J Neurophysiol 105: 2150 2156, 2011.

First published February 23, 2011; doi:10.1152/jn.00781.2010.

Modulation of motor cortex neuronal networks by rTMS: comparison of local

and remote effects of six different protocols of stimulation
V. Di Lazzaro,1,2 M. Dileone,1 F. Pilato,1 F. Capone,1 G. Musumeci,1 F. Ranieri,1 V. Ricci,3 P. Bria,3
R. Di Iorio,1 C. de Waure,4 P. Pasqualetti,2 and P. Profice1
1
Institute of Neurology, Universita` Cattolica, Rome; 2Department of Neuroscience, AFaR-Fatebenefratelli Association
for Biomedical Research, San Giovanni Calibita-Fatebenefratelli Hospital, Isola Tiberina, Rome; 3Institute of Psychiatry,
Universita` Cattolica, Rome; and 4Institute of Hygiene, Catholic University of the Sacred Heart, Rome, Italy

Submitted 10 September 2010; accepted in final form 15 February 2011

Di Lazzaro V, Dileone M, Pilato F, Capone F, Musumeci G,

Ranieri F, Ricci V, Bria P, Di Iorio R, de Waure C, Pasqualetti P,
Profice P. Modulation of motor cortex neuronal networks by rTMS:
comparison of local and remote effects of six different protocols of
stimulation. J Neurophysiol 105: 2150 2156, 2011. First published
February 23, 2011; doi:10.1152/jn.00781.2010.Repetitive transcranial magnetic stimulation (rTMS) of human motor cortex can produce
long-lasting changes in the excitability of excitatory and inhibitory
neuronal networks. The effects of rTMS depend critically on stimulus
frequency. The aim of our present study was to compare the effects of
different rTMS protocols. We compared the aftereffects of 6 different
rTMS protocols [paired associative stimulation at interstimulus intervals of 25 (PAS25) and 10 ms (PAS10); theta burst stimulation
delivered as continuous (cTBS) or intermittent delivery pattern
(iTBS); 1- and 5-Hz rTMS] on the excitability of stimulated and
contralateral motor cortex in 10 healthy subjects. A pronounced
increase of cortical excitability, evaluated by measuring the amplitude
of motor evoked potentials (MEPs), was produced by iTBS (56%)
and PAS25 (45%). Five-hertz rTMS did not produce a significant
increase of MEPs. A pronounced decrease of cortical excitability was
produced by PAS10 (31%), cTBS (29%), and 1-Hz rTMS
(20%). Short-interval intracortical inhibition was suppressed by
PAS10. Cortical silent period duration was increased by 1-Hz stimulation. No significant effect was observed in the contralateral hemisphere. Head-to-head comparison of the different protocols enabled us
to identify the most effective paradigms for modulating the excitatory
and inhibitory circuits activated by TMS.
brain stimulation; repetitive transcranial magnetic stimulation; brain
plasticity; long-term potentiation; long-term depression

(TMS) techniques can

noninvasively activate the human brain, evoking artificial activity in cortical neuronal networks (Hallett 2007). Technical
advances have offered the possibility of delivering repetitive
TMS (rTMS), and it has been observed that rTMS may induce
changes in brain excitability that outlast the stimulation period.
The aftereffects of rTMS might relate to activity-dependent
changes in the effectiveness of synaptic connections between
cortical neurons, reflecting plasticity mechanisms of the brain
(see Fitzgerald et al. 2006, Hoogendam et al. 2010, and
Ziemann et al. 2008 for review). Low-frequency rTMS (stimulus rates of 1 Hz or less) produces a lasting decrease in motor
cortex excitability (Chen et al. 1997), whereas high-frequency
rTMS (stimulus rates of 5 Hz or higher) (Berardelli et al. 1998;

TRANSCRANIAL MAGNETIC STIMULATION

Address for reprint requests and other correspondence: V. Di Lazzaro,

Istituto di Neurologia, Universita` Cattolica, L.go A. Gemelli 8, 00168 Rome,
Italy (e-mail: vdilazzaro@rm.unicatt.it).
2150

Maeda et al. 2000; Peinemann et al. 2000) promotes a shortterm increase in cortical excitability. More recently, two novel
protocols of rTMS that resemble experimental models of induction of long-term potentiation (LTP) and long-term depression (LTD) of synaptic activity have been introduced: the first
is the so-called paired associative stimulation (PAS) (Stefan et
al. 2000), and the second is termed theta burst stimulation
(TBS) (Huang et al. 2005). PAS is based on the Hebbian
concept of spike timing-dependent plasticity: two inputs, the
first arising from electrical peripheral nerve stimulation and the
second delivered over the motor cortex using TMS, are paired
to activate brain networks at approximately the same time. If
the TMS pulse is applied at an interstimulus interval (ISI)
slightly longer (25 ms; PAS25) or slightly shorter (10 ms;
PAS10) than the time needed for the afferent inputs, generated
by peripheral nerve stimulation, to reach the cerebral cortex
and if a sufficient number of pair of stimuli is delivered, the
excitability of the sensory-motor cortex increases or decreases,
respectively. TBS employs brief bursts of high-frequency (50
Hz), low-intensity stimuli. Different patterns of delivery of
TBS (continuous vs. intermittent) produce opposite effects on
the excitability of the stimulated motor cortex (Huang et al.
2005). The intermittent TBS (iTBS) paradigm produces a
prolonged LTP-like increase of motor cortex excitability
(Huang et al. 2005), whereas the continuous TBS (cTBS)
paradigm produces a prolonged LTD-like decrease of motor
cortex excitability.
The majority of previous studies investigated the effects of
rTMS on cortical excitatory circuits by measuring the amplitude of motor evoked potentials (MEPs) elicited by singlepulse TMS before and after rTMS; only a few studies have
evaluated the effects of rTMS on cortical inhibitory circuits
(Daskalakis et al. 2006; Fitzgerald et al. 2006). The modulatory
effects of rTMS are not limited to the cortical area targeted by
rTMS but also may occur at distant interconnected sites in the
brain and, in particular, in the contralateral nonstimulated
motor cortex (Di Lazzaro et al. 2008; Gilio et al. 2003;
Schambra et al. 2003; Suppa et al. 2008; Wassermann et al.
1998). Only a limited number of studies have investigated this
aspect. Moreover, because almost all of the studies employed
a single rTMS protocol and because there is a high interindividual variability of the aftereffects of rTMS (Maeda et al.
2000), at least in part related to genetic features (Cheeran et al.
2009), it is difficult to compare the results reported for different
paradigms, and it is still unclear which are the most effective
protocols in modulating specific cerebral cortex circuits. In

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LOCAL AND REMOTE EFFECTS OF rTMS

only one study were the effects of TBS on threshold and

amplitude of MEPs compared with those produced by 1- and
5-Hz rTMS (Zafar et al. 2008). The aim of the present study
was to compare in a group of healthy subjects the effects of
different protocols of rTMS on the excitability of the excitatory
and inhibitory circuits of the stimulated and contralateral motor
cortex.
METHODS

Subjects
Ten healthy volunteers [mean age 26.6 4.1 (SD) yr] participated
in the experiments; eight of the subjects were nonsmokers. All gave
their written informed consent. The study was performed according to
the Declaration of Helsinki and was approved by the ethics committee
of the Medical Faculty of the Catholic University of Rome.
TMS Measures
TMS was performed with a high-power Magstim 200 stimulator
(Magstim, Whitland, UK). A figure-of-eight coil, with external loop
diameters of 9 cm, was held over the motor cortex at the optimum
scalp position to elicit MEPs in the contralateral first dorsal interosseous (FDI) muscle. The optimum scalp position to elicit MEPs in
contralateral FDI was determined on each session and was marked on
the scalp with a felt-tip pen to assure a stable coil placement throughout the session. The experimenter performing TMS was the same
across all six sessions. The induced current in the brain flowed in a
posterior-to-anterior direction. MEPs were recorded via two 9-mmdiameter Ag-AgCl surface electrodes with the active electrode over
the motor point of the left FDI and the reference on the metacarpophalangeal joint of the index finger. The electromyogram (EMG) was
amplified and filtered (bandwidth 3 Hz-3 kHz) by D360 amplifiers
(Digitimer, Welwyn Garden City, UK). Data were collected on a
computer with a sampling rate of 10 kHz per channel and stored for
later analysis using a CED 1401 analog-to-digital converter (Cambridge Electronic Design, Cambridge, UK). Resting motor threshold
(RMT) was defined as the minimum stimulus intensity that produced
a liminal MEP (50 V in at least 5 of 10 trials) at rest. Active motor
threshold (AMT) was defined as the minimum stimulus intensity that
produced a small MEP (200 V in 5 of 10 trials) during isometric
contraction of the FDI at 20% of maximum voluntary strength. A
constant level of voluntary contraction was maintained with reference
to an oscilloscope display of the EMG signal in front of the subject.
Auditory feedback of the EMG activity was also provided. RMT and
AMT are given as percentages of maximum stimulator output
(%MSO).
MEP amplitude was evaluated using a stimulus intensity of 120%
RMT with the muscle at rest. Ten sweeps of the data were collected,
and the mean peak-to-peak amplitude of the MEPs was calculated.
Contralateral silent periods (cSP) were elicited while subjects held a
tonic voluntary contraction of 50% of MVC of the FDI contralateral
to the stimulated hemisphere. Five stimuli at 200% AMT were given.
The duration of cSP was measured from the end of MEP to the
reappearance of sustained EMG activity. Ipsilateral SP were elicited
whilst subjects held a tonic voluntary contraction of 50% of maximum voluntary contraction (MVC) of the FDI ipsilateral to the
stimulated hemisphere. Five stimuli at 200% AMT were given. The
ipsilateral cortical silent period was measured according to the objective graphical method described by Garvey et al. (2001). This method
allows an automated and objective estimation of onset and offset
points based on statistical analysis of variation of the baseline EMG
activity (Garvey et al. 2001). EMG signal was sampled at 5 kHz. We
analyzed 100 ms of rectified averaged prestimulus EMG signal (that
is, 500 data points) to calculate the mean EMG level and the mean
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consecutive difference of the data points. Ipsilateral SP onset was the

