Brain Stimulation 6 (2013) 424e432

Contents lists available at SciVerse ScienceDirect

Brain Stimulation
journal homepage:

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 modifications. 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 modifications, 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 first stimulation
(during after-effects; 3, or 20 min interval), or after the after-effects of the first 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 specific time window is able to induce l-LTP-like plasticity in the
human motor cortex.
Ó 2013 Elsevier Inc. All rights reserved.

Transcranial direct current stimulation
Motor cortex
Late phase LTP

Long term potentiation (LTP), the enduring functional
enhancement of synaptic connections, or structural modification 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 defined, 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 sufficient, for l-LTP generation
* Corresponding author. Tel.: þ49 551399571; fax: þ49 551398621.
E-mail address: (M.A. Nitsche).
These authors contributed equally.
1935-861X/$ e see front matter Ó 2013 Elsevier Inc. All rights reserved.

in brain slices two or more stimulation sessions within a critical
time window of about 30 min after the first 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 Ca2þ/calmodulin-dependent kinases (CaMKs),
which control the trafficking 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 modifications [9,10]. Similar mechanisms have been identified for the
formation of long term memory, which is defined as the permanent
storage of acquired information [11,12], for which l-LTP might be
a candidate mechanism.
Although first explored in animals, at present, non-invasive
brain stimulation allows the induction of related neuroplastic

because this stimulation duration induces motor-cortical excitability enhancements of about 60 min duration [28]. DMO has been shown to block sodium and calcium channels at very high dosages. The motor-cortical electrode (the anode) was placed over the representational field of the right abductor digiti minimi muscle (ADM) as identified by transcranial magnetic stimulation (TMS). we hypothesized that the resulting excitability diminution might be caused by a calcium overflow-caused neuronal counter-regulation.2. 3. This stimulation duration has been shown to elicit a motor cortex excitability enhancement lasting for about 1 h in former experiments [28]. / Brain Stimulation 6 (2013) 424e432 excitability alterations in humans [13].K. 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. Direct current stimulation of the motor cortex In all three experiments.20]. however. and thus does not affect monitoring of cortical excitability without the presence of plasticity induction protocols. Two hours after oral intake.1. Repetitive transcranial magnetic stimulation (rTMS). is available from learning experiments.32]. Furthermore. an NMDA receptor antagonist. We chose these intervals. were included in experiment 2 (26 min continuous anodal tDCS under calcium channel block). although the duration of the inter-session intervals differ to that of the animal slice experiments [19. being in favor for a cumulative effect of spaced stimulation. see below) with an intensity of 1. results in improved performance [21e27]. who agreed to participate in another experimental session including administration of a calcium channel blocker. Indirect evidence for a beneficial effect of spaced.0 mA. 3 females). . We aimed to induce l-LTP-like plasticity in the human motor cortex by periodical tDCS. was administered the day after the application of the 20 min interval tDCS protocols 2 h before the next morning MEP measurement. which might share some similarities with l-LTP. Dyfed. as shown above. The dosage and timing of drug application were chosen according to the pharmacokinetics [33] to ensure a sufficient plasma level [17]. For the 26 min contiguous stimulation condition. because protocols inducing l-LTP-like plasticity are heterogeneous. and 3 and 24 h (after the aftereffects of the first stimulation). All gave written informed consent. The coil was held tangentially to the skull.16]. To test this hypothesis. Six subjects (mean age 26. which is in the range of e-LTP. the latter effects are not present at the chosen dosage [17]. 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. and 20 min (during aftereffects of the first stimulation).5 kHz. was orally administered. as well as two sessions of 13 min anodal tDCS with different inter-stimulation intervals to induce l-LTP-like plasticity. peak magnetic field ¼ 2. and calcium overflow can result in a reduction of cortical excitability [29]. since the after-effects of tDCS are calcium-dependent [15]. and the experiments conformed to the principles laid down in the Declaration of Helsinki. on glutamatergic mechanisms [14e18]. Single pulse TMS was conducted by a Magstim 200 magnetic stimulator (Magstim Company. Material and methods Monitoring of motor-cortical excitability Subjects Fifteen healthy right-handed subjects (mean age 25. and the after-effects are NMDA receptor-dependent. which might be due to the induction of l-LTPlike processes in humans. where it was shown that spaced learning e typically with short intervals of a few minutes. In experiment 3. 425 probed. and transcranial direct current stimulation (tDCS) are able to induce cortical excitability alterations. direct currents were transferred via a pair of saline-soaked surface sponge electrodes (35 cm2) fixed to the scalp and delivered by a specially developed battery-driven constant current stimulator (Schneider Electronic. which depend. Monte-Silva et al.2  1. paired associative stimulation (PAS). The optimal position was defined as the site where stimulation resulted consistently in the largest MEPs. a sufficient plasma level of FLU is achieved [30]. Whiteland. UK) with a figure-of-eight magnetic coil (diameter of one winding ¼ 70 mm. repeated tDCS without pharmacological intervention). to explore selectively the NMDA receptor-dependency of the l-LTP-like effects. The signals were amplified and filtered with a time constant of 10 ms and a low-pass filter of 2. similar to LTP in animals. tDCS induces neuroplasticity by subthreshold neuronal membrane resting potential modification through application of direct currents. Germany) with a maximum output of 2 mA. and thus will not affect monitoring of cortical excitability without the presence of plasticity induction protocols. We conducted this control experiment because tDCS is known to induce plasticity of the glutamatergic system [15. periodical stimulation with an interval of 24 h showed enhanced efficacy of the second stimulation protocol with regard to motor cortex excitability alterations. The inter-stimulation intervals were zero (temporally contiguous stimulation for 26 min). Although beyond its NMDA receptor antagonistic effects.5  3.2 T). with the handle pointing backwards and laterally at an angle of 45 from midline. We chose a single anodal tDCS duration of 13 min.10]. Anodal tDCS of the left primary motor cortex was performed for 13 min (or 26 min in one of the conditions. In some studies. a calcium channel antagonist penetrating into the central nervous system. Surface EMG was recorded from the right ADM with AgeAgCl electrodes in a belly-tendon montage. 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 3.6. Gleichen. 2 h before tDCS 10 mg flunarizine (FLU). the duration of these effects is similar to that of e-LTP.6  3. and the respective dosage elicits prominent effects in the central nervous system [15. 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. we applied a calcium channel antagonist in this stimulation condition in another control experiment. Five of the subjects from experiment 1 (mean age 24. but does not affect single pulse TMS-elicited MEP amplitudes [15]. and the other electrode (the cathode) was located contralaterally above the right orbit. The study was approved by the ethics committee of University of Goettingen. as compared to massed intervention. and l-LTP involves NMDA receptors in animal experiments [9. 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. Pharmacological intervention In experiment 2.31. a single oral dose of 150 mg dextromethorphan (DMO). We applied one control session of a single session of 13 min tDCS. 3 females). This dosage does not affect motor-evoked potential amplitudes (MEP) elicited by single pulse transcranial magnetic stimulation without prior plasticity induction [34]. For most protocols.

the intensity of the magnetic cortical stimulus was adjusