Modulation of Cortical Oscillatory Activity During Transcranial Magnetic Stimulation

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1 Human Bain Mapping 29: (2008) Modulation of Cotical Oscillatoy Activity Duing Tanscanial Magnetic Stimulation Deboa Bignani, 1,2 Paolo Manganotti, 2 Paolo M. Rossini, 1,3 and Calo Miniussi 1,4 * 1 Unità di Neuoscienze Cognitive, IRCCS Cento S. Giovanni di Dio Fatebenefatelli, Bescia, Italy 2 Dipatimento di Scienze Neuologiche e della Visione, Univesità di Veona, Veona, Italy 3 Dipatimento di Neuoscienze, AFaR S. Giovanni Calibita Fatebenefatelli & Clinica Neuologica, Univesità Campus Bio-medico, Roma, Italy 4 Dipatimento di Scienze Biomediche e Biotecnologie, Univesità di Bescia, Bescia, Italy Abstact: Tanscanial magnetic stimulation (TMS) can tansiently modulate cotical excitability, with a net effect depending on the stimulation fequency (1 Hz inhibition vs. 5 Hz facilitation, at least fo the moto cotex). This possibility has geneated inteest in expeiments aiming to impove deficits in clinical settings, as well as deficits in the cognitive domain. The aim of the pesent study was to investigate the on-line effects of low fequency (1 Hz) TMS on the EEG oscillatoy activity in the healthy human bain, focusing paticulaly on the outcome of these modulatoy effects in elation to the duation of the TMS stimulation. To this end, we used the event-elated desynchonization/synchonization (ERD/ERS) appoach to detemine the pattens of oscillatoy activity duing two consecutive tains of sham and eal TMS. Each tain of stimulation was deliveed to the left pimay moto cotex (MI) of healthy subjects ove a peiod of 10 min, while EEG hythms wee simultaneously ecoded. Results indicated that TMS induced an incease in the powe of bain hythms that was elated to the peiod of the stimulation, i.e. the synchonization of the a band inceased with the duation of the stimulation, and this incease was invesely coelated with moto-evoked potentials (MEPs) amplitude. In conclusion, low fequency TMS ove pimay moto cotex induces a synchonization of the backgound oscillatoy activity on the stimulated egion. This induced modulation in bain oscillations seems to incease coheently with the duation of stimulation, suggesting that TMS effects may involve shot-tem modification of the neual cicuity sustaining MEPs chaacteistics. Hum Bain Mapp 29: , VC 2007 Wiley-Liss, Inc. Key wods: TMS; electoencephalogaphy; event-elated desynchonization/synchonization; ERD/ ERS; TMS/EEG coegistation; moto-evoked potentials; MEP INTRODUCTION Contact gant sponsos: Ministeo della Sanità, Conto capitale 2002, Telecom Italia Mobile. *Coespondence to: Calo Miniussi, Dipatimento di Scienze Biomediche e Biotecnologie, Sezione di Fisiologia, Univesità di Bescia, Viale Euopa 11, Bescia. miniussi@med.unibs.it Received fo publication 16 Januay 2007; Accepted 17 Apil 2007 DOI: /hbm Published online 7 June 2007 in Wiley InteScience (www. intescience.wiley.com). Tanscanial magnetic stimulation (TMS) is an electophysiological technique, which allows the investigation of the functional state of the human ceebal cotex (Helle and Van Hulsteyn, 1992). By means of a pulsed magnetic field ceated by a ound o eight-shaped coil positioned next to the scalp, electic cuents ae induced in the bain and these, in tun, poduce tansynaptic depolaization of neuons located in the supeficial cotical layes (Helle and Van Hulsteyn, 1992). When deliveed ove the pi- VC 2007 Wiley-Liss, Inc.

