Neuroscience Letters 455 (2009) 13

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Electrical and magnetic repetitive transcranial stimulation of the primary motor

cortex in healthy subjects
Francesca Gilio c , Elisa Iacovelli a , Vittorio Frasca a , Maria Gabriele a , Elena Giacomelli a ,
Carlo De Lena a , Anna Maria Cipriani c , Maurizio Inghilleri a,b,
a
b
c

Department of Neurological Sciences, Sapienza University of Rome, Italy

S. Raffaele Institute IRCCS, Sapienza University of Rome, Italy
Neurology and Neurophysiopathology Unit, S. Pertini, Rome, Italy

a r t i c l e

i n f o

Article history:
Received 17 December 2008
Received in revised form 18 January 2009
Accepted 9 March 2009
Keywords:
Repetitive transcranial magnetic
stimulation
Repetitive transcranial electric stimulation
Cortical excitability
Primary motor cortex

a b s t r a c t
Repetitive transcranial magnetic stimulation (rTMS) delivered in short trains at 5 Hz frequency and
suprathreshold intensity over the primary motor cortex (M1) in healthy subjects facilitates the motorevoked potential (MEP) amplitude by increasing cortical excitability through mechanisms resembling
short-term synaptic plasticity. In this study, to investigate whether rTES acts through similar mechanisms we compared the effects of rTMS and repetitive transcranial electrical stimulation (rTES) (10
stimuli-trains, 5 Hz frequency, suprathreshold intensity) delivered over the M1 on the MEP amplitude.
Four healthy subjects were studied in two separate sessions in a relaxed condition. rTMS and anodal rTES
were delivered in trains to the left M1 over the motor area for evoking a MEP in the right rst dorsal
interosseous muscle. Changes in MEP size and latency during the course of the rTMS and rTES trains
were compared. The possible effects of muscle activation on MEP amplitude were evaluated, and the
possible effects of cutaneous trigeminal bre activation on corticospinal excitability were excluded in a
control experiment testing the MEP amplitude before and after supraorbital nerve repetitive electrical
stimulation. Repeated measures analysis of variance (ANOVA) showed that rTES and rTMS trains elicited
similar amplitude rst MEPs and a similar magnitude MEP amplitude facilitation during the trains. rTES
elicited a rst MEP with a shorter latency than rTMS, without signicant changes during the course of the
train of stimuli. The MEP elicited by single-pulse TES delivered during muscle contraction had a smaller
amplitude than the last MEP in the rTES trains. Repetitive supraorbital nerve stimulation left the conditioned MEP unchanged. Our results suggest that 5 Hz-rTES delivered in short trains increases cortical
excitability and does so by acting on the excitatory interneurones probably through mechanisms similar
to those underlying the rTMS-induced MEP facilitation.
2009 Elsevier Ireland Ltd. All rights reserved.

Transcranial magnetic stimulation (TMS) is a noninvasive tool commonly used for investigating mechanisms of cortical excitability in
humans [3,9,13,17]. Repetitive TMS (rTMS) applied in short trains
at high frequency and suprathreshold intensity over the primary
motor cortex (M1) elicits changes in cortical excitability, recorded as
a progressive increase in motor evoked potential (MEP) amplitude,
through intracortical mechanisms resembling short-term synaptic
plasticity [4,11,14,18]. By recording the corticospinal activity during high-frequency suprathreshold rTMS in a man with epidural
electrodes implanted, Di Lazzaro et al. [8] directly demonstrated
the increase in the number and amplitude of indirect (I) waves
during rTMS trains underlying the phenomenon of MEP amplitude
facilitation [8].

