several neuroimaging studies have investigated neural responses to art , aesthetic judgment and beauty ( cela - conde et al . , 2004 ; kawabata and zeki , 2004 ; vartanian and goel , 2004 ) .
on the contrary , little is known about neuronal circuits engaged during observation of implied motion ( i m ) in a work of art .
recent studies using transcranial magnetic stimulation ( tms ) demonstrated that observation of i m in visual images and works of art , alongside with the activation of movement processing mt / v5 areas ( david and senior , 2000 ; kourtzi and kanwisher , 2000 ) , modulates primary motor cortex ( m1 ) excitability as well as related intracortical inhibitory and excitatory circuits ( battaglia et al . , 2011 ) .
it has been proposed that similar circuits encode implied and real motion processing in the onlooker .
consequently , the activation of the mirror neuron system has been indicated as a mechanism allowing an individual to extract dynamic information from a still image with i m ( battaglia et al . , 2011 ; sbriscia - fioretti et al . , 2013 ;
mirror neurons , first discovered in macaque monkey premotor and parietal cortices ( gallese et al . , 1996 ) , fire during both action observation and execution .
subsequently , brain stimulation and neuroimaging studies demonstrated a similar pattern of activation ( mirror neuron - like activity ) in the human brain ( buccino et al .
recently , evidence has been presented supporting the facilitatory effects of action observation on congruent motor execution , motor learning ( brass et al . , 2000 ) and cortical long - term potentiation ( ltp)-like plasticity ( lepage et al . , 2012 ; sale and mattingley , 2013 ) . on the whole ,
these data support the hypothesis that action observation training might be used to augment traditional rehabilitation paradigms and speed the recovery of motor functioning ( mulder , 2007 ) .
a recent meta - analysis suggests that the dorsal part of the premotor cortex , rather than the posterior inferior frontal gyrus and adjacent ventral premotor cortex , is consistently activated in functional magnetic resonance imaging studies of imitation ( molenberghs et al . , 2009 ) .
thus , it is conceivable that frontal regions which extend beyond the classical mirror neuron network are crucial for imitation . in healthy subjects , repetitive tms ( rtms ) studies
have been used to investigate functional connectivity and plasticity between pmd and primary motor cortex ( m1 ) ( bumer et al .
no studies have explored the effects of observation of i m in artistic images on the strength of pmd  m1 connections . since its first description in 1945 ( adrian , 1945 ) art therapy has been shown to improve physical , mental health and social functioning in a variety of medical conditions ( miller and hou , 2004 ; secker et al . , 2007 ) .
therefore , the effects of art as a valuable adjunct to conventional rehabilitation should be evaluated with rigorous experiments .
given that current rehabilitation treatments are capable of changing brain connectivity and inducing functional reorganization ( dimyan and cohen , 2011 ) , the present study aims to provide evidence that observation of i m in a works of art can modulate neuroplasticity in fundamental motor circuits .
this will further our understanding of the foundation underlying the use of art in rehabilitation .
we studied 12 healthy volunteers ( mean agestandard deviation , 328 years ; 5 females ) .
all participants were right - handed according to the edinburgh inventory 2550 ( oldfield , 1971 ) and gave written informed consent to participate in the experiment according to the declaration of helsinki .
the study was approved by the local ethics committees ( new york college of podiatric medicine ) . during the experiment subjects
were seated comfortably in an armchair with both arms relaxed and were instructed to keep their eyes open in front of a computer monitor .
the electrophysiological experiments were performed either during the observation of a painting with i m or during observation of a  static 
stimuli were presented on a computer monitor using the presentation software ( neurobehavioral systems inc . ,
first the participants were instructed to focus their attention on the computer monitor ( appearance of a plus sign in the center of the monitor ) .
the videos of the paintings started after 5 sec and the images were presented continuously throughout the tms paradigms .
for the i m condition we selected michelangelo s expulsion from paradise ( sistine chapel , 15081512 ) , with its depiction of the gesture which adam makes with his extended right hand to keep the sword - bearing angel at bay .
for the no - im condition we selected michelangelo s creation of adam in which adam leans on his right forearm in a clearly static posture ( sistine chapel , 15081512 ) ( fig .