2 Bignani et al. may moto cotex (M1) with adequate intensity, magnetic stimuli induce neual effeent volleys along the coticospinal pathway and tigge electomyogaphic esponses, named moto-evoked potentials (MEPs) which can be ecoded fom the muscles contalateal to the site of stimulation (Bake et al., 1985). Amplitudes and latencies of MEPs ae paametes, which allow the evaluation of the functional state of the coticospinal pathway, thus poviding valuable infomation about the functioning of moto pathways in both physiological and pathological conditions (Bake et al., 1986; Rossini and Rossi, 1998). In geneal, moto esponses induced by TMS ae the esult of a combination of excitatoy/inhibitoy events occuing at diffeent neual levels along the moto pathway and the elative contibution of these events is fa fom being entiely claified. Technical advances in the ealy 1990s intoduced a novel type of TMS able to delive tains of epetitive stimuli (TMS) opening new eseach diections. Since TMS has been intoduced, it has become evident that the effects of cotical stimulation may outlast the specific stimulation peiod, and this possibility has geneated inteest in expeiments aiming to impove deficits in the cognitive domain (Cotelli et al., 2006) as well as in clinical applications in the field of neuopsychiaty (e.g. teatment of depession) and fo teating movement disodes (Geoge et al., 1999; Miniussi et al., 2005; Wassemann and Lisanby, 2001). Above all, the possibility of inducing long-lasting changes in cotical excitability might explain the beneficial esults obtained in depessed patients (Siebne and Rothwell, 2003), suggesting that TMS may induce modulations, o even a eaangement, of synaptic efficiency within a given netwok. Nevetheless, the mechanisms undelying these changes in cotical function emain unclea. It has been shown that seveal paametes, such as fequency, duation, and intensity of stimulation, influence the effects of TMS on cotical excitability. A low fequency stimulation (stimulus ates of 1 Hz o less) of the pimay moto cotex is epoted to lead to a tansient decease in coticospinal excitability (Chen et al., 1997), while highe fequencies (stimulus ates of moe than 5 Hz) may pomote a shot-tem incease in cotical excitability (Beadelli et al., 1998; Di Lazzao et al., 2002; Maeda et al., 2000; Pascual-Leone et al., 1998; Peinemann et al., 2000). With egad to the duation of supatheshold TMS effects, Pascual-Leone et al. (1994b) demonstated a 3 4 min peiod of inceased excitability afte 10 pulses of 20 Hz TMS. Beadelli et al. (1998) obseved an incease in coticospinal excitability up to 900 ms afte one tain of 5 Hz TMS and an incease in ceebal blood flow was obseved at 10 min afte 1 Hz stimulation to the moto cotex (Fox et al., 1997). All these studies suggest that the modulatoy effects of TMS on coticospinal excitability can vay fom milliseconds to minutes, depending on fequency, stimulus intensity, intetial inteval, and duation of the TMS. Nevetheless, stimulating the ceebal cotex has played an impotant ole in theapeutic applications of TMS. Theefoe, the possibility to veify on line its inhibitoy o facilitatoy effects on bioelectical activities of the stimulated cotex, as well as of cotical aeas well outside the moto cotex, is of geat inteest to eseach and clinical application. Studying the modulations of ongoing oscillatoy EEG activity by TMS may be a key to veifying such effects. In geneal, voluntay movements ae accompanied by a modulation in the a and b powe bands, which is chaacteized by a decement (event-elated desynchonization o ERD) stating about 1 3 s befoe the onset of a self-paced finge o hand movement ove contalateal sensoimoto aeas and becoming bilateal when the movement begins; an incement (event-elated synchonization o ERS) occuing ealie fo the b than fo the a band can be obseved afte the movement execution (Deambue et al., 1993; Leocani et al., 1997; Manganotti et al., 1998; Pfutschelle and Beghold, 1989; Pfutschelle and Lopes da Silva, 1999; Stancak and Pfutschelle, 1996). Thee is a geneal ageement that deceases in EEG powe eflect oscillatoy aspects of cotical activation (i.e. aousal) while inceases of EEG powe have been associated with pedominantly inhibitoy activities (Chen et al., 1998; Hummel et al., 2002; Pfutschelle et al., 1996). Even though it has been peviously demonstated that TMS can modulate the ongoing oscillatoy EEG activity, only a limited numbe of studies have investigated this topic. Recently, Stens et al. (2002) have evaluated the effects of TMS in the a band afte a tain of 1,500 low fequency (1 Hz) stimuli deliveed ove the pimay moto cotex at a subtheshold intensity. Recodings wee taken pio to, immediately afte, 25 min afte, and 50 min afte TMS. Powe deceased by 6% duing the active compaed to the est state, but thee was no