Professor Thomas Müller
email: thomas.mueller@ruhr-uni-bochum.de

MD Siegfried Muhlack

Prior exercise improves reactivity and movement in patients with Parkinson's disease

Keywords: endurance exercise improves reactivity and movement Parkinson's disease, levodopa, exercise

Background

Following cued levodopa (LD) intake, endurance exercise showed a beneficial effect on scored motor performance in patients with Parkinson's disease (PD) in comparison with rest. This may result from an exercise induced increase of endogenous dopamine synthesis. As a result, beneficial effects on movement and reactivity may occur.

Objectives

To measure reactivity and motor performance in a repeated fashion with instrumental tasks after cued administration of soluble 200 mg LD/50 mg benserazide.

Design

PD patients consecutively performed paradigms, which assess reactivity and movement performance, after a standardised period of rest or of age related, heart rate adapted endurance exercise on two consecutive days in random order.

Results

Reactivity and execution of simple and complex motion series were significant better following exercise than after rest.

Discussion

Endurance exercise has a beneficial effect on reactivity and movement behavior in PD patients following cued application of LD probably due to an augmented synthesis of dopamine respectively other catecholamines and release in the prefrontal cortex, the nucleus accumbens and the basal ganglia. Small changes in catecholamine modulation of prefrontal cortex cells can have profound effects on the ability of the prefrontal cortex to guide behaviour. Previous exercise may also improve pedunculopontine nucleus function, which is involved in motor related attention processes.

Introduction

Treated patients with Parkinson's disease (PD) report a reduced exercise capacity due to hypothetical limited effect of dopaminergic drugs or an increased need for them. Clinical trials excluded possible causes like central dopamine deficiency, impaired gastrointestinal absorption and bioavailability of levodopa (LD), mitochondrial muscle dysfunction. But in PD patients, endurance exercise had a beneficial effect on not blinded scored motor symptoms following cued LD intake compared with rest. [START_REF] Muhlack | Exercise improves efficacy of levodopa in patients with Parkinson's disease[END_REF] However even blinded rating is questionable, when comparing an exercise -with a rest condition due to onset of vegetative epiphenomena, i.e. sweating. This observed improved motor performance after endurance exercise may be due to release of endogenous dopamine, which contributes to regulation of reactivity and of movement. 2;3 Conventional PD rating tools may be not sensitive and specific enough to capture these subtle changes of behavior, which may be determined by simple instrumental tasks. Their execution depends on endogenous dopamine release in nigrostriatal and prefrontal brain structures in healthy humans, whereas an equal cued, dopaminergic stimulation is necessary to enable identical conditions in a cohort of PD patients to investigate whether endogenous dopamine release following exercise improves reaction-and movement behavior. [START_REF] Vingerhoets | Which clinical sign of Parkinson's disease best reflects the nigrostriatal lesion?[END_REF] Various types of movement series exist. There are complex ones like peg insertion which ask for additional involvement of i.e. prefrontal areas. They are similar to performed motion series of simple reaction time tasks, since they require execution of aimed movements. In contrast tapping procedures are simple movement sequences, which are automatic and repetitive. They are more influenced by velocity with involvement of basal ganglia structures only. Objective of this exploratory, pilot trial was to assess reactivity and motion behaviour after cued dopaminergic stimulation and an interval of rest or of exercise in PD patients.

Subjects and Methods

Subjects

Design

PD patients were put off their regular PD drug therapy for 12 hours over night. They had a standardised breakfast (300 kcal) in the morning at 7 am. Soluble LD/benserazide (200mg/50mg, Madopar LT®) was applied after prior domperidone (Motilium®, 40 mg) intake to avoid nausea 30 minutes later. A simple reaction time paradigm (SRT) was performed 30 minutes before (point I) and 60, 90, 120 150 minutes after LD application (moments II -V). Conditions were identical except rest or exercise, both of which were performed in random order (randomization to condition sequence with sealed envelopes: Tanja Steiner) between moments I and II on two consecutive days in each participant (Figure 1). The patient did not know in advance, whether they would perform the exercise or the rest session on day 1, when performing the baseline investigation. One patient was tested at each session only. PD patients were trained with the use of the cycle ergo meter on the day before start of the trial. The patients took their remaining drug regime after the study interval to reduce a bias of prior long-term dopamine substitution.

Exercise condition

Cycle ergo meter exercise for the lower extremities was employed using a heart rate adaptive training program (Cateye Ergociser Modell EC-1600). By pedalling on the bike ergo meter, the target heart rate was set in correlation to the age. The heart frequency was measured by ear-clip every 30 seconds and automatically relayed to the cycle workload.

After reaching the age adjusted, required heart rate (formula: 200 -age) in the 15 minutes lasting warm up period, a steady state of endurance exercise within totally one hour lasting interval of exercise between moments I and II was performed with continuous, automatic adaptation of the bike pedal resistance.

Rest session

The patients were in a lying position.

Simple reaction time paradigm (SRT)

The response to a visual SRT task (manufactured by Schuhfried Ges.m.b.H., Austria) was measured. The apparatus consisted of a 31 cm x 42 cm rectangular surface with two stimulus lights (red and yellow), each coupled to the reaction button electrode 1 cm in diameter 15 cm equidistant from a central start button electrode. SRT performance did not depend on the red light, which was not presented and employed during the whole test procedure. The subject pressed with the index finger of the dominant (right-: N = 19, left-: N = 1, both-handed: N = 2) hand the central start button. After the appearance of the yellow stimulus light the subject had to switch off the light as quickly as possible by moving his finger from the central start button to the reaction button. Reaction time (RT) was considered as elapsed interval between onset of the yellow stimulus light and release of the start button. Movement time (MT) was the period between release of the start button and the pressing of the reaction button. Thus this paradigm only asks the subject to detect one stimulus and to produce the same response on every trial. [START_REF] Muller | Delayed motor response correlates with striatal degeneration in Parkinson's disease[END_REF] Since the more affected hand of PD patients presents slower SRT performance than the other less affected one, PD patients were asked only to use the right dominant hand for this withinsubjects comparison. [START_REF] Rafal | Preparation of manual movements in hemiparkinsonism[END_REF] RT and MT were assessed by a computer to millisecond accuracy. 28 RT trials were run. We used out of 26 right answers a truncated mean of values, which excludes measures greater than or less than 2 standard deviations (SD) of the mean value, for statistical analysis. In this trial, no participant fell within the 2 SD cut off.

Peg insertion

In order to execute the peg insertion procedure (manufactured by Schuhfried Ges.m.b.H., Austria) subjects were asked to transfer 25 pegs (diameter 2.5 mm, length 5 cm) from a rack into one of 25 holes (diameter 2.8 mm) in a computer-based contact board individually and as quickly as possible. The distance between rack and appropriate holes was exactly 32 cm. The board was positioned in the middle and the task was carried out on each side. When transferring each peg from rack to hole, elbows were allowed to be in contact with the table. The interval between inserting of the first and the last pin initially with the right -and then the left hand was assessed. Then the data for the right and left hand were averaged to reduce the amount of data. The period for this task was measured by a computer in seconds with an accuracy below 100 ms. [START_REF] Muller | Tapping and peg insertion after levodopa intake in treated and de novo parkinsonian patients[END_REF] Tapping In order to execute the tapping test (manufactured by Schuhfried Ges. m. b. H., Mödling Austria), we instructed the individuals to tap as quickly as possible on a computer based contact board (3 cm x 3 cm) with a contact pencil for a period of 32 seconds after the initial flash of a yellow stimulus light. We did not control for peak height reached by the pencil. The board was positioned in the middle. When performing the task, elbows were allowed to be in contact with the table. We registered the number of contacts by means of a computerized device. First we measured the tapping rate with the right hand and then with the left. [START_REF] Muller | Tapping and peg insertion after levodopa intake in treated and de novo parkinsonian patients[END_REF] All participants were asked to familiarize with the applied instrumental tests for an interval of 1 minute to reduce or avoid learning and training effects on test performance on the day before. Figure 2C compares the computed differences of baseline and each assessment point (formula see statistics section) between both conditions. Its outcomes support the observed RT reduction after exercise and the RT increase following rest.

