Brain Stimulation 6 (2013) 424e432

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Brain Stimulation
journal homepage: www.brainstimjrnl.com

Original Articles

Induction of Late LTP-Like Plasticity in the Human Motor Cortex by Repeated

Non-Invasive Brain Stimulation
Katia Monte-Silva 1, Min-Fang Kuo 1, Silvia Hessenthaler, Shane Fresnoza, David Liebetanz, Walter Paulus,
Michael A. Nitsche*
Georg-August-University, Dept. Clinical Neurophysiology, Robert-Koch-Strasse 40, 37099 Goettingen, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 27 October 2011
Received in revised form
20 February 2012
Accepted 24 April 2012
Available online 12 June 2012

Background: Non-invasive brain stimulation enables the induction of neuroplasticity in humans,

however, with so far restricted duration of the respective cortical excitability modications. Conventional
anodal transcranial direct current stimulation (tDCS) protocols including one stimulation session induce
NMDA receptor-dependent excitability enhancements lasting for about 1 h.
Objective: We aimed to extend the duration of tDCS effects by periodic stimulation, consisting of two
stimulation sessions, since periodic stimulation protocols are able to induce neuroplastic excitability
alterations stable for days or weeks, termed late phase long term potentiation (l-LTP), in animal slice
preparations. Since both, l-LTP and long term memory formation, require gene expression and protein
synthesis, and glutamatergic receptor activity modications, l-LTP might be a candidate mechanism for
the formation of long term memory.
Methods: The impact of two consecutive tDCS sessions on cortical excitability was probed in the motor
cortex of healthy humans, and compared to that of a single tDCS session. The second stimulation was
applied without an interval (temporally contiguous tDCS), during the after-effects of the rst stimulation
(during after-effects; 3, or 20 min interval), or after the after-effects of the rst stimulation had vanished
(post after-effects; 3 or 24 h interval).
Results: The during after-effects condition resulted in an initially reduced, but then relevantly prolonged
excitability enhancement, which was blocked by an NMDA receptor antagonist. The other conditions
resulted in an abolishment, or a calcium channel-dependent reversal of neuroplasticity.
Conclusion: Repeated tDCS within a specic time window is able to induce l-LTP-like plasticity in the
human motor cortex.
2013 Elsevier Inc. All rights reserved.

Keywords:
Transcranial direct current stimulation
Human
Motor cortex
Plasticity
Late phase LTP

Introduction
Long term potentiation (LTP), the enduring functional
enhancement of synaptic connections, or structural modication of
neuronal connectivity, is an important neurophysiological underlying mechanism of learning and memory processes [1e4]. The
most important kind of cortical LTP involves the glutamatergic
system. Based on in vitro slice electrical stimulation experiments,
different forms of LTP have been dened, dependent on the duration of the accomplished excitability enhancements.
Early LTP (e-LTP) is discerned from late LTP (l-LTP) by excitability
alterations lasting for more than 3 h [5,6]. Whereas for induction of
e-LTP a single stimulation session is sufcient, for l-LTP generation
* Corresponding author. Tel.: 49 551399571; fax: 49 551398621.
E-mail address: mnitsch1@gwdg.de (M.A. Nitsche).
1
These authors contributed equally.
1935-861X/$ e see front matter 2013 Elsevier Inc. All rights reserved.
doi:10.1016/j.brs.2012.04.011

in brain slices two or more stimulation sessions within a critical

time window of about 30 min after the rst stimulation are usually
necessary [6]. Spaced stimulation protocols have been shown to
induce long-lasting plasticity not only in animal slice preparations,
but also in in vivo animal studies, although with different time
windows [7]. E-LTP depends on activation of calcium-dependent
kinases, e.g. the Ca2/calmodulin-dependent kinases (CaMKs),
which control the trafcking of AMPA, and NMDA receptors to the
subsynaptic membrane [2]. l-LTP requires gene expression and
protein synthesis to accomplish alterations of synaptic strength
[5,8], also involving AMPA and NMDA receptor activity modications [9,10]. Similar mechanisms have been identied for the
formation of long term memory, which is dened as the permanent
storage of acquired information [11,12], for which l-LTP might be
a candidate mechanism.
Although rst explored in animals, at present, non-invasive
brain stimulation allows the induction of related neuroplastic