appaent diffeence between the diffeent active peiods. Moeove, changes occued on the hemisphee ipsilateal but not in the one contalateal to the stimulation. In a coegistation EEG-TMS study, Paus et al. (2001) epoted that single-pulse TMS induced a highly synchonous oscillation in the b ange (15 30 Hz) that lasted fo seveal hunded milliseconds. Moeove, they obseved that the pobability of potentiating such hythmicity was linked to the intensity of stimulation: the only two subjects with a minimal oscillatoy esponse wee those with the lowest stimulation intensity. Fuggetta et al. (2005) showed that the magnetic stimulation applied to M1 poduced a synchonization both in a and in b hythms, which inceased linealy with TMS intensity. In addition, this effect was clealy shot-lasting because it occued within the fist 500 ms afte the magnetic stimulation. The TMSinduced oscillations obseved in Paus et al. (2001) and Fuggetta et al. (2005) have been moe linked to the esetting of the ongoing oscillatoy activity (poduced by extenal magnetic stimulation of the bain) than to an idling state of the bain (Pfutschelle et al., 1996). Resetting activity might be established in cotical netwoks o might be diven by a common thalamic pacemake (Destexhe et al., 1999; Steiade and Amzica, 1996). 604

3 Modulation of Cotical Oscillatoy Activity by TMS In the pesent study, we investigated fo the fist time the immediate effects of TMS on the ongoing EEG oscillatoy activity in the healthy human bain, with paticula focus on the elationship of such vaiations to the duation of the stimulating pocedue. In pactice, we divided 10 min of continuous eal low fequency TMS into thee consecutive peiods and compaed the cotical esponse fom the fist block of stimulation (fom 0 to 3.33 min) to the second (fom 3.34 to 6.66 min) and to the thid (fom 6.67 to 10 min) block of stimulation, using the ERD/ERS appoach to detemine the pattens of oscillatoy activity duing these thee stimulation peiods. One of the basic featues of ERD/ERS measuements is that the EEG powe of an inteval of inteest (active peiod) is displayed elative to (i.e. as a pecentage of) the powe of the same EEG leads ecoded duing a efeence peiod. In this study, the powe in a and b fequency bands computed in the 480 ms following a low fequency TMS (1 Hz) was compaed with two diffeent efeence peiods: a standad efeence peiod 480 ms peceding each single pulse of the magnetic stimulation (standad efeence) and a sham efeence that was collected 480 ms following each single pulse of sham magnetic stimulation (sham efeence) collected in a 10 min session just befoe eal TMS. The standad efeence was chosen accoding to the common pocedue of using the few seconds befoe the event of inteest (i.e. TMS pulse) as efeence peiod, while the sham efeence was chosen to bette addess the modulation of cotical oscillatoy activity ove time as well as the eventual effect of the acoustic event epesented by the stimulato s noise. In fact, if modulations of cotical oscillatoy activity induced by TMS pesist ove time, this effect should affect both the inteval peceding and following each magnetic stimulation pulse in a tain of pulses. Thus, small changes should be detected by compaing the pe- and post-tms pulse peiods, but lage changes should emege when compaing the EEG powe induced by eal TMS with that induced by sham TMS. MATERIALS AND METHODS Pocedue and Subjects Six healthy ight-handed voluntees (thee males and thee females, mean age 34 yeas) wee enolled afte giving witten infomed consent. None had histoy of neuological disode o head injuy. All expeimental potocols had been appoved by the local Ethics Committee. Real and sham TMS was applied ove the left M1 simultaneously with EEG data collection. Each subject undewent an expeiment consisting of two 10-min sessions, a sham TMS and a eal TMS session espectively, sepaated by some minutes stimulus-fee inteval to allow eplacement of the coil (fom sham coil used in the fist session to eal coil used in the second session). Fo each session of stimulation, a tain of 600 magnetic stimuli wee deliveed at 110% esting moto theshold with 1 Hz epetition ate. Subjects woe ea plugs and wee seated in a comfotable amchai in an electically-insulated and sound-poof oom with thei hands ponated in a elaxed position and eyes open. Stimulation TMS was caied out by a Magstim SupeRapid magnetic stimulato connected to fou booste modules and a standad figue-of-eight shaped coil with an oute winding diamete of 70 mm (Magstim Company, Whitland, UK) that geneates 2.2 T as a maximum output. In the pesent potocol individual biphasic stimuli wee employed. The coil was placed tangentially to the scalp with the handle pointing backwads and lateally at about a 458 angle away fom the midline. The cuent flow of the initial inging phase of the biphasic