Corresponding author at: Department of Neurological Sciences, Sapienza University of Rome, Viale dellUniversit, 30. 00185 Rome, Italy. Tel.: +39 06 49914485.
E-mail address: maurizio.inghilleri@uniroma1.it (M. Inghilleri).
0304-3940/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2009.03.035

The human cortex can also be stimulated noninvasively using

transcranial electrical stimulation (TES) [19]. Ample evidence from
studies using direct recording from the pyramidal tract in cats and
primates shows that low-intensity anodal TES delivered over M1
recruits a single descending volley in the pyramidal tract, namely a
direct (D) wave, by directly activating the cortico-spinal tract. Conversely, at the higher intensities needed to induce a MEP at rest, the
early wave is followed by I waves reecting indirect activation of the
pyramidal cells through cortico-cortical connections [1,7,9,19,20].
Even though ample evidence shows that TES activates the corticospinal tract trans-synaptically, TES has been applied in study
protocols designed to test spinal excitability without considering
that intracortical circuits are also activated [2,4,12,16].
We designed this study, to nd out whether rTES, like rTMS,
produces a MEP amplitude facilitation by acting on intracortical circuits through mechanisms involving short-term synaptic plasticity.
To do so, we compared changes in MEP amplitudes and latencies in
response to rTES and rTMS delivered in trains over the M1 in healthy

F. Gilio et al. / Neuroscience Letters 455 (2009) 13

subjects at rest. Control experiments were run to exclude the effects

of muscle contraction and cutaneous rTES-induced trigeminal bre
activation on MEP amplitudes.
Four healthy subjects, among the authors, participated in the
study (two men, two women, mean age 35 5 years). Written consent was obtained from each participant and the experimental
procedures were approved by the local ethical committee.
TMS and rTMS were delivered through a high-frequency magnetic stimulator (Magstim Rapid, Magstim Company Ltd., Whitland,
South West Wales, UK) connected to a gure-of-eight coil placed
over the optimal position for evoking a MEP in the right rst dorsal
interosseous (FDI) muscle. Motor threshold (RMT) was calculated
as the lowest intensity able to evoke a MEP of more than 50 V in
at least ve out of ten consecutive trials. Four trains of 10 stimuli
at 5 Hz frequency were delivered with the subjects at rest. Stimulation intensity was chosen to obtain a rst MEP in the train similar
to that evoked by the rst stimulus in the rTES train. The inter-train
interval was 2 min.
TES and rTES were delivered through an electric stimulator [13]
connected to a pair of surface Ag/AgCl electrodes. Anodal cortical
stimulation was delivered with the cathodal electrode located over
the frontal region (Fz) and the anodal electrode located 7 cm lateral to the vertex and 2 cm in front of the interaural line in the left
hemisphere. A single shock was also delivered during voluntary
contraction of the FDI muscle (30% of the maximal contraction).
Four trains of 10 stimuli at 5 Hz frequency were delivered with the
subjects at rest. Stimulation intensity was set at an intensity able
to evoke a MEP of 0.4 mV in the FDI muscle. The inter-train interval
was 2 min.
To exclude the effects of cutaneous trigeminal bre activation on
corticospinal excitability, in a separate session all subjects underwent a control experiment to test MEP amplitudes before and after
repetitive electrical stimulation to the supraorbital (SO) nerve. The
SO nerve was stimulated at its emergence by means of surface
electrodes placed longitudinally 2 cm apart (square-wave pulses,
duration of 0.2 ms, constant current stimulator, intensity set at 10
times the perception threshold) and 5 trains of 10 electrical stimuli were delivered at 5 Hz frequency with an inter-train interval
of 2 min. Five MEPs evoked by single anodal cortical stimuli were
collected 200 ms after the repetitive nerve stimulation ended (conditioned responses). Five unconditioned MEPs were also randomly
collected. TES was delivered at an intensity able to evoke an unconditioned MEP of 0.4 mV in the FDI muscle.
The electromyographic (EMG) activity was recorded through
a pair of surface disk electrodes placed over the right FDI muscle in a belly-to-tendon conguration. EMG signals were recorded
and ltered with a Digitimer D360 (Digitimer Ltd., UK) (bandwidth 2 Hz1 kHz, sampling rate 3 kHz) and analyzed off-line with
a personal computer through a 1401 plus A/D laboratory interface
(Cambridge Electronic Design, UK). Subjects were asked to control
the FDI muscle activity with the aid of EMG acoustic and visual
(Tektronic 5103N oscilloscope) feedback.
The presence of MEPs throughout the train of stimuli was evaluated by visual inspection. The size of MEPs evoked by rTMS, rTES
and TES stimuli was measured peak-to-peak. The latency of MEP
was measured from the onset of the stimulus artefact and the onset
of the MEP. Traces with EMG activity were rejected. In the rTES and
rTMS trains the ratio of the MEP amplitude evoked by the last and
the rst stimulus was calculated (X/I MEP ratio) [15].
The latency and size of the rst MEP and the X/I MEP ratio in
the rTES and rTMS trains were analysed with a repeated-measures
analysis of variance (ANOVA) with stimulation (rTMS vs. rTES) as
main factor. The size of the last MEP in the rTES trains and the size of
the MEP evoked by single TES during muscle contraction were compared in a one-way ANOVA with condition (relaxation vs. muscle
contraction) as main factor. Changes in MEP amplitude and latency