K. Monte-Silva et al. / Brain Stimulation 6 (2013) 424e432

excitability alterations in humans [13]. Repetitive transcranial

magnetic stimulation (rTMS), paired associative stimulation (PAS),
and transcranial direct current stimulation (tDCS) are able to induce
cortical excitability alterations, which depend, similar to LTP in
animals, on glutamatergic mechanisms [14e18]. For most protocols,
the duration of these effects is similar to that of e-LTP. In some
studies, however, periodical stimulation with an interval of 24 h
showed enhanced efcacy of the second stimulation protocol with
regard to motor cortex excitability alterations, being in favor for
a cumulative effect of spaced stimulation, which might share some
similarities with l-LTP, although the duration of the inter-session
intervals differ to that of the animal slice experiments [19,20].
Indirect evidence for a benecial effect of spaced, as compared to
massed intervention, which might be due to the induction of l-LTPlike processes in humans, is available from learning experiments,
where it was shown that spaced learning e typically with short
intervals of a few minutes, results in improved performance
[21e27].
We aimed to induce l-LTP-like plasticity in the human motor
cortex by periodical tDCS. tDCS induces neuroplasticity by
subthreshold neuronal membrane resting potential modication
through application of direct currents, and the after-effects are
NMDA receptor-dependent. We chose a single anodal tDCS duration
of 13 min, because this stimulation duration induces motor-cortical
excitability enhancements of about 60 min duration [28], which is
in the range of e-LTP. We applied one control session of a single
session of 13 min tDCS, as well as two sessions of 13 min anodal
tDCS with different inter-stimulation intervals to induce l-LTP-like
plasticity. The inter-stimulation intervals were zero (temporally
contiguous stimulation for 26 min), 3, and 20 min (during aftereffects of the rst stimulation), and 3 and 24 h (after the aftereffects of the rst stimulation). We chose these intervals, because
protocols inducing l-LTP-like plasticity are heterogeneous, as
shown above. For the 26 min contiguous stimulation condition, we
hypothesized that the resulting excitability diminution might be
caused by a calcium overow-caused neuronal counter-regulation,
since the after-effects of tDCS are calcium-dependent [15], and
calcium overow can result in a reduction of cortical excitability
[29]. To test this hypothesis, we applied a calcium channel antagonist in this stimulation condition in another control experiment.
Furthermore, the determinants of l-LTP-like plasticity induced by
the 20 min break condition were explored by NMDA receptor block
during the after-effects in another control experiment. We conducted this control experiment because tDCS is known to induce
plasticity of the glutamatergic system [15,16], and l-LTP involves
NMDA receptors in animal experiments [9,10].

425

probed. All gave written informed consent. The study was approved
by the ethics committee of University of Goettingen, and the
experiments conformed to the principles laid down in the Declaration of Helsinki.
Direct current stimulation of the motor cortex
In all three experiments, direct currents were transferred via
a pair of saline-soaked surface sponge electrodes (35 cm2) xed to
the scalp and delivered by a specially developed battery-driven
constant current stimulator (Schneider Electronic, Gleichen,
Germany) with a maximum output of 2 mA. The motor-cortical
electrode (the anode) was placed over the representational eld
of the right abductor digiti minimi muscle (ADM) as identied by
transcranial magnetic stimulation (TMS), and the other electrode
(the cathode) was located contralaterally above the right orbit.
Anodal tDCS of the left primary motor cortex was performed for
13 min (or 26 min in one of the conditions, see below) with an
intensity of 1.0 mA. This stimulation duration has been shown to
elicit a motor cortex excitability enhancement lasting for about 1 h
in former experiments [28].
Pharmacological intervention
In experiment 2, 2 h before tDCS 10 mg unarizine (FLU),
a calcium channel antagonist penetrating into the central nervous
system, was orally administered. Two hours after oral intake,
a sufcient plasma level of FLU is achieved [30], and the respective
dosage elicits prominent effects in the central nervous system
[15,31,32], but does not affect single pulse TMS-elicited MEP
amplitudes [15], and thus does not affect monitoring of cortical
excitability without the presence of plasticity induction protocols.
In experiment 3, a single oral dose of 150 mg dextromethorphan
(DMO), an NMDA receptor antagonist, was administered the day
after the application of the 20 min interval tDCS protocols 2 h
before the next morning MEP measurement, to explore selectively
the NMDA receptor-dependency of the l-LTP-like effects. The
dosage and timing of drug application were chosen according to the
pharmacokinetics [33] to ensure a sufcient plasma level [17]. This
dosage does not affect motor-evoked potential amplitudes (MEP)
elicited by single pulse transcranial magnetic stimulation without
prior plasticity induction [34], and thus will not affect monitoring of
cortical excitability without the presence of plasticity induction
protocols. Although beyond its NMDA receptor antagonistic effects,
DMO has been shown to block sodium and calcium channels at very
high dosages, the latter effects are not present at the chosen
dosage [17].

Material and methods

Monitoring of motor-cortical excitability
Subjects
Fifteen healthy right-handed subjects (mean age 25.5  3.6; 9
females) who were not under the effects of any acute or chronic and
potentially interfering pharmacological treatment participated in
the main experiment (experiment 1, repeated tDCS without pharmacological intervention). Five of the subjects from experiment 1
(mean age 24.6  3.1; 3 females), who agreed to participate in
another experimental session including administration of a calcium
channel blocker, were included in experiment 2 (26 min continuous
anodal tDCS under calcium channel block). Six subjects (mean age
26.2  1.2; 3 females), who showed long-lasting excitability
enhancement in the during after-effect (20 min interval) condition (3 of them participated already in experiment 1) agreed to
participate in experiment 3, in which the impact of NMDA receptor
block on the after-effects of this plasticity induction protocol was