MT only significantly (exercise: F (4,84) = 3.17, p < 0.02 [Figure 3A]; rest: F (4,84) = 1.86, n.s. [Figure 3B]) reduced following exercise. Baseline values between the rest and exercise session were not significant different (MT: exercise vs rest [p = 0.8]). Figure 3C describes the computed MT differences of baseline and each assessment point between both conditions. The number of right answers did not significantly change after exercise (F (4,84) = 0.12, ns) (data not shown), but decreased after rest (F (4,84) = 6.89, p < 0.001; post hoc analysis: I [27.9 ± 0.29; mean ± SD] vs II [27.59 ± 0.59] p < 0.001; I vs III; ns.; I vs IV; ns.; I vs V ns.).

Peg insertion

The interval for the peg insertion task significantly reduced after exercise (right: F (4,84) = 16.04, p < 0.001; Figure 4A; left: F (4,84) = 13.91, p < 0.001; Figure 4B) and rest (right: F (4,84) = 4.90, p = 0.0013; Figure 4C; left: F (4,84) = 6.85, p < 0.001; Figure 4D). Baseline values were not significant different (peg insertion interval: exercise vs rest [right: p = 0.16; left: p = 0.38]). Figures 4E and4F compare the computed differences of baseline and each assessment point between both conditions. Their results demonstrate the significant better improvement of peg insertion performance after exercise in particular with the right hand (Figure 4E), but not with the left one (Figure 4F).

Tapping

The tapping rate significantly increased after exercise (right: F (4,84) = 5.70, p < 0.001; Figure 5A; left: F (4,84) = 5.48, p < 0.001; Figure 5B), but not after rest (right: F (4,84) = 0.73, ns; Figure 5C; left: F (4,84) = 2.48, ns; Figure 5D). Baseline values did not significantly differ (right: p = 0.35; left: p = 0.46). Analogue to the previous analyses, significant differences between rest and exercise were found following moments IV and V (right hand, Figure 5 E), respectively moment V (left hand, Figure 5 F). This supports the finding of an improved tapping rate after exercise. There was no impact of the set covariates in the whole analysis and no complaints of PD patients before and after exercise.

Discussion

Exercise and reactivity

The improved SRT performance following exercise may result from an augmented endogenous dopamine synthesis and release in various brain structures. [START_REF] Muller | Delayed motor response correlates with striatal degeneration in Parkinson's disease[END_REF] Dopamine improves the nucleus accumbens function, which represents an interface between limbic and motor structures. The nucleus accumbens plays a major role in control of goaldirected actions and receives a dopaminergic input from the ventral tegmental area.

Behavioural studies consistently showed that the nucleus accumbens supports instrumental behaviours elicited by cues associated with drugs. Indirect stimulation of dopamine receptors in the nucleus accumbens by d-amphetamine shortened reaction times. Thus stimulation of the nucleus accumbens with dopamine is involved in guiding the speed of instrumental responding. 8;9 The impaired SRT execution after rest confirms previous outcomes on reactivity in PD patients with an exact timing of both LD intake and test performance. [START_REF] Muller | Delay of simple reaction time after levodopa intake[END_REF] An animal study also showed an impaired RT after cued dopaminergic stimulation in a particular species of fast reacting rats. [START_REF] Wilcox | Apomorphine doses impair the reaction time of fast reacting but not slow reacting rats[END_REF] Sedative effects of LD may play a role and release of fatigue counteracting brain norepinephrine is lower during rest than exercise. [START_REF] Dishman | Treadmill exercise training augments brain norepinephrine response to familiar and novel stress[END_REF] Therefore the number of correct answers also worsened in the SRT paradigm after rest.

Exercise and motor performance

Execution of the peg insertion task was better after exercise than rest in particular with the right dominant hand. This outcome confirms the prior observed behavioural pattern with the SRT paradigm with better test execution after exercise and that instrumental determination of disturbed movement performance is more valuable on the dominant side in PD patients. [START_REF] Van Hilten | Where to record motor activity: an evaluation of commonly used sites of placement for activity monitors[END_REF] The peg insertion paradigm asks for execution of complex movement sequences with a complex interplay of additional need for visual and spatial cognition, self-elaboration of internal strategies, sorting and planning. All these processes are influenced by the modulating role of striatal dopamine levels on association areas of the prefrontal cortex and the basal ganglia. 4;14 Prior exercise supported endogenous dopamine synthesis and release in these brain areas. 2;15 It is known, that small changes in catecholamine modulation of prefrontal cortex cells can have profound effects on the ability of the prefrontal cortex to guide behaviour. We assume that these hypothetical higher prefrontal catecholamine levels following exercise supported the better peg insertion performance as they also improved the execution of the attention related components of this task. [START_REF] Arnsten | Neurobiology of executive functions: catecholamine influences on prefrontal cortical functions[END_REF] The tapping procedure only asks for performance of an automated, repetitive motion series without need of attention and concentration related cognition load. Therefore it depends more on dopamine dependent basal ganglia function alone. Thus tapping outcomes also improved due to an exercise increased endogenous striatal dopamine release. Generally, one may also hypothesize, that prior exercise additionally resulted in a better pedunculopontine nucleus function, which is involved in motor related attention processes. [START_REF] Androulidakis | Oscillatory activity in the pedunculopontine area of patients with Parkinson's disease[END_REF] Our study results also confirm findings that exercise provides benefit in PD patients in terms of overall motor function, in particular bradykinesia. [START_REF] Dibble | High intensity eccentric resistance training decreases bradykinesia and improves quality of life in persons with Parkinson's disease: A preliminary study[END_REF] This was also found with improvement of UPDRS scores and of the control respectively coordination of grasping forces during the performance of a functional bimanual dexterity task. In particular, when PD patients were forced to perform exercise with a certain pedal rate and resistance 30 % above their preferred voluntary one, a better motor performance and control was observed. [START_REF] Ridgel | Forced, not voluntary, exercise improves motor function in Parkinson's disease patients[END_REF] In our trial, pedal rate and resistance was also continuously adapted to the heart rate. Therefore we assume that the exercise condition within our trial was more similar to a more forced exercise paradigm and to an aerobic exercise condition. [START_REF] Bergen | Aerobic exercise intervention improves aerobic capacity and movement initiation in Parkinson's disease patients[END_REF] Forced exercise may lead to a shift in motor control strategy, from feedback to a greater reliance on feed forward processes. This even suggests that forced exercise may alter central motor control processes. [START_REF] Ridgel | Forced, not voluntary, exercise improves motor function in Parkinson's disease patients[END_REF] This in line with trial outcomes, which describe dosedependent benefits of exercise in early PD patients and suggest that even high-intensity exercise can normalize corticomotor excitability. [START_REF] Fisher | The effect of exercise training in improving motor performance and corticomotor excitability in people with early Parkinson's disease[END_REF] Our results with a significant better performance of instrumental motor tests following exercise support this hypothesis to a certain extent. We asked our participants to perform exercise above the voluntary level but however not with high intensity.

Limitations

Our explorative pilot trial did not investigate, whether PD patients without any dopamine substitution show the same behavior, i.e. turn out to react different after a period of rest or exercise. Generally, test outcomes were better at baseline of the exercise condition. We cannot explain this phenomenon with dopamine related reward-or expectation mechanisms related to exercise, [START_REF] Guthrie | A neurocomputational model of tonic and phasic dopamine in action selection: a comparison with cognitive deficits in Parkinson's disease[END_REF] since we performed a crossover design with 11 patients starting with exercise on day 1 and the remaining ones on day 2. However all participants knew the test condition to be performed on the second day. This may reduce expectation and accordingly cause less release of endogenous dopamine and other catecholamines, since the participants were more adapted to the whole situation. Therefore one may also hypothesize, that the more dopamine related motor tests may be more sensitive to this hypothetical phenomenon than the reaction time paradigm. This was not the case, when the right and left handed execution of the motor tests were analyzed separately and accordingly no significant differences appeared concerning the SRT outcomes at baseline.

