the neural mechanisms that underlie l - dopa - induced dyskinesias in 
 parkinson s disease ( pd ) remain poorly understood [ 14 ] .
dyskinesias have 
 been associated with pulsatile stimulation of dopamine receptors , downstream 
 changes in genes and proteins , and changes in nondopamine transmitter systems 
 [ 46 ] .
all these changes lead to alterations in the firing patterns within the 
 basal ganglia , causing overactivations of the primary motor cortex and premotor 
 cortical areas [ 7 , 8 ] .
activation studies in pd patients ( h2  o 
 positron emission tomography ( pet ) and xe single photon emission 
 computed tomography ( spect ) ) are in line with these results , and indicate that 
 l - dopa - induced dyskinesias are associated with inappropriate overactivity of 
 striatofrontal projections both at rest and during volitional actions [ 2 , 9 ] . 

comparison of rest with dyskinesias versus rest without dyskinesias showed a 
 relative activation of the motor cortex , the lateral and mesial premotor areas 
 including the supplementary motor area ( sma ) , the dorsal prefrontal cortex , and 
 the basal ganglia .
the comparison of dyskinetic versus nondyskinetic states 
 during voluntary movement revealed additional activation of the same brain 
 regions .
unfortunately , in the pet study of brooks et al .   mentioned 
 above , regional cerebral blood flow ( rcbf ) in pd patients with and without 
 l - dopa induced dyskinesias was not compared to that of healthy subjects , and 
 therefore , it is difficult to speculate whether the relative increases in rcbf 
 observed in patients with dyskinesias were in fact above or still below that of 
 controls in all the reported areas including the dlpfc .
repetitive transcranial magnetic stimulation ( rtms ) can produce 
 changes in excitability of cortical circuits that outlast the period of 
 stimulation , opening the possibility of intervening directly with the 
 mechanisms of cortical plasticity in the human cortex .
rtms is also a suitable tool to investigate plasticity 
 within a distributed functional network since conditioning effects of rtms are 
 not limited to the stimulated area but give rise to functional changes in 
 interconnected cortical areas .
the magnitude and the direction of rtms - induced 
 plasticity depend on extrinsic factors ( i.e. , the variables of stimulation such 
 as intensity , frequency , and total number of stimuli ) and intrinsic factors 
 ( i.e. ,   the functional state of the cortex targeted by rtms ) .
high frequencies 
 of rtms applied over the motor cortex lead to facilitatory aftereffects on 
 corticospinal excitability while low - frequency stimulations 
 lead to opposite ( i.e. , inhibitory ) aftereffects ( for review , see 10 ) .  
the main 
 problem remains , that the basic mechanisms mediating the effects of rtms are 
 still poorly understood .
recenly , several studies have suggested the therapeutic efficacy of 
 rtms in pd while other studies have found no clinical improvement of motor 
 performance in pd   [ 1115 ] .
using rtms over the sma , an italian group 
 showed that 900 pulses of 1  hz rtms with an intensity set at 90% of the resting 
 motor excitability threshold markedly reduced drug - induced dyskinesias in 8 
 advanced pd patients . in spite of this promising result , a year later the same 
 group   discovered that 5 repeated sessions of the 1  hz rtms failed to 
 enhance and/or prolong the beneficial effects of the procedure , and thus could 
 not be considered clinically useful .
very similar results were reported by a toronto 
  group   who 
 used the same stimulation parameters but targeted the primary motor cortex 
 contralateral to the side with more severe dyskinesias in 6 pd patients . 
 although the benefit was evident at day 1 after a two - week course of rtms , 
 the authors have not managed to obtain more sustained clinical improvement 
 after the treatment .
we recently reported the results of a pilot study that investigated 
 whether 5 repeated sessions of high frequency rtms at 10  hz applied over the 
 motor cortex ( mc ) of the relevant leg or over the left dorsolateral prefrontal 
 cortex ( dlpfc ) might result in a modification of 
off-related freezing of 
 gait and motor symptoms of pd in 6 advanced pd patients .
the selection of 
 mc and dlpfc targets for rtms was based on dopamine release in the putamen and 
 caudate , respectively [ 19 , 20 ] .
the target selection was further supported by 
 the results of lomarev et al .   who had reported positive cumulative benefit 
 of high - frequency rtms applied over the same targets for improving gait , as 
 well as reducing upper limb bradykinesia in pd patients . in our study ,
rtms was 
 well tolerated . despite a reported trend towards improvement of the stroop test 
 interference after rtms over the left dlpfc ,
interestingly , patients who had undergone the dlpfc treatment 
 reported a subjective improvement of their dyskinesias . therefore , in this 
 paper , we present a post hoc analysis of the data from the same group of 
 patients that focuses on cortical excitability and on the possible modification 
 of l - dopa - induced dyskinesias by repeated sessions of rtms over the dlpfc .
we studied 6 
 patients ( 5 men and 1 woman , mean age 67.37.7 years ) with advanced pd 
 according to the uk parkinson s disease brain bank criteria [ 21 , 22 ] , 
 a hoehn - yahr stage   between 2.5 and 4 while in the  off  state , and 
 without dementia ( mini - mental state examination   score > 24 ) .
a montgomery - asberg depression 
 rating scale   cut - off score of 14 was used to exclude patients with 
 depression .
patients with a predominant tremor form of pd were also excluded 
 from the study .
the daily l - dopa 
 dose equivalent was 1145.8413.2  mg ; all patients were on l - dopa  entacapone plus a dopamine 
 receptor agonist .
the antiparkinsonian medication was stable for at least 4 
 weeks prior to the study commencement and during the study .
all patients signed 
 the informed consent form , which was approved by the ethics committee of saint anne s hospital in 
 brno .
rtms was applied 
 over the left dlpfc ( 5  cm anterior to the optimum scalp position for activation 
 of the contralateral first dorsal interosseus muscle ) or over the optimal 
 position for the tibialis anterior muscle contralateral to the foot which was 
 most frequently used by the patient to make the first step after freezing of 
 gait ( i.e. , mc ) , using the magstim super rapid stimulator and a figure - of - eight 
 coil with a mean diameter of 7  cm .
the coil was placed tangentially to the 
 scalp with the handle pointing backwards and laterally at 45 angle away from 
 the midline inducing a posterior - anterior current in the brain .
nowadays , 
 the frameless stereotaxy has been used in order to target the region of brain 
 corresponding to cytoarchitectonic area 9/46 as defined by petrides and pandya 
 .
nevertheless , this part of the brain was found to overlap with the region 
 targeted by the standard procedure ( rektorova and paus , unpublished data ) , used 
 in most studies .
rtms was 
 delivered in the  on  state ( i.e. , when pd medication was working and 
 providing benefit ) without dyskinesias according to the individual patients 
 diaries , at the same time of day . one treatment consisted of 5 sessions over 5 
 consecutive days .
we chose a repeated sessions design , 
 and our patients were stimulated while being in their  on  state since both 
 conditions have been associated with better rtms outcomes in literature [ 12 , 14 ] .
one 
 session consisted of 1350 pulses delivered at 10  hz frequency , at an intensity 
 of 90% of the resting motor excitability threshold ( for the right 
 first dorsal interosseus muscle when stimulating the left dlpfc , and the 
 appropriate tibialis anterior muscle when stimulating the mc ) .
although we aimed at a crossover design , 
 only two patients completed both treatments ( with a one - month interval between 
 them ) . in order to 
 ensure the method was safe with regard to cognitive functions
, a brief 
 neuropsychological battery of tests was administered prior to , immediately 
 after , and one week after each rtms treatment .
experienced neurologists who were 
 blinded to the stimulation site performed a neurological evaluation prior to , 
 immediately after , and one week after appropriate rtms treatment .
the 
 evaluation was conducted during the patients relative  off  state ( i.e. , 
 when pd medication was not improving pd symptoms resulting in a lack of 
 mobility ) according to the individual patients diaries , at the same time of 
 day .
it consisted of the updrs , part iii 
 ( motor examination ) and part 
 iv ( treatment complications ) .
a more detailed evaluation and analysis of 
 gait and  off-related freezing of gait has been reported elsewhere .
single - pulse and paired - pulse tms 
 was performed while the patient was  on  to study the effects of rtms 
 treatments on cortical excitability .
we used the paired - pulse tms paradigm of 
 ridding et al .   at interstimulus intervals ( isi )
we evaluated motor evoked potentials ( meps ) elicited by single and 
 paired - pulse tms pulses over the mc contralateral to either the right first dorsal interosseus muscle ( prior to , immediately after , and one week after the rtms 
 treatment over the left dlpfc ) or the appropriate tibialis anterior muscle 
 ( prior to , immediately after , and one week after the rtms treatment over the 
 appropriate mc ) .  
