Premotor transcranial direct current stimulation (tdcs) affects primary motor excitability in humans

Transcription

1 European Journal of Neuroscience, Vol. 27, pp , 2008 doi: /j x Premotor transcranial direct current stimulation (tdcs) affects primary motor excitability in humans Klára Boros, 1 Csaba Poreisz, 1 Alexander Münchau, 2 Walter Paulus 1 and Michael A. Nitsche 1 1 Department of Clinical Neurophysiology, Georg-August University, Robert Koch Straße 40, Göttingen, Germany 2 Department of Neurology, University Medical Centre Hamburg-Eppendorf, Germany Keywords: dorsolateral prefrontal cortex (DLPFC), premotor cortex (PM), short intracortical inhibition and facilitation (SICI ICF), transcranial direct current stimulation (tdcs), transcranial magnetic stimulation (TMS) Abstract Recent studies have shown that repetitive transcranial magnetic stimulation (rtms) over the premotor cortex (PM) modifies the excitability of the ipsilateral primary motor cortex (M1). Transcranial direct current stimulation (tdcs) is a new method to induce neuroplasticity in humans non-invasively. tdcs generates neuroplasticity directly in the cortical area under the electrode, but might also induce effects in distant brain areas, caused by activity modulation of interconnected areas. However, this has not yet been tested electrophysiologically. We aimed to study whether premotor tdcs can modify the excitability of the ipsilateral M1 via corticocortical connectivity. Sixteen subjects received cathodal and anodal tdcs of the PM and eight subjects of the dorsolateral prefrontal cortex. Premotor anodal, but not premotor cathodal or prefrontal tdcs, modified selectively short intracortical inhibition intracortical facilitation (SICI ICF), while motor thresholds, single test-pulse motor-evoked potential and input output curves were stable throughout the experiments. Specifically, anodal tdcs decreased intracortical inhibition and increased paired-pulse excitability. The selective influence of premotor tdcs on intracortical excitability of the ipsilateral M1 suggests a connectivity-driven effect of tdcs on remote cortical areas. Moreover, this finding indirectly substantiates the efficacy of tdcs to modulate premotor excitability, which might be of interest for applications in diseases accompanied by pathological premotor activity. Introduction The premotor (PM) and the primary motor cortex (M1) are densely interconnected, as demonstrated in animals and humans (Morecraft & van Hoesen, 1993; Fink et al., 1997; Ghosh & Porter, 1998; Tokuno & Nambu, 2000). In humans, the connectivity between PM and M1 has been explored electrophysiologically by single-pulse transcranial magnetic stimulation (TMS) (Civardi et al., 2001), paired-pulse TMS (Koch et al., 2007) and repetitive transcranial magnetic stimulation (rtms) (Gerschlager et al., 2001; Münchau et al., 2002; Rizzo et al., 2004). Civardi et al. (2001) demonstrated that lowintensity stimulation of PM at short interstimulus intervals (6 ms) reduces the size of muscle-evoked potentials (MEPs) elicited from M1, but high-intensity TMS leads to an increase of excitability (at intervals of 6 and 15 ms). This complex PM M1 interaction was substantiated by rtms studies. rtms of 1 Hz at 90% active motor threshold (AMT) over the left PM caused a lasting reduction in excitability of the corticospinal system (Gerschlager et al., 2001), whereas low-frequency rtms (1 Hz) at a low intensity (80% AMT) had no after-effects on single test-pulse MEP, but selectively enhanced intracortical excitability at an interstimulus interval of 7 ms in a TMS double stimulation paradigm (Münchau et al., 2002). High-frequency rtms (5 Hz) resulted in opposite effects (Rizzo et al., 2004). This suggests that depending on stimulation intensity and frequency TMS might predominantly affect different premotor motor neuronal Correspondence: Dr K. Boros, as above. klara.boros@med.uni-goettingen.de Received 28 June 2007, revised 17 December 2007, accepted 10 January 2008 populations. Influencing M1 excitability through PM stimulation might be of future clinical relevance. In patients with Parkinson s disease, abnormal modifiability of the premotor motor connectivity