In conclusion, endurance exercise has a beneficial effect on reactivity and movement behavior in PD patients following cued application of LD probably due to an augmented synthesis and release of endogenous dopamine in various brain structures. The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JNNP and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence

  LD/Dopadecarboxylase inhibitor (N = 22, 450 ± 112.5 mg [mean ± SD]; Entacapone N = 9, 1000 ± 200 mg; bromocriptine N = 2, 30 mg each; cabergoline N = 2, 2 mg, 4 mg; pergolide N = 2, 2.25 mg, 8 mg; pramipexole N = 11, ropinirole N = 2, 9 mg, 15 mg; rotigotine N = 1, 8 mg; rasagiline N = 3, 1 mg each, selegiline N = 3, 7.5 mg each; amantadine N = 1, 300 mg).

  StatisticsT-tests for comparisons between rest and exercise, respectively ANCOVA (covariates: age, body weight, UPDRS) with repeated measures design within one condition were employed for this statistical exploratory analysis. The Least Significant Difference t-test was used for post hoc comparisons. The level of significance was p < 0.05. To minimise the effect of various baseline values, the differences (formula: value [moment I] minus value [II, III, IV, V each] during the rest-and within the exercise session were calculated.EthicsAll participants gave written informed consent. The study was approved by the local ethic committee of the university.ResultsSRTRT (F (4,84) = 2.5, p < 0.05; Figure2A[see Figure for post hoc analysis]) reduced after exercise and increased after rest (F (4,84) = 2.55, p < 0.05; Figure2B). There were no significant differences of the baseline values (RT: exercise versus [vs] rest: p = 0.1).

Figure 1

 1 Figure 1 -5 sent as extrafiles

  22 treated idiopathic PD patients, diagnosed according to the UK Brain Bank criteria, (age: 61.05 ± 2.08 [mean ± SD] years; duration of PD: 4.21 ± 0.61 years; UPDRS: 34.19 ± 2.62; UPDRS I: 1.91 ± 0.34; UPDRS II: 10.33 ± 1.18; UPDRS III: 19.29 ± 1.82; UPDRS IV: 2.57 ± 0.62; HYS: 1.91 ± 1.58; Mini Mental State Examination: 28.39 ± 1.97) without unpredictable motor fluctuations were consecutively enrolled. PD patients, who had other medical conditions, which may affect the outcomes of the performed assessments, did not participate. There concomitant drug treatment consisted of

Acknowledgement

We thank Tanja Steiner and Christiane Stamm for technical assistance. We thank the participating patients.

Competing Interest: None declared.


Dr Annemieke J Videler
email: a.j.videler@amc.uva.nl

PhD Anita Beelen

PhD Ivo N Van Schaik

PhD Camiel Verhamme

MD Leonard H Van Den Berg

PhD Marianne De Visser

PhD Frans Nollet

Tripod pinch strength and thumb opposition are the major determinants of

Keywords: Peripheral neuropathy, Charcot-Marie-Tooth disease, Hereditary motor and sensory neuropathy, Hand, Clinical evaluation, Rehabilitation CMT1A, Charcot-Marie-Tooth disease type 1A, MMN, multifocal motor neuropathy, AROM, active range of motion, PROM, passive range of motion, SWM, Semmes-Weinstein monofilaments, SHT, Sollerman hand function test

INTRODUCTION

Charcot-Marie-Tooth disease type 1A (CMT1A), the most frequent form of CMT is caused by a duplication of the gene for peripheral nerve myelin protein 22 (PMP22) on chromosome 17p11.2 (MIM 118220) and characterized by slowly progressive length-dependent distal muscle weakness, atrophy and sensory loss. [START_REF] Harding | The clinical features of hereditary motor and sensory neuropathy types I and II[END_REF][START_REF] Birouk | Charcot-Marie-Tooth disease type 1A with 17p11.2 duplication. Clinical and electrophysiological phenotype study and factors influencing disease severity in 119 cases[END_REF][START_REF] Raeymaekers | Duplication in chromosome 17p11.2 in Charcot-Marie-Tooth neuropathy type 1a (CMT 1a). The HMSN Collaborative Research Group[END_REF][START_REF] Vance | Linkage of Charcot-Marie-Tooth neuropathy type 1a to chromosome 17[END_REF] Symptoms and signs are most pronounced in feet and hands. Although it is generally assumed that the hands of CMT1A patients are involved less frequently and later in the disease as compared to the lower extremities, a recent study among young children with CMT1A showed that hand weakness and dysfunction may be present from the earliest stages of the disease. [START_REF] Burns | Hand involvement in children with Charcot-Marie-Tooth disease type 1A[END_REF] In CMT1A, the intrinsic hand muscles are primarily affected resulting in a (mild) clawing position of the fingers, loss of thumb opposition, and impaired grip and pinch strengths. [START_REF] Harding | The clinical features of hereditary motor and sensory neuropathy types I and II[END_REF][START_REF] Miller | The hand in Charcot-Marie-Tooth disease[END_REF][START_REF] Videler | Hand strength and fatigue in patients with hereditary motor and sensory neuropathy (types I and II)[END_REF] Sensory loss commonly involves decreased sense of vibration and touch. [START_REF] Harding | The clinical features of hereditary motor and sensory neuropathy types I and II[END_REF] Manual dexterity, defined as the ability to move the hands easily and skilfully and to work with hands is of utmost importance in performing daily activities and known to be frequently impaired in CMT1A. [8;9] Identifying the underlying determinants is important to develop intervention strategies that can effectively maintain or improve manual dexterity in CMT1A. Loss of thumb and finger mobility, grip and pinch strengths and sensory functions may all impact manual dexterity in CMT1A and were therefore chosen as potential determinants in this study. We hypothesized that both motor and sensory impairments would contribute to the loss of manual dexterity in CMT1A and therefore compared the CMT1A results with those of patients with multifocal motor neuropathy (MMN), chosen as a reference group in which motor impairments are the only determinants of manual dexterity. MMN is a rare acquired immunemediated disorder characterized by slowly progressive, asymmetric, predominantly distal weakness of one or more limbs without sensory loss usually starting in the hand or forearm. [START_REF] Van Asseldonk | Multifocal motor neuropathy[END_REF] In this study we determined the impact of hand strength, joint mobility and sensory function on manual dexterity in both CMT1A and MMN patients.

METHODS

Patients

All (n = 63) DNA-proven CMT1A patients between 18 and 70 years old known at the departments of Rehabilitation and Neurology at the Academic Medical Center (AMC) were invited to participate. Additionally, a convenience sample of 20 patients with MMN, diagnosed on clinical, laboratory and electrophysiological characteristics [START_REF] Van Asseldonk | Multifocal motor neuropathy[END_REF], aged between 18 and 70 years old and known at the departments of Neurology at the AMC and the University Medical Centre Utrecht were asked to participate.

Patients were excluded if they had difficulty comprehending the Dutch language or with a history of comorbidity that might interfere with upper limb function in CMT1A or MMN, including stroke, plexopathy, radiculopathy, upper limb surgery, alcohol abuse or a psychiatric disorder. All subjects gave informed consent to the procedures, which were approved by the Medical Ethical Committees of the AMC.

Assessments

Clinical information was obtained by one examiner (AV) in a standardized manner and included medical history taking with the general question whether patients noticed hand involvement and if so which symptoms they perceived, a detailed clinical assessment of the motor and sensory functions of the hand, and evaluation of manual dexterity.