first point to fall below the lower variation limit if 50% or more of the
data points in the following 5-ms window were also below the lower
variation limit. Ipsilateral SP offset was the first point to fall above the
lower variation limit if 50% or more of the data points in the following
5-ms window were also above the lower variation limit. To automate
the procedure, we used a self-made function for the MATLAB
software (The MathWorks). Ipsilateral SP were measured before and
after rTMS using the same stimulus intensity. All measurements were
performed with two magnetic stimulators connected to a figure-ofeight coil through a Y-shaped cable (Magstim) with the use of a single
stimulator for single-pulse and short-latency afferent inhibition studies and two stimulators for paired-pulse paradigms.
Short-interval intracortical inhibition. Short-interval intracortical
inhibition (SICI) was studied using the technique of Kujirai et al.
(1993). Two magnetic stimuli were given through the same stimulating coil over the motor cortex at an ISI of 2 ms, and the effect of the
first (conditioning) stimulus on the second (test) stimulus was investigated. Five single-pulse stimuli and five paired stimuli at 2-ms ISI
were delivered. The conditioning stimulus was set at an intensity of
5% MSO below AMT. The intensity of the test stimulus was adjusted
to elicit an unconditioned test MEP in the relaxed FDI of 1 mV in
peak-to-peak amplitude. Subjects were provided with audiovisual
feedback of the EMG at high gain (50 V/D) to assist in maintaining
complete relaxation, since slight activation of the target muscle may
result in significant SICI reduction (Ridding et al. 1995). The amplitude of the conditioned MEP was expressed as a percentage of the
amplitude of the unconditioned test MEP.
Intracortical facilitation. We also evaluated intracortical facilitation (ICF) by analyzing the facilitatory interaction that occurs between
pairs of magnetic stimuli given over the motor cortex at 15-ms ISI.
Five single-pulse stimuli and five paired stimuli at 15-ms ISI were
delivered. Subjects were given audiovisual feedback at high gain to
assist in maintaining complete relaxation. The amplitude of the
conditioned MEPs was expressed as a percentage of the amplitude of
the test MEPs.
Short-latency afferent inhibition. Short-latency afferent inhibition
(SAI) was studied using the technique that we have described previously (Tokimura et al. 2000). Conditioning electrical pulses (constantcurrent square-wave pulses; duration 200 s) were applied through a
bipolar electrode to the median nerve at the wrist (cathode proximal).
The intensity of the conditioning stimulus was set to evoke a just
visible twitch of the thenar muscles. The intensity of the TMS test
pulse over the motor cortex was adjusted to evoke an unconditioned
MEP in the relaxed FDI of 1 mV in peak-to-peak amplitude.
The conditioning stimulus to the median nerve preceded the TMS
test pulse by ISIs that were related to the individual latency of the N20
component of the median nerve somatosensory evoked potential. To
record somatosensory evoked potentials, the active electrode was
attached 3 cm posterior to C4 or C3 (according to the 10 20
international EEG system) and the reference was 3 cm posterior to C3
or C4 , respectively. Five hundred responses were averaged to identify
the latency of the N20 peak. ISIs corresponding to the N20 latency
plus 2, 3, and 4 ms were investigated (Tokimura et al. 2000) with five
repeats per ISI in a pseudorandomized order. Subjects were given
audiovisual feedback of the EMG signal at high gain (50 V/D) to
assist in maintaining complete relaxation of the FDI. The mean
amplitudes of the conditioned MEPs at the various ISIs were expressed as a percentage of the mean amplitude of the unconditioned
test MEP. These data were averaged across all ISIs to obtain a grand
mean single value of SAI.
Repetitive TMS
Repetitive TMS was delivered over the right motor cortex hot
spot for MEPs in the contralateral FDI muscle. The coil was held