Fig. 1. Changes in motor evoked potential (MEP) amplitude, expressed as a percentage of the rst MEP in the train, during the course of repetitive transcranial magnetic
stimulation (rTMS, continuous line) and repetitive transcranial electrical stimulation
(rTES, dashed line).

during the course of the train were analysed in a two-way ANOVA

with stimulation and number of stimuli as main factors. The
effects of repetitive SO nerve stimulation on the MEP amplitude
were tested in a one-way ANOVA with condition (conditioned
vs. unconditioned MEP) as main factor. All data are expressed as
means S.E. p values less than 0.05 were considered to indicate
statistical signicance.
None of the rTES or rTMS procedures caused any of the participants to experience adverse effects. All subjects complained of
pain during rTES. The TES and rTES intensity was 60.5 2.0% of the
stimulator maximal output.
No signicant difference was found in the size of the rst MEP
in the rTES and rTMS trains (0.36 0.07 mV vs. 0.39 0.09 mV;
F(1,3) =1.28; p = 0.33). Conversely, the rst MEP in the rTES trains was
signicantly shorter in latency than the rst MEP in the rTMS trains
(19.7 0.17 ms vs. 22.8 0.4 ms; F(1,3) = 23.9; p = 0.016).
Two-way ANOVA of MEP amplitudes during the course of rTES
and rTMS showed a signicant effect of factor number of stimuli
(F(9,27) = 4.32; p = 0.0014), without a signicant two-way interaction
(F(9,27) = 0.08, p = 0.99). rTES and rTMS increased the MEP size to a
similar extent (X/I MEP ratio 3.36 1.02 vs. 3.07 0.79; F(1,3) = 0.03;
p = 0.85) (Fig. 1).
The last MEP in the rTES trains was signicantly larger than
the MEP evoked by single-pulse TES delivered during the target
muscle contraction (1.09 0.21 mV vs. 0.54 0.08 mV; F(1,3) = 11.3,
p = 0.043).
Two-way ANOVA of MEP latencies during the course of rTES
and rTMS showed a signicant effect of factor stimulation
(F(1,3) = 36.8; p = 0.008), without a signicant two-way interaction
(F(9,27) = 0.7, p = 0.6). Post hoc analysis showed that MEP latency was
shorter during rTES trains than during rTMS trains, without signicant changes during the course of train of stimuli (last MEP latency:
rTES 19.7 0.17 ms; rTMS 22.5 0.4 ms; F(9,27) = 0.4; p = 0.8).
The perception threshold ranged between 1.7 and 2 mA. All
subjects complained of pain during repetitive SO nerve stimulation. ANOVA showed that repetitive SO nerve stimulation left the
conditioned MEP unchanged in amplitude (unconditioned MEP:
0.3 0.06 mV; conditioned MEP: 0.3 0.04 ms; F(1,4) = 0.2; p = 0.4).
Our data in healthy subjects show that MEP amplitudes increase
to a similar extent during rTMS and rTES trains. Although the rst
MEP in the train has a similar amplitude during rTMS and rTES
trains its latency is signicantly shorter during rTES than during
rTMS trains and remains unchanged throughout the train.
We took several precautions to avoid technical problems related
to stimulation and recording techniques. We can exclude the possibility that MEP facilitations arose from pain produced by rTES
because studies showed that nociceptive CO2 -laser stimulation