Standard goniometric measurements of both the passive range of motion (PROM) and active range of motion (AROM) of the fingers joints were used and classified as impaired or normal. [START_REF] Ca | Range-of-motion measurement of the hand[END_REF] Thumb opposition was evaluated according to the Kapandji opposition score which defines ten stages of opposition, with full opposition scored as stage 10. [START_REF] Kapandji | Clinical evaluation of the thumb's opposition[END_REF] Three sensory functions were evaluated: the threshold for touch, tactile discrimination and vibration sense. The threshold for touch was determined at seven locations divided over the hand and fingers using Semmes-Weinstein monofilaments (SWM). [START_REF] Weinstein | Fifty years of somatosensory research: from the Semmes-Weinstein monofilaments to the Weinstein Enhanced Sensory Test[END_REF] The set of monofilaments ranged from 2.38 to 6.65, which represent the values of the logarithmic transformation of the force needed to buckle the filament. The finest filament felt at each location was recorded and the average of the seven locations on the hand was calculated. We used data of a Dutch reference population to determine the cut-off for normal threshold for touch. [START_REF] Hage | Difference in sensibility between the dominant and nondominant index finger as tested using the Semmes-Weinstein monofilaments pressure aesthesiometer[END_REF] We calculated the mean + 1.96 standard deviation (SD) with these reference data, resulting in filament 3.61 as cut-off for normal threshold for touch. Testing with monofilaments has good to excellent intra-and interobserver reliability in CMT. [START_REF] Schreuders | Sensory evaluation of the hands in patients with charcot-marie-tooth disease using semmes-weinstein monofilaments[END_REF] Tactile discrimination was evaluated with static two-point discrimination (s2PD) using a Disk-Criminator at the tip of the index finger. [START_REF] Lundborg | The two-point discrimination test. Time for a re-appraisal?[END_REF] For s2PD a distance of less than 6 mm was taken as cutoff for normal tactile discrimination in the finger pulp. [START_REF] Lundborg | The two-point discrimination test. Time for a re-appraisal?[END_REF] The vibration threshold was assessed using a Rydel-Seiffer tuning fork at the dorsum of the distal interphalangeal joint of the index finger. The average of three repeated readings was calculated as the vibration threshold. Results can be compared with reference values and graded as normal (grade 0) or disturbed (grade 1 to 4). A disturbed vibration sense at the dorsum distal interphalangeal joint of the index finger is classified as grade 1; abnormal sense at the ulnar styloid process as grade 2; at the medial humerus epicondyle as grade 3; and at the acromioclavicular joint as grade 4. [START_REF] Pestronk | Sensory exam with a quantitative tuning fork: rapid, sensitive and predictive of SNAP amplitude[END_REF][START_REF] Merkies | Reliability and responsiveness of a graduated tuning fork in immune mediated polyneuropathies. The Inflammatory Neuropathy Cause and Treatment (INCAT) Group[END_REF] Good inter-and intraobserver agreements and high responsiveness values were demonstrated for this tuning fork. [START_REF] Pestronk | Sensory exam with a quantitative tuning fork: rapid, sensitive and predictive of SNAP amplitude[END_REF][START_REF] Merkies | Reliability and responsiveness of a graduated tuning fork in immune mediated polyneuropathies. The Inflammatory Neuropathy Cause and Treatment (INCAT) Group[END_REF] While muscle strength is commonly measured subjectively, using the MRC scale, hand-held dynamometry is regarded as more precise, sensitive and objective. [START_REF] Escolar | Clinical evaluator reliability for quantitative and manual muscle testing measures of strength in children[END_REF] Therefore we assessed maximal isometric grip and pinch (two-point, tripod, and lateral pinch) strength using digital handgrip dynamometers (Lode Medical Technology) according to a standardized testing procedure. [START_REF] Mathiowetz | Grip and pinch strength: normative data for adults[END_REF] Patients were seated on a height-adjustable chair and received verbal encouragement during strength measurements. For grip strength testing, the handle of the dynamometer was set in the second position, recommended to test maximal grip strength irrespective of age, weight or hand dimensions. [START_REF] Firrell | Which setting of the dynamometer provides maximal grip strength?[END_REF] The mean force (in Newtons) of three trials was taken for all strength measurements. The use of hand-held dynamometry to measure hand strength has been shown to be highly reliable and valid in adults with CMT. [START_REF] Videler | Hand strength and fatigue in patients with hereditary motor and sensory neuropathy (types I and II)[END_REF][START_REF] Svensson | Hand function in Charcot Marie Tooth: test-retest reliability of some measurements[END_REF] To examine manual dexterity, the standardized Sollerman hand function test (SHT) was used, consisting of 20 subtests (scale 0 to 4) assessing unilateral and bilateral handgrip function, and reflecting the most common handgrips used during daily activities. [START_REF] Sollerman | Sollerman hand function test. A standardised method and its use in tetraplegic patients[END_REF] Both hands were evaluated. Subjects with normal manual dexterity should achieve a total of 80 points with their dominant hand and 77-79 points with the non-dominant hand.

Statistical analysis

Data from clinical examination were analyzed using descriptive statistics and compared with normal values. Published reference values of healthy subjects, stratified by age, gender and hand dominance, were recalculated into Newtons and used to determine the impact of CMT1A on hand strength (grip, two-point pinch, tripod and lateral pinch). [START_REF] Mathiowetz | Grip and pinch strength: normative data for adults[END_REF] Differences between dominant and non-dominant hand function variables were analyzed with the Wilcoxon signed ranks test. The scores on the Sollerman test were log-transformed (Log ((80score) +1)) to yield a normal distribution as they were skewed to the left. Associations between manual dexterity (log-transformed SHT sum score) and continuous hand function variables (thumb opposition, clawing position, the threshold for touch, two-point discrimination, grip strength, two-point pinch, tripod pinch and lateral pinch strength) were investigated using Pearson product moment correlation coefficients (r). Associations between manual dexterity and dichotomous variables (AROM, PROM, vibration sense) were investigated with point biserial correlation coefficients (r pb ). In the CMT1A group multiple linear regression analysis was performed to determine the independent contribution of hand function variables on manual dexterity. Variables with a univariate p-value < 0.10 were entered in a multiple stepwise linear regression analysis to obtain a set of mutually independent determinants. Considering the relatively large number of independents for the multiple regressions and a sample size of 63 CMT1A patients at most, three separate models were built for manual dexterity with determinants on joint mobility (model 1), sensory functions (model 2) and grip and pinch strength (model 3) with 3 to 4 potential determinants in each model. A final model for manual dexterity was made using the independent factors that were significant in the three regression models. Diagnostic tests were used to check for violations of the assumptions inherent in linear regression models. Given the small sample size, only univariate analyses were used in MMN. All statistical analyses were performed using SPSS software package for Windows version 16.0. A P value of < 0.05 was considered to be statistically significant.

RESULTS

Patient characteristics

From the 63 eligible CMT1A patients, 53 agreed to participate. Four patients were excluded: 3 with comorbidity (Dupuytren disease, recent shoulder surgery, psychiatric disorder) and 1 with history of alcohol abuse. Sixteen MMN patients were willing to participate. One MMN patient was excluded because of hand surgery. The final study samples comprised of 49 patients with CMT1A and 15 patients with MMN (Table 1). All MMN patients reported hand involvement, whereas this was the case in 38 (78%) of the CMT1A patients. All but one MMN patient perceived loss of hand strength compared to 35 (71%) of the CMT1A patients.

The percentage of patients who perceived hand fatigue, pain, cramps and tremor varied between 41 and 73% in both groups. One MMN patient mentioned symptoms of sensory loss.

Descriptive data on hand function and manual dexterity

Group values for dominant and non-dominant hand function and manual dexterity in CMT1A and MMN patients are given in Table 2. Differences between the dominant and non-dominant hand were found in CMT1A for grip strength (p = 0.035), two-point discrimination (p = 0.007) and for the threshold for touch (p = 0.049).

Limited active range of motion presenting as a clawing position of the fingers was found in 27% of the CMT1A patients. The extent of clawing varied between involvement of all four fingers n = 7(14%) and solely the fifth finger n = 2 (4%). Loss of thumb opposition was found in more than half of the patients.

An impaired threshold for touch was found in 51% of the CMT1A patients. Thirteen patients (27%) scored outside the normal value range (< 6 mm) for static two-point discrimination. Disturbed vibration sense was found in 20% of the patients; five (10%) with grade 1, one (2%) with grade 2 and four patients (8%) with grade 4.

A large variation in grip strength (range 42 and 544 Newton) was found (Table 2).

Compared with reference values of healthy subjects grouped by gender and age, maximal isometric grip and pinch (two-point, tripod, and lateral pinch) strength was lower in CMT1A patients (Table 3). Mean grip strength in CMT1A reached 65% of the normal reference value. Two-point, tripod and lateral pinch strengths of CMT1A patients were reduced to approximately 50%.