pulse in the TMS coil induces a cuent flowing fom posteio to anteio in the undelying moto cotex. To establish the moto hot spot and the esting moto theshold, the coil was moved in steps of 0.5 cm in the fonto-cental egion of the scalp. The optimal position ( hot spot ) was functionally defined as the point whee a specific TMS pulse induced a maximum evoked moto esponse fom the abducto pollicis bevis (APB) muscle of the ight hand. At this point, to assist in the position of the TMS ove the subject s head, the coil was stabilized in the same position, with espect to the site of stimulation, by means of a mechanical suppot that consisted of a holding am (Magic am Manfotto, with two lage clamps) and a heavy duty tipod. Once the coil was immobilized, the esting moto theshold was detemined as the lowest stimulus intensity, which poduced in the APB muscle at least five MEPs of 50 lv out of 10 consecutive stimuli (Rossini et al., 1994). Fo the sham-tms condition, the Magstim Placebo Coil System was used. This is a device specially designed to eplicate the standad figue-of-eight coil; it poduces dischage noise without stimulating cotical tissue, since its magnetic field output is about ten-times lowe compaed to that deliveed by the standad coil. The expeimental set-up was theefoe simila in both the sham and eal TMS sessions. EEG Recodings TMS-compatible EEG equipment (BainAmp 32MRplus, BainPoducts GmbH, Munich, Gemany) was used fo ecoding TMS-evoked potentials fom the scalp. The EEG activity was continuously acquied fom 19 scalp sites using electodes mounted on an elastic cap, positioned accoding to the Intenational system. Additional electodes wee used as gound and efeence. The gound electode was placed in the midoccipital position (OZ). The left and ight mastoid seved as efeence fo all electodes. A continuous ecoding mode without any sample 605

4 Bignani et al. and hold cicuits was chosen. The design of new amplifies allows appopiate selection of amplifie sensitivity and opeational ange that is adapted to the TMS stimulus magnitude (Bonato et al., 2006). This obviates the need to wait fo the signal to ecove afte the TMS pulse. The signal was digitized at a sampling ate of 2.5 khz, using a 16 bit A/D-Convete with 0.1 lv/bit sensitivity. Data wee ecoded with a band-pass filte of Hz. To minimize oveheating of the electodes located in the vicinity of the stimulating coil, magnetic field-compatible Ag/AgCl-coated electodes wee used. Skin/electode impedance was measued with the dedicated BainVision module and was confimed to be 5 ko. Hoizontal and vetical eye movements wee detected by ecoding the electooculogam (EOG). The voltage diffeence between two electodes located to the left and ight of the extenal canthi ecoded hoizontal eye movements. The voltage diffeence between efeence electodes and electodes located beneath the ight eye ecoded vetical eye movements and blinks. EMG activity and MEPs fom the ight APB wee ecoded via suface electodes in belly-tendon montage; the signal was band-pass filteed at 50 1,000 Hz with all the othe paametes as fo the EEG signal. EEG Analysis To chaacteize the cotical oscillatoy activity, EEG data wee analyzed offline with a commecial softwae (Scan 4.3, Compumedics Neuoscan). Since the fist few milliseconds following the TMS pulse contained lage and tansient signals pobably due to cuents induced by the magnetic field, the EEG tace analyses began at 20 ms afte magnetic stimulation. Epochs of 480 ms wee obtained fo the active peiod fom 20 to 500 ms afte the eal TMS, fo the standad efeence fom 500 to 20 ms peceding the eal TMS, and fo the sham efeence fom 20 to 500 ms afte the sham TMS pulse. Fo each type of peiod, the total 600 epochs wee divided into thee blocks of stimulation, each containing 200 tials (fist: magnetic stimuli; second: magnetic stimuli; thid: magnetic stimuli). All the epochs wee visually inspected and those with excessively noisy EEG (i.e. due to EMG contamination) o eye-movement atifacts (blinks o saccades) wee ejected fom the analyses. Oveall, the numbe of accepted epochs fo each block anged between 65 and 194. Fo each subject and fo each epoch/sweep, the powe specta was estimated fo the a (8 12 Hz) and b (12 30 Hz) fequency bands by means of the Fast Fouie tansfom (Hamming window; fequency esolution ¼ 2,000 Hz). The mean band powe was then obtained by aveaging the powe values of the sweeps fo each block of stimulation. To quantify the EEG powe changes induced by TMS, event-elated ERD/ERS wee computed in accodance with the standad fomula: [(band powe in active peiod) (band powe in efeence peiod)/(band powe in efeence peiod) 3 100] (Pfutschelle and Lopes da Silva, 1999). Two diffeent ERD/ERS wee computed depending on the 480 ms efeence peiod used (standad efeence, sham efeence). The ERD/ERS tansfomation is