TMS-elicited motor-evoked potentials (MEPs) were recorded to

measure excitability changes of the motor cortex representation
area of the right ADM. Single pulse TMS was conducted by a Magstim 200 magnetic stimulator (Magstim Company, Whiteland,
Dyfed, UK) with a gure-of-eight magnetic coil (diameter of one
winding 70 mm, peak magnetic eld 2.2 T). The coil was held
tangentially to the skull, with the handle pointing backwards and
laterally at an angle of 45 from midline. The optimal position was
dened as the site where stimulation resulted consistently in the
largest MEPs. Surface EMG was recorded from the right ADM with
AgeAgCl electrodes in a belly-tendon montage. The signals were
amplied and ltered with a time constant of 10 ms and a low-pass
lter of 2.5 kHz, then digitized at an analog-to-digital rate of 5 kHz
and further relayed into a laboratory computer using the Signal
software and CED 1401 hardware (Cambridge Electronic Design,

426

K. Monte-Silva et al. / Brain Stimulation 6 (2013) 424e432

Cambridge, UK). The intensity was adjusted to elicit baseline MEPs

of, on average, 1 mV peak-to-peak amplitude and was kept constant
for the post-stimulation assessment. We chose this stimulation
intensity, because it allows obtaining motor cortex excitability
enhancements or reductions without the danger of bottom or
ceiling effects.

stimuli before the respective after-effect measures. The EMG

electrode and coil positions were marked with a water-proof pen to
guarantee identical positions during the whole course of the
experiments. Baseline MEP amplitudes and percentage of maximal
stimulator output did not show major differences between sessions
(Table 1). Experimental procedures are summarized in Fig. 1.

Experimental procedures

Experiment 2
In this control experiment, the procedure was identical to that of
26 min contiguous tDCS with the exception that unarizine (FLU)
was administered 2 h before the plasticity induction procedure.
Two hours after intake of FLU, a second baseline (baseline 2) was
determined to control for a possible inuence of the drug on
cortical excitability and adjusted if necessary (baseline 3). Aftereffects of tDCS on excitability were monitored for 120 min after
the end of stimulation.

Experiment 1
Experiment 1 included 6 experimental conditions which were
conducted in a repeated measures, randomized complete crossover
design. This means that all subjects completed all 6 sessions of this
experiment. The experimental sessions were separated by at least
one week to exclude interference between the stimulation conditions. They took place in an air-conditioned laboratory with
a constant temperature of 20  C to guarantee constant skin
temperature throughout the experimental sessions. The volunteers
were seated in a comfortable chair, with head and arm rests. The
left motor-cortical representational eld of the right abductor digiti
minimi muscle (ADM) was identied using TMS (the coil position
which produced the largest MEPs in the resting ADM). Identication of the hot-spot took about 20e30 min in each subject. For all
stimulation protocols, the intensity of the magnetic cortical stimulus was adjusted to elicit MEPs with a peak-to-peak amplitude of
on average 1 mV. Determination of baseline TMS intensity took
5e10 min for each subject. 25 MEPs were recorded after stable MEP
amplitudes were obtained at a frequency of 0.25 Hz for baseline
determination. The motor cortex tDCS electrode (anode) was xed
on the ADM hot-spot and the other (the cathode) was xed at the
contralateral forehead above the orbit. Afterwards motor-cortical
tDCS was performed as follows:
(a) continuous application of one or two 13 min anodal tDCS
sessions (13e0e0 or 13e0e13);
(b) two 13 min long blocks of tDCS with a short interval of 3 or
20 min (during after-effects; 13e3e13 or 13e20e13);
(c) two 13 min long blocks of tDCS with a long interval of 3 or 24 h
(after after-effects; 13e3 he13 or 13e24 he13).
Immediately after the last tDCS session, 25 MEPs were recorded
every 5 min at 0.25 Hz for half an hour, and then every 30 min up to
2 h after the end of each stimulation. The TMS recordings were also
performed at four additional time points: same day evening (se,
mean interval between the 120 min and the se measure 236  88
(standard deviation) min), next morning (nm), next afternoon (na),
and next evening (ne). Since we did not expect longer-lasting aftereffects induced by the single 13 min anodal tDCS session, and
a control experiment (see Suppl. material, Fig. S1) show no effects of
a single 13 min session of tDCS the day after stimulation, here the
after-effects were evaluated until same evening after tDCS. To
guarantee optimal coil positioning during the course of the experiment, it was regularly checked if the actual coil position in threedimensional space elicited the largest MEPs during the after-effect
measures. This required usually obtaining about ve to seven TMS

Experiment 3
The protocol of two consecutive tDCS with 20 min interval was
chosen, and subjects took DMO 2 h before MEP measurement next
morning, the time point where a trend of peak excitability
enhancement was observed in Experiment 1 (Fig. 2B).
Data analysis and statistics
Experiment 1
MEP amplitude means were calculated for each time bin covering
baseline and post-stimulation values. The post-stimulation MEPs
were normalized intra-individually and are given as baseline ratios.
An analysis of variance (ANOVA) model for repeated measures for
the time bins up to the evening of the day of tDCS application was
calculated with the dependent variable MEP amplitude, and with
time course, and tDCS condition as within-subjects factors. Additionally Fishers LSD tests were used to test whether baseline MEP
amplitudes differed signicantly between the tDCS protocols,
whether the MEP amplitudes after tDCS differed signicantly from
the pre-DC amplitudes, and whether the MEP amplitudes of the
repeated tDCS conditions differed from the 13e0e0 protocol. A P
value of <0.05 was considered signicant for all statistical analyses.
All results are given as mean and standard error of the mean (SEM).
The Mauchly test of sphericity was conducted and GreenhouseGeisser correction was performed when appropriate.
Experiment 2 and 3
The procedures were identical to that described for experiment
1 with the exception that we conducted a two-factorial repeated
measures ANOVA with the within-subject factors time, and
medication (with vs. without FLU or DMO).
Results
With the exception of a slight itching sensation under the tDCS
electrodes mentioned by some participants during stimulation, no
other side effects of tDCS or medication were reported.