the experiments were block randomized and there was an interval of 5 days between the blocks .
surface electromyography ( emg ) was recorded from the right extensor carpi radialis ( ecr ) muscle with disposable adhesive disk electrodes placed in a tendon - belly arrangement on the skin overlying the ecr muscle .
the signal was amplified , filtered ( bandpass 2 hz to 5 khz ) , digitized ( micro 1401 , cambridge electronics design , cambridge , uk ) and stored in a laboratory computer for off - line analysis .
tms was performed with a standard figure - of - eight coil and magstim 200 stimulators ( magstim co. , whitland , uk ) .
the coil was placed at the optimal position for eliciting meps from the right ecr muscle (  hot spot  ) .
the coil was held tangentially to the skull with the handle pointing backwards and laterally at an angle of 45 to the sagittal plane .
thus , the electrical current induced in the brain was approximately perpendicular to the central sulcus .
this orientation of the induced electrical field is thought to produce a predominantly transsynaptic activation of the corticospinal neurons ( rothwell et al . , 1999 ) . during the experiments
emg activity was continuously monitored by visual ( oscilloscope ) and auditory ( speakers ) feedback to ensure complete relaxation .
resting motor threshold ( rmt ) was determined as the minimum stimulator intensity ( to the nearest 1% ) to produce an mep of 50 v in five of 10 trials .
mean peak - to - peak mep amplitudes were determined with 20 monophasic magnetic stimuli delivered to the motor hot spot of the ecr muscle at rate of 0.1 hz at a stimulation intensity of 120% rmt .
tms parameters were tested according to published guidelines for the use of tms in clinical neurophysiology ( rothwell et al . , 1999 ) .
high and low frequency pmd were performed according to a previous described methodology ( rizzo et al . , 2004 ) .
in brief , rtms was delivered using a standard figure - of - eight coil connected to a magstim rapid stimulator ( magstim co. ) .
first , we searched for the optimum site for stimulating the ecr motor hot spot and determined individual active motor threshold ( amt ) .
amt was defined as the minimum stimulus intensity that produced a liminal motor evoked response ( about 100 v in 50% of 10 trials ) . for pmd stimulation
the 5-hz rtms session consisted of five trains of 300 stimuli separated by an intertrain interval of 1 min ( 10 min in total ) .
the 1-hz rtms session consisted of five trains over the left pmd , 300 stimuli each , with a pause of 1 min between train .
the intensity of premotor rtms was set at 90% of amt because this is the optimum intensity to modulate cortical excitability in ipsilateral m1 ( gerschlager et al . , 2001 ) .
the stimulation intensity was matched during the two experimental conditions in order to obtain an mep of 0.40.5 mv .
we recorded ten meps at baseline , 15 , and 30 min after conditioning ( t15 and t30 ) . in an additional experiment subjects
were required to observe the movement for 20 min ; we then tested the effect of 5-hz pmd conditioning protocol on mep amplitude . in this way
the stimulation protocols were in accordance with published safety guidelines for rtms ( wassermann , 1998 ) .
rmt , amt , and mep amplitude recorded during observation of i m and no - im were analyzed with analysis of variance ( anova ) ( main effects  condition  ) .
the effects of 5- and 1-hz rtms conditioning on pmd  m1 connectivity and plasticity were assessed using a mixed model repeated - measure design with main effect  time  as within subject ( three levels t0 , t15 , t30 ) ; main effect  stimulation 
( two levels , 5- and 1-hz rtms ) and  condition  ( two levels , i m and no - im ) as between subjects factors . a greenhouse  geisser correction was used when appropriate .
when an f value was significant , post hoc paired - sample t - tests were performed .
we studied 12 healthy volunteers ( mean agestandard deviation , 328 years ; 5 females ) .
all participants were right - handed according to the edinburgh inventory 2550 ( oldfield , 1971 ) and gave written informed consent to participate in the experiment according to the declaration of helsinki .
the study was approved by the local ethics committees ( new york college of podiatric medicine ) .