Manual dexterity of the dominant hand was impaired in all but 3 (94%) patients with CMT1A (Table 2). The most difficult Sollerman subtests were: picking up coins and putting them into a purse, opening and closing a zipper, picking up nuts and putting them on bolts, doing up buttons, putting a paperclip on a envelope, and pouring water from a cup. A more detailed description of the SHT data in this study sample has been published previously. [START_REF] Videler | Manual dexterity in Hereditary Motor and Sensory Neuropathy type 1a: severity of limitations and feasibility and reliability of 2 assessment instruments[END_REF] On all aspects of joint mobility and hand strength, MMN patients scored worse than CMT1A patients (Table 2). Impaired sensory function was found in 1 MMN patient. Manual dexterity of the dominant hand was limited in 87% of the MMN patients. The average SHT sum score was substantially lower for both the dominant and non-dominant hand in MMN patients compared to CMT1A patients. The SHT subtests that caused most difficulty in MMN patients were comparable with those of CMT1A patients, but additionally included activities that require a strong grip such as pouring water from a jug, lifting an iron and unscrewing a lid from a jar.

Associations between hand function and manual dexterity

Associations between hand-function impairments and manual dexterity of CMT1A and MMN patients are shown in Table 4. In CMT1A, all variables of hand function were significantly associated with manual dexterity, with the strongest associations found for tripod pinch and two-point pinch strength (Figure 1). In MMN, tripod pinch strength showed the strongest association with manual dexterity scores. Sensory functions were not related to manual dexterity in MMN patients. In both patient groups manual dexterity was not related to age and gender.

In table 5 the results of the multiple linear regression analysis are given. The final model showed that in CMT1A, only tripod pinch strength (57% explained variance)(online Figure 2) , thumb opposition (10% added explained variance) and to a lesser degree vibration sense (2% added explained variance) were independently associated with manual dexterity (69% explained variance).

DISCUSSION

The main finding of this study is that tripod pinch strength and thumb opposition are the major determinants of impaired manual dexterity in CMT1A. Together with impaired vibration sense, they explained 69% of the total variance in manual dexterity scores. Inclusion of the MMN patients corroborated that motor impairment is the main determinant of decreased manual dexterity in CMT1A.

The sensory impairments appeared to have some but limited consequences for manual dexterity. Among the sensory variables, only vibration sense was independently associated with manual dexterity. In the literature, impaired sensory functions of the hand are reported in 14-64% of the CMT patients, but results are difficult to compare due to different measurement methods and cut-off values. [START_REF] Harding | The clinical features of hereditary motor and sensory neuropathy types I and II[END_REF][START_REF] Birouk | Charcot-Marie-Tooth disease type 1A with 17p11.2 duplication. Clinical and electrophysiological phenotype study and factors influencing disease severity in 119 cases[END_REF][START_REF] Schreuders | Sensory evaluation of the hands in patients with charcot-marie-tooth disease using semmes-weinstein monofilaments[END_REF][START_REF] Thomas | The phenotypic manifestations of chromosome 17p11.2 duplication[END_REF][START_REF] Krajewski | Neurological dysfunction and axonal degeneration in Charcot-Marie-Tooth disease type 1A[END_REF][START_REF] Shy | Reliability and validity of the CMT neuropathy score as a measure of disability[END_REF][START_REF] Verhamme | Clinical disease severity and axonal dysfunction in hereditary motor and sensory neuropathy Ia[END_REF] Tripod pinch strength was the most discriminative factor explaining manual dexterity in both groups. Several explanations can be given. Firstly, this grip pattern in particular may become affected in CMT1A and MMN patients as a result of the weak intrinsic thenar muscles. Secondly, a tripod pinch grip is one of the most frequently used grip patterns in daily activities. [START_REF] Sollerman | Sollerman hand function test. A standardised method and its use in tetraplegic patients[END_REF][START_REF] Aaron | Development of the Functional Dexterity Test (FDT): construction, validity, reliability, and normative data[END_REF] Finally, the manual dexterity subtests that were the most difficult to perform almost all required pinch grips like the tripod pinch. The 9% explained variance, added by loss of thumb opposition in CMT1A, is more difficult to understand as the thumb is positioned and stabilized by the same thenar muscles used during pinch grip. An explanation may be that some patients could not perform the required tripod grip for the dynamometer. In these patients tripod pinch strength was recorded as 0 Newton but a differentiation in opposition score could still be given. With the multivariable model a large percentage of the variance in manual dexterity could be explained in CMT1A. However, other non investigated factors such as loss of proprioception and coordination may have contributed to manual dexterity.

Hand function and manual dexterity were impaired in the majority of this large group of CMT1A patients. Previously we showed that impaired manual dexterity was associated with limitations in upper limb related activities and restricted participation in CMT1A. [START_REF] Videler | Manual dexterity and related functional limitations in Hereditary Motor and Sensory Neuropathy. An explorative study[END_REF][START_REF] Videler | Limited upper limb functioning has impact on restrictions in participation and autonomy of patients with Hereditary Motor and Sensory Neuropathy 1A[END_REF] Hand involvement was present in 78% of our patients which is more frequent than other CMT studies reporting upper limb involvement in one-half to two-third. [START_REF] Harding | The clinical features of hereditary motor and sensory neuropathy types I and II[END_REF][START_REF] Verhamme | Clinical disease severity and axonal dysfunction in hereditary motor and sensory neuropathy Ia[END_REF] This may be explained by the fact that our study addresses various aspects of hand function. Loss of thumb opposition and impaired threshold for touch were present in more than half of the patients.

Disturbed discrimination and vibration sense in approximately a quarter. Grip and pinch strength in CMT1A reached 65% and 50% of the normal reference value. Although 27% of the CMT1A patients presented with clawing of the fingers, restricted passive range of motion was found in only 6%. Apparently, in most CMT1A patients there is little contracture of the fingers and thus preventive measures are generally not needed.

Recently, potential differences between dominant and non-dominant hand strength in CMT have been discussed and contradictory findings have been reported. [START_REF] Vinci | Overwork weakness in Charcot-Marie-Tooth disease[END_REF][START_REF] Van Pomeren | The hypothesis of overwork weakness in Charcot-Marie-Tooth: a critical evaluation[END_REF][START_REF] Carter | Profiles of neuromuscular diseases. Hereditary motor and sensory neuropathy, types I and II[END_REF] In a cohort of 106 CMT patients, the dominant hand was found to be weaker than the non-dominant hand and ascribed to overwork weakness. [START_REF] Vinci | Overwork weakness in Charcot-Marie-Tooth disease[END_REF] In contrast, we found a significant difference between dominant and non-dominant grip strength in favour of the dominant hand which refutes the overwork hypothesis.

Manual dexterity was limited more often and more severely in patients with MMN compared to CMT1A. Activities, that were difficult to perform for MMN patients, were comparable with those of CMT1A patients but limited dexterity in MMN was also due to impaired grip strength. Tripod pinch strength also appeared to be the most explaining factor of manual dexterity in MMN. Hand function and manual dexterity have not yet been studied in MMN; therefore, our findings cannot be compared with those of others.

This study evaluated hand function and manual dexterity in a large sample of CMT1A patients. Some selection bias may have occurred. Patients with advanced upper limb involvement could have been more willing to participate than those with less impairment.

Nonetheless, a high percentage of the known CMT1A patients at our departments participated and upper limb involvement of the participating patients ranged from unaffected to severely affected.

In the absence of Dutch norm values for grip and pinch strength, we used normative data from the USA collected with a comparable, but non-digital hand-held dynamometer (Jamar). [START_REF] Mathiowetz | Grip and pinch strength: normative data for adults[END_REF] Applying normative data internationally is not ideal. However, we do believe that the used values are currently the most appropriate because they were obtained from a large study sample, a broad range of socioeconomic and occupational groups were included and the same standardized testing procedure and data analysis was applied (subject position, handle setting, patient instructions, mean of three trials).