Table 1
Baseline MEP amplitudes and TMS stimulation intensity of experiment 1.
Protocol

13e0e0

13e0e13

13e3e13

13e-20e13

13e3 he13

13e24 he13

MEP (mV)
%MSO

0.947  0.10
45.7  0.07

1.003  0.10
43.9  0.07

0.947  13
45.1  0.06

0.890  10
44.3  0.08

0.951  0.13
44.5  0.06

0.999  0.12
44.3  0.06

Baseline MEP amplitudes were close to 1 mV in all experimental conditions. Slight but signicant differences were obtained between the 13e20e13 protocol on the one hand,
and the 13e0e13, and 13e24 he13 on the other, which did however not affect the main analyses. Stimulation intensity to induce MEP amplitude sizes of about 1 mV
(in percentage of maximal stimulator output (%MSO) did not differ between all experimental sessions (Fishers LSD tests, P > 0.05). Values are given as means  standard
deviation (sd).

K. Monte-Silva et al. / Brain Stimulation 6 (2013) 424e432

Current Stimulation

Baseline MEPs
Stimulation
Parameters

427

Single Pulse TMS

Post tDCS MEPs

Repeated
Anodal tDCS

0.25 Hz

Single Pulse TMS

0.25 Hz

1mA

1mV

1mV

13-0-0 protocol
13 min
tDCS

Short intervals

13-0-13 protocol
13 min

13 min

tDCS

tDCS

13-3-13 protocol

BASELINE

Experiment 1

No intervals

13 min

3min

tDCS

13 min
tDCS

break

13-20-13 protocol
13 min

20 min

13 min

tDCS

break

tDCS

13-3h-13 protocol
13 min

3 hours

tDCS

Long intervals

13 min

24 hours

10 15 20 25 30

60

90

120

g)
enin

t ev

g)

o on
t er n

(ne
x

(nex

t af

orn
in

na

ne

(s a
me

tDCS

2 hours

nm

13 min

tDCS

eve

13-0-13 protocol
13 min

(ne
xt m

nin
g)

tDCS

break

se

B ASE LI NE 3

B ASE LI NE 2

B ASE LI NE 1

tDCS

10 mg of FLU

tDCS

13-24h-13 protocol
13 min

Experiment 2

13 min

break

Time after tDCS (minutes)

Figure 1. Experimental course. In experiment 1, the effect of different intervals of 13 min anodal tDCS on motor cortex excitability, as monitored via single pulse TMS, was explored.
Specically, 13 min stimulation only (13e0e0 protocol), and 26 min continuous stimulation (13e0e13) were compared with two 13 min stimulation protocol separated by short
(3 min (13e3e13); 20 min (13e20e13)) or long (3 h (13e3 he13); 24 h (13e24 he13)) intervals. With the exception of the 13e0e0 protocol, where excitability measures were
terminated the evening of the day of tDCS application, excitability was monitored up to the evening after the (second) DC stimulation. A complete crossover design was performed.
Experimental sessions were separated by at least one week from each other, and the different tDCS protocols were applied in randomized order. In experiment 2, we explored the
effect of unarizine, a calcium channel blocker, on the excitability diminution induced by 26 min continuous tDCS.

Experiment 1
Baseline MEP amplitudes did not differ between the respective
tDCS conditions, with the exception that there was a slight, but
signicant, difference between the 13e20e13 (MEP amplitude size
0.9 mV) condition on the one hand, and the 13e0e13 (MEP
amplitude size 1.0 mV), and 13e24 he13 (MEP amplitude size
1.0 mV) on the other (pairwise comparisons between all combinations of conditions, Table 1). Baseline TMS intensity in percentage
of maximum stimulator output was identical between conditions
(Table 1; pairwise comparisons between all combinations of
conditions). Since the MEP differences were small, TMS intensities
between conditions did not differ, and the MEP amplitudes of the
directly compared (see below) 13e0e0 and the 13e20e13 condition did not differ signicantly, this should not have a relevant
impact on the results of this study.
The ANOVA resulted in a signicant interaction of tDCS
condition  time course, and a signicant main effect of tDCS
condition (Table 2). As shown by the post-hoc tests, the single
session of anodal tDCS for 13 min resulted in an excitability
enhancement of about 20% versus baseline which was signicant
for up to 60 min after stimulation (signicant results of the posthoc tests for contrasts comparing baseline with post-tDCS data