during the experiment subjects were seated comfortably in an armchair with both arms relaxed and were instructed to keep their eyes open in front of a computer monitor .
the electrophysiological experiments were performed either during the observation of a painting with i m or during observation of a  static 
stimuli were presented on a computer monitor using the presentation software ( neurobehavioral systems inc . ,
first the participants were instructed to focus their attention on the computer monitor ( appearance of a plus sign in the center of the monitor ) .
the videos of the paintings started after 5 sec and the images were presented continuously throughout the tms paradigms . for the i m condition we selected michelangelo s expulsion from paradise ( sistine chapel , 15081512 ) , with its depiction of the gesture which adam makes with his extended right hand to keep the sword - bearing angel at bay .
for the no - im condition we selected michelangelo s creation of adam in which adam leans on his right forearm in a clearly static posture ( sistine chapel , 15081512 ) ( fig .
the experiments were block randomized and there was an interval of 5 days between the blocks .
surface electromyography ( emg ) was recorded from the right extensor carpi radialis ( ecr ) muscle with disposable adhesive disk electrodes placed in a tendon - belly arrangement on the skin overlying the ecr muscle .
the signal was amplified , filtered ( bandpass 2 hz to 5 khz ) , digitized ( micro 1401 , cambridge electronics design , cambridge , uk ) and stored in a laboratory computer for off - line analysis .
tms was performed with a standard figure - of - eight coil and magstim 200 stimulators ( magstim co. , whitland , uk ) .
the coil was placed at the optimal position for eliciting meps from the right ecr muscle (  hot spot  ) .
the coil was held tangentially to the skull with the handle pointing backwards and laterally at an angle of 45 to the sagittal plane .
thus , the electrical current induced in the brain was approximately perpendicular to the central sulcus .
this orientation of the induced electrical field is thought to produce a predominantly transsynaptic activation of the corticospinal neurons ( rothwell et al . , 1999 ) . during the experiments
emg activity was continuously monitored by visual ( oscilloscope ) and auditory ( speakers ) feedback to ensure complete relaxation .
resting motor threshold ( rmt ) was determined as the minimum stimulator intensity ( to the nearest 1% ) to produce an mep of 50 v in five of 10 trials .
mean peak - to - peak mep amplitudes were determined with 20 monophasic magnetic stimuli delivered to the motor hot spot of the ecr muscle at rate of 0.1 hz at a stimulation intensity of 120% rmt .
tms parameters were tested according to published guidelines for the use of tms in clinical neurophysiology ( rothwell et al . , 1999 ) .
high and low frequency pmd were performed according to a previous described methodology ( rizzo et al . , 2004 ) .
in brief , rtms was delivered using a standard figure - of - eight coil connected to a magstim rapid stimulator ( magstim co. ) .
first , we searched for the optimum site for stimulating the ecr motor hot spot and determined individual active motor threshold ( amt ) .
amt was defined as the minimum stimulus intensity that produced a liminal motor evoked response ( about 100 v in 50% of 10 trials ) . for pmd stimulation
the 5-hz rtms session consisted of five trains of 300 stimuli separated by an intertrain interval of 1 min ( 10 min in total ) .
the 1-hz rtms session consisted of five trains over the left pmd , 300 stimuli each , with a pause of 1 min between train .
the intensity of premotor rtms was set at 90% of amt because this is the optimum intensity to modulate cortical excitability in ipsilateral m1 ( gerschlager et al . , 2001 ) .
the stimulation intensity was matched during the two experimental conditions in order to obtain an mep of 0.40.5 mv .
we recorded ten meps at baseline , 15 , and 30 min after conditioning ( t15 and t30 ) . in an additional experiment subjects
were required to observe the movement for 20 min ; we then tested the effect of 5-hz pmd conditioning protocol on mep amplitude . in this way
the stimulation protocols were in accordance with published safety guidelines for rtms ( wassermann , 1998 ) .