was normalized by l-dopa (Buhmann et al., 2004). Transcranial direct current stimulation (tdcs) is a new non-invasive paradigm to induce neuroplastic cortical excitability alterations. Anodal tdcs increases and cathodal tdcs diminishes excitability (Nitsche & Paulus, 2000). tdcs can alter cortical excitability for up to 1 h (Nitsche & Paulus, 2001; Nitsche et al., 2003a). Direct functional effects of tdcs are spatially restricted (Nitsche et al., 2003b, 2007). However, in a recently conducted positron emission tomography (PET) study, widespread cerebral activity changes were observed after motor cortex prefrontal tdcs (Lang et al., 2005). These might be due to connectively driven activity changes of remote areas, which have not been tested directly thus far. Thus, the aim of our study was to examine if tdcs of one cortical area (PM) produces excitability changes in an interconnected area (M1). We hypothesized that tdcs of PM induces excitability changes in the ipsilateral M1 via cortico-cortical connections. In contrast, tdcs of the dorsolateral prefrontal cortex (DLPFC) should not influence M1 excitability, because these areas are not directly interconnected. Cortico-spinal excitability of M1 was studied by using active and resting motor thresholds, single test-pulse TMS and input output curves (Chen, 2000; Nitsche et al., 2005). M1 inhibition and facilitation were examined using a TMS double-stimulation protocol (Kujirai et al., 1993). To rule out that the effects of premotor tdcs were caused by spreading of current flow to M1, we added a control experiment.

2 tdcs-driven premotor motor cortex interactions 1293 Methods and materials Subjects Seventeen healthy subjects participated in the study. Sixteen participants (20 29 years old, mean age ¼ ± 2.31 years, seven men) were included in the PM tdcs study, whereas eight (20 26 years old, mean age ¼ ± 2.00 years, three men) subjects participated in the DLPFC tdcs condition. Seven subjects participated in both experiments. Eight subjects (21 41 years old, mean age ¼ ± 6.35 years, two men) participated in the control experiment. None was on regular or acute medication. Participants gave informed written consent and were paid. The investigation was approved by the Ethics Committee of the University of Göttingen, and conforms to the Declaration of Helsinki. Experimental design Subjects were seated in a comfortable reclining chair with a mounted headrest during the experiments. All subjects participated in two or four experimental sessions on separate days 1 week apart to avoid carryover effects. In healthy young subjects repeated 1-Hz rtms of the left dorsal PM can prolong the aftereffects on M1 excitability when given on consecutive days, but not when applied with an interval of 1 week (Bäumer et al., 2003). They received anodal and cathodal tdcs in a counterbalanced order. Resting motor threshold (RMT), active motor threshold (AMT), the intensity to evoke MEP of 1 mv peak-to-peak amplitude, single test-pulse MEP, input output curve (I O curve) and short interval intracortical inhibition intracortical facilitation (SICI ICF) were recorded with TMS before tdcs and three times after the stimulation, i.e. approximately 0 min after tdcs (post1), 30 min after tdcs (post2) and 60 min after the end of DC stimulation (post3). After the tdcs session the protocols (SICI ICF and I O curve) were applied in randomized order interindividually (except the threshold determination, which had to be performed first to determine the stimulation intensities for the remaining protocols). Stimulus intensities (as percentage of maximal stimulator output) of TMS were determined at the beginning of each experiment. In the control experiment the primary motor cortex representation of the abductor digiti minimi muscle (ADM) was stimulated by a small tdcs electrode (3.5 cm 2 ), and motor cortical excitability was determined for the ADM and for the adjacent first dorsal interosseus (FDI) muscle representation, which was not covered by the tdcs electrode. We hypothesized that in this case tdcs would affect ADM-, but not FDI-excitability, if there is no spread of current flow. First, the M1 representation of the ADM and