from ve to 25 min after tDCS, and 60 min after tDCS). 26 min

contiguous stimulation-induced a signicant reduction of motor
cortex excitability of about 20% compared to baseline lasting
approximately equally long (signicant results of the post-hoc tests
for contrasts comparing baseline with post-tDCS data immediately
after, and 5 min after tDCS, 15e25 min, 60 and 120 min after tDCS;
signicant results of the post-hoc tests for contrasts comparing the
13e0e0 with the 13e0e13 condition for time points immediately
after stimulation up to 60 min after stimulation). If 13 min tDCS was
repeated during the after-effects of the rst stimulation (13e3e13
or 13e20e13), an initial excitability enhancement of about 10e20
percent compared to baseline MEP amplitudes was seen only
trendwise during the rst 30 min after tDCS (non-signicant results
of the post-hoc tests for contrasts comparing baseline with posttDCS data), with the exception of a signicant excitability
enhancement immediately after tDCS in the 13e3e13 condition,
and completely disappeared 30 (13e3e13), or 90 min (13e20e13
condition) after stimulation. Some hours later however, at the
evening of the stimulation day, excitability was again signicantly
enhanced for up to about 30 percent as compared to baseline MEPs,
and MEP values of the 13e0e0 condition at the same time point
(signicant results of the post-hoc tests for comparisons of 13e0e0
condition versus 13e3e13, and 13e20e13 conditions). This

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K. Monte-Silva et al. / Brain Stimulation 6 (2013) 424e432

Figure 2. Inter-stimulation interval determines the effects of anodal tDCS on motor cortex excitability. As can be seen from the results of the study, interval duration determines the
effects of repeated anodal tDCS on motor cortex excitability. (A) A single session of 13 min anodal tDCS (13e0e0) enhances excitability up to 60 min after DC stimulation. (B)
Prolonging stimulation duration for 26 min (13e0e13) however converts the after-effects into excitability diminution. An inter tDCS-interval of a few minutes (13e3e13, 13e20e13
protocols) primarily diminishes the efcacy of tDCS to enhance excitability, which however is present trendwise up to about 90 min after tDCS. From the evening of the stimulation
day on for up to the next evening, however, motor cortex excitability is again enhanced signicantly. This late excitability enhancement is more prominent for the 20 min interval
condition. (C) Prolonging the inter tDCS-intervals for 3 or 24 h (13e3 he13, 13e24 he13 protocols) however abolishes the excitability enhancement, and turns it slightly into
inhibition. Filled symbols indicate signicant differences from baseline MEPs, asterisks signicant differences of the effects of 13 min only tDCS on MEP amplitudes at identical time
points (P  0.05), and error bars standard deviation of the mean (SEM). se same evening; nm next morning, na next afternoon, ne next evening.

excitability enhancement remained signicant until the evening

after the day tDCS was applied (results of the post-hoc tests for
contrasts comparing baseline with post-tDCS data next morning,
afternoon and evening for the 13e3e13 condition, and next afternoon and evening for the 13e20e13 condition), and thus for more
than 24 h. For the long interval conditions (second stimulation after
the after-effects of the rst stimulation), no signicant excitability
enhancement was seen for the whole course of the experiments.

Instead, a slight, but signicant reduction of excitability (about 10%

as compared to baseline values) took place after the second
stimulation (signicant results of the post-hoc tests for contrasts
comparing baseline values with post-tDCS MEP amplitudes for the
20 min time point in the 13e3 he13, and for the 5 min time point in
the 13e24 he13 condition, signicant results of the post-hoc test
for contrasts comparing the 13e0e0 with the 13e3 he13 condition
for time points 5, 20, 30, and 60 min after tDCS, and for the

K. Monte-Silva et al. / Brain Stimulation 6 (2013) 424e432

Experiment 3

Table 2
Results of the ANOVAs.

Experiment 2

Experiment 3

Time course
tDCS condition
Time course  tDCS condition
Time course
Medication
Time course  Medication
Time course
Medication
Time course  Medication

D.f.

F-value

P-value

11
5
11
10
1
10
14
1
14

1.392
4.239
1.638
1.704
30.634
1.176
2.461
1.331
3.059

0.182
0.002a
0.003a
0.114
0.005a
0.335
0.007a
0.301
0.001a

In experiment 1, the ANOVA resulted in a signicant main effect of time course,

and a signicant tDCS condition  time course interaction. The ANOVA reveals
a signicant main effect of medication in experiment 2. In experiment 3, the ANOVA
shows a signicant main effect of time course and a signicant interaction of
medication  time course.
a
Indicates signicant effects (P  0.05).

contrasts comparing the 13e0e0 with the 13e24 he13 condition

for time point 5 min after tDCS) (Fig. 2AeD).
Experiment 2

MEP amplitude normalized by pretDCS baseline

The ANOVA revealed a signicant main effect of medication

(Table 2). FLU abolished the induction of inhibitory after-effects by
26 min anodal stimulation. As can be seen in Fig. 3, 26 min anodal
tDCS-induced a signicant excitability diminution without medication (signicant results of the post-hoc tests for the contrasts
comparing baseline with post-tDCS values from 5 to 90 min after
tDCS), which was not present under calcium channel block by FLU
(no signicant results in the respective post-hoc tests comparing
baseline with post-tDCS MEP values). Consequently, MEP values
differed signicantly between FLU, and placebo medication conditions for the time points 5, 20, and 25 min, as revealed by the
respective post-hoc contrasts. FLU did not affect MEP baseline values
(baseline 1 1.002 mV  0.143 sd; baseline 2 1.008 mV  0.105 sd;
two-tailed paired Students t-test P 0.913), and baseline MEPs
were identical between the experimental sessions with and without
FLU (baseline 3 with FLU 1.008 mV  0.105; baseline without FLU
1.092 mV  0.058; two-tailed paired Students t-test P 0.233).

1.5

FLU

1.3

none

1.2
1.1
1.0

0.8

Discussion
This study shows that periodical anodal tDCS is able to induce
long-lasting, l-LTP-like excitability enhancements of the primary
motor cortex. This effect critically depends on the duration of the
interval between tDCS applications. If the second stimulation was
performed during the after-effects of the rst one (as it was the case
with an interval of 3 or 20 min), the combined after-effects of the
two blocks were present for more than 24 h after tDCS. In contrast,
an interval of 3 or 24 h abolished the after-effects of tDCS. These
excitability alterations obtained by spaced tDCS cannot be caused
by total stimulation duration alone, because two contiguous blocks
of 13 min of anodal tDCS, i.e., the same total stimulation duration

Medication

tDCS condition 13-0-13

1.4

0.9

The ANOVA showed a signicant interaction of time course 

medication, and a signicant main effect of time course (Table 2).
Cortical excitability was trendwise increased after repetitive anodal
tDCS with 20 min break for up to 120 min after tDCS (no signicant
results in the respective post-hoc tests comparing baseline with
post-tDCS MEP values with the exception of the 15 min time point),
and was signicantly enhanced the same evening until next day
noon without medication (signicant results in the respective posthoc tests comparing baseline with post-tDCS MEP values for the time
points same evening, next morning, and next noon). A similar
effect of this tDCS protocol was observed in the DMO condition
before the drug was applied (no signicant results in the
respective post-hoc tests comparing baseline with post-tDCS MEP
values with the exception of the time points 25, and 60 min after
tDCS; no signicant results for contrasts comparing no medication
and DMO for the respective time points). When given the morning
after tDCS, DMO abolished the long-lasting excitability enhancement. Post-hoc tests revealed that the blocking effect was signicant
for the MEP measurement of next morning, 2 h after DMO intake,
and lasted until next noon (no signicant results in the respective
post-hoc tests comparing baseline with post-tDCS MEP values in the
DMO condition; signicant results for contrasts comparing no
medication and DMO for the respective time points next morning
and next noon) (Fig. 4).

*
*

0.7
0.6

MEP amplitude normalized by pre-tDCS

baseline

Experiment 1

429

3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4

tDCS condition 13-20-13

Medication
DMO
None

*
*
0

5 10 15 20 25 30 60 90 120

se nm na ne

0.5
0

10

15

20

25

30

60

90

120

Time course (min)

Figure 3. The reduction of cortical excitability induced by 26 min anodal tDCS depends
on calcium channel activity. In a subgroup of participants, 26 min continuous anodal
tDCS was performed under the calcium channel blocker unarizine (FLU). As can be
seen by the results, FLU abolishes the excitability diminution induced by the prolonged
stimulation. Filled symbols indicate signicant differences between baseline and
post-tDCS MEPs, asterisks signicant differences between the effects of FLU on MEP
amplitudes at identical time points (P  0.05; error bars: SEM).

Time course (min)

Figure 4. The NMDA receptor antagonist DMO abolishes the late-onset excitability
enhancement induced by repeated tDCS with a short interval. A late-onset, long-lasting
enhancement of cortical excitability was observed when a second tDCS was applied
within the effective time window after the rst tDCS (13e20e13 protocol).
Dextromethorphan (DMO) blocked this l-LTP-like excitability enhancement when
administered 2 h before the surge of MEP next morning. Filled symbols indicate
signicant differences from baseline MEPs, and asterisks represent signicant
differences between the conditions with and without DMO (P  0.05; error bars: SEM).
se same evening; nm next morning, na next afternoon, ne next evening.

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K. Monte-Silva et al. / Brain Stimulation 6 (2013) 424e432