the motor cortical sites were chosen to specifically 
 accommodate the primary goal of our freezing of gait protocol design .
mep size reflects more globally the corticospinal 
 input - output balance , excitatory inputs from high - threshold glutamatergic 
 pathways to the motor cortex lead to intracortical facilitation , whereas 
 inhibitory inputs from low - threshold gaba - a - mediated pathways lead to 
 intracortical inhibition at short isi .
the effects of 
 rtms treatments on single mep amplitudes and updrs before and after the rtms 
 treatments were analysed by a two - tailed paired - sample t test . a 
 two - factor repeated measures anova , with the factors of  time  ( 3 levels : 
 baseline , immediately after the rtms treatment , and 1 week after the rtms 
 treatment ) and 
isi  ( 3 levels : averaged data from the inhibiting isi ( 1 and 
 3 milliseconds ) , the facilitating isi ( 15 milliseconds ) , and the intermediate 
 isi ( 7 milliseconds ) ) was performed to evaluate the effects of the rtms 
 treatment applied over the mc and the dlpfc , respectively .
despite our aim to perform a 
 randomised crossover design , the study was stopped prematurely as several 
 patients withdrew the consent form before completing both treatments .
a 
 subjective lack of treatment effect on freezing of gait ( i.e. , the primary 
 outcome ) was the reason for the study withdrawal .
this study presents the 
 results from four patients that completed the rtms treatment over the mc and 
 four patients that completed the treatment over the dlpfc . despite the lack of 
 effect of rtms on freezing of gait and motor symptoms of pd , the patients reported 
 a decreased frequency and intensity of dyskinesias after 5 consecutive sessions 
 of rtms targeted over the left dlpfc .
these changes were reflected by the mean 
 updrs iv decrease immediately after 5 sessions of the stimulation as compared 
 with the baseline scores ( see table 1 ) .
the result did not reach statistical 
 significance ( p=.06 ) but could be suggestive of a mild improvement of 
 dyskinesias .
there was no cumulative effect of 
 the rtms treatment over the mc on corticospinal excitability as measured by mep 
 responses produced by single tms pulses over the left mc contralateral to the 
 appropriate tibialis anterior muscle ( see table 2 ) .
however , we found that 5 
 consecutive sessions of high frequency rtms of the left dlpfc produced a 
 significant decrease in the amplitude of mep responses produced by single tms 
 pulses over the left mc contralateral to the right first 
 dorsal interosseus 
 muscle ( see table 2 ) .
there 
 were no effects of any rtms treatments on the time course of intracortical inhibition or intracortical facilitation in the 
 appropriate motor cortex ; this was verified with a two - factor repeated measures 
 anova with the factors  time  and  isi  : f(4,26 ) = 0.72 , p=.6 for rtms 
 treatment over the dlpfc ; f(4,10 ) = 2.4 , p=.1 for rtms treatment over the mc .
the results of our study point to a 
 possible impact of repeated sessions of rtms applied over the left dlpfc on l - dopa - induced 
 dyskinesias in advanced pd patients .
the mean change in the updrs iv scores did 
 not reach a statistical significance , but we did not evaluate dyskinesias by 
 more appropriate standardized scales since the assessment of dyskinesias was 
 not the primary outcome of our pilot study . on the other hand ,
updrs iv was 
 used to test dyskinesias in a pet study by brooks et al . .
there are a 
 number of hypothetical mechanisms that might underlie the possible effects of 
 rtms of the dlpfc on l - dopa - induced dyskinesias in advanced pd .
it appears that changes of the 
 excitability of the primary motor cortex may be more efficiently performed 
 through stimulation of premotor and/or other more anterior brain regions , for 
 review , see .
we found that 5 consecutive sessions of high frequency rtms 
 of the left dlpfc produced a significant decrease in the amplitude of mep 
 responses produced by single tms pulses over the left mc for the right first 
 dorsal interosseus muscle ( i.e. , decrease of the cortico - spinal excitability ) . 

these results are in accord with the results of rollnick et al .   who also 
 demonstrated that stimulation of the dlpfc via subthreshold 5  hz rtms induces 
 motor cortex depression in healthy subjects .
it was posited by the authors that 
 this finding might be explained by the fact that prefrontal brain areas 
 contribute to motor cortex inhibition through antagonisms between frontal and 
 parietal lobes [ 32 , 33 ] but the exact mechanisms are not fully understood . 