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over the motor cortex with the handle pointing posteriorly and
approximately perpendicular to the central sulcus.
For 1- and 5-Hz rTMS and for TBS, we used a MagPro stimulator
(Medtronic, Copenhagen, Denmark) connected to a figure-of-eight
coil (MCF B65; Medtronic). The initial direction of the current
induced in the brain was anterior to posterior. The magnetic stimulus
had a biphasic waveform with a pulse width of 280 s and
maximum magnetic field strength of 1.5 T. The stimulation intensity
was defined in relation to AMT or RMT evaluated using the MagPro
stimulator.
Five-hertz rTMS. The intensity of rTMS was set at 90% RMT.
Three rTMS trains of 300 pulses, each separated by 2 min, were
delivered.
One-hertz rTMS. One-hertz rTMS was performed at 110% RMT.
Nine hundred stimuli were delivered in a single train.
Intermittent TBS. An intensity of 80% AMT was used. We used the
iTBS protocol in which 10 bursts of high-frequency stimulation (3
pulses at 50 Hz) are applied at 5 Hz every 10 s for a total of 600 pulses
(Huang et al. 2005).
Continuous TBS. An intensity of 80% AMT was used. We used the
cTBS protocol in which 3 pulses of stimulation are given at 50 Hz,
repeated every 200 ms for a total of 600 pulses (Huang et al. 2005).
Paired Associative Stimulation
We used a high-power Magstim 200 (Magstim) connected to a
figure-of-eight coil, with external loop diameters of 9 cm, held over
the right motor cortex at the optimum scalp position to elicit MEPs in
the contralateral FDI. The induced monophasic current in the brain
flowed in a posterior-to-anterior direction.
The intervention consisted of single electrical stimuli delivered to
the left ulnar nerve at the wrist at 300% of the perceptual threshold,
followed by TMS at intensities sufficient to produce an unconditioned
response amplitude of 1 mV in the resting FDI. For the PAS25
protocol, 90 pairs were delivered at 0.05 Hz over 30 min at an ISI of
25 ms, because this interval had been shown in previous experiments
to be effective in increasing cortical excitability (Stefan et al. 2000).
For the PAS10 protocol, 90 pairs were delivered at 0.05 Hz over 30
min at an ISI of 10 ms, because this interval had been shown in
previous experiments to be effective in decreasing cortical excitability
(Wolters et al. 2003).
Experimental Design
In all subjects, rTMS was performed on the nondominant hemisphere and all TMS measures were evaluated bilaterally at baseline
(T0, before rTMS) and at two time points after rTMS (T1 and T2).
Measurements at T1 were performed immediately after rTMS; measurements at T2 were performed 30 min after the end of rTMS. The
order of measurement of different parameters and the order of the
hemisphere (stimulated or contralateral) was pseudorandomized and
counterbalanced across subjects and rTMS protocols but remained
constant in pre- and/or postevaluation for each session. All subjects
were tested using all rTMS protocols in a randomized crossover
design. The intersession interval for a given subject was at least 1 wk
to exclude interactions between sessions.
For MEP amplitude measurement, the same stimulus intensity was
used before and after rTMS. For the paired-pulse TMS protocols
(SICI and ICF), the intensity of the test stimulus after rTMS was
adjusted whenever necessary to ensure that the test MEP matched the
amplitude to the baseline test MEP before rTMS, and the conditioning
stimulus intensity was adjusted relative to the post-rTMS AMT. For
SP evaluation, the stimulus intensity after rTMS was adjusted relative
to the post-rTMS AMT. The time of testing was around 12:00 PM and
was the same across all sessions.
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Statistics
All data are means SD. The effects of different rTMS protocols
were tested separately for each TMS parameter (RMT, AMT, MEP
amplitude, cSP, iSP, SAI, SICI, ICF) using a repeated-measures
analysis of variance (rmANOVA) with rTMS protocol (6 levels: 1 Hz,
5 Hz, cTBS, iTBS, PAS25, PAS10), hemisphere (2 levels: stimulated,
nonstimulated), and time (T0, T1, T2) as within-subject factors. The
Mauchly test was used to evaluate the sphericity assumption, and the
Greenhouse-Geisser correction was employed when necessary to
correct for nonsphericity. The Kruskal-Wallis test was performed to
test differences between the values of each TMS parameter at baseline; the test was carried out separately for stimulated and nonstimulated hemispheres. The post hoc analyses between times were performed by comparing T0 vs. T1 and T0 vs. T2 for each TMS
parameter, protocol, and hemisphere. The level of significance was set
at P 0.05, and Sidak correction was applied for post hoc comparisons.
To evaluate which was the most effective facilitatory protocol and
the most effective inhibitory protocol on cortical excitability as
evaluated by measuring MEP amplitude, we performed a further post
hoc analysis comparing the effects produced by the three excitatory (5
Hz, iTBS, PAS25) and the three inhibitory protocols (1 Hz, cTBS,
PAS10) by means of the Sidak procedure. The Sidak procedure is
slightly more powerful than the Bonferroni method. Such a procedure
is available in many popular statistical software applications (such as
SPSS). The alpha adjustment is made by S 1 (1 )1/k instead
of the Bonferroni approach, B /k, where k is the number of
comparisons between means (Edwards and Berry 1987). We used
SPSS software version 12.0 to perform analyses.
RESULTS

Results are summarized in Tables 1, 2, 3 and 4.

Baseline Analysis
The Kruskal-Wallis test showed no differences between
baseline parameters.
Motor Threshold (RMT and AMT)
The rmANOVA with rTMS protocol (6 levels: 1 Hz, 5 Hz,
cTBS, iTBS, PAS25, PAS10), hemisphere (stimulated, nonstimulated), and time as within-subject factors showed a significant interaction between time, protocol, and hemisphere
(F10,90 2.99, P 0.03) for RMT. No significant results or
interactions were observed for AMT. Post hoc analysis showed
a significant increase in RMT between T0 and T1 for PAS10
(P 0.041) and a significant decrease between the same two
time points for PAS25 (P 0.047) for the stimulated hemisphere.
MEP amplitude
The rmANOVA with rTMS protocol, hemisphere, and time
as within-subject factors showed a significant association with
time (F2,18 7.31, P 0.01) and hemisphere (F1,9 9.94,
P 0.01) and a significant interaction between protocol and
hemisphere (F5,45 2.99, P 0.05) and between time,
protocol, and hemisphere (F10,90 4.49, P 0.01). Post hoc
analysis showed a significant increase in MEP amplitude between T0 and T1 for iTBS (P 0.015) and PAS25 (P 0.021)
and between T0 and T2 for PAS25 (P 0.027) for the
stimulated hemisphere (Fig. 1). Moreover, post hoc analysis
showed a significant decrease in MEP amplitude between T0

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Table 1. Facilitatory protocols: study of the stimulated hemisphere

Five Hertz

iTBS

PAS25

Measure

T0

T1

T2

T0

T1

T2

T0

T1

T2

RMT
AMT
MEP
cSP
iSP
SICI
ICF
SAI

45.7 12.2
35.9 8.4
0.7 0.4
153 43
33.9 42.4
39 14.2
128 33
44.4 14.9

45.3 12.1
35.3 8.6
0.7 0.4
146 27
31.5 38.5
50.4 28.3
157.1 95.3
43 26.8

47.1 11.9
36.1 7.5
0.7 0.4
143 49
41.1 45.5
34.7 21.3
134.1 44.4
61.1 23.3

46.4 12
36.4 8.1
0.64 0.2
149 49
37.1 13.6
37.1 19
134.3 37.2
41.4 17.4

45.7 11.9
35.7 8.9
1 0.4
154 36
30.9 14.9
36 20
146.4 49.9
46.4 17.7

46.4 12
35.8 7.7
0.8 0.3
154 50
33.6 20.1
32.8 14.8
120.9 37.1
40.2 12.8

46 12.3
34.9 8.9
0.67 0.35
144 22
33.9 13.7
38.9 20.2
129.4 52.3
41.7 11.1

44.8 12.5
34.7 9.3
0.97 0.39
149 15
27 22.1
39.7 22.1
114.4 56.9
44.2 9.8

45.2 12.5
34.4 8.9
0.92 0.27
150 15.5
35.1 18.3
44.4 28.5
113.5 31.4
48.5 15.5

Motor thresholds (% of maximum stimulator output), MEP amplitude (mV), contralateral and ispilateral silent period (ms), short interval intracortical inhibition
(% of test MEP), intracortical facilitation (% of test MEP), and short latency afferent inhibition (% of test MEP) before (T0) and after (T1T2) different protocols
of rTMS (means SD). Significant changes vs. T0 (P 0.05) are reported in bold. RMT, resting motor threshold; AMT, active motor threshold; MEP, motor
evoked potential; cSP, contralateral silent period; iSP, ipsilateral silent period; SICI, short interval intracortical inhibition; ICF, intracortical facilitation; SAI,
short latency afferent inhibition.

and T1 for 1 Hz (P 0.027), cTBS (P 0.015), and PAS10

(P 0.021) for the stimulated hemisphere (Fig. 1).
Comparison of the effects of the three different excitatory
protocols on the amplitude of MEPs evoked by the stimulated
hemisphere showed that iTBS (P 0.010) and PAS (P
0.035) produced a significantly larger increase in MEP amplitude than 5-Hz rTMS. No difference was found between iTBS
and PAS25 (P 0.946).
Comparison of the effects of the three different inhibitory
protocols on the amplitude of MEPs evoked by the stimulated
hemisphere showed that the larger suppression of MEPs was
found with PAS10, and this approached statistical significance
compared with 1-Hz rTMS (P 0.070). No evidence of
difference was found for the other comparisons (PAS vs.
cTBS and cTBS vs. 1 Hz, consistently P 0.40).

protocols, and hemispheres and no significant interaction between the within-subject factors.
Short-Interval Intracortical Inhibition
The rmANOVA with rTMS protocol, hemisphere, and time
as within-subject factors showed a significant effect of time
(F2,18 8.83, P 0.01). Post hoc analysis showed a significant decrease between T0 and T2 (P 0.021) for PAS10 for
the stimulated hemisphere.
Intracortical Facilitation
The rmANOVA with rTMS protocol, hemisphere, and time
as within-subject factors showed no significant effect of time,
protocols, and hemispheres and no significant interaction between the within-subject factors.

Contralateral Silent Period

The rmANOVA with rTMS protocol, hemisphere, and time
as within-subject factors showed a significant effect of time
(F2,18 5.97, P 0.02) and hemisphere (F1,9 7.31, P
0.02). Post hoc analysis showed a significant increase in cSP
duration between T0 and T1 only for 1-Hz rTMS (P 0.038)
for the stimulated hemisphere.

Short-Latency Afferent Inhibition

The rmANOVA with rTMS protocol, hemisphere, and time
as within-subject factors showed no significant effect of time,
protocols, and hemispheres and no significant interaction between the within-subject factors.
DISCUSSION

Ipsilateral Silent Period

The rmANOVA with rTMS protocol, hemisphere, and time
as within-subject factors showed no significant effect of time,

The results of our present study confirm that it is possible to

produce lasting effects on cortical excitability in both the
stimulated and contralateral motor cortex by using different

Table 2. Inhibitory protocols: study of the stimulated hemisphere

One Hertz

cTBS

PAS10

Measure

T0

T1

T2

T0

T1

T2

T0

T1

T2

RMT
AMT
MEP
cSP
iSP
SICI
ICF
SAI

49.1 18.6
36.4 11.4
0.76 0.39
137 28
36.3 28.4
34.8 17.4
120.8 32.2
41.7 17.7

49.9 19.2
37 10.7
0.61 0.29
158 49
37.4 21.5
35.4 15.9
135.2 28.8
58.4 15.3

49.6 18.8
36.1 11.3
0.84 0.49
145 34
37.6 17.2
37.2 27.1
124.4 42.6
47.4 15.6

46.4 11.5
34.9 7
0.85 0.32
140 35
34.3 17.5
42.6 21.1
147 30
35.2 12.7

47 11.7
35 6.7
0.6 0.3
141 30
35.4 22.9
38.7 20.5
131 39.9
31.8 16.3

47 11.7
34.8 6.7
0.9 0.4
151 38
34.1 20.9
41.2 20.7
131.8 32
45 19.7

46 14.7
35 9.3
0.7 0.2
155 49
30.9 24.1
35 15.2
135.8 59.4
47.9 10.5

47 14.7
35.5 10
0.48 0.3
159 55
36.1 32.1
49.2 22.2
127.6 28.1
50.2 22.9

46.9 14.6
35.8 9.4
0.65 0.39
163 59
45.1 48
46.9 18.2
133.6 37
45.7 21.5

Motor thresholds (% of maximum stimulator output), MEP amplitude (mV), contralateral and ispilateral silent period (ms), short interval intracortical inhibition
(% of test MEP), intracortical facilitation (% of test MEP), and short latency afferent inhibition (% of test MEP) before (T0) and after (T1T2) different protocols
of rTMS (means SD). Significant changes vs. T0 (P 0.05) are reported in bold.
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Table 3. Facilitatory protocols: study of the nonstimulated hemisphere

Five Hertz

iTBS

PAS25

Measure

T0

T1

T2

T0

T1

T2

RMT
AMT
MEP
cSP
iSP
SICI
ICF
SAI

44.8 7.6
34.6 6.2
1 0.6
148 49
30.6 17.2
31.6 8.9
125.4 48.7
43.5 16.8

45.2 7.8
34.5 5.8
1.2 0.7
135 29
21.6 22.6
45.7 19.9
142.8 57.7
48.1 21.2

45.5 7.6
34.8 5.6
1 0.3
135 22
30.3 22.7
33.4 18.1
139 52.7
41.3 17

47.3 9.7
35.5 6.3
0.79 0.29
131 23
27.1 13.4
37.2 17.6
150.7 79.7
40.7 18.5

47.9 9.7
35.8 5.7
0.66 0.23
145 51
34 12.1
34.9 11
139.3 64.8
37.8 13.3

T0

47.7 9.4
45.4 9.2
34.8 5.4
34.9 6.9
0.8 0.3
0.87 0.35
143 39
149 23
31.3 14.9 24.1 21.3
40 16.2
27 19.1
152.6 83.5 141.6 55
38.5 22
38.1 12.8

T1

T2

45.7 8.7
34.3 6.4
0.78 0.5
144 22
37.7 23.1
41.5 16.9
134.8 46.3
41.3 17.9

45.6 8.8
34.5 6.1
0.82 0.34
145 18
31.5 17.9
35.9 19.1
152.4 94.9
44.7 21.6

Motor thresholds (% of maximum stimulator output), MEP amplitude (mV), contralateral and ispilateral silent period (ms), short interval intracortical inhibition
(% of test MEP), intracortical facilitation (% of test MEP), and short latency afferent inhibition (% of test MEP) before (T0) and after (T1T2) different protocols
of rTMS (means SD).