rmt , amt , and mep amplitude recorded during observation of i m and no - im were analyzed with analysis of variance ( anova ) ( main effects 
the effects of 5- and 1-hz rtms conditioning on pmd  m1 connectivity and plasticity were assessed using a mixed model repeated - measure design with main effect  time  as within subject ( three levels t0 , t15 , t30 ) ; main effect  stimulation 
( two levels , 5- and 1-hz rtms ) and  condition  ( two levels , i m and no - im ) as between subjects factors . a greenhouse  geisser correction was used when appropriate . when an f value was significant , post hoc paired - sample t - tests were performed .
rmt and amt recorded in the right ecr muscle did not differ between conditions ( rmt : i m , 41%3.6% ; no - im , 40.1% 2.8% , f [ 1 , 22]=0.9 , p=0.8 ; amt : i m , 32.2%2.7% ; no - im , 33.7%3.2% ; f [ 1 , 22]=1.1 ; p=0.9 ) .
observation of the picture with i m induced an increase in mep size ( i m , 0.820.04 mv ; no - im , 0.610.038 mv ; f [ 1 , 22]=7.2 ; p=0.006 ) ( data not showed ) . regarding the pmd  m1 connectivity and plasticity paradigm , three - way repeated measure anova yielded a significant main effect of  stimulation 
( f [ 1 , 44]=7.4 , p<0.009 ) and  time  ( f [ 1 , 44]=7.5 , p<0.001 ) without a  stimulation  x  condition  x  time  interaction ( f [ 2 , 88]=2.5 , p<0.08 ) . the assumption of sphericity was not violated .
post hoc analysis indicated that meps recorded 15 and 30 min after pmd conditioning were significantly different from baseline ( p=0.005 and p=0.0004 , respectively ) .
we then performed paired samples t - tests follow - up analysis to ascertain the effect of 5- and 1-hz rtms pmd on meps recorded during observation of i m and no - im .
the 5-hz rtms paradigm increased meps amplitude during both i m and no - im condition compared to t0 ( i m : t15 [ p=0.00 02 ] , t30 [ p=0.001 ] ; no - im : t15 [ p=0.001 ] , t30 [ p=0.004 ] ) . furthermore , there was no difference in baseline mep amplitude between the two condition ( f [ 1 , 22]=1.1 , p=0.3 ) ( fig .
similar results were obtained in a control experiment using a longer exposure to the conditions ( total duration 25 min ) ( i m : to , 0.78 0.03 mv ; t15 , 1.360.05 mv ; t30 , 1.120.05 mv ; no - im : to , 0.820.04 mv ; t15 , 1.290.05 mv ; t30 , 1.360.04 mv ( main effect  condition 
f [ 2 , 28]=28.2 , p<0.0001 ;  condition  x  time  interaction : f [ 2 , 44]= 2.01 , p=0.31 ) ( data not showed ) . on the contrary , the 1-hz rtms conditioning decreased meps amplitude only during the i m condition compared to t0 ( i m ; t15 [ p=0.01 ] , t30 [ p=0.008 ] ; no - im : t15 [ p=0.8 ] , t30 [ p= 0.4 ] ) ( fig .
3 ) . on the whole , the data indicate that observation of i m in a work of art modulated connectivity and plasticity of pmd  m1 inhibitory circuits .
in this study we investigated whether i m information from a static image of a painting affects cortical excitability , connectivity and plasticity . our results demonstrated that observation of an image with i m increases mep size .
furthermore , using rtms , we showed that i m in a work of art modulates pmd 
first we replicated earlier findings indicating that mep amplitude increases upon observation of pictures with i m ( battaglia et al . , 2011 ; urgesi et al . , 2006 ) .
this suggests that motor resonance to i m in paintings might be due to the activation of mirror neuron / action observation - execution and motor imagery networks ( battaglia et al . , 2011 ) .
furthermore , using a well - characterized paradigm to explore functional connectivity and plasticity ( mnchau et al .