Preservation of upper limb functioning of CMT1A patients is a major issue but there is no evidence about effective interventions. This study shows that tripod pinch strength and thumb opposition should be the focus of intervention strategies that aim to preserve or enhance manual dexterity in CMT1A. Surgical interventions such as tendon transfer procedures may restore thumb opposition and tripod pinch strength, but literature on this subject is limited. [START_REF] Wood | Treatment of the upper limb in Charcot-Marie-Tooth disease[END_REF][START_REF] Brown | Evaluation and management of upper extremity neuropathies in Charcot-Marie-Tooth disease[END_REF] Orthotic management, assistive devices and exercise programs have only been described for the lower limbs. [START_REF] Vinci | Poor compliance with ankle-foot-orthoses in Charcot-Marie-Tooth disease[END_REF][START_REF] Shy | Therapeutic strategies for the inherited neuropathies[END_REF][START_REF] Chetlin | Resistance training effectiveness in patients with Charcot-Marie-Tooth disease: recommendations for exercise prescription[END_REF][START_REF] Lindeman | Strength training in patients with myotonic dystrophy and hereditary motor and sensory neuropathy: a randomized clinical trial[END_REF] The results of this study justify the need for therapeutic and surgical intervention studies aimed at improving hand function, manual dexterity, and ultimately daily life functioning of patients with CMT1A.

Acknowledgment

We would like to thank the staff of the department of Neurology of the University Medical Centre in Utrecht, and in particular Dr. R. van de Berg-Vos, for their support in the recruitment of MMN patients.

which resulted in a score of 0 Newton. Associations between manual dexterity (log-transformed SHT sum score) and continuous hand function variables were investigated using Pearson product moment correlation coefficients (r). * P < 0.05, **P < 0.01; Associations between manual dexterity and dichotomous variables were investigated with point biserial correlation coefficients; The results of the dominant hand were taken for all associations; AROM = active range of motion; PROM = passive range of motion.


Irene Fumiaki Y Yoshida

Alek Martinez-Torres

Etienne Pogosyan

Erika Holl

Chiung Chu Petersen

Tomas Chen

Patricia Foltynie

Ludvic U Limousin

Marwan I Zrinzo

Hariz

Pogosyan

Fumiaki Yoshida

Irene Martinez-Torres

Alek Pogosyan

Etienne Holl

Erika Petersen

Chiung Chu Chen

Tom Foltynie

Patricia Limousin

Ludvic U Zrinzo

Marwan I Hariz

Peter Brown
email: p.brown@ion.ucl.ac.uk

VALUE OF SUBTHALAMIC NUCLEUS LOCAL FIELD POTENTIALS RECORDINGS IN PREDICITING STIMULATION PARAMETERS FOR DEEP BRAIN STIMULATION IN PARKINSON´S DISEASE

Keywords: Deep brain stimulation, subthalamic nucleus, Parkinson's disease. Local field potentials

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

High-frequency 'deep brain stimulation' (DBS) of the subthalamic nucleus (STN) can be a highly effective treatment in Parkinson's disease. [START_REF]The Deep-Brain Stimulation for Parkinson's Disease Study Group. Deep-Brain Stimulation of the Subthalamic Nucleus or the Pars Interna of the Globus Pallidus in Parkinson's Disease[END_REF][START_REF] Benabid | Deep brain stimulation for Parkinson's disease[END_REF][START_REF] Krack | Five-year follow-up of bilateral stimulation of the subthalamic nucleus in advanced Parkinson's disease[END_REF] However, therapeutic efficacy is limited by difficulties in consistently and correctly targeting the STN, particularly the 'motor' domain of this nucleus, while side effects may, in part, relate to poor positioning of electrodes. We have previously reported that there is a local increase in beta LFP activity when the DBS electrode penetrates the dorsal STN as suggested by correlation with intra-operative clinical effect and post-operative imaging data. [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF] This is consistent with similar findings made using semi-microelectrodes in 6-OHDA midbrain lesioned parkinsonian rodents [START_REF] Sharott | Dopamine depletion increases the power and coherence of beta-oscillations in the cerebral cortex and subthalamic nucleus of the awake rat[END_REF][START_REF] Mallet | Disrupted dopamine transmission and the emergence of exaggerated beta oscillations in subthalamic nucleus and cerebral cortex[END_REF] and microelectrodes in parkinsonian patients. [START_REF] Levy | Dependence of subthalamic nucleus oscillations on movement and dopamine in Parkinson's disease[END_REF][START_REF] Kuhn | The relationship between local field potential and neuronal discharge in the subthalamic nucleus of patients with Parkinson's disease[END_REF][START_REF] Weinberger | Beta oscillatory activity in the subthalamic nucleus and its relation to dopaminergic response in Parkinson's disease[END_REF][START_REF] Trottenberg | Frequency-dependent distribution of local field potential activity within the subthalamic nucleus in Parkinson's disease[END_REF] Nevertheless, whether intra-operative recordings from DBS electrodes help predict stimulation settings and which electrode contact

METHODS

Patients and surgery

The patients (n = 31, mean age 57.0 ± 7.4 years, range 38 -68, 17 males) participated with informed consent and the permission of the local ethics committee. The pattern of DBS recordings made intra-operatively was previously reported in seven of the patients but without data from chronic treatment (cases 1, 2 and 5-9 in [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF]). All patients had advanced idiopathic PD with motor fluctuations and/or dyskinesia. The mean disease duration was 11.7 years (range 5 -28 years). The mean Unified Parkinson's Disease Rating Scale (UPDRS) motor score was 44.8 ± 14.5 off drugs and 13.9 ± 10.0 after levodopa treatment (p < 0.001, two tailed paired t-test). Implantation of bilateral STN DBS electrodes was performed sequentially in the same surgical session under local anaesthesia in all but one patient who was implanted unilaterally. Patients were operated on after overnight withdrawal of their levodopa medication and after dopamine agonists had been tapered down and terminated during the two weeks before surgery. The DBS electrode used was model 3389 (Medtronic Neurological Division, Minneapolis) with four platinum-iridium cylindrical surfaces (1.27 mm diameter and 1.5 mm length) and a centre-to-centre separation of 2mm. Contact 0 was the lowermost and contact 3 was the uppermost. Fast acquisition T2 weighted axial and coronal stereotactic MRI scans using Leksell's Frame (Elekta, Sweden) were performed with contiguous slices of 2 mm thickness. This visualised the STN and especially its medial border. [START_REF] Hariz | A quick and universal method for stereotactic visualization of the subthalamic nucleus before and after implantation of deep brain stimulation electrodes[END_REF] The images were imported into dedicated stereotactic planning software (Framelink, Medtronic, Minneapolis). Initial anatomical targeting was performed on an axial section at the level of the maximal rubral diameter. In this plane, a line joining the anterior borders of the red nuclei defines the centres of the subthalamic nuclei. [START_REF] Bejjani | Bilateral subthalamic stimulation for Parkinson's disease by using three-dimensional stereotactic magnetic resonance imaging and electrophysiological guidance[END_REF] The target point within the STN was defined at the level of this line. The trajectory towards the target point was refined such that the maximum number of electrode contacts would reside within the visualized STN. The double oblique trajectory to the target was planned on reconstructed images to avoid the ventricles. Coordinates of the target point were cross-checked manually on enlarged MRI film copies. Microelectrode recordings were not performed. At surgery, impedance recording with a radiofrequency (RF) electrode allowed distinction between grey and white matter to a point 4 to 6 mm above the planned target. The rigid RF electrode also created a track that was followed by the DBS electrode which itself was introduced to 4-6 mm above the planned target with a stylet in place that rendered it more rigid. The DBS electrode was subsequently advanced through 'virgin' brain in 2 mm steps from the point 4 to 6 mm above to a point 2 to 4 mm below the intended surgical target. Intra-operative monopolar test stimulation was performed at 3 selected levels (usually intended target and/or level of any "stun" effect, as well as one to two levels ventrally) to detect clinical benefit as well as any side effects once the DBS electrode had completed its trajectory. Stimulation was only performed once all LFP recordings had taken place. Once the optimum target point for stimulation was identified, the electrode was advanced 2 mm, in order for the contacts to "encompass" the optimal target point before it was fixed in position with the Medtronic burrhole cap or the stimloc system (Medtronic, Minneapolis, USA). The same procedure was then repeated for the other side. All patients received immediate post-operative stereotactic MRI using fast spin-echo T2 weighted axial and coronal MRI scans with contiguous slices of 2 mm thickness, while the Leksell frame was still applied to the head documenting the location of the DBS electrode. Electrodes were thereafter connected to a battery-operated programmable pulse generator (Kinetra 7428, Medtronic). Post-operative contact localization was performed using Framelink software (Medtronic inc. Minneapolis, MN). The coordinates of each contact were transposed onto the preoperative stereotactic MR images [START_REF] Kuhn | Pathological synchronisation in the subthalamic nucleus of patients with Parkinson's disease relates to both bradykinesia and rigidity[END_REF]. A neurosurgeon (EH, EP or LZ) assessed the anatomical position of each contact in relation to the visualized STN in the axial and coronal planes. The contact from each electrode with the best anatomical position was determined and classified into one of two groups. In Group I the contact was inside or, at worst, touching the most superior part of STN. Group II essentially consisted of the contact that was closest to the superior part of the STN in those electrodes not fulfilling the above criteria. The depths of the contacts of each electrode were also described with reference to the surgical target point.