FDI were determined with TMS and the positions were marked with a skin marker. At the beginning, RMT, AMT and the TMS intensity necessary to evoke MEP of 1 mv peak-to-peak amplitude of both muscles were determined. Single test-pulse MEP and SICI ICF were recorded from both muscles (ADM, FDI) separately before anodal tdcs of the M1 ADM and twice after tdcs, i.e. approximately 0 min (post1) and 30 min after the end of DC stimulation (post2). Transcranial magnetic stimulation (TMS) TMS was performed using a standard double ( figure-of-eight ) 70-mm coil connected to two biphasic Magstim Rapid stimulators (Magstim Company, Whiteland, Dyfed, UK) via a bistim module. The coil was placed tangentially to the scalp, with the handle pointing posterolaterally at a 45 angle from the midline. The TMS coil was connected with a current reversal cable, thus enabling an initial anterior posterior current flow in the brain followed by posterior anterior current flow direction. The optimum position was defined as the site where TMS resulted consistently in the largest MEP in the resting muscle. The optimal presentation of the M1 was marked with an eyeliner. Surface electromyography was recorded from the right ADM by use of Ag AgCl electrodes. In the control experiment electromyography was recorded additionally from the right FDI in a belly tendon montage. The active electrode was placed over the muscle belly, the reference electrode over the tendon. The signals were amplified and filtered (1.59 Hz to 1 khz, sampling rate of 5 khz), digitized with a micro 1401 AD converter (Cambridge Electronic Design, Cambridge, UK), and recorded by a computer using SIGNAL software (Cambridge Electronic Design, version 2.13). Data were analysed offline on a personal computer. Complete muscle relaxation was controlled online via auditory and visual feedback of electromyography activity. Measurement of corticospinal and intracortical excitability RMT was defined as the lowest stimulus intensity that elicited a peakto-peak MEP amplitude of 50 lv or more in the resting muscle in at least three out of six recordings. AMT was the minimum intensity eliciting an MEP of a superior size compared with moderate spontaneous muscular background activity (15% of the maximum muscle strength) in at least three out of six trials (Rothwell et al., 1999; Nitsche et al., 2005). The intensity of the stimulator output for the single test-pulse MEP was adjusted so that stimulation led to an average MEP amplitude of about 1 mv peak-to-peak before the DC stimulation. The I O curves were determined using increasing stimulus intensities (100, 110, 130 and 150% of RMT), each with 20 stimuli per block (Nitsche et al., 2005). For two subjects 150% RMT exceeded the maximal stimulator output, and thus MEP was recorded with intensities of 130% RMT in these subjects. SICI ICF were measured using a TMS double-stimulation protocol including 2-, 3-, 5-, 7-, 10- and 15-ms interstimulus intervals. The first three ISIs represent inhibitory and the last two ISIs facilitatory intervals (Kujirai et al., 1993; Ziemann et al., 1996; Nitsche et al., 2005). The exact interval between the paired-pulses was randomized (4 ± 0.4 s). In this protocol, a subthreshold conditioning stimuli precedes the test stimulus. The pairs of stimuli were organized in blocks, in which each ISI and one additional single test pulse was represented once. The blocks were repeated 12 times and the order of the different pulses was randomized between blocks. To avoid any floor or ceiling effect, the intensity of the conditioning pulse was set to a relatively low value of 70% of AMT (Nitsche et al., 2005). The single test-pulse TMS intensity was adjusted to the intensity to evoke an MEP of 1 mv peak-to-peak amplitude. The mean peak-to-peak amplitude of the conditioned MEP at each ISI was expressed as a percentage of the mean peak-topeak size of the unconditioned test pulse. Voluntary contraction of the target muscle decreases SICI ICF. Therefore, blocks in which the target muscle was not relaxed were discarded from the analysis (Ridding et al., 1995). At least eight from 12 blocks were analysed in every subject in each time block. None of the blocks lasted longer then 10 min continuously. Transcranial direct current stimulation (tdcs) tdcs was delivered by a battery-driven constant-current stimulator (NeuroConn GmbH, Ilmenau, Germany) through conductive-rubber