applied continuously without any tDCS-free interval in between,

resulted in reduced motor cortex excitability.
Induction of l-LTP-like plasticity by tDCS with an inter tDCS block
interval of minutes
If two anodal DC stimulations were spaced by 3 or 20 min,
a two-phasic response on MEP amplitudes resulted. An initial
excitability enhancement was present for up to 90 min after stimulation. Motor cortex excitability was at baseline level 2 h after
stimulation, but was enhanced again in the later excitability
measures. This late excitability enhancement, which might be
larger for the 20 min interval condition, was stable up to the
evening after the day of tDCS, and thus for over 24 h. Whereas the
early excitability enhancement induced by spaced tDCS is similar to
that induced by a single stimulation session, however somewhat
smaller, the later one differs relevantly. Moreover, the reduction of
motor cortex excitability between both MEP enhancements is
compatible with discernable mechanisms. The early excitability
enhancements might follow similar mechanisms as those induced
by a single tDCS session. These effects depend on NMDA receptors
and calcium channel activity [15,16], and thus share similarities
with glutamatergic early LTP, as described in animal slice preparations [2]. The slight reduction of their magnitude, as compared to
the single stimulation session, might be caused by homeostatic
effects (see below). For the later occurring excitability enhancements, these include at least some features of l-LTP. They last
considerably longer than 3 h, and are induced by repeated stimulation with an inter-stimulation interval of some minutes [5,8,35].
In contrast with e-LTP, l-LTP requires protein kinase A activity,
protein synthesis, and RNA synthesis [5], amongst others, to
stabilize enhancements of synaptic connectivity, in animal slice
studies. Furthermore, NMDA receptors are involved in the induction and maintenance of l-LTP [10], as suggested also in the present
study, where DMO, an NMDA receptor antagonist, abolished l-LTPlike plasticity. Interestingly, in the latter experiment (experiment
3), the after-effects of tDCS on MEP amplitudes show not the
reduction of excitability present in experiment 1 (13e20e13
condition) 120 min after stimulation. Experiment 3 only included
subjects showing l-LTP-like excitability changes, whereas the
respective condition in experiment 1 included subjects with and
without l-LTP-like after-effects. It could thus be speculated that the
respective reduction of excitability after 120 min can be at least
partially dedicated to the subjects showing no late phase plasticity.
Moreover, it might be that the biphasic excitability enhancement is
caused by different mechanisms of action. Protein and RNA
synthesis, which are essential features of l-LTP in animal slice
experiments, start with a delay of up to 4 h after plasticity induction
[6], and the excitability enhancement accomplished by these
mechanisms might only partially overlap with the early ones. The
presence of the respective excitability alterations the day after
stimulation might be furthermore supported by sleep-dependent
processes, since plasticity is known to be consolidated during
sleep [36,37].
Although these long-lasting alterations of synaptic efcacy share
some physiological similarities with late phase LTP, as obtained in
animal slice experiments, such as the relevance of a specic time
windows of some minutes for the induction of the after-effects, the
duration of the after-effects for more than 24 h, and their NMDA
receptor-dependency [6,9,10], the induction procedures in humans
and animal slices differ for some aspects, and the excitability
measure via monitoring of MEP amplitudes does not cover all
aspects of LTP. Thus the relationship between both phenomena
should be explored to a larger extent in future studies. Taking into
account the proposed relevance of l-LTP for long term memory

formation, the results of the present study are moreover in principal

accordance with the observation that periodical training with break
intervals of typically some minutes improves some forms of long
term motor memory in which the primary motor cortex is involved,
including adaption and consolidation [21e27]. However, although
it has been proposed that l-LTP is the physiological basis of long
term memory formation and consolidation, and both phenomena
share certain similarities such as NMDA receptor-dependency
[9,10,38,39], direct evidence in humans is missing at present, and
should be explored more directly in future studies.
Modulation of LTP-like plasticity by an inter tDCS block interval of
hours
In contrast to the effect of short intervals on plasticity, inter
tDCS-intervals of hours prevented any plasticity induction by the
second DC stimulation. The abolishment of facilitatory plasticity
might be due to homeostatic mechanisms, which is the alteration of
the efcacy of a stimulus to induce neuroplasticity dependent on
prior activity of the cortical network, neuron, or synapse, which
helps to avoid runaway excitation, and thus preserves the stability
of the network. Multiple cellular mechanisms of homeostatic
effects have been identied in animal and slice experimentation
[40]. Since tDCS induces calcium channel- and NMDA receptordependent plasticity, a calcium-mediated alteration of glutamatergic receptors [41] is an attractive candidate mechanism,
however the exact mechanism of action awaits further exploration.
Whereas homeostatic mechanisms have been identied in humans
before by combined application of different non-invasive brain
stimulation techniques [13,42e45], these were usually obtained by
an inter-stimulation interval of some minutes. Here much longer
intervals induce homeostatic effects. Interestingly, these occur
hours after the overt effects of tDCS on the MEP amplitude have
vanished. Future studies should explore if these are due to intracortical excitability changes, which are not necessarily paralleled by
modications of corticospinal excitability [46,47].
Conversion of the after-effects of tDCS by prolonged stimulation
The categorically different effects of 26 min continuous tDCS as
compared to spaced stimulation of the same total duration shows
that the effects of stimulation depend on timing, and not on total
stimulation duration. They are in good accordance with the observation that spaced learning results in better effects than massed
learning [22,27,48]. One likely mechanism of action of the conversion of the after-effects of tDCS is a neuronal counter-regulation,
which prevents over-excitation. Since the effects of tDCS on
cortical excitability are calcium-dependent, the activation of
hyperpolarizing potassium channels, which depends on intraneuronal calcium concentration [29], is a candidate mechanism.
Accordingly the conversion of the tDCS-induced excitability
diminution was abolished by calcium channel block via FLU.
General remarks
Some limiting aspects of the present study should be taken into
account. For experiment one, in the 13e0e0 condition, MEP
measures were terminated at the end of the rst day, and not
continued to the next day. This might hamper direct comparison
between the experimental conditions to some extent. In accordance
with the results of previous experiments [28], and a control
experiment (Supplemental material, Fig. S1), which show no
alteration of cortical excitability versus baseline after 13 min anodal
tDCS the day after plasticity induction), we do not expect a reoccurrence of after-effects more than 1 h after these had

