EEG study

Brain Research Bulletin 69 (2006) 86–94

A direct demonstration of cortical LTP in humans: A combined TMS/EEG study S.K. Esser, R. Huber, M. Massimini, M.J. Peterson, F. Ferrarelli, G. Tononi ∗ Department of Psychiatry, University of Wisconsin, 6001 Research Park Boulevard, Madison, WI 53719, USA Received 20 September 2005; received in revised form 31 October 2005; accepted 3 November 2005 Available online 1 December 2005

Abstract Repetitive transcranial magnetic stimulation (rTMS) is increasingly being used to promote cortical reorganization, under the assumption that it can induce long-term potentiation (LTP) of neural responses. This assumption is supported by several lines of indirect evidence. For example, rTMS of motor cortex can induce a potentiation of muscle motor evoked potentials that outlasts the stimulation by several minutes. In animal models, a direct demonstration of LTP is typically obtained by high-frequency electrical stimulation coupled with local field recordings of population responses. In this study, we exploited a new approach based on combined rTMS/high-density electroencephalography (hd-EEG) to obtain direct, noninvasive evidence for LTP in humans. Cortical responses to single TMS pulses were measured with hd-EEG before and after applying rTMS to motor cortex (5 Hz, 1500 pulses). The results demonstrate that, after rTMS, EEG responses at latencies of 15–55 ms were significantly potentiated. A topographic analysis revealed that this potentiation was significant at EEG electrodes located bilaterally over premotor cortex. Thus, these findings provide a direct demonstration in humans of LTP induced by rTMS. © 2005 Elsevier Inc. All rights reserved. Keywords: Motor cortex; Premotor cortex; Plasticity; rTMS; 5 Hz; Evoked potential

1. Introduction The delivery of repetitive transcranial magnetic stimulation (rTMS) has been introduced as a tool for treating a number of neuropsychiatric disorders, including depression, schizophrenia and anxiety disorders [23]. The efficacy of such treatment has varied from case to case, suggesting that a refinement of techniques in use is necessary to develop the full clinical potential of rTMS. To achieve this refinement in a principled fashion, it is essential to understand how rTMS affects the cerebral cortex. The clinical use of rTMS is predicated on the assumption that rTMS can achieve a reorganization of cortical circuitry. A growing body of evidence supports this assumption, including a number of studies demonstrating that the amplitude of motor evoked potentials (MEPs) produced by TMS delivered to motor cortex are increased following rTMS. Specifically, it has been shown that the delivery of 5 Hz rTMS to motor cortex can lead

∗

Corresponding author. Tel.: +1 608 263 6063; fax: +1 608 263 0265. E-mail address: [email protected] (G. Tononi).

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to an increase in MEP amplitude. This increase becomes significant after the delivery of 900 pulses [31] and lasts for at least 30 min following delivery of 1800 pulses of rTMS [30]. Conversely, the application of low-frequency rTMS (1 Hz) can reduce MEP amplitude [14,24], with effects lasting almost 1 h following delivery of 1500 pulses [35]. Some studies suggest that these effects are the result of changes in the cerebral cortex. For example, TMS paired-pulse paradigms designed to measure short-term inhibition and facilitation at the cortical level show altered responses following rTMS [7,31]. In addition, Di Lazzaro et al. have used recordings from the cervical epidural space to show that descending motor cortical activity evoked by TMS is increased after 20 pulses of 5 Hz rTMS, with an effect lasting for at least 2 min [6]. Imaging studies have also examined changes induced by rTMS. Using PET, it has been demonstrated that high-frequency rTMS delivered to left motor cortex results in increased levels of resting regional cerebral blood flow and glucose metabolism in bilateral motor cortex and supplementary motor area [32,33]. It has also been shown that targeting the hand area of motor cortex with 1 Hz stimulation results in reduced coupling between premotor and motor cortex [21].