3 1294 K. Boros et al. electrodes, placed in two saline-soaked sponges. For the left PM cortex stimulation the size of the active electrode was 3 11 cm. We used this specific electrode to stimulate the premotor cortex, but to avoid covering of adjacent areas by the tdcs electrode. The conventional 5 7 cm sponge was used for the left DLPFC polarization. In both cases, the reference electrode (5 7cm) was placed above the contralateral orbit. Left PM was defined as being 2.5 cm anterior to the left M1 motor area (Fink et al., 1997; Picard & Strick, 2001; Rizzo et al., 2004). The middle of the active electrode was placed 2.5 cm anterior to the motor hot spot of the ADM. The electrode was positioned with its long axis approximately parallel to the sulcus centralis. To stimulate the DLPFC, the active electrode was placed over F3 according to the international system for EEG electrode placement (Gerloff et al., 1997; Fregni et al., 2005; Iyer et al., 2005). This has been confirmed as a relatively accurate method for localizing the DLPFC by neuronavigation techniques (Herwig et al., 2003). The electrodes were fixed by elastic bands. Anodal tdcs was performed for 13 min and cathodal tdcs for 9 min, because these tdcs conditions had resulted in excitability changes in previous experiments stable for about 1 h (Nitsche & Paulus, 2000, 2001; Nitsche et al., 2003a). tdcs was applied with a current strength of 1 ma. The current was always ramped up or down over the first and last 8 s of stimulation. All of the subjects felt a mild local tingling sensation under the electrodes by both polarities. Subjects were blinded for tdcs conditions. In the control experiment a reduced active (3.5 cm 2 ) electrode size was used (Nitsche et al., 2007), and the size of the reference electrode was 5 7 cm (35 cm 2 ). The small anode was placed over the M1 representation of the right ADM, whilst the cathode was positioned over the contralateral orbit. The duration of anodal stimulation was 13 min, and current strength was reduced according to the small electrode size to 0.1 ma to keep current density constant, as described in a former experiment (Nitsche et al., 2007). Data analysis Peak-to-peak amplitudes (mv) of each MEP were measured off-line, and mean MEP amplitudes were calculated for each stimulation condition and for each time bin covering the recordings before and after tdcs. For the SICI ICF paradigm, means of conditioned MEP amplitudes were normalized by calculating the quotient of the conditioned MEP unconditioned MEP separately for each time-point of measure. For each measure (RMT, AMT, single test-pulse MEP, I O curve, SICI ICF) and for each stimulation site (PM and DLPFC tdcs), we performed separate repeated-measure analyses of variance (anova). Mean MEP amplitudes or TMS intensities from each subject served as the dependent variable. For the motor thresholds and single test-pulse MEP, DC INTERVENTION (anodal vs. cathodal tdcs) and TIME (before, post1, post2, post3) served as an independent variable. For the I O curves, INTENSITY, and for SICI ICF, ISI served as additional independent variables. In the control experiment repeatedmeasures anovas were calculated separately for each parameter (single test-pulse MEP and SICI ICF). Mean MEP amplitudes from each subject served as the dependent variable whilst the MUSCLE (ADM or FDI), TIME (before, post1, post2) and ISI (SICI ICF only) served as an independent variable. The Greenhouse Geisser correction was used when necessary to correct for non-sphericity. Fisher s post-hoc tests (level of significance 0.05) were calculated to compare the time course of MEP amplitudes and TMS intensities and to compare the effect of tdcs polarity for a given time-point in the case of significant results in the anova. Furthermore the same post-hoc tests were used to exclude baseline differences between the different stimulation conditions. Results None of the subjects reported any adverse effects during and after conduction