 years of cross-species studies[END_REF]O'Doherty, 2007). This reversal action, reflecting flexibility, guides selection of the most advantageous choices considering potential positive and negative consequences.

Then the ventral frontal cortex could act to signal the task transition and readjust between the dorsal and ventral attentional networks, to priorize goal or stimulus according to the situation [START_REF] Shulman | Reactivation of networks involved in preparatory states[END_REF][START_REF] Corbetta | The reorienting system of the human brain: from environment to theory of mind[END_REF]. It also could be involved in the transitions between task boundaries as complex gait should not be considered as a continuous activity but as a series of event with interruptions and terminations requiring updating [START_REF] Bouret | Network reset: a simplified overarching theory of locus coeruleus noradrenaline function[END_REF][START_REF] Zacks | Event segmentation[END_REF][START_REF] Corbetta | The reorienting system of the human brain: from environment to theory of mind[END_REF].

It is also important to note that when disease duration and gender were added as covariates, the hypometabolism of temporopolar area after gait decreased in the FoG group -suggesting that the hypometabolism of this area observed in the FoG group is more related to more advanced disease.

Sensory integration: the parietal pole

The greater activation of parietal areas in patients with FoG may reflect an increased need to rely on external cues because internal integration is impaired [START_REF] Hallett | The intrinsic and extrinsic aspects of freezing of gait[END_REF]. It may also reflect difficulty in resisting external interference [START_REF] Naismith | The specific contributions of set-shifting to freezing of gait in Parkinson's disease[END_REF][START_REF] Vandenbossche | Freezing of Gait in Parkinson Disease Is Associated With Impaired Conflict Resolution[END_REF]. Increased brain activation in the FoG group appears to reflect increased processing of sensory inputs rather than increase in inputs per se (which are identical for the two groups). Indeed, if proprioceptive afferences intensity are similar between groups during the FoG-trajectories, the level of deregulation could be the cortical integration of body weight load [START_REF] Mensink | Additional weight load increases freezing of gait episodes in Parkinson's disease; an experimental study[END_REF] or proprioception [START_REF] Tan | Proprioceptive deficits in Parkinson's disease patients with freezing of gait[END_REF]. Exaggerated processing of sensory inputs might explain the "transient disruptions of locomotor circuitry leading to a motor block" [START_REF] Nutt | Freezing of gait: moving forward on a mysterious clinical phenomenon[END_REF] when the sensory integration overloads the locomotor network. Usually, the sensitivity to sensory changes sensitivity is controlled by the task-relevance with insensitivity to task-irrelevant perceptual salient stimuli [START_REF] De Fockert | Neural correlates of attentional capture in visual search[END_REF][START_REF] Kincade | An event-related functional magnetic resonance imaging study of voluntary and stimulus-driven orienting of attention[END_REF]. In case of automatic gait for example, the vestibular and somatosensory cortex showed deactivation, which thus prevents adverse interactions with the spinal pattern and sensory signals [START_REF] Jahn | Brain activation patterns during imagined stance and locomotion in functional magnetic resonance imaging[END_REF]. This multisensory inhibition, operating during unhindered locomotion, seems impaired in freezers patients. Freezers could display disorders in the process of categorizing stimuli, according to task-relevance or not, showing poor goal-directed and abnormal stimulus-driven activation. FoG could then be related to an impairment in discriminating what is task (gait) relevant and consider as relevant all distracters.

4.1.3.

Premotor differences between non-FoG and FoG groups after gait.

In both the whole-brain analysis and the ROI analysis, the premotor cortex and SMA were less activated in the FoG group than in the non-FoG group after gait. This finding is in line with literature reports [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF][START_REF] Herman | Neuroimaging as a window into gait disturbances and freezing of gait in patients with Parkinson's disease[END_REF]Shine et al., 2013a).

Differences in this region might be due to several different mechanisms -especially those affecting attentional networks and those involved in externally guided (visually guided) movements [START_REF] Corbetta | Control of goal-directed and stimulus-driven attention in the brain[END_REF]. In line with our present observations, preferential use of the premotor area in PD has also been described for alternating finger movements [START_REF] Samuel | Evidence for lateral premotor and parietal overactivity in Parkinson's disease during sequential and bimanual movements. A PET study[END_REF] and "paradoxical gait" (Hanakawa et al., 1999a), in which visual stimuli improved the PD patients' gait parameters. The FEF that adjusts the visuospatial exploration during gait (by guiding the eye and head movements) might also have a key role here [START_REF] Pierrot-Deseilligny | Cortical control of ocular saccades in humans: a model for motricity[END_REF][START_REF] Koyama | Functional magnetic resonance imaging of macaque monkeys performing visually guided saccade tasks: comparison of cortical eye fields with humans[END_REF][START_REF] Brown | Relation of frontal eye field activity to saccade initiation during a countermanding task[END_REF], as suggested by the correlation between the percent time with FoG and FEF activation. The SMA is classically considered as comprising an anterior region (the pre-SMA, involved in the early stages of motor processing such as motor selection and preparation) and a posterior region (the SMA proper, involved in later stages such as initiation and execution of the motor program) [START_REF] Matsuzaka | A motor area rostral to the supplementary motor area (presupplementary motor area) in the monkey: neuronal activity during a learned motor task[END_REF][START_REF] Passingham | Functional organization of the motor system[END_REF][START_REF] Lee | Subregions within the Supplementary Motor Area Activated at Different Stages of Movement Preparation and Execution[END_REF]. The difference between FoG and non-FoG patients highlighted here (Figure 2) mainly concerns the posterior SMA; this may reflect that freezers have greater difficulties in initiation and execution than in motor selection. This is suggested (for example) by endless repetition of the motor program during trembling in place -a phenomenon frequently observed during gait initiation (Jacobs et al., 2009b). However, the coupling between preparation and execution is also included in pathophysiological hypotheses (Jacobs et al., 2009a;[START_REF] Nutt | Freezing of gait: moving forward on a mysterious clinical phenomenon[END_REF], making it difficult to distinguish between these two components in an analysis of FoG. Lastly, the respective involvements of the preSMA and/or SMA proper might vary as a function of the subtype of freezing (freezing during initiation vs. freezing when turning, for example). One can hypothesize that (i) the preSMA is more involved in gait initiation failure and (ii) the SMA proper is more involved in freezing during movement execution (which was more frequent here).

However, our paradigm was unable to distinguish between the metabolic patterns respectively associated with these two subtypes of FoG -although it was noteworthy that freezing in our FoG group occurred mainly when turning.

Pathophysiological hypotheses

Deregulation of the frontoparietal network in freezers might explain the dual nature of external cueing in FoG. Under ecological conditions, external cues (such as dual tasks) are often disruptive and trigger FoG -probably by overloading the basal ganglia (according to Shine et al.'s model (2011a)) and making gait less automatic. In contrast, external cues can also improve gait parameters (i.e. by increasing step length and lowering step cadence) [START_REF] Azulay | Influence of visual cues on gait in Parkinson's disease: contribution to attention or sensory dependence?[END_REF] and might thus decrease FoG (at least temporarily, until the repeated use of the strategy wears off). Hence, external cues (whether auditory or visual) may also help the patient to overcome FoG episodes, when the cues' function is to normalize gait parameters [START_REF] Nutt | Freezing of gait: moving forward on a mysterious clinical phenomenon[END_REF]. One can hypothesize that the premotor area's role is particularly enhanced when visuomotor modulation is intense. Indeed, when the cues are similar to the standardized gait program, visuomotor coordination is enough to produce relatively normal gait in the FoG group (due to the maintenance of parietal inputs). In contrast, cues that are unrelated to the internal motor program (i.e. external cues that distract attention from redefined motor patterns) will oblige the premotor area to detect motor errors (i.e. mismatch between the executed motor program and the environment, in terms of speed or direction) and correct them by adapting the motor program via the modulation of the downstream structures. We suggest that this latter mechanism is inefficient in the FoG group.

Furthermore, it is important to note that the brain regions in which metabolic activity was correlated with FoG were largely those correlated with gait impairments in general (Table 6). More severe impairment was related to greater metabolic activity in the FEFs, cerebellum and basal ganglia activity and lower metabolic activity in the premotor and orbitofrontal areas. This finding suggests that there is a continuum between gait disorders and freezing, which might be related to an imbalance between compensatory frontal mechanisms and more basic locomotor networks.

Correlations for time spent with FoG were less unicist (with a positive correlation with FEF and a negative correlation with inferior frontal gyrus, Table 6). Maybe this difference could be explained by the smaller effective (only FoG patients were included here) or could reflect different mechanisms, both impairment and compensation attempt when gait without FoG restart (restart of the dorsal way).

4.2.

The ROI analysis: FoG vs. non-FoG differences in activation of the basal ganglia activation.

In our study, the basal ganglia (namely the putamen) were activated after gait (compared with rest) in the FoG group but not in the non-FoG group. The FoG group showed greater activation of the globus pallidus and thalamus, which may lead to deregulation of the MLR [START_REF] Lewis | A pathophysiological model of freezing of gait in Parkinson's disease[END_REF]Shine et al., 2011a).

Use of the basal ganglia's thalamocortical circuits may manifest itself via "jamming motor execution" in FoG (as seen for trembling in place [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF]) or by a total arrest in motor execution (as in start hesitation) [START_REF] Schaafsma | Characterization of freezing of gait subtypes and the response of each to levodopa in Parkinson's disease[END_REF].

After gait with FoG, our results evidenced (i) greater activation of the putamen and thalamus (ii) and significant deactivation of the basal ganglia's outputs (the globus pallidus and MLR). The MLR is functionally and anatomically impaired in PD, and the impairment increases with disease progression [START_REF] Karachi | Cholinergic mesencephalic neurons are involved in gait and postural disorders in Parkinson disease[END_REF]. Functional imaging studies with various paradigms have shown that the MLR is involved in FoG [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF]Shine et al., 2013a;[START_REF] Maillet | Neural substrates of levodopa-responsive gait disorders and freezing in advanced Parkinson's disease: A kinesthetic imagery approach[END_REF]. Although we did not observe intergroup differences in MLR activation in the whole-brain analysis, the ROI analysis showed that this area was significantly deactivated after complex gait in the FoG group. The lack of a significant difference in the present study and the trend to deactivation (in contrast to the study by [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF] and [START_REF] Maillet | Neural substrates of levodopa-responsive gait disorders and freezing in advanced Parkinson's disease: A kinesthetic imagery approach[END_REF]) may be due to methodological differences. Activation of the MLR may be more related to mental imagery of gait initiation, which requires inhibition of the engaged motor program (Jahn et al., 2008b) because motor execution was imagined rather than performed. Furthermore, our study focused on the underlying locomotor pattern associated with FoG and not solely on the narrow time window within which FoG occurs (Shine et al., 2013a). Indeed, gait initiation episodes are much less frequent during actual gait trajectories than during mental imagery paradigms (every 4 to 12 seconds for 25 minutes, for example, in the study by [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF]. It is noteworthy that prefrontal cortex hypoactivation and the absence of MLR hyperactivation were recently described in patients with progressive supranuclear palsy (of which FoG is a cardinal feature) during a modulated gait paradigm (as used in the present study) [START_REF] Zwergal | Functional disturbance of the locomotor network in progressive supranuclear palsy[END_REF].

As also reported by Shine et al. (2013a), we observed greater cortical activation in patients with FoG after gait (relative to rest, Table 4). This activation involved not only the primary motor cortex (as in non-FoG patients) but also the premotor area and the posterior parietal cortex. These observations are also suggestive of cortico-subcortical decoupling. When comparing gait with rest within each group, cortical activation was more widespread (i.e.

spatially extended) in patients with FoG (although no intergroup differences were noted for the resting condition). This probably corresponds to an attempt by patients with FoG to (i) compensate for impairment of the basal ganglia and (ii) use the external environment to adapt the gait parameters. However, when comparing the two groups after activation by real gait trajectories (i.e. the intergroup contrast), overall activation of the premotor area was less intense in patients with FoG than in patients without FoG. Indeed, cortical activation in patients with FoG was greater in the parietal part of the frontoparietal network (which handles sensory inputs). In contrast, the frontal pole (which handles motor outputs)

is poorly activated and is unable to directly shunt the basal ganglia. These observations may explain why our metabolic results (with glucose uptake averaged over 30 minutes of gait) highlighted relatively low activation in the premotor area in patients with FoG, whereas studies exploring perfusion during mental imagery of gait (with the equivalent of FoG-like episodes) reported hyperactivation of the premotor area [START_REF] Maillet | Neural substrates of levodopa-responsive gait disorders and freezing in advanced Parkinson's disease: A kinesthetic imagery approach[END_REF].

The premotor activation during FoG or FoG-like episodes may also exist in freezers but is less intense than that observed in non-freezers. These results are consistent with a hypothesis in which the external loop has a compensatory role (Hanakawa et al., 1999a(Hanakawa et al., , 1999b;;[START_REF] La Fougère | Real versus imagined locomotion: a [18F]-FDG PET-fMRI comparison[END_REF] but is not sufficiently effective in PD patients with FoG.

Our present results highlighted (i) the low overall activation of the premotor area after gait in PD patients with FoG but (ii) a positive correlation between the percent time with FoG and the metabolism of the FEFs (as similarly shown during upper limb motor blocks by Vercruysse et al. (2014a)). These observations suggest that PD patients with FoG attempt to use this parietofrontal network but that the latter does not effectively adapt the motor program.

Is the cerebellar network also involved in gait with

freezing?

Intergroup differences in the frontal and parietal regions might also reflect the involvement of neocerebellar circuits via frontopontine and parietopontine fibres. Indeed, metabolism in the paracentral lobule, superior frontal gyrus and neocerebellar lobes was correlated with the percent time with FoG during the gait task preceding the PET acquisition. These areas belong to the neocerebellar network, which processes sensory information monitors and optimizes movements by using sensory feedback [START_REF] Jueptner | A review of differences between basal ganglia and cerebellar control of movements as revealed by functional imaging studies[END_REF] Even after gender and disease duration were added as covariates, we again observed hyperactivation of the cerebellum, primary visual cortex and claustrum in freezers after gait (reflecting the preferential use of basic rhythmic loops [START_REF] Tanné-Gariépy | Projections of the claustrum to the primary motor, premotor, and prefrontal cortices in the macaque monkey[END_REF][START_REF] Smith | Rat Claustrum Coordinates but Does Not Integrate Somatosensory and Motor Cortical Information[END_REF].

Putative brain lateralization in FoG

Our findings are somewhat limited by the question of laterality: in freezers, left-side premotor hypometabolism was observed when studying the effect of group, whereas rightside premotor hypometabolism was highlighted when studying the effect of gait condition.

Bilateral involvement is probable but was not unambiguously demonstrated here -perhaps due to the small sample size. Literature studies of paradoxical gait in PD have shown hyperperfusion of the right premotor cortex (Hanakawa et al., 1999), whereas a mental imagery task revealed hyperperfusion of the left supplementary motor cortex in nonfreezers (relative to freezers) [START_REF] Snijders | Gait-related cerebral alterations in patients with Parkinson's disease with freezing of gait[END_REF]. The left premotor cortex is sometimes considered to have a preferential role in goal-directed movement [START_REF] Schluter | Temporary interference in human lateral premotor cortex suggests dominance for the selection of movements. A study using transcranial magnetic stimulation[END_REF][START_REF] Schluter | Cerebral dominance for action in the human brain: the selection of actions[END_REF][START_REF] Rushworth | The left parietal and premotor cortices: motor attention and selection[END_REF], although this may have been due to the preferential use of right-hand motor paradigms. In contrast, [START_REF] Bartels | Brain imaging in patients with freezing of gait[END_REF] suggested that FoG is caused by neuronal circuitry dysfunctions in the right parietal-lateral premotor area [START_REF] Crémers | Hemispheric specialization during mental imagery of brisk walking[END_REF][START_REF] Fling | Asymmetric pedunculopontine network connectivity in parkinsonian patients with freezing of gait[END_REF][START_REF] Maillet | Neural substrates of levodopa-responsive gait disorders and freezing in advanced Parkinson's disease: A kinesthetic imagery approach[END_REF]Peterson et al., 2014a). In freezers, hypometabolism has been detected in the left premotor area and in the right parietal cortex [START_REF] Bartels | Striatal dopa and glucose metabolism in PD patients with freezing of gait[END_REF], showing that the both sides of the cortex are affected (albeit in different areas). In fact, there is no clear evidence of laterality of the premotor cortex in action selection (whether in response to visuospatial signals or not) [START_REF] Wise | Premotor and parietal cortex: corticocortical connectivity and combinatorial computations[END_REF]. For bilateral movements, action selection is probably underpinned by a bilateral network [START_REF] Horenstein | Comparison of unilateral and bilateral complex finger tapping related activation in premotor and primary motor cortex[END_REF] within which the left and right premotor cortices interact [START_REF] O'shea | Functionally specific reorganization in human premotor cortex[END_REF]. The laterality of FoG merits further investigation.

Study limitations

The present study had several limitations. Firstly, the sample size was small for such a complex disease phenomenon; the lack of statistical power explains why the effect of group was not corrected for multiple comparisons. Secondly, and even though the FoG and non-FoG groups did not differ significantly in terms of age and motor scores, the disease duration was longer in the FoG group (as is generally observed in this type of patient [START_REF] Perez-Lloret | Prevalence, Determinants, and Effect on Quality of Life of Freezing of Gait in Parkinson Disease[END_REF]). This disparity might explain in part the regional metabolic differences between the groups.

Thirdly, our study was unable to discriminate between the paroxysmal mechanisms corresponding to the various components associated with FoG (such as gait hypokinesia, sequence effects and sudden motor blocks) and the different subtypes of FoG. The injection of perfusion radiotracers (such as 99m Tc-bicisate) at the time at which each specific subtype of FoG occurs (e.g. FoG during turns) might enable characterization of the corresponding brain perfusion patterns. Lastly, axial symptoms are generally more frequent in freezers [START_REF] Giladi | Freezing of gait in PD: prospective assessment in the DATATOP cohort[END_REF] -even though the axial subscore was similar in our FoG and non-FoG groups-and may be related to the same pathophysiological mechanisms as FoG itself [START_REF] Park | Gait freezing and speech disturbance in Parkinson's disease[END_REF][START_REF] Vervoort | Which Aspects of Postural Control Differentiate between Patients with Parkinson's Disease with and without Freezing of Gait?[END_REF]. Axial symptoms (such as postural stability) are involved in motor control processes related to balance during locomotion.

Indeed, axial symptoms form part of the spectrum of gait disturbances encompassing paroxysmal FoG [START_REF] Heremans | Freezing of gait in Parkinson's disease: where are we now?[END_REF], slow execution speed and low step length.

Hence, axial symptoms may also account for intergroup differences in brain metabolism after gait [START_REF] Karachi | Cholinergic mesencephalic neurons are involved in gait and postural disorders in Parkinson disease[END_REF]. These confounding variables must be systematically monitored in future studies.

In PD patients performing arm movements, high movement velocity was associated with hyperperfusion of the premotor and parietal areas [START_REF] Turner | The functional anatomy of parkinsonian bradykinesia[END_REF], whereas low movement velocity was associated with hypoperfusion of the left dorsolateral prefrontal and premotor cortices [START_REF] Carbon | Correlates of movement initiation and velocity in Parkinson's disease: A longitudinal PET study[END_REF]. In patients with progressive supranuclear palsy, post-gait differences in brain metabolism (vs. healthy controls) were still significant after adjustment for gait velocity [START_REF] Zwergal | Functional disturbance of the locomotor network in progressive supranuclear palsy[END_REF]. Taken as a whole, these data suggest that the premotor hypometabolism observed in freezers may be partly due to a more general gait impairment (including a lower gait speed outside FoG episodes).

Conclusion

The present study is the first to demonstrate a characteristic, FoG-associated cortical pattern of metabolic activation in a paradigm including real gait performed by PD patients.

In the FoG group, we notably observed (i) hypoactivation of the frontal premotor cortex, (ii) hyperactivation of the parietal cortices and (iii) deregulation of the basal ganglia output (globus pallidus and MLR). Further multimodal imaging studies may help to elucidate the mechanisms underlying FoG in PD patients and prompt the development of techniques for modulating the affected brain networks (such as the frontoparietal and/or neocerebellar networks) involved in visually guided movements (like complex gait) in PD. In parkinsonian patients, the parieto-premotor network could be considered as a compensatory network, that could be overloaded in freezers in different situations such as when they had to reduce their step length (looking at visual cues consisted in white strips placed on the floor) leading to FoG [START_REF] Iansek | The sequence effect and gait festination in Parkinson disease: contributors to freezing of gait?[END_REF][START_REF] Chee | Gait freezing in Parkinson's disease and the stride length sequence effect interaction[END_REF]. This task could be further investigated specifically by brain imaging techniques. Data are quoted as the mean (standard deviation).

The axial motor subscore was defined as the sum of items 18, 27, 28, 29 and 30 of the Unified Parkinson's Disease Rating Scale part III (speech, rising from a chair, posture, gait and postural stability). The "Cluster" column indicates the number of voxels in the significant area. Coordinates (x; y; z) are presented in Talairach space.

Threshold of at least 30 contiguous voxels were applied for clusters, with a two-tailed p value of 0.005. Local maxima more than 8 mm apart were reported only if the corresponding area was different.

First, in greyed out are represented results of gait activation that is hypermetabolic regions during gait by comparing with rest (distinctly between groups, see Figure 1). Secondly are represented the major results of our study, that is differences between FoG-group and non-FoG group (see Figure 2). Coordinates are given in the Talairach space.

BA: Brodmann area / unc: uncorrected

  reconstructed [ 18 F]-FDG images were first recorded in Digital Imaging and Communications in Medicine format and then transformed into the Neuroimaging Informatics Technology Initiative format for further processing. Imaging data were processed and statistically analyzed with SPM5 (Wellcome Department of Cognitive Neurology, London, UK) implemented in MATLAB 7 (Mathworks Inc., Sherborn, MA, USA). Reconstructed [ 18 F]-FDG brain PET images were spatially normalized against the Montreal Neurological Institute (MNI) template (McGill

Figure 1 :Figure 2 :

 12 Figure 1: schema of the PET acquisitions

Figure 3 :

 3 Figure 3: Significant differences (p<0.005) in metabolism when comparing gait in FoG and

Figure 4 :

 4 Figure 4: Correlations between brain metabolism after the gait session (taking account all

  Table 1 about here Intergroup comparisons did not reveal any FoG vs. non-FoG differences in demographic and cognitive data in general or the patients' age, gender, Mini Mental State Examination scores and Mattis Dementia Rating Scale score in particular. There was a non-significant trend toward higher off-drug UPDRS III scores (p=0.124) and a longer time since disease

onset in the FoG group, when compared with the non-FoG group (p=0.007). The two groups differed regarding the gait subscore in off drug (UPDRS III) and freezing subscore (UPDRS II). FoG scores were correlated with motor axial symptoms, as has been observed

Table 2 about here 3.2. Brain metabolism: [ 18 F]-FDG PET at rest and after the FoG trajectory (Table 3) Please insert Table 3 about here 3.2.1. Brain glucose metabolism at rest in patients in the FoG and non-FoG groups

 2 At rest, there was no difference in brain glucose metabolism between the FoG and non-

	FoG groups.
	3.2.2.	Brain glucose metabolism after gait (vs. rest) in
	patients (Figure 2)
	3.2.2.1.	The non-FoG group

Table 1 -

 1 A: The demographic, neuropsychological and clinical characteristics of the study subgroups, and statistical comparisons.

	FoG group	non-FoG group	p

Freezing item (out of 4) -14

	Dual task with counting: number of	62	(37)	10	(3)	<0.001
	steps for 6 meters
	Dual task with counting: time for 6	28	(14)	8	(3)	<0.001
	meters (in sec)
		1.2	(0.6)	0.4	(0.8)	0.001
	Time since disease onset (years)	11.0	(2.6)	8.1	(1.9)	0.007
	FoG Questionnaire score (out of 24)	12.9	(4.4)	0.2	(0.4) <0.001
	FOG-TRAJECTORIES DATA
	FoG episodes
	Total time with FoG as a proportion of
	the FoG trajectories	0.39	(0.23)	0	(0.00) <0.001
	Gait parameters between FoG episodes
	Passage through a narrow gap: number
	of steps for 6 meters	23	(16)	10	(3)	<0.001
	Passage through a narrow gap: time for	13	(14)	8	(2)	<0.001
	6 meters (in sec)

Table 1 -

 1 B: Antiparkinsonian treatments of the two groups (FoG and non-FoG group). For levodopa equivalent dose daily, the results were presented as mean (standard deviation) and for others treatments, % of patients.

	FoG group	non-FoG group

FoG>FoG in the resting condition No clusters FoG>Non-FoG in the resting condition

	No clusters
	Non-FoG>FoG IN THE GAIT CONDITION
							Superior Temporal
	225	3.86	<0.001	46	16	-36 BA38	Gyrus	Temporopolar area
							Superior Frontal
	232	3.73	<0.001	10	56	-24 BA11	Gyrus	Orbitofrontal area
							Middle Frontal
	77	2.97	0.001	26	14	48 BA8	Gyrus	Frontal eye fields
	FoG>Non-FoG in THE GAIT CONDITION
								Somatosensory
	125	3.28	0.001	-24	-38	44 BA5	Paracentral Lobule	associative cortex

ACKNOWLEDGEMENTS

We thank David Fraser (Biotech Communication) for editorial assistance.

Abbreviations