defined as the pecentage decease/incease of instant powe density at the event compaed to a pe-event baseline. Theefoe, event-elated powe deceases (cotical activation state) ae expessed as negative values, while event-elated powe inceases (cotical idling state) ae expessed as positive values. Fo each of the two fequency bands of inteest (a 8 12 Hz; b Hz), fou factos wee tested within subjects, using ANOVAs: efeence peiod (standad efeence vs. sham efeence), stimulation block (fist, second, thid), egion [fontal (F3, Fz, F4), cental (C3, Cz, C4), paietal (P3, Pz, P4)], and side [ight (F4, C4, P4), midline (Fz, Cz, Pz), left (F3, C3, P3)]. The Huynh Feldt e coection facto was applied whee appopiate to compensate fo possible effects of nonspheicity in the measuements compaed. The coection facto educes the degee of feedom of the usual F-test; only the coected pobability values ae epoted. We used Statistica Data Analysis Softwae (Statsoft) to pefom all the statistical analyses. In all conditions, the nomal distibution was tested applying the Kolmogoov Sminov test (fo all P > 0.2). Post-hoc tests wee pefomed to investigate significant effects, by means of t tests, using the Bonfeoni coection as appopiate in the case of multiple compaisons. MEP Analysis The MEPs ecoded fom the ight APB wee computed as the absolute amplitude between the two lagest peaks of opposite polaity afte 20 ms fom the TMS pulse. MEPs amplitude was measued peak-to-peak fom the initial down-going deflection to the following up-going one (Fig. 3). Mean MEP peak-to-peak amplitudes (mv) wee nomalized and calculated fo each block of stimulation. To veify whethe thee was any coespondence between the modulatoy effects of TMS on the amplitude of the MEPs and the modulatoy effects of TMS on the event-elated synchonization, a Peason s coelation (P < 0.05) coefficient was calculated between the changes in the MEPs and the changes in the event-elated synchonization ove C3 and P3 though the thee blocks of stimulation. RESULTS Subjects did not epot any advese side effects duing the couse of the expeiment. Mean moto theshold was 62%, anging fom 58 to 65%, theefoe the mean stimulation intensity was 68% of the maximum output of the stimulato. In both the fequency bands, TMS induced a geneal incease in EEG powe oscillations (ERS), which eached lage amplitudes in the a compaed to the b band independently of the baseline. 606

5 Modulation of Cotical Oscillatoy Activity by TMS 6SD ¼ 16.23) (P ¼ 0.02). Thee was no diffeence between the ight side and the midline (P ¼ 1.0). Finally, a fou-way inteaction [efeence peiod 3 stimulation block 3 egion 3 side: F(8, 40) ¼ 2.76, P ¼ 0.01] showed a diffeence in the synchonization amplitude powe between the fist and the thid stimulation blocks on C3 (mean ¼ 64.62%; 6SD ¼ vs. mean ¼ %; 6SD ¼ 46.19) and P3 (mean ¼ 26.78%; 6SD ¼ vs. mean ¼ 92.79%; 6SD ¼ 23.09) electodes paticulaly when the sham TMS efeence was used (Figs. 1A and 2). As a matte of fact, the post hoc analysis evealed significant diffeences between the two blocks diectly fo C3 (P < 0.001) and P3 (P < 0.001) electodes with espect to the sham TMS efeence. The same compaison using the standad efeence showed a significant diffeence between the fist and the thid stimulation blocks on P3 (P ¼ 0.048), but not on C3 (P ¼ 1.0) electodes. This esult was indicative of an inceasing modulatoy effect elated to the duation of the stimulation that was also patly efeence-specific. b Band Figue 1. Aveage data of the event-elated powe modulations induced by 1 Hz TMS fo the a fequency band (Panel A) and fo the b fequency band (Panel B) using the sham TMS efeence. The data ae shown as a function of thee successive stimulation blocks: fist in white fom tial 1 to 200; second in gay fom tial 201 to 400; thid in black fom tial 401 to 600. On the x-axis the analyzed ecoding electodes ae epoted. Bas coespond to the standad eo of mean. The statistical analysis pefomed on the b fequency band showed a significant two-way inteaction [egion 3 side: F(4, 20) ¼ 4.17, P ¼ 0.01] indicating that the synchonization powe was lage ove the C3 electode (ipsilateal to the TMS stimulation) compaed to all the fontal electodes (all P < 0.013), to the contalateal C4 (P < 0.001) and to the paietal Pz and P4 electodes (all P < 0.005). Thee was a tend fo this diffeence to be lage when the sham TMS efeence was consideed with espect to the standad efeence [efeence peiod 3 egion 3 side: F(4, 20) ¼ 2.72, P ¼ 0.058] (Fig. 1B). Moeove, the main facto of efeence peiod appoached significance [F(1, 5) ¼ 6.13, P ¼ 0.056], evealing an inteesting tend wheeby eal TMS induced a lage synchonization powe elative to the sham TMS efeence (mean ¼ 47.01%; 6SD ¼ 26.93) than to the standad (i.e., pe eal TMS pulse) efeence (mean ¼ 8.79%; 6SD ¼ 10.75). a Band The statistical