of the study with the exception of a slight tingling sensation under the electrodes. Premotor tdcs The two-way anovas conducted for RMT and AMT showed no significant main effect for DC INTERVENTION and TIME, nor for the interaction between both factors (Table 1, Fig. 1). Similarly, the repeated-measures anova calculated for the single-test pulse amplitude resulted in no significant main effects or interaction (Table 1, Fig. 1). For the I O curve, the anova shows significant main effects of INTENSITY (Table 1, Fig. 2). With regard to SICI ICF, the repeated-measure anova displays significant main effects of ISI and DC intervention (Table 1). According to the post-hoc Fisher s LSD test, this is due to significantly reduced inhibition at ISIs of 2 and 3 ms and enhanced facilitation at an ISI of 7, 10 and 15 ms after anodal tdcs for up to 30 min after intervention. There was a trend for enhanced MEP amplitudes at an Table 1. Results of the anovas for premotor transcranial direct current stimulation (tdcs) d.f. F-value P-value Resting motor threshold (RMT) DC INTERVENTION 1, TIME 3, DC INTERVENTION TIME 3, Active motor threshold (AMT) DC INTERVENTION 1, TIME 3, DC INTERVENTION TIME 3, Single test-pulse TMS DC INTERVENTION 1, TIME 3, DC INTERVENTION TIME 3, I O curve DC INTERVENTION 1, TIME 3, GG INTENSITY 3, < 0.001* DC INTERVENTION TIME 3, GG DC INTERVENTION INTENSITY 3, GG TIME INTENSITY 9, GG DC INTERVENTION TIME INTENSITY 9, GG Short intracortical inhibition intracortical facilitation (SICI-ICF) DC INTERVENTION 1, * TIME 3, GG ISI 5, < 0.001*,GG DC INTERVENTION TIME 3, GG DC INTERVENTION ISI 5, GG TIME ISI 15, GG DC INTERVENTION TIME ISI 15, Two-way repeated-measure anovas were used to analyse RMT, AMT and single test-pulse TMS. Three-way repeated-measures anovas were calculated for I O curve and SICI ICF. *P <0.05; GG The adjusted P-values after Greenhouse Geisser correction; d.f., degrees of freedom.

4 tdcs-driven premotor motor cortex interactions 1295 Fig. 1. Motor thresholds and single test-pulse MEPs. RMT, AMT and single test-pulse MEP were recorded with TMS before tdcs and three times after the stimulation, i.e. approximately 0 min (post 1), 30 min (post 2) and 60 min after the end of tdcs (post 3). Anodal or cathodal tdcs were applied over the left premotor cortex (A and C) and over the left DLPFC (B and D). Thresholds are given in percentage of stimulator intensity (A and B) and the single test-pulse MEPs in peak-to-peak amplitude (C and D). DLPFC and premotor cortex tdcs resulted in no significant changes of RMT, AMT or single test-pulse MEP. Error bars are SEM. ISI of 5 ms following anodal tdcs. However, this change was not significant (Fig. 3). Because single-pulse MEP amplitude and motor thresholds were not affected by tdcs, TMS intensity did not need to be adjusted. The post-hoc test showed no significant differences between anodal and cathodal conditions for the respective baseline values for all parameters (P > 0.05). However, at an ISI of 7 ms baseline MEP amplitudes looked somewhat smaller in the anodal tdcs condition than in the remaining conditions, although the difference was not significant. To test the robustness of the results for this ISI, we excluded the four subjects with the lowest baseline values and re-calculated the results. The anova showed almost the same significance level as with 16 subjects (STIM P ¼ 0.013, F 1,11 ¼ 8.646; ISI P ¼ 0.001, F 5,55 ¼ 9579). The post-hoc test resulted in significant differences in the anodal PM tdcs condition for ISI of 7 ms before vs. post1 (P ¼ 0.04) and before vs. post2 (P ¼ 0.019). The mean values for ISI of 7 ms were ± for baseline, ± for post1, ± for post2 and ± for post3. DLPFC tdcs The anovas performed for RMT, AMT and single test-pulse MEP did not result in significant main effects or interactions (Table 2, Fig. 1). For the I O curve, the main effect of INTENSITY was significant due to larger MEPs caused by higher TMS intensities, but which were not affected by tdcs (Table 2, Fig. 2). With regard to SICI ICF, only the main effect ISI was significant in the respective anova, caused by the specific influence of the respective ISIs on MEP amplitudes (Table 2, Fig. 3).