and 5-Hz rTMS (Di Lazzaro et al. 2002; Fierro et al. 2007;
Koch et al. 2008; Quartarone et al. 2005; Wu et al. 2000).
These discrepancies might be explained be either a high variability of all these effects that makes them less robust or a short
duration of these aftereffects. This latter point appears relevant,
because we measured a large number of TMS parameters and
several minutes were required to complete all the tests. For this
reason, short-lived changes such as the facilitation of MEPs
after 5-Hz rTMS might be difficult to demonstrate in the
present experimental setting. Moreover, stimulus parameters
might have influenced our results. It has been demonstrated
that TBS effects on SICI are highly intensity-dependent, and
intensities lower than those used in the present study may be
more effective in modulating SICI (McAllister et al. 2009).
Finally, it should be considered that although some of the
changes observed in the present study did not reach statistical
significance, there was a clear tendency toward a change in the
same direction as reported in previous studies. This is the case
for the effects of iTBS on MEPs evoked by stimulation of the
contralateral hemisphere, which were significantly suppressed
in two previous studies (Di Lazzaro et al. 2008; Suppa et al.
2008) and showed a consistent decrease (16%) in the present
study, and for the suppression of SICI after 5-Hz rTMS (Di
Lazzaro et al. 2002; Fierro et al. 2007; Koch et al. 2008;
Quartarone et al. 2005; Wu et al. 2000), which also was
observed in the present study, although the change did not
reach statistical significance. These findings demonstrate that
by using different protocols of rTMS, it is possible to target
specific cortical excitatory and inhibitory networks. MEP amplitude mainly reflects excitatory glutamatergic neurotransmis-

protocols of rTMS. As previously demonstrated, iTBS and

PAS25 produced a pronounced increase of MEP amplitude
(Huang et al. 2005; Stefan et al. 2000), whereas 1-Hz rTMS
(Chen et al. 1997), cTBS (Huang et al. 2005), and PAS10
(Wolters et al. 2003) produced a pronounced suppression of
MEP amplitude in the stimulated hemisphere. This was associated with a slight decrease in RMT for PAS25 and a slight
increase in RMT for PAS10; the most prolonged effect on MEP
amplitude was found for the PAS25 protocol. As previously
reported (Russmann et al. 2009), PAS10 produced a decrease of
SICI of the stimulated hemisphere; however, in our study this
effect was slightly delayed. Moreover, as reported by Romeo et
al. (2000) and Daskalakis et al. (2006), 1-Hz rTMS increased
cSP duration after stimulation of the stimulated hemisphere.
The head-to-head comparison of the different rTMS protocols
in a group of subjects enabled us to overcome the limitation of
interindividual variability and to identify the protocols with the
most pronounced effects on the excitability of intracortical
excitatory and inhibitory circuits. The most pronounced increase in MEP amplitude was produced by iTBS (56%) and
PAS25 (45%), whereas the most pronounced MEP suppression was produced by PAS10 (31%) and cTBS (29%).
Several previously reported aftereffects of rTMS were not
consistently observed in present study: 1) an increase in MEP
amplitude after 5-Hz rTMS (Berardelli et al. 1998; Quartarone
et al. 2005); 2) a decrease in the amplitude of MEPs evoked by
stimulation of the nonstimulated hemisphere after iTBS (Di
Lazzaro et al. 2008; Suppa et al. 2008); and 3) a change in SICI
after iTBS (Huang et al. 2007; Suppa et al. 2008), cTBS
(Huang et al. 2007; McAllister et al. 2009; Suppa et al. 2008),
Table 4. Inhibitory protocols: study of the nonstimulated hemisphere
One Hertz