analysis pefomed on the a fequency band evealed a significant effect of egion [F(2, 10) ¼ 4.40, P ¼ 0.042]. Planned t tests poved that the synchonization ove the cental electodes (mean ¼ 52.69%; 6SD ¼ 24.90) was lage compaed to that ecoded ove the fontal electodes (mean ¼ 17.97%; 6SD ¼ 13.02) (P ¼ 0.042), while no diffeence emeged between cental and paietal electodes (P ¼ 0.44). A significant effect of side [F(2, 10) ¼ 10.55, P ¼ 0.01] also emeged, indicating that the left hemisphee (ipsilateal to the TMS stimulation) showed a lage synchonization amplitude (mean ¼ 56.48%; 6SD ¼ 24.48) compaed to the ight hemisphee (mean ¼ 21.37%; 6SD ¼ 13.80) (P ¼ 0.004) and to the midline (mean ¼ 29.09%; Figue 2. Scalp distibution maps of the aveage ERD/ERS induced by the eal TMS fo the a fequency band, with sham as efeence, epesented sepaately fo the thee stimulation blocks. Red colo epesents maximum elative synchonization. 607

6 Bignani et al. was computed using the sham TMS efeence (efeence peiod 3 stimulation block 3 egion 3 side), a coelation analysis was pefomed between the changes of the MEPs and of the ERS in the thee intevals (a band; sham TMS efeence). The decease in the MEPs amplitude coelated with the incease of the powe synchonization ove the C3 electode (Fig. 3B). As a matte of fact, a significant coelation emeged between the diffeence in the amplitude of the MEPs between the fist and the thid stimulation blocks and the diffeence in the powe synchonization ecoded ove the electode C3 between the two blocks ( ¼ 0.88, P ¼ 0.02) (Fig. 3C). A coelation appoaching significance was also obseved between the fist and the second stimulation blocks ( ¼ 0.79, P ¼ 0.063). No coelation was found between the decease of the MEPs and the incease of the synchonization amplitude ove the P3 electode. DISCUSSION Figue 3. Panel A: Moto-evoked potential ecoded fom the ight APB; gand aveaged data elicited duing eal TMS of the left MI in thee successive stimulation blocks: the fist: magnetic stimuli (solid line), the second: magnetic stimuli (thin line), and the thid: magnetic stimuli (dashed line). Panel B: Mean amplitude of the moto-evoked potential (on the left) and of the powe synchonization ecoded ove the electode C3 (on the ight) elicited duing the fist and the thid blocks of stimulation. Bas coespond to the standad eo of mean. Panel C: The scatteplot shows the significant coelation between the changes in the amplitude of the MEPs, i.e. a decease, on the y-axis and the changes in the powe synchonization ecoded ove the electode C3, i.e. an incease, on the x-axis, between the fist and the thid stimulation blocks. The dotted lines epesent 95% confidence limits. MEPs As can be seen in Figue 3A, the mean amplitude of the MEPs deceased in the second and thid blocks of stimulation in compaison to the fist block. Nevetheless this diffeence did not appoach a significant value in statistical analysis (P ¼ n.s.; fist block lv vs. thid block lv). Since a diffeence between the fist and the thid stimulation blocks emeged in the analysis of a band synchonization ove C3 and P3 electodes when the ERS The pesent study was designed to exploe EEG powe modulations induced by low fequency TMS in the a and b fequency bands. Accoding to pevious studies (Fuggetta et al., 2005; Paus et al., 2001), a widespead synchonization of a and b activity has been obseved afte magnetic stimulation. In self-paced movements, the powe synchonization typically emeges afte the onset of the movement and it has been linked to an idling (Pfutschelle et al., 1996) o nil-woking state (Mulholland, 1995) o to an inhibitoy contol of neuonal activity (Hummel et al., 2002; Pfutschelle and Andew, 1999; Suffczynski et al., 1999), while desynchonization is pesent duing self-paced movement and is coelated with the activation of moto aeas (Pfutschelle, 1992). Since in the pesent study subjects wee in a elaxed state and had no pocess to contol, it is most likely that the synchonization obseved afte the cotical stimulation eflects esetting of the oscillatos, as peviously suggested (Fuggetta et al., 2005; Paus et al., 2001). Nevetheless, it has ecently been suggested that a ERS may stem pincipally fom hythmic fluctuations of inhibitoy neuons (Klimesch et al., 2007), and theefoe it may play an active ole in the inhibitoy contol of cotical pocessing as evidence against the idling hypothesis. Thee was a slight diffeence in the powe modulation pattens between a and b bands. In geneal, a lage synchonization was eached in the a hythm than in the b hythm. Futhemoe, in the a band, lage amplitude was obseved ove the stimulated hemisphee than to the contalateal one, while in the b band only the cental paietal egion showed a focal diffeence. This esult is in line with findings, which state that the effect of TMS is stongest whee the induced electic field is stongest (Rothwell, 1991), in this case in the left moto aea. The lage involvement of the posteio egions elative to the cental stimulated aea in b, and patly also in the a band, could be explained by the close connections between moto and 608