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Collectively, these studies demonstrate that rTMS can produce a cortical reorganization in humans. However, technical reasons have previously prevented a direct demonstration of long-term potentiation (LTP) or long-term depression (LTD) induction. In animals, direct demonstration of LTP was provided by Bliss and Lomo [3] in a classic set of experiments by electrically stimulating hippocampal fiber tracts at frequencies ranging from 5 to 15 Hz. They assessed LTP as changes in the amplitude of the population response to electrical stimulation, recorded using extracellular electrodes. The recent combination of TMS and EEG provides a means of approximating this protocol. TMS can be substituted for electrical stimulation to allow for the safe and noninvasive activation of the human brain, while surface potentials recorded using EEG can be used in place of extracellular population recordings to provide a direct means of assessing cortical responses to stimulation. Using these techniques, we have for the first time directly measured cortical responses to TMS before and after high frequency (5 Hz) rTMS. We present these results here, which provide evidence of cortical LTP induction in humans. 2. Materials and methods The basic experimental design we employed consisted of recording EEG responses to TMS in test phases before and after the delivery of real and sham rTMS, as depicted in Fig. 1A and B. Seven right-handed male subjects (mean age 26, range 19–35) participated in the study. All participants gave written informed consent prior to study procedures. The experiment was approved by the University of Wisconsin Human Subjects Committee, and was in compliance with national legislation and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (Declaration of Helsinki). Prior to the experiment a neurological screening was performed to exclude subjects with conditions that could predispose them to potential adverse effects of TMS.

2.1. TMS delivery 2.1.1. Targeting TMS TMS was targeted to the hand area of motor cortex throughout the experiment (Fig. 1A). In order to ensure precision and reproducibility of stimulation, we employed a navigated brain stimulation (NBS) system (Nexstim Ltd., Helsinki FI). The NBS device locates (with an error <4 mm) the relative positions of the subject’s head and the TMS coil by means of an optical tracking system. For targeting purposes, the NBS calculates the distribution of the electric field on the cortical surface using a 3D reconstruction of the subject’s brain. The brain reconstruction was created prior to the experiment from a T1 weighted MR image (resolution .5 mm) of the subject’s head acquired with a 3T GE Signa scanner. Using this system, the coil target as well as the position, direction and angle of the stimulator were monitored in real-time throughout the session to ensure targeting consistency. This device also allowed us to digitize the location of the EEG electrodes in each subject. The coil was initially positioned over motor cortex according to anatomical landmarks and then moved in .5 cm increments over this area while stimulating. Motor evoked potentials were recorded from the first dorsal interosseous muscle during stimulation. The location where the largest response was evoked was chosen for use in the experiment. 2.1.2. Stimulation parameters A Magstim standard rapid rate stimulator with an air-cooled figure of eight coil (outer diameter 10 cm) was used for stimulation. EEG responses to TMS were assessed in test phases before and after conditioning. Stimulus intensity was set relative to resting motor threshold (RMT), which was identified in the relaxed first dorsal interosseous using a maximum-likelihood threshold hunting

Fig. 1. (A) The experimental setup: recordings were made from scalp EEG and EMG from the first dorsal interosseous muscle of the right hand. In the real rTMS experiment, stimulation was delivered to left motor cortex, while in the sham experiment, stimulation was delivered with the coil rotated 90◦ and separated from the scalp by a spacer cube. (B) Graphical depiction of the experimental protocol. Two hundred TMS pulses were delivered in test phases before and after a conditioning phase of 1500 pulses. In the conditioning phase, rTMS pulses were delivered at a frequency of 5 Hz with pauses interspersed according to safety guidelines. (C) Evoked potentials obtained with and without noise masking are compared while the coil, connected to the scalp by a plastic cube (to preserve bone conduction), was placed above the vertex (Cz). The auditory N100-P200 component was abolished by noise masking.

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procedure [1]. The test phases consisted of 200 TMS pulses delivered, one every .5–.7 s at 90% RMT. The conditioning phase consisted of 1500 TMS pulses delivered at 90% RMT. rTMS pulses were delivered at a base frequency of 5 Hz with pauses in stimulation determined according to safety guidelines [37]. Specifically, pulses were organized into bursts of 50 pulses delivered at 5 Hz. Bursts were organized into trains of six bursts, with each separated by 5 s. A total of five trains were delivered, each separated by 1 min (Fig. 1B). While motor evoked responses were not the main focus of this study, we assessed changes in MEP amplitude in a few subjects in a MEP test phase before the pre rTMS test phase and after the post rTMS test phase (that is, at the very beginning and very end of the experiment). MEPs were generated by delivering 20 TMS pulses, one every 10 s at 130% RMT.

reported after the experiment that he could not hear the tone. In the remaining six subjects, response latency was unchanged (pre .46 s, post .54 s, p > .05, two-tailed paired t-test).

2.5. Analysis of data The EEG responses to TMS were analyzed first by rejecting noisy channels and epochs (−100 to 300 ms) containing eye movements or other artifacts. TMS stimulation can produce baseline shifts in the EEG response that take several milliseconds to recover. If present, data from these sections were not included in the analysis, as noted in the Results (the shifts lasted at most 15 ms). The data was then average referenced, baseline corrected (100 ms prestimulus), band pass filtered (5–100 Hz) and averaged for each subject.

2.2. Recording responses to TMS We recorded the EEG responses to TMS by means of a cap with 60 carbon electrodes and a specifically designed TMS-compatible amplifier (Nexstim Ltd., Helsinki, FI). The artifact induced by TMS was gated and saturation of the amplifier was avoided by means of a proprietary sample-and-hold circuit that kept the analog output of the amplifier constant from 100 ms pre- to 2 ms poststimulus [36]. To help produce a high signal-to-noise ratio, the impedance at all electrodes was kept below 3 K
. The EEG signals, referenced to an additional electrode on the forehead, were filtered (.1–500 Hz) and sampled at 1450 Hz with 16 bit resolution. To record MEPs, two extra sensors were used to record the electromyogram from the first dorsal interosseous muscle.

2.3. Control experiment Subjects also underwent a control experiment separated by at least 1 week from the potentiation experiment. The control experiment used the methods described above except that sham rTMS was delivered in the conditioning phase. For sham rTMS, the coil was rotated 90◦ about the axis of the handle and separated from the head using a 2 cm plastic spacer cube (Fig. 1A). This ensured that brain stimulation would not occur, but that the subject still felt the vibrations produced by the click of the TMS coil.

2.4. Reduction of confounding factors In addition to the magnetic artifact, the evoked response to TMS is affected by sensory and attentional factors that can confound the interpretation of EEG data. 2.4.1. Auditory stimulation The click associated with the coil’s discharge propagates through air and bone and can elicit an auditory N1–P2 complex at latencies of 100–200 ms [28]. In order to prevent contamination of TMS-evoked potentials, a procedure was adopted to completely eliminate the subject’s perception of the coil’s click. The waveform of the TMS click was digitized and processed to produce a continuous audio signal that captures its specific time-varying frequencies. Prior to the experiment, we delivered regular TMS test pulses to the subjects while the masking noise was played through inserted earplugs. At this time we adjusted the masking volume (always below 90 dB) until the subjects reported that the TMS click was not perceptible. Bone conduction was attenuated by placing a thin layer of foam between the coil and the EEG cap. In a separate experiment, we were able to demonstrate that this procedure eliminated the auditory evoked potential produced by the TMS click (Fig. 1C). 2.4.2. Attentional effects A subject’s attentional state can modulate the amplitude and latency of evoked potentials [16]. Therefore, the subject’s attentional load was controlled with a signal detection task during the experiment. Brief tones were periodically played through the subject’s headphones. These tones were rare compared to the number of TMS pulses delivered (10 tones during each of the response test phases and 20 tones during the conditioning phase). The subject was instructed to respond to these tones with a button press using their nonstimulated hand and their response latency was measured. Data is omitted from one subject who

2.5.1. Global mean field power analysis Total EEG activity was assessed using the global mean field power (GMFP), calculated as:




GMFP(t) =

k i

(Vi (t) − Vmean (t))2 K

 ,

where t is time, K the number of channels, Vi the voltage in channel i averaged across subjects and Vmean is the mean of the voltages in all channels [22]. In each subject, the amplitudes of the first five peaks in the GMFP calculated in the two test phases were identified for further analysis. ANOVA for repeated measures was performed on the peaks observed in the GMFP with the factors ‘condition’ (sham or real rTMS), ‘phase’ (pre or post rTMS) and ‘peak’ (peak number). Data from the first GMFP peak was omitted from this analysis due to the small number of available samples. Where significant interactions were found, peaks were compared by post hoc two-tailed paired t-tests. 2.5.2. Topographic analysis of activation To plot the data topographically, in each subject values in channels that were rejected were replaced using nearest neighbor interpolation. The data was then spatially normalized for possible differences in EEG cap positioning. Using data recorded from the NBS system, the location of each electrode relative to the position of the maximum induced electric field (located in motor cortex in each subject) was determined. The electrode grid was then realigned so that in all subjects the electrodes were in the same position relative to the maximum induced electric field. This resulted in a left–right or anterior–posterior shift of at most one electrode in each subject. To remove potential magnetic artifacts, the data was zero padded over the range 0–15 ms. Peaks in the grand average of all subjects were identified as local maxima or minima that exceeded three times the standard deviation of the prestimulation activity. Corresponding peaks in individual subjects were chosen as the maximum or minimum value occurring within 10 ms of the grand average peak. The peak amplitudes from individual subjects were then compared between the pre and post rTMS test phases using two-tailed paired t-tests. The total activation produced by TMS across the scalp was determined by rectifying the signal recorded in each channel and calculating its average over the time range 20–80 ms (the artifact free time period during which the significantly changed peaks in the GMFP occurred in all subjects). Statistical nonparametric mapping with the suprathreshold cluster analysis for multiple comparisons described in Nichols and Holmes [27] was used to compare the values in each channel in the pre and post rTMS test phases across subjects. 2.5.3. Source localization To produce a specific localization of where the TMS evoked activity was occurring, the averaged responses, the MRI sets and the electrode coordinates were input to the software package Curry 5.0. Analysis was performed as in Massimini et al. [26]. Briefly, a realistic Boundary Element Model of each subject’s head was calculated from individual MRIs. Current density on the cortical surface was then estimated using the minimum norm least squares (MNLS) method. A detailed description of the assumptions and methods used for MRI processing and source reconstruction can be found elsewhere [4,11,12]; see also Curry 5.0 User guide).