5 1296 K. Boros et al. Fig. 2. Input output curve before and after premotor and DLPFC tdcs. The I O curve was recorded with TMS before tdcs and three times after the stimulation, i.e. approximately 0 min (post 1), 30 min (post 2) and 60 min after the end of DC stimulation (post 3). The mean MEP amplitudes are presented at 100, 110, 130 and 150% stimulator intensity of RMT. Anodal tdcs was applied for 13 min over the PM and DLPFC (A and B) and cathodal DC for 9 min (C and D). Error bars are SEM. Again, the post-hoc test showed no significant differences between anodal and cathodal conditions for the respective baseline values for all parameters (P > 0.05). Control experiment The repeated-measures anova calculated for the single test-pulse amplitude resulted in a significant main effect of TIME (Table 3, Fig. 4). Anodal tdcs of the ADM selectively enhanced the MEP amplitude of the ADM significantly, but not of the FDI (Fisher s LSD, P < 0.01). With regard to SICI ICF, only the main effect of ISI was significant in the respective anova, caused by the specific influence of the ISIs on MEP amplitudes (Table 3, Fig. 4). The post-hoc test showed no significant differences between ADM and FDI measurements for the respective baseline values for all parameters (P > 0.05). Discussion The present results show that PM tdcs selectively influences intracortical excitability of the ipsilateral M1, supporting the concept that these cortical areas are densely interconnected. In a broader context they also substantiate that tdcs produces specific, connectively driven changes in cortical areas at a distance of the stimulation site. In our experiments, PM tdcs had a specific influence on intracortical inhibition and facilitation. Anodal tdcs significantly reduced intracortical inhibition at ISIs of 2 and 3 ms, enhanced intracortical facilitation at ISIs of 10 and 15 ms and the MEP amplitude of ISI of 7 ms. For ISI of 7 ms, the results might, however, be exaggerated by the relative noisy baseline measures. Interestingly, cathodal tdcs of the PM did not change intracortial excitability. This effect on intracortical excitability is similar to the results of a former experiment, in which we stimulated the primary motor cortex (Nitsche et al., 2005), but seems to be more specific, as it affects not all ISIs

6 tdcs-driven premotor motor cortex interactions 1297 Fig. 3. Intracortical inhibition and facilitation. The single-pulse standardized double-stimulation MEP amplitudes are illustrated for each ISI. SICI ICF was recorded with TMS before tdcs and three times after the stimulation, i.e. approximately 0 min after tdcs (post 1), 30 min after tdcs (post 2) and 60 min after the end of DC stimulation (post 3). Anodal tdcs (A and B) or cathodal tdcs (C and D) were applied to the left premotor cortex and over the left DLPFC. Selectively anodal tdcs significantly increased the MEP amplitude at an ISI of 2, 3, 7, 10 and 15 ms immediately after (post 1) and 30 min after tdcs (post 2) compared with the baseline. * and # indicate significant differences between the baseline and post 1 measure for a specific ISI (*P < 0.05; # P < 0.001); and between the baseline and post 2 condition ( P < 0.05; P < 0.001). Error bars represent SEM. modulated in the former experiments and here only anodal tdcs was effective. This might be caused by a more selective influence of M1 inhibitory and facilitatory systems by an indirect, connectivity-driven stimulation via premotor motor connections in the present study. However, when compared with results from rtms studies, tdcs influenced a broader range of ISIs. As with previous rtms studies (Münchau et al., 2002; Bäumer et al., 2003; Rizzo et al., 2004), an ISI of 7 ms was affected by anodal tdcs. However, the excitability curve was modulated also at shorter and longer ISIs, i.e. physiological inhibition was reduced and facilitation was increased, in our experiment. This might be caused by