7 Modulation of Cotical Oscillatoy Activity by TMS somatosensoy egions, the so called sensoy-moto aea. The use of supatheshold intensity induced muscle twitches that could modulate cental pocessing via sensoy affeents. It has been shown that incease of b hythm, contalateal to the stimulated hand, can be found in sensoimoto aeas following peipheal somatosensoy stimulation (fo a eview, see Neupe et al., 2006); this obsevation would suppot ou esults, accounting fo the b hythm synchonization. Howeve, these studies also suggest an associated desynchonization of the a hythm (Neupe et al., 2006) and this is inconsistent with ou esults. Theefoe, the effect due to the affeent input (muscle twitches) could account fo synchonization of the b, but not of the a hythm. a hythm includes what is called the l hythm (10 Hz), that tends to be associated with moto activity, and seveal expeiments suggest that the synchonization of the l hythm is associated with cotical inhibition of the moto cotex (Pfutschelle et al., 1996). Finally, the coil position o its oientation could also account fo these diffeences in the pattens of a and b bands (Amassian et al., 1989; Pascual-Leone et al., 1994a; Ruohonen et al., 1996). In contast with ou esults a pevious study by Stens et al. (2002) found a decease of a powe immediately afte subtheshold 1 Hz TMS. This study diffes fom ous since we did use an on-line ecoding and the stimulation intensity was 110% of the individual moto theshold. Such diffeences in the expeimental setting can account fo diffeent esults. In fact othe studies (Fuggetta et al., 2005; Paus et al., 2001) suggest that the intensity of stimulation detemines the incease of powe induced by TMS and that these effects ae shot lasting. To chaacteize a possible stengthening of the effects induced by the TMS on cotical activity, we compaed EEG powe modulations, as well as MEPs amplitude, in thee consecutive peiods duing a tain of stimulation. An incease in the powe synchonization fom the fist to the thid block of stimulation appeaed only in the a band and invesely coelated with the MEPs amplitude. This esult suggests that the inceasing modulatoy effect of TMS on EEG activity ove the couse of the stimulation may elate to the amount of enegy tansfeed to the bain. Changes in the a band may epesent activities elated to souce geneatos in moto aeas as measued by movement-elated EEG signal powe and coheence modulation (Geloff et al., 1998; Leocani et al., 1997; Manganotti et al., 1998; Pfutschelle et al., 1994; Salmelin and Hai, 1994; Too et al., 1994). In addition, the a band has been documented to be moe eactive than boad b band to movement pogamming and execution (Manganotti et al., 1998) and to single pulse TMS (Fuggetta et al., 2005). Moe significant diffeences wee obseved between the fist, second and thid blocks of stimulation when the eal TMS condition was compaed with the sham baseline than when it was compaed with the pe-tms baseline. The TMS likely affected cotical activity ove the entie session of stimulation by inceasing the band powe synchonization both in the active (post-tms pulse) and in the efeence peiod (pe-tms pulse). Consequently, the atio between them emained the same and did not eveal evident powe modulations elated to TMS duation. On the contay, the sham magnetic stimulation did not poduce any powe alteations, theefoe epesenting the ideal efeence peiod. This intepetation is suppoted by the study of Fuggetta and colleagues (2005) in which they demonstated that the sham condition did not poduce any effect on the oscillatoy EEG activity. Seveal consideations should be made in elation to the use of the sham condition and possible potential confounds. The fist is elated to the ode of the sham condition in the pesent expeiment (i.e. sham always peceded to eal stimulation) and the second is that indeed sham stimulation by itself is not an ideal contol condition in the sense that it does not epoduce the skin sensation that one gets with eal TMS. As egads the fist point, the choice of this ode aises the possibility that the incease in a powe thoughout the 10-min session could be a esult of changes in subjects aousal duing the eal stimulation. Nevetheless, the choice of this expeimental pocedue was based on the fact that it was not possible to countebalance the sham and eal sessions since the possibility of a lasting effect afte TMS may have influenced a subsequent sham ecoding. Moeove, the specificity of the esults, that wee localized and latealized, cannot account fo the explanation of a geneal aousal