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Fig. 2. Responses to TMS before and after 5 Hz conditioning in a single subject: (A) Average of 20 MEPs produced by stimulation at 130% RMT. MEPs were significantly increased in amplitude following rTMS, as indicated by the asterisk. (B) Responses in EEG channels across the scalp to stimulation at 90% RMT. The green ‘×’ indicates the location of the coil target. (C) Activity in six channels located bilaterally in front of the site of stimulation. (D) Total activation produced by TMS as measured by the GMFP. The first five peaks in the response are labeled as P1–P5. (E) Source localization of the activity occurring during each peak in the GMFP. The top 20% of current produced is shown. The green ‘×’ indicates the location of the coil target. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

3. Results In our initial analysis we looked at a single subject’s responses to TMS before and after conditioning. Fig. 2A shows that motor responses to TMS (measured as the amplitude of MEPs) were significantly increased following 5 Hz rTMS (p < .05, twotailed paired t-test, n = 20 trials). Cortical responses to TMS are depicted in Fig. 2B and C. These figures show that TMS pro-

duced large deflections in scalp voltage primarily near the site of stimulation and to a lesser extent at distant sites. This activity lasted throughout the duration we consider here (the first 100 ms following TMS). A visual comparison of the pre and post rTMS responses suggests that certain peaks of the EEG deflections were increased following 5 Hz rTMS. Next, we determined the time course of the total EEG response to TMS by calculating the GMFP. Fig. 2D shows

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Table 1 Talairach coordinates and functional areas of maximum activation in the source localization calculated at each peak of pre and post rTMS GMFP in one subject Phase

Peak

X

Y

Z

Area

Pre

1 2 3

48 42 50

−17 −1 −9

58 57 54

4 5

44 28

−28 −22

62 69

Left sensorimotor cortex Left premotor cortex Left sensorimotor/premotor cortex border Left sensorimotor cortex Left sensorimotor cortex

1 2 3 4 5

55 34 57 34 26

−10 −5 −5 −5 −2

43 65 46 65 68

Left sensorimotor cortex Left premotor cortex Left premotor cortex Left premotor cortex Left premotor cortex

Post

that the GMFP contained distinct peaks, which we sequentially labeled P1–P5. These peaks had similar latencies when compared between the pre and post rTMS test phases. The amplitudes of P2–P5 were increased to various degrees following rTMS in this subject. To determine the brain areas most strongly activated during each of these peaks, we performed source localization at the time of each peak. The strongest activation was initially produced in sensorimotor cortex, and during later peaks was generated in either sensorimotor cortex or premotor cortex (Table 1).

We next used several of these measures to examine how cortical responsiveness changed following rTMS in all seven of the subjects we examined. To provide an initial impression of the data in all subjects, we first examined the grand average (Fig. 3). It was clear that the EEG response to TMS included several large deflections in channels near the site of the stimulation with less activity occurring further away. Furthermore, we found that five electrodes contained deflections that were significantly increased in amplitude following rTMS (p < .05, uncorrected t-test). These increases were located in electrodes located bilaterally in front of the coil target. To determine the timing of the EEG components potentiated by rTMS, we measured the first five peaks in each subject’s GMFP before and after sham rTMS and real rTMS (Fig. 4). Magnetic artifacts prevented earlier peaks from being measured in certain subjects (the number of subjects used was 2, 6, 7, 7 and 7 for P1–P5, respectively, in the sham experiment and 3, 5, 7, 7 and 7 for P1–P5 in the real rTMS experiment). There was no significant difference between peak latencies when compared across conditions (ANOVA for repeated measures). The peaks occurred with latencies of 5 ± 0, 18 ± 1, 35 ± 3, 55 ± 4 and 84 ± 6 ms (mean ± standard error). A comparison of peak amplitudes revealed a significant interaction of the factors ‘condition’ and ‘phase’ (F(1,4) = 39.87, p < .01, ANOVA for repeated measures). Post hoc t-tests revealed that the amplitudes of P2,