F. Gilio et al. / Neuroscience Letters 455 (2009) 13

reduces rather than facilitates the MEP amplitude [21] suggesting

that nociceptive system activation inhibits MEPs. Nor did the MEP
facilitation reect the muscle contraction induced by pain during
rTES because subjects were asked to completely relax, trials with
EMG activity were rejected and the rst MEP in the rTES and rTMS
trains had similar amplitudes. Finally, evidence arguing against
the possibility that the rTES-induced MEP facilitation depended on
pain-related muscle contractions is that painful repetitive trigeminal nerve stimulation left the MEP amplitude unchanged. Moreover,
the amplitude of the MEP evoked during FDI contraction was higher
than the amplitude of the rst MEP in the rTES train but smaller than
the last MEP in the train. We can also exclude effects on MEP facilitation depending on higher attentional levels during rTES than during
rTMS. A recent study in our laboratory showed that attentional processes increase the MEP size facilitation when subjects were asked
to focus their attention on the stimulated hand [6]. However, the
control experiment using repetitive electrical SO-nerve stimulation inducing a similar cutaneous sensation failed to increase the
conditioned MEP amplitude.
Other possibilities are that MEP amplitude facilitation depended
on an increased amount and distribution of electrical current
induced in the brain by subsequent electrical stimuli or increased
excitability of the cortico-spinal axons, or both mechanisms. These
hypotheses nevertheless seem unlikely because we would then
expect not only an increase in the MEP size during rTES trains
but also a progressive reduction in the MEP latency due to
spread to more distal parts of the cortical axons [13]. Moreover,
previous studies have shown that the 5 Hz-rTMS-induced MEP facilitation does not depend on increased cortico-spinal excitability
[4].
Our nding that MEP amplitudes increased to a similar extent
during rTMS and rTES trains delivered over M1 in healthy subjects at rest provides new information suggesting that at stimulus
intensities needed to induce a MEP at rest, rTES probably facilitates intracortical circuits similarly to rTMS [4,11,14]. TES activates
corticospinal bres directly at the axon-hillock thus producing
D waves [1,9,19,20]. Studies on the descending volleys after TES
have shown that in healthy subjects at rest low stimulation intensity TES produces an early D wave along the corticospinal tract,
without producing a motor response [7,9,19]. At the intensities
needed to induce a MEP at rest, the early wave is followed
by later I waves, suggesting that TES also activates the corticospinal tract trans-synaptically. When TES is used in experimental
study protocols designed to test subcortical activation of the
pyramidal tract [2,4,12,16] the ndings therefore need interpreting with caution because cortical synaptic inuences cannot be
excluded.
Previous studies on the MEP amplitude facilitation during short
rTMS trains delivered at high frequency over the M1 have shown
that rTMS increases cortical excitability mainly through mechanisms resembling those of the short-term synaptic enhancement
described in animal experiments [4,5,10,14]. Short-term synaptic
enhancement involves several processes, including synaptic potentiation, augmentation and post-tetanic potentiation, lasting from
milliseconds to tens of seconds, and depending on mechanisms
acting at post-synaptic level on NMDA-receptor-mediated components of the excitatory post-synaptic potentials [5,10,14]. These
short-term processes enhance synaptic strength promoting the
subsequent expression of long-term synaptic potentiation [5]. The
rTMS-induced MEP facilitation over the M1 also reects inuences
from distant areas [6].

Finally, because TES and TMS applied to the motor cortex

in humans may activate the corticospinal neurons directly and
indirectly with a different threshold for evoking the various components of the descending volley (D-wave, I1-wave and later I-waves)
a further hypothesis to take into consideration is that rTES, similarly
to rTMS, increases cortical excitability by modifying the threshold
of structures evoking later waves [17,19].
In conclusion, our ndings suggest that, similarly to rTMS, rTES
modulates the excitability of intracortical circuits and probably
does so by acting on synaptic mechanisms.
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