effect. In the same vein, an incease of synchonization should also have been pesent ove the fist 10 min in the sham condition, since subjects wee in a elaxed state and had no pocess to contol skin sensation o twitches induced by TMS; nevetheless, no diffeences wee pesent between the fist and the last block in the sham condition. As egads the second point, a diffeent contol condition, like eal TMS on occipital cotex, might have been a supeio appoach instead of sham. Nevetheless, to ty to ensue that changes in pefomance ae specifically attibutable to the effects that TMS induces upon the bain, it was necessay to have a contol condition fee fom influences of specific TMS effects, since the stimulation of othe aeas as contol condition could have poduced an unbalanced baseline condition inducing modifications of the geneal bain esponse. The main finding of the pesent expeiment is the coelation between the pogessive decease of MEPs amplitude and the simultaneous incease in EEG synchonization, which in itself povides additional infomation on plasticity in the human bain. It has been known fo some time that MEPs amplitude tends to decease pogessively duing ecuent TMS at a slow epetition-ate; this phenomenon has been ascibed to self-defending mechanisms against the impact of stimulation deliveed fom the outside within the inta-cotical cicuity (Rossini et al., 1991). Howeve this explanation is unlikely fom a phylogenic point of view since thee ae no easons that such a mechanism could be useful. A moe pobable explanation is 609

8 Bignani et al. that such modulation can be ascibed to the activation of a fom of neuonal gain-contol, suggesting an active inhibitoy mechanism fo the contol of cotical pocessing, conguent also with the idea that a ERS may stem pincipally fom hythmic fluctuations of inhibitoy neuons. Recently it has been documented that low fequency TMS (1 Hz) given at subtheshold (95% esting theshold) o supatheshold (110% esting theshold) intensity poduce a tansient decease in coticomoto excitability that lasts seconds to minutes (Chen et al., 1997; Maeda et al., 2000; Muellbache et al., 2000; Touge et al., 2001). In addition, low fequency (1 Hz) TMS ove the moto cotex poduced an incease in ipsilateal cotico-cotical coheence immediately afte the TMS (Stens et al., 2002). It has been suggested that TMS is able to excite cotical inteneuones, theeby acting tansynaptically on pyamidal cells. Subtheshold low fequency TMS has also been shown to decease egional ceebal blood flow, consistent with the idea of TMS-induced activation of local inhibitoy mechanisms (Paus et al., 1998; Spee et al., 2000). Moeove, using evoked potentials, it has been shown that 1 Hz TMS of human pimay moto cotex changes cotical excitability at the site of stimulation as well as in ipsilateal somatosensoy cotex, pobably via cotico-cotical pathways between moto and sensoy cotex (Enomoto et al., 2001). Thus, in ageement with pevious publications, we can hypothesize that TMS poduces changes in cotical inhibitoy mechanisms esponsible fo the development of cotical oscillations and inceased connectivity (Conteas et al., 1997; Paulus et al., 1999; Rubin and Teman, 2000). The decease in MEPs amplitude duing 1 Hz TMS stimulation is consistent with the potentiation of inhibitoy mechanisms elated to this kind of stimulation. On the othe hand, the eduction in MEPs amplitudes can be elated to a eduction in synaptic efficacy unde the stimulated site; in this case, thee is less postsynaptic efficacy fo a fixed excitatoy input diven by the magnetic pulse and theefoe less pemoto neuon (i.e. pyamidal cells) activity, esulting in a educed moto esponse (Lee et al., 2003). Although the magnitude of this effect was clea, so fa we can only say that TMS poduces changes in cotical excitability at the site of stimulation as well as in coelated aeas. Some data indicate that the effects of cotical stimulation may not only induce a modification of cotical excitability at the site of stimulation, but thee is also the possibility of a subcotical contibution to its effects. A few studies have analyzed possible activation of subcotical aeas o changes of neuoactive substances afte TMS in humans, showing modulation at distant levels (Stafella et al., 2001, 2003; but see Shaul et al., 2003 in human neuoblastoma cells; Szuba et al., 2001 in thyoid homone; Zangen and Hyodo, 2002 in neuotansmitte). Indeed, the possibility to disentangle focal fom distant effects induced by TMS upon diffeent stuctues of the cental nevous system may have valuable implications, and combining EEG ecoding with TMS is a fascinating way to study these aspects. In summay, slow TMS ove pimay moto cotex induces a synchonization of a and b bands that pefeentially affects the stimulated hemisphee. 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