a less focal stimulation of the larger tdcs electrode as compared with the rtms studies, which might have covered not only the dorsal, but also parts of the ventral premotor cortex. With regard to the time course, anodal PM tdcs induced M1 excitability changes for 30 min, which is in the range of the efficacy of M1 stimulation in former studies (Nitsche & Paulus, 2001; Nitsche et al., 2003a). In contrast to anodal PM tdcs, cathodal tdcs did not alter M1 excitability. This might have a biological basis, i.e. PM M1 connections are not sensitive to tdcs, or could have methodological reasons. In particular, because experiments were carried out at rest, PM M1 connections were probably inactivated to an extent that further inhibition was not possible. Conditioning pulse intensities in the paired-pulse paradigm were relatively low (70% AMT), which might have resulted in the missing inhibitory effect at an ISI of 5 ms in our experiments (Nitsche et al., 2005). This should be explored in future studies using different conditioning pulse intensities to determine thresholds for inhibition and facilitation (Orth et al., 2003). In the current study, we used stimulators delivering biphasic pulses instead of the conventionally applied monophasic ones. Although the effects

7 1298 K. Boros et al. Table 2. Results of the anovas for DLPFC tdcs d.f. F-value P-value Resting motor threshold (RMT) DC INTERVENTION 1, TIME 3, DC INTERVENTION TIME 3, Active motor threshold (AMT) DC INTERVENTION 1, TIME 3, DC INTERVENTION TIME 3, Single test-pulse TMS DC INTERVENTION 1, TIME 3, DC INTERVENTION TIME 3, I O curve DC INTERVENTION 1, TIME 3, GG INTENSITY 3, < 0.001*,GG DC INTERVENTION TIME 3, GG DC INTERVENTION INTENSITY 3, GG TIME INTENSITY 9, GG DC INTERVENTION TIME INTENSITY 9, GG Short intracortical inhibition intracortical facilitation (SICI-ICF) DC INTERVENTION 1, TIME 3, ISI 5, < 0.001* DC INTERVENTION TIME 3, DC INTERVENTION ISI 5, TIME ISI 15, DC INTERVENTION TIME ISI 15, Two-way repeated-measure anovas were used to analyse RMT, AMT and single test-pulse TMS. Three-way repeated-measures anovas were calculated for I O curve and SICI ICF. *P <0.05; GG The adjusted P-values after Greenhouse Geisser correction; d.f., degrees of freedom. Table 3. Results of the anovas for M1 (ADM) tdcs d.f. F-value P-value Single test-pulse TMS MUSCLE 1, TIME 2, *,GG MUSCLE TIME 2, GG Short intracortical inhibition intracortical facilitation (SICI-ICF) MUSCLE 1, TIME 2, GG ISI 5, *,GG MUSCLE TIME 2, GG MUSCLE ISI 5, GG TIME ISI 10, GG MUSCLE TIME ISI 10, Two-way repeated-measure anova was used to analyse single test-pulse TMS. Three-way repeated-measure anova was calculated for SICI ICF. *P <0.05; GG The adjusted P-values after Greenhouse Geisser correction; d.f., degrees of freedom. of monophasic and biphasic stimulation on SICI ICF have not been compared in any single study directly thus far, comparing the actual results with those of a former study, where monophasic Magstim 200 stimulators were used (Nitsche et al., 2005), shows that these deliver comparable SICI ICF curves. Thus, it is unlikely that the different results of the current study, when compared with the foregoing rtms experiments, are caused by this factor. Fig. 4. Single-pulse MEPs and intracortical inhibition and facilitation after M1 (ADM) tdcs. The single-pulse MEPs (A) and standardized doublestimulation MEP amplitudes (B and C) are illustrated for both muscles (ADM, FDI). Single-pulse MEPs and SICI ICF were recorded with TMS before tdcs and twice after the stimulation, i.e. approximately 0 min (post 1) and 30 min (post 2) after the end of tdcs. Anodal tdcs was applied to the left motor cortex over the representation of the ADM. Anodal tdcs significantly increased the single-pulse MEP amplitude of the ADM immediately after (post 1) stimulation compared with baseline. SICI ICFs were not affected by tdcs. *P <0.01, comparing the baseline and post 1 measure.