Recordings

Recordings were made from every 2 mm step in the electrode descent while the patients were awake, with eyes open and at rest. Each depth was recorded for 60-65 s. STN LFPs were recorded bipolarly from the four adjacent contacts of each DBS electrode (contact pairs 01, 12, 23). Signals were amplified, pass band filtered between 1 and 80 Hz and sampled at 184 Hz in 23 patients, 500 Hz in two patients and 1600 Hz in three patients (Biopotential Analyzer Diana, St Petersburg, Russia) or pass band filtered between 1 and 80 Hz and sampled at 1024 Hz in three patients (Porti Amplifier, TMSI international, Enschede, the Netherlands). The optimum sampling rate was 184 Hz, as higher rates do not afford any advantage given that the pass-band of interest was under 35 Hz. Purpose written software (available from A.P. on request) saved the original time series on a portable PC and displayed on-line the evolving patterns of beta band power from contact pairs 01, 12 and 23 as the DBS electrode was advanced. [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF] Thereafter LFPs were interpolated to a sampling rate of 184 Hz, where necessary, and examined off-line in Spike2 software (Cambridge Electronics Design, Cambridge, UK). Spectra of LFP power were estimated in Spike2 using the discrete Fourier transform. Spectral resolution was 0.72 Hz. Analysis excluded periods of recording while the electrode was moved.

A beta frequency band peak in the power spectra was operationally defined as a local maximum between 11 and 35 Hz in which the mean power over the 5 contiguous frequency bins centred on the peak frequency exceeded 180 % of the mean power over the five contiguous bins of lower and higher frequency. We considered recordings from contact 01 at all depths and used the depth with the highest peak power over the frequency band of interest to define the centre frequency of the peak. The mean (absolute rather than relative) power over the five frequency bins centred on this peak was then estimated for each recording depth of the electrode. We chose five bins to allow for any minor change in peak frequency between depths. We identified a discrete peak between 11 and 35 Hz in power spectra of contact 01 in all but two sides. A step change in peak beta power was operationally defined as at least a 100% increase in mean beta power at contact 01 between successive depths as the electrode was advanced in 2 mm increments, or, where the maximum peak lay at the most superficial depth tested (4 sides), there was at least a 100% drop in mean beta power at contact 01 when it was advanced a further 2 mm. For example, if the mean power of the peak doubled when the electrode was moved from 4 mm to 2 mm above the anatomical target point then the step change and site of the local beta generator were considered to be at 2 mm. Here we define the beta generator as the local electrical source of beta activity, acknowledging that this may be driven by input from elsewhere. If there was more than one step change in beta power between depths, then we only considered the step change that involved the highest mean beta power. For example, if the mean power of the peak doubled when the electrode was moved from 4 mm to 2 mm above the anatomical target point, but then double again when the electrode was moved from 2 mm to 0 mm then the step change and site of the local beta generator were considered to be at 0 mm, the depth with the highest peak mean power. In a previous study we used the LFP power across the beta band rather than the power concentrated in any peak within this frequency band. [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF] We elected to focus on peak power as this afforded a better signal to noise ratio and given the recent emphasis on spectral peaks rather than broad band power changes in correlations between LFP power and clinical state. [START_REF] Kuhn | Pathological synchronisation in the subthalamic nucleus of patients with Parkinson's disease relates to both bradykinesia and rigidity[END_REF] Note that absolute and not relative or normalised power was analysed.

Clinical assessments of efficacy of chronic DBS

Clinical assessment of the efficacy of chronic DBS could not be performed in two of the 31 patients. One patient developed unexplained obtundation post-operatively and the DBS electrodes were therefore removed in the immediate post-operative period. One patient succumbed to cancer few months after surgery. Post-operative clinical assessments and programming were performed by a neurologist blinded to the results of the intra-operative recordings. Stimulation parameters and UPDRS motor scores were determined a minimum of 6 months after surgery (median 19, range 6 -51 months). Each contact was screened in turn to assess both therapeutic efficacy and unwanted side effects. Monopolar stimulation with increasing voltage was used with a note made of effectiveness at stepwise intervals. A pulse width of 60 µS and frequency of 130 Hz was used. The contact with the greatest efficacy at the lowest voltage and the least side effects was chosen for each hemibody. A poor stimulation efficacy on symptoms prompted exploration of wider pulse widths (broadest pulse width was 90 µS and this was used on 4 out of the 57 sides), higher stimulation frequency (145 Hz on 4 sides, 150 Hz on 2 sides, 160 Hz on 2 sides, 180 Hz on 5 sides and 185 Hz on 2 sides) and in case of persistent side effects, bipolar stimulation was used (5 sides). Total electrical energy delivered (TEED) was calculated assuming an impedance of 1kOhm. [START_REF] Moreau | STN-DBS frequency effects on freezing of gait in advanced Parkinson disease[END_REF] Overall, the motor UPDRS fell from 44.7 ± (SEM) 2.9 off medication without BDS to 21.2 ± 1.5 off medication with DBS. The depths of the contacts of each electrode used for chronic stimulation were also described with reference to the surgical target point (the same reference as used for the depth of the beta generator). In those 5 cases with bipolar stimulation, stimulation depth was assumed to lie midway between the respective contacts.

Statistical analysis

Correlations were performed using Spearman's Rho so as to accommodate the non-parametric distribution of our data and to avoid any spurious correlations due to outlying values, although this has the disadvantage that correlation coefficients cannot be used to estimate the proportion of the variance of one signal linearly predicted by another.

RESULTS

We identified a discrete peak between 11 and 35 Hz in power spectra of the LFP activity recorded at contact 01 in all but two sides (both on the left). As previously reported, [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF] the power of this peak was clearly modulated during electrode descent, except on one side. Thus three (5 %) out of 57 sides did not have an obvious peak or step change in LFP activity along the electrode trajectory. There was no difference in the efficacy of DBS between the 54 sides with evidence of a local beta generator (mean stimulation voltage 2.8 ± 0.1 [SEM] V, pulse width 60 µS [90 µS on 4 sides], frequency 139 ± 2 Hz, improvement in contralateral UPDRS hemibody motor scores during DBS off-medication 8.2 ± 0.7 or 51.3 ± 4.2 % of pre-operative score), and those three sides without such evidence, although the latter group was very small (stimulation voltage 1.7, 3.8 and 1.9 V, pulse width 60 µS, frequency 130 Hz, improvement in contralateral UPDRS hemibody motor scores during DBS off-medication 13 [97 %], 3 [50%] and 10 [62%], respectively). However, those sides with evidence of a local beta generator tended to have more optimal anatomical targeting (88 % in group I) than those without evidence of a local beta generator (33% or 1 side in group I; Fisher Exact test, p = 0.054).

Among the 54 sides in whom there was a focal beta peak in the subthalamic LFP peaks were fairly well distributed across 14-34 Hz (Fig 1). The median power of the peak LFP activity recorded at the initial step in beta activity, i.e. the depth considered to be that of the local beta generator, was 3.3 µV (interquartile range 1.1 to 7.2 µV; Kolmogorov-Smirnov test for normal distribution, p = 0.004). Two mm above (below on four sides) this, the median power over the frequencies of the peak was 0.5 µV (interquartile range 0.1 to 1.2 µV; Kolmogorov-Smirnov test for normal distribution, p = 0.003). The median percentage change between the two levels was 500 % (z = 6.393, p < 0.001).There was no difference between the depth of the local beta generator recorded intra-operatively (mean 0.83 mm and median 0 mm, respectively, depth defined relative to the surgical target point) and the depth of the contact independently chosen for chronic DBS (1.01 and 0.5 mm; Wilcoxon signed ranks test, z = -0.571, p = 0.568). However, both differed from the depth of the target point aimed at during surgery (Wilcoxon signed ranks tests, z = -2.973, p = 0.003 and z = -4.161, p < 0.001, respectively).