Fig. 3. Grand average of seven subjects responses to TMS before and after 5 Hz rTMS, zero padded over 0–15 ms. The data in each subject was adjusted prior to calculating the grand average to account for differences in net location (see Section 2). The green ‘×’ indicates the location of the coil target. The circles containing the colored traces indicate peaks where a significant difference exists between pre and post rTMS values (uncorrected two-tailed paired t-test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 4. Amplitude and standard error bars of the first five peaks observed in the GMFP, labeled P1–P5, for the pre and post rTMS test phases in the sham and real rTMS experiments. Asterisks indicate peaks showing a significant increase following rTMS (p < .05). Table 2 Location of electrodes belonging to significant cluster of channels X

Y

Z

Brodmann area

−35 −2 30 22 40

−1 −2 −1 24 1

62 70 66 61 57

Left premotor cortex Left premotor cortex Right premotor cortex Right premotor cortex Right premotor cortex

P3 and P4 were significantly increased following real (p < .05), but not after sham rTMS. Topographically, total activity in the time range 20–80 ms (see Section 2) was evoked most strongly in electrodes over and just frontal to the site of stimulation (Fig. 5). Using a nonparametric analysis, we found that the TMS evoked activity was significantly increased in a cluster of five electrodes located just frontal to the site of stimulation (p < .05, statistical nonparametric mapping, suprathreshold cluster test controlling for multiple comparisons). The electrodes in this cluster were located over left and right premotor cortex (Table 2). 4. Discussion The research presented here demonstrates that the EEG response to TMS pulses delivered to motor cortex is increased in amplitude following rTMS. The increase in response was observed in electrodes located bilaterally over premotor cortex. As noted above, studies measuring MEP amplitude have previously provided indirect evidence that 5 Hz rTMS produces lasting effects in cortex [30,31]. Other research has found increases in regional cerebral blood flow and glucose metabolism in resting motor areas following high-frequency rTMS [32,33]. In addition, a limited number of studies have provided direct demonstrations of potentiation in humans. Wolters et al. found that pairing electrical stimulation of the median nerve with TMS delivered at specific latencies to the postcentral gyrus results in an increase of the EEG response to median nerve stimu-

Fig. 5. A topographic depiction of the voltage in each channel rectified and averaged over the time range 20–80 ms, averaged for all subjects. The data in each subject was adjusted prior to this calculation to account for differences in net location (see Section 2). The circle indicates the area the coil was targeted to in all seven subjects, following this adjustment. Top: data is depicted for responses in the pre and post rTMS test phases. Bottom: a depiction of the post rTMS–pre rTMS data, masked to show only significant clusters.

lation alone [38]. Other work has shown that the high-frequency presentation of a visual or auditory stimulus leads to a potentiation of the EEG response to single presentations of the same stimulus [5,34]. The research presented in this paper is the first