8 tdcs-driven premotor motor cortex interactions 1299 In clear difference to tdcs of M1, single-pulse MEP amplitudes and the I O curve, as well as RMT and AMT were not specifically modulated by PM tdcs. To rule out the possibility that the effects of premotor tdcs on M1 excitability were not connectively driven, but caused by a spread of current flow from PM to the adjacent M1, we conducted a control experiment. Here we stimulated the primary motor cortical hot spot of the ADM with tdcs using a small tdcs electrode (Nitsche et al., 2007) and monitored single-pulse MEP amplitude as well as intracortical inhibition and facilitation for the cortical representations of the ADM and FDI. The latter was not covered by the small tdcs electrode. Interestingly, the results show a selective enhancement of the single-pulse MEP amplitude after anodal tdcs of the ADM, while the single-pulse MEP amplitude of the FDI was not influenced. Singlepulse MEP amplitude is a measure of cortico-spinal excitability and might resemble excitability of intrinsic primary motor cortical neurons. Intracortical inhibition and facilitation, which resemble cortico-cortical excitability, and thus depend on stimulation of motor cortical afferents, were not influenced by selective tdcs of the hot spot of the ADM. This pattern of results clearly differs from the excitability modification induced by premotor tdcs. For the singlepulse MEP amplitude, these results are not compatible with a relevant spread of current flow beyond the cortical area covered by the electrode, i.e. from the small electrode covering the ADM to the FDI representation. This result (i) is thus in accordance with our former study (Nitsche et al., 2007) and (ii) makes it improbable that the premotor tdcs electrode, which was used in the main experiment, and has a larger distance to M1, had a relevant influence on M1 excitability caused by current spread. Interestingly, intracortical inhibition and facilitation were also not modified in this control experiment by the small tdcs electrode placed over M1, in contrast to the main experiments. In this connection, it is important to note that in another study, tdcs performed with a large electrode (35 cm 2 ) over M1 modified intracortical excitability and single-pulse MEP amplitude (Nitsche et al., 2005). The most plausible explanation for this dissociation of results is that tdcs with the large M1 electrode affected premotor areas and or afferents (cortico-cortical excitability) and M1 neurons (cortico-spinal excitability), whereas the much more restricted small M1 electrode was not sufficiently sized to influence the afferents. Accordingly, PM tdcs would have selectively affected the M1 afferents also covered by the large M1 electrode in a former study (Nitsche et al., 2005). This argument explains on the one hand the effect of the large M1 electrode on single-pulse MEP amplitudes and intracortical excitability, and on the other hand the selective effects of the small M1 electrode on single-pulse MEP amplitude and the premotor tdcs electrode on intracortical excitability. All in all, these results are not in accordance with a spreading effect of premotor stimulation to M1, but with a specific, connectively driven influence of premotor stimulation on M1 excitability. In other words, premotor tdcs may modulate premotor motor cortex synaptic connections, without overtly influencing the excitability of the primary motor cortical target neuron. The fact that tdcs over the DLPFC did not alter M1 excitability lends further support to a connectivity-driven effect of PM tdcs on M1 excitability, because the M1 is not directly connected with the DLPFC. Moreover, it argues against a nonspecific, e.g. arousal-driven, effect of tdcs on excitability. However, the results of our study cannot definitely exclude an effect of premotor tdcs on M1 excitability caused by a spread of current. Taken together, the results of our study suggest that anodal tdcs of a given cortical area has specific effects on the excitability of interconnected cerebral regions, which differ from effects of direct stimulation of these regions. tdcs might thus be used to explore the functional connectivity of the human brain. Moreover, it might be an interesting tool to modulate pathologically altered connectivity in diseases. Acknowledgements This study was supported by the Walter und Ilse Rose-Stiftung. We would like to thank Leila Chaieb for suggested corrections to the English text. 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