was found 
 by means of pet and functional magnetic resonance imaging ( fmri ) while studying 
 simple or complex motor hand tasks [ 34 , 35 ] .
it was also observed in spect and 
 pet studies of pd patients with l - dopa - induced dyskinesias [ 2 , 9 ] . 

electrophysiological data furhter support this notion : increased mep aplitudes 
 at rest were found in pd patients with l - dopa induced dyskinesias [ 36 , 37 ] . 

neuroimaging studies have shown that subthalamic nucleus stimulation acts 
 through the reduction of abnormal overactivity in the motor system at rest   [ 34 , 38 ] .
even though dyskinesias were not systematically monitored in 
 these studies , it has been well known that subthalamic nucleus stimulation 
 surgery is able to reduce l - dopa - induced dyskinesias .
taken together , 
 normalizing ( i.e. , reducing ) the excitability of the primary motor cortex , for 
 example , by rtms of the dlpfc might be one possible factor in improving l - dopa - induced 
 dyskinesias in advanced pd patients .
rtms applied over the left dlpfc can 
 induce significant increases of rcbf in the stimulated area .
functional 
 imaging studies ( pet and fmri ) report decreases in rcbf and mri signal in the 
 dlpfc and the rostral sma in advanced pd patients compared to controls [ 354145 ] . the dlpfc is connected with the rostral sma .
after 
 unilateral pallidotomy , glucose metabolism increased in the dlpfc , in addition 
 to metabolic changes in other cortical and subcortical areas .
rcbf 
 increases were reported in the dlpfc and the rostral sma during apomorphine 
 pumps .
again , pallidotomy , subthalamic nucleus stimulation and continuous 
 apomorphine are able to reverse dyskinesias [ 394951 ] . 

therefore , the direct involvement of the dlpfc and the degeneration of 
 mesofrontal dopaminergic afferents might also be hypothesized to play some role 
 in the development of dyskinesias . finally , 
 involvement of the dorsolateral 
the prefrontal circuit encompasses the dorsal prefrontal cortex , the dorsal 
 caudate nucleus , the dorsal medial globus pallidus , and the ventral anterior 
 nucleus of the thalamus .
have shown that high frequency rtms 
 of the dlpfc leads to dopamine release in the ipsilateral caudate nucleus [ 19 , 20 ] .
we used the same parameters as strafella et al . with regard to frequency 
 and intensity of stimulation
we have shown for the first time that repeated sessions of 
 subthreshold 10  hz rtms of the dlpfc induced significant reductions of the 
 ipsilateral motor cortex excitability in advanced pd population , and might 
 possibly be suggestive of an improvement of l - dopa - induced dyskinesias .
it has 
 to be pointed out that this was a small observational study not specifically 
 designed to assess dyskinesias in pd .
dlpfc stimulation was not controlled by 
 the placebo rtms and therefore a possible placebo effect can not be excluded 
 either . on the other hand , the dlpfc stimulation was controlled by yet 
 another active stimulation over the mc .
improvement of dyskinesia could not 
 have been expected by patients since changes in the motor complications score 
 were not included in the primary outcomes of our pilot study .
further 
 research is warranted to explore more precisely whether the unilateral or 
 bilateral stimulation of the dlpfc via repeated sessions of high frequency rtms 
 induces beneficial effects on l - dopa - induced dyskinesias in advanced pd 
 patients , and whether it could be considered clinically useful .
 we studied whether five sessions of 10  hz repetitive transcranial magnetic stimulation ( rtms treatment ) applied over the dorsolateral prefrontal cortex ( dlpfc ) or the primary motor cortex ( mc ) in advanced parkinson 's disease ( pd ) patients would have any effect on l - dopa - induced dyskinesias and cortical excitability . 
 we aimed at a randomised , controlled study . 
 single - pulse transcranial magnetic stimulation ( tms ) , paired - pulse transcranial magnetic stimulation , and the unified parkinson 's disease rating scale ( updrs parts iii and iv ) were performed prior to , immediately after , and one week after an appropriate rtms treatment . 
 stimulation of the left dlpfc induced a significant motor cortex depression and a trend towards the improvement of l - dopa - induced dyskinesias . 
1. INTRODUCTION
2. PATIENTS AND METHODS
3. STATISTICAL ANALYSIS
4. RESULTS
5. DISCUSSION