, 2002 ; passingham , 1985 ; rizzo et al . , 2004 ) , our results revealed that long - term decrease in mep amplitude induced by 1-hz rtms pmd conditioning are prevented during observation of a painting with i m .
thus , i m observation facilitates pmd  m1 functional connectivity through a specific effects on inhibitory connections .
pmd is not considered part of the mirror system in humans and primates ( rizzolatti and craighero , 2004 ) .
furthermore , pmd neurons play a pivotal role in externally cued arm movements ( wise , 1985 ) and exhibits similar activity patterns during both action observation , motor imagery and performance ( cisek and kalaska , 2004 ; szameitat et al . , 2007 ) .
in addition , single - unit activity within both m1 and pmd is similar during observation and execution of a familiar task ( tkach et al . , 2007 ) .
it is noteworthy that our findings do not imply the presence of classical mirror neurons in pmd but rather illustrate a modulation of the pmd 
. however , some words of caution are required when interpreting the proposed sequence of effective connectivity networks during processing of i m in a painting .
pmd is part of a rather large frontoparietal network that transforms sensory information into actions ( chouinard et al . , 2003 ;
it has been reported that 1-hz rtms of the left pmd induces compensatory excitatory activity in both contralateral and ipsilateral hemisphere ( oshea et al . , 2007 ) .
it is thus conceivable that plastic changes induced by observation of a painting with i m might take place in a more extensive connectivity network still to be investigated .
our results are in accordance with previous findings reporting modulation of premotor - motor connectivity during movement observation .
observation of whole hand grasp and precision grip modulate ventral - premotor  m1 connectivity probed with a paired - pulse paradigm ( davare et al . , 2008 ;
koch et al . , 2010 ) . by using a different paradigm we showed that observation of i
m in a work of art induces a similar modulation in a different subregion of premotor cortex .
it has been suggested that it may be related either to long - term depression / ltp ( chen et al . , 1997 ) and/or changes of cortical excitability ( touge and takeuchi , 2001 ) .
it is likely that the after - effects induced by pmd rtms conditioning are linked to long - lasting changes in synaptic efficacy rather that changes in membrane resting potential ( rizzo et al . , 2004 ) .
m1 connectivity is particularly relevant for motor recovery and residual motor output after stroke ( fridman et al . , 2004 ; johansen - berg et al . , 2002 ) and
successful rehabilitative treatments are associated with strengthening of these connections ( rehme et al . , 2012 ) .
hence , our results further point toward the use of art with i m as plasticity - based intervention in rehabilitation .
future studies are needed to investigate whether observation of paintings with i m movements induces a similar modulation in intrinsic m1 inhibitory circuits recruited with m1 1-hz rtms conditioning .
we would be the first to acknowledge that factors such as expertise in art , prior exposure , individual temperament and particular psychological traits can influence the aesthetic and emotional responses to the artwork .
moreover , it is clear that the extraction of dynamic information from still images entails visual perception , association of the represented form with an action and mental imagery .
thus , individual mental imagery abilities and personal memories of the execution of an action might have affected neural responses to i m ( mizokami et al . , 2014 ) .
limitations like these will need to be addressed in future studies in order to ascertain the degree to which our finding might be generalized to other forms of dynamic art ( abstract vs. figurative art , different artists and periods , stylistic and structural properties , emotional content ) .
the fact that our results highlight a complex modulation of premotor - motor networks during observation of a painting with i m support the hypothesis that observation of art might be used to augment traditional rehabilitation paradigms and speed the recovery of motor functioning .
 following the discovery of mirror neurons , much attention has been de - voted to understanding the neural responses evoked by observation of implied motion in works of art . 
 neuroimaging studies have demonstrated that dorsal premotor cortex ( pmd ) is commonly involved during observation of movements but the role of the inhibitory and excitatory connections between pmd and primary motor cortex ( m1 ) during observation of implied motion remains uncertain . in this study , using high and low frequency repetitive transcranial magnetic stimulation ( rtms ) , we examined pmd  m1 connectivity and plasticity during observation of michelangelo s frescos with and without implied motion ( sistine chapel , 15081512 ) . 
 we found that observation of implied motion in a painting specifically reduces the activity of inhibitory pmd  m1 connections . on the contrary pmd  
 m1 facilitatory connections , as examined by means of 5-hz rtms , were not modulated during observation of the painting . 
 our data suggest that observation of implied motion in a painting modulates pmd  m1 connectivity and plasticity . 
 these results are consistent with the hypothesis that art with implied motion might be used as a plasticity - based intervention in rehabilitation . 