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to provide direct evidence of LTP in humans by using a close approximation of the traditional LTP paradigm [3]. Our results provide some indication as to the location of TMS activation and LTP induction. By examining the mean rectified EEG response to TMS in a 60 ms window following stimulation, we found that TMS delivered to motor cortex evoked activity most strongly in electrodes located over left premotor cortex both before and after conditioning. This fits with previous imaging research, which has found that subthreshold TMS delivered to motor cortex produces a strong activation in ipsilateral premotor cortex and other motor areas, but not motor cortex [2,10]. Why would peak activation occur at some distance from the stimulation site? Modeling work has demonstrated that TMS directly produces a large, simultaneous activation of both excitatory and inhibitory neurons at the site of stimulation. This artificial perturbation results in inhibitory activity silencing local neural firing within a few milliseconds of TMS [9]. Areas distant from the site of stimulation, however, are activated indirectly as the TMS induced perturbation propagates through excitatory long-range pathways. This likely leads to activity that lasts for a longer duration in these circuits, resulting in an overall larger EEG response at distant sites. The results from our source localization, performed in one subject from whom data was recorded with sufficient quality for an accurate source estimation, supports this possibility. We found that activity was produced at the site of stimulation immediately following TMS (with a latency of 5 ms), and primarily in ipsilateral premotor cortex at later time points. This suggests a rapid termination of activity in motor cortex, while activity propagates to and persists in premotor cortex. As we found that TMS delivered to motor cortex predominantly produced activation in electrodes over left premotor cortex, it should not be surprising that it was also in these electrodes that we observed a significant increase in activity following rTMS. This result builds upon previous research showing that glucose metabolism is increased in premotor cortex following 5 Hz rTMS delivered to motor cortex [32]. A relatively weak, but demonstrable, response was evoked in right premotor cortex, consistent with previous source localization studies [18]. We found that this signal was also markedly potentiated after rTMS. We found that the second, third and fourth peaks of the GMFP were significantly increased in amplitude following rTMS. Source localization showed that these peaks primarily correspond to activity in premotor cortex, which fits well with our finding that the total evoked activity occurring in the time range of these GMFP peaks was increased in electrodes located over premotor cortex following rTMS. The first peak in the GMFP (occurring at 5 ms) was not increased following 5 Hz rTMS. Source localization revealed that this peak primarily corresponded to activity in motor cortex, where potentiation was not observed. It is of interest that the cortical activity underlying MEP generation occurs within the first few milliseconds of TMS delivery [8] and it is believed that MEP potentiation is not purely a spinal phenomenon [7,31]. This calls into question how MEPs can be potentiated while the cortical activity we observed in the same time window was not. One explanation for this apparent discrepancy is that the

EEG response test pulses in this experiment were delivered below motor threshold so as to reduce the potential for artifacts in the EEG and avoid somatosensory feedback resulting from peripheral motor activation. These test pulses may have evoked a response in motor cortex distinct from that activated by the suprathreshold test pulses that produce MEPs. Indeed, modeling work has suggested that increasing levels of stimulation recruit additional cortical populations [9]. The subthreshold circuit therefore might not be potentiated immediately at the site of stimulation, while the suprathreshold circuit does experience such changes. Another possibility is that the responsiveness of excitatory cells may have increased, while responsiveness of inhibitory cells may have decreased, as suggested by Quartarone et al. [31]. EEG does not discriminate between excitatory and inhibitory activity, so the same total response to TMS might be recorded by EEG, while the activity produced actually has a greater excitatory affect. We also found that while earlier components of the EEG response to TMS were potentiated, the late activity we observed in P5 (occurring on average with a latency of 84 ms) was not affected. This suggests that the later activation may have a different source than the earlier activity. For example, the earlier activity may be generated by evoked activity in various cortical areas, while the later components may be the result of partial phase resetting of ongoing cortical oscillations, similar to alpha ringing that is observed in visual evoked potentials [25]. Supporting this possibility, TMS/EEG work has indicated that ongoing cortical oscillations are modulated by TMS [13]. It still remains to be determined how long the EEG response to TMS is increased following rTMS. MEPs are increased for at least 30 min following protocols similar to what we employed [30,31], suggesting that the cortical potentiation is a relatively long-term change. In this study, we only measured EEG responses within 10 min following completion of rTMS during a period of quiet wakefulness. This was because, in a concatenated study, we proceeded to examine EEG responses in the same subject during a subsequent sleep episode (Huber et al., in preparation). Remarkably, our preliminary data indicates that the affects on the sleep EEG lasted for up to 2 h after the termination of rTMS. Future experiments using TMS/EEG could be employed to determine whether low-frequency rTMS is able to induce a depression of cortical responses, as has been demonstrated with motor evoked potentials [35]. In addition, new experimental protocols have recently been developed using theta-burst stimulation to produce depression or potentiation of MEPs using only 600 rTMS pulses [17]. It would be of interest to determine whether this new protocol increases EEG responses to TMS in a similar fashion to the protocol employed here. The use of EEG to assess brain responses to TMS rather than muscle output allows the effects of rTMS to be investigated in nonmotor brain areas that may be linked to various neuropsychiatric disorders. For example, to treat depression, a number of clinical studies have targeted high-frequency rTMS to left prefrontal cortex or low-frequency rTMS to right prefrontal cortex [15,19,20,29]. As we have demonstrated here, potentiation may occur at sites distant to the site of stimulation to a larger extent

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than where stimulation is delivered. The technique presented here could be used to assess where potentiation or depression is induced, and to what extent, by stimulation of specific areas. This tool could then be used to help determine the ideal stimulation location for inducing potentiation or depression in desired cortical targets.

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