K. Monte-Silva et al. / Brain Stimulation 6 (2013) 424e432

vanished. We did not perform a separate experimental session

without stimulation to control for a drift of MEP amplitudes,
because it has been shown recently that MEP amplitudes elicited by
single pulse TMS are stable for at least 24 h [49]. For the pharmacological control experiments, the number of subjects is relatively
small, however, the results look reasonable clear and stable.
Furthermore, DMO is not a pure NMDA receptor antagonist, but has
additional ion channel-blocking effects. A relevant effect of DMO on
ion channels is however improbable at the present dosage [17].
Since the pharmacological control studies were planned and
conducted after completion of experiment one, it was not possible
to conduct them in a randomized, double-blinded design, as far as
volunteers already involved in experiment one were tested. As
subjects were not aware of specic hypotheses, the occurrence of
expectancy effects is improbable in our opinion. An order effect for
these experiments cannot be ruled out completely, but is improbable, because most of the subjects included in these experiments
had already participated in six tDCS sessions before (experiment 1),
and the respective control experiments were conducted months
after experiment 1 in most of the subjects. Finally, although time
course and pharmacological characteristics of the neuroplastic
alterations accomplished by anodal stimulation with an interval of
a some minutes resemble some characteristics of late LTP, from the
results of the present study it cannot be concluded that these are
identical to late LTP, as obtained in animal experiments.
The results show similarities with another study of our group, in
which the effects of repeated excitability-diminishing cathodal
tDCS were explored [50]. Similarly, in that study conduction of the
second stimulation protocol during the after-effects of the rst one
enhanced efcacy of stimulation, although to a somewhat minor
extent, as compared to the results of the present study, whereas
a break duration of hours diminished the effects of tDCS. These
results are compatible with similar mechanisms of action for both,
anodal and cathodal, tDCS with regard to the effects of repeated
stimulation on prolongation of after-effects. However, doubling the
stimulation duration of cathodal tDCS without a break did not
convert the after-effects, but resulted in a prolongation. The reason
for this dissociation might be that cathodal tDCS at the present
intensity is expected to result in much lower calcium inux than
anodal tDCS, and thus even prolonged stimulation might not sufce
to enhance intracellular calcium to a level inducing LTP-like effects.
The results of the current study show brain stimulation-induced
l-LTP-like effects in the human motor cortex. They demonstrate that
this kind of plasticity is induced by periodical stimulation, and that
a specic time window is critical for its induction. Future experiments should explore the specic mechanisms of action to a larger
extent. It might also be speculated that optimal timing of plasticity
induction is suited to improve learning in humans, assuming that
l-LTP is one important physiological underlying mechanism of long
term memory formation. Moreover, the results might help to
improve the efcacy of tDCS as a therapeutic tool for the treatment
of neuropsychiatric diseases accompanied by pathological alterations of brain activity. Specically, the efcacy of tDCS to improve
motor functions after stroke, or depressive symptoms [51e53],
might be enhanced, if periodical stimulation protocols, as proposed
by the current study, are applied instead of stimulation once daily,
which is currently applied most often in clinical studies. However,
a one-to-one transferability of the present results obtained on
plasticity to behavioral and clinical effects is not self-evident. It
might be the case that combination of plasticity induction with
performance or in patients follow different rules of consolidation of
the effects than plasticity induction in the resting healthy brain.
Indeed, once daily stimulation has been shown to result in e in
some studies cumulative e effects on clinical symptoms [54,55].
Moreover, in the present study we accomplished only the

431

combination of two tDCS sessions, whereas in most clinical studies

tDCS was repeated on ve consecutive days or more, which might
also have an impact on the results. We only explored global
corticospinal excitability alterations induced by tDCS. The transferability of the results to stimulation protocols aiming to modify
behavior, or clinical symptoms, might be limited, because effects of
tDCS on other parameters not explored in the present experiments,
e.g. on intracortical facilitation, inhibition, i-wave facilitation [47],
the GABAergic system [56], or other molecular or cellular alterations, might contribute, and be not in complete accordance with
the global excitability measures. Future experiments should
explore these parameters, and their importance for the respective
functional alterations, to a larger extent. Finally, the results of the
13e0e13 tDCS sessions hints for a non-linear connection between
stimulation duration, and plasticity duration, and direction, in our
healthy volunteers group. This might mean that also for clinical
application, prolongation of stimulation duration might not result
in higher efcacy in each case. However, due to additional physiological differences between the subjects participating in the present
experiment, and patients suffering from neuropsychiatric diseases,
the transferability of these results e especially with regard to
specic stimulation parameters - to patients is hypothetical, and
should be explored directly in future studies.
Financial disclosures
MAN is member of the advisory board of Neuroelectrics. The
other authors declare no conict of interest.
Acknowledgments
KM-S was supported by CAPES, Brazil. This project was further
supported by the German Ministry for Education and Research,
Bernstein-Center for Computational Neuroscience, Goettingen, and
the Rose Foundation.
Supplementary material
Supplementary data related to this article can be found online at
doi:10.1016/j.brs.2012.04.011.
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