cTBS

PAS10

Measure

T0

T1

T2

T0

T1

T2

RMT
AMT
MEP
cSP
iSP
SICI
ICF
SAI

48 12.2
36.1 8.9
0.7 0.3
145 42
39.7 21.8
33.2 19.4
124 30.9
41 15.7

47.6 11.8
36.1 8.9
0.9 0.27
150 47
34.8 17.5
40.2 20.9
132 36.2
53.1 23

47 11.3
35.4 8.8
0.94 0.4
154 60
37.4 18.5
42 18.4
146.2 87.9
42.4 18.8

45.5 7
34.9 4.7
0.82 0.4
141 17
33.5 23.6
38.8 17.4
131.8 25.4
39.7 14.8

45.7 6.1
34.7 4
0.93 0.47
146 33
24.8 24.8
45.4 16.8
123.3 39.9
42.7 15.8

45.5 6.6
34.6 4.2
0.82 0.28
151 32
33.9 23.6
46 23.3
143 59.9
49.6 19

T0
45.5 9.2
34.5 6.8
0.76 0.32
150 44
23.9 22.1
37.4 14.3
105.4 49.9
43.2 15.6

T1

T2

45.2 8.5
34 7.5
0.95 0.53
151 47
25.1 17.2
50.5 21.1
130.6 40.7
51.7 26.5

45.1 8.4
34 6.3
0.88 0.3
147 33
31.9 25.8
55.6 33.4
126.4 30.4
39 18

Motor thresholds (% of maximum stimulator output), MEP amplitude (mV), contralateral and ispilateral silent period (ms), short interval intracortical inhibition
(% of test MEP), intracortical facilitation (% of test MEP), and short latency afferent inhibition (% of test MEP) before (T0) and after (T1T2) different protocols
of rTMS (means SD).
J Neurophysiol VOL

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LOCAL AND REMOTE EFFECTS OF rTMS

2155

Fig. 1. Effects of paired associative stimulation at 25-ms

interstimulus interval on the stimulated (PAS25SH) and
non-stimulated (PAS25nSH) hemisphere motor evoked
potential (MEP) amplitude, paired associative stimulation at 10-ms interstimulus interval on the stimulated
(PAS10SH) and non-stimulated (PAS10nSH) hemisphere MEP amplitude, intermittent theta burst stimulation on the stimulated (iTBS-SH) and non-stimulated
(iTBS-nSH) hemisphere MEP amplitude, continuous
theta burst stimulation on the stimulated (cTBS-SH) and
non-stimulated (cTBS-nSH) hemisphere MEP amplitude, one Hz repetitive stimulation on the stimulated
(1Hz-SH) and non-stimulated (lHz-nSH) hemisphere
MEP amplitude, five Hz repetitive stimulation on the
stimulated (5Hz-SH) and non-stimulated (5Hz-nSH)
hemisphere MEP amplitude. Repetitive transcranial
magnetic stimulation (rTMS) was performed on the
nondominant hemisphere, and all TMS measures were
evaluated bilaterally at baseline (T0, before rTMS) and
at 2 time points after rTMS (TI and T2) Values are
means and 95% confidence intervals of the after-before
differences in mean amplitudes immediately after rTMS
(T1-T0: A) and 30 min after the end of rTMS (T2-T0:
B). *P 0.05.

sion at the level of the motor cortex, SICI reflects GABAA

intracortical activity, and cSP mainly reflects GABAB intracortical activity (Paulus et al. 2008). Thus our results confirm
that by using rTMS, it is possible to modulate excitatory glutamatergic neurotransmission in both directions, to suppress intracortical inhibitory GABAA activity, and to enhance intracortical
GABAB activity. Interestingly, the time course of the effects of
rTMS might be different for excitatory and inhibitory circuits. We
observed a delayed and isolated suppression of intracortical inhibitory activity after PAS10. This confirms the notion that excitatory and inhibitory circuits may be modulated independently
(McAllister et al. 2009). Head-to-head comparison of the effects
produced by different paradigms on specific cortical circuits could
provide valuable information for the development of therapeutical
J Neurophysiol VOL

strategies based on neuromodulation in neuropsychiatric disorders

characterized by abnormal excitability of cortical circuits that can
be targeted and modulated by rTMS.
A main limitation of the present study is that we studied only
the more commonly used protocols of stimulation, but there are
many other protocols, such as paired-pulse rTMS (Thickbroom
et al. 2006) and quadripulse rTMS (Hamada et al. 2008), that
were not analyzed. Moreover, it should be considered that
although we attempted to test the effects of the more commonly used protocols, the parameters of these protocols used in
different studies are quite variable in that several studies have
suggested that higher intensities and longer duration of stimulation will produce stronger effects, also that higher frequencies of rTMS such as 10 or 20 Hz seem to have a stronger

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2156

LOCAL AND REMOTE EFFECTS OF rTMS

effect, and, finally, that the direction and the phases of the
induced current in the brain influence the changes produced in
brain excitability. There are also several PAS protocols with
variations in the intensity of median nerve stimulation, repetition rates, ISIs, and number of paired stimuli. Therefore, the
present study does not provide general conclusions regarding
the relative effects of the protocols evaluated but only on those
commonly used. However, the comparison performed in the
present study identifies the most effective protocols; thus new
protocols can be tested against these and the results easily
compared. Another main limitation is that we only evaluated
the effects on young healthy subjects; older healthy subjects or
patients with neurological disorders may respond differently to
rTMS. A further limitation is represented by our selective
study of the motor cortex; other brain areas may represent more
effective targets of neuromodulation in conditions such as
movement and psychiatric disorders. Finally, it should be
considered that our present findings are mainly confirmatory in
that the effects of individual rTMS protocols have been evaluated in several previous studies.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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