There was a correlation between the depth of the local beta generator recorded intra-operatively and the depth of the electrode contact independently chosen for chronic DBS (Spearman's Rho = 0.35, p = 0.01, n = 54; Fig 2). A potential association between the depth of the beta generator and the optimal site for amelioration of parkinsonism was strengthened by considering those sides in which there was a disparity between the depth of the generator and that of the contact chosen for chronic stimulation. There was a weak correlation between the absolute disparity in mm and the stimulation voltage used for chronic stimulation, such that a bigger difference between depths was associated with a higher stimulation voltage or TEED (Rho = 0.322, p = 0.017, n = 54 and Rho = 0.308, p = 0.024, respectively). There was no such tendency between the stimulation voltage or TEED employed for chronic stimulation and the absolute difference between the surgical target point depth and that of the contact chosen for chronic stimulation (Rho = 0.103, p = 0.468 and Rho = 0.127, p = 0.368, respectively). This suggests more stimulation voltage had to be employed if a depth was selected for chronic stimulation that differed from that of the local beta generator, but not if it differed in depth from the surgical target point. This relationship between the depth of the generator and stimulation intensity was maintained even if we controlled for the clinical effect of DBS. To this end we derived a measure of the normalised efficacy of DBS by dividing the DBS-induced improvement in contralateral hemibody UPDRS motor scores by the TEED. There was a negative correlation between the normalised chronic DBS efficacy and the absolute difference between the depth of the local beta generator and that of the contact chosen for chronic stimulation (Rho = -0.315, p = 0.021), so that chronic stimulation at the depth of the intra-operatively defined generator was associated with more effective long term stimulation.

As before, however, there was no correlation between the normalised chronic DBS efficacy and the absolute difference between the surgical target point depth and that of the contact chosen for chronic stimulation (Rho = -0.116, p = 0.411), so that the depth of the surgical target point was a relatively poor predictor of stimulation efficacy. The scale of the effect of disparity between the depths of the generator and contact chosen for chronic stimulation is highlighted in Fig 3 . The normalized efficacy of DBS was more than halved when the contact was two or more mm from the depth of the generator.

Finally, there was a positive correlation between the depth of the local beta generator and that of the optimally anatomically placed contact on those 46 sides (Group I) in which a contact was inside or, at worst, touching the most superior part of STN (Rho = 0.379, p = 0.011). As expected, there was no such correlation in the remaining sides where the best situated contact was neither inside nor abutted the superior part of the STN (Group II), although the numbers were much smaller (n = 8, Rho = 0.226, p =0.55 )

DISCUSSION

We have shown that functional localization of the STN by on-line spectral analysis of LFPs recorded from the DBS electrode may provide information that helps predict optimal stimulation settings during long term follow-up. In a previous study we showed that the same signals were quick to record and analyse intra-operatively, and correlated with successful targeting with respect to intra-operative stun effects and post-operative imaging. [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF] The latter was also confirmed in the present, larger sample. Together, these results point to the utility of the intra-operative spectral analysis of LFPs recorded from the DBS electrode in aiding the functional localization of the STN.

We found no evidence for significant peri-operative brain shift, in so far as there was no difference between the mean depth of the local beta generator and the depth of the contact independently chosen for chronic stimulation, although there was some individual variability. The limited delays introduced by our intra-operative functional localisation technique may have helped avoid significant subdural air collection. This contrasts with more prolonged microelectrode techniques following which brain shift may be a problem. [START_REF] Miyagi | Brain shift: an error factor during implantation of deep brain stimulation electrodes[END_REF][START_REF] Halpern | Brain shift during deep brain stimulation surgery for Parkinson's disease[END_REF] Furthermore, there were no peri-operative haemorrhages in our series.

One of the problems that besets studies in this field is the absence of a gold-standard with which to compare one targeting technique against another. Imaging can detect whether a DBS contact is likely to be within or abutting STN, but electrode artefact means that localisation is somewhat indirect and the relationship to the motor territories of the STN presumptive. We elected to pursue a more empirical approach to validation of recordings made directly from the DBS electrode which involved an assessment of how well the intra-operative LFP recordings could predict the chronic contacts and voltages independently selected for chronic stimulation. However, clinical validation is also imperfect in so far as not all electrode contacts and energy combinations can be assessed, and the parameters for chronic stimulation are selected not only for their efficacy in improving parkinsonism but also for their restriction of side-effects. Accordingly, even an excellent intra-operative marker of motor STN would afford incomplete prediction of chronic stimulation contacts and voltages. This problem may have been exacerbated in our study by the assessment of patients up to 51 (median 19) months after surgery.

It is important to note that the depth of the surgical target point was a relatively poor predictor of the depth of the local beta generator or the contact selected for chronic stimulation. We conclude that although anatomical targeting based on pre-operative stereotactic MRI may be very good at selecting an appropriate electrode trajectory towards and through the nucleus it is relatively poor in identifying the precise rostro-caudal depth of the stimulation target, whether the latter is functionally defined as the local beta generator, or clinically defined, as the site of the best contact for chronic stimulation. Indeed, one has to remember that the target point is nothing but a point aimed at, and not necessarily the exact point in space where the best stimulation effect is. There was even evidence that the local beta generator may be more indicative of the optimal depth for ameliorating parkinsonism than the contact level chosen for therapeutic stimulation over long-term follow-up, perhaps because the latter represents a compromise between efficacy and side-effects and may not be selected after evaluation of all possible contact combinations. Thus more stimulation voltage and energy tended to be delivered if a depth was selected for chronic stimulation that differed from that of the local beta generator. In contrast, disparities in the depth selected for chronic stimulation and the depth of the surgical target point had no significant effect on stimulation voltage or energy.

Our previous findings [START_REF] Chen | Intra-operative recordings of local field potentials can help localize the subthalamic nucleus in Parkinson's disease surgery[END_REF] suggested that the intra-operative recording of local field potential activity directly from the DBS electrode may potentially provide an alternative to microelectrode recordings for identifying the depth of the STN, with attendant advantages in terms of the duration of the operation and possible reduction in the risks of intra-operative haemorrhage. Nevertheless, the technique relies on minimal error in the anterior-posterior plane, as a single trajectory cannot provide information about localisation in these dimensions, and the utility and safety of exploration of alternative trajectories with the DBS electrode remains uncertain. Thus the technique must be combined with accurate targeting in the anterior-posterior plane using stereotactic pre-operative MRI. Nevertheless, the present data provide further support for the clinical relevance of the local beta activity and suggest that the technique whereby LFPs are recorded from the DBS electrode also helps predict the optimal stimulation contact for use in chronic DBS.

Fig 1 .

 1 Fig 1. Distribution of spectral peaks, as recorded intra-operatively from the DBS electrode (n = 54, one peak per side).

Fig 2 .Fig 3 .

 23 Fig 2. Correlation between the depth of the local beta generator recorded intra-operatively and the depth of the contact independently chosen for chronic DBS. The size of circle indicates the number of sides (incidence) that shared these graphical coordinates. Depths are relative to the surgical target point. Spearman's Rho = 0.35, p = 0.01, n = 54.

Funding: Peter Brown was supported by the Medical Research Council of Great Britain and the DANA Foundation, Patricia Limousin, Tom Foltynie, Ludvic Zrinzo and Marwan Hariz by the Parkinson's appeal, Irene Martinez-Torres by a grant from Fondo de Inversion Sanitaria (FIS), Instituto de Salud Carlos III, Spanish Department of Science and Innovation (FI08/00108), and Fumiaki Yoshida by Uehara Memorial Fundation.

Competing interests: Peter Brown is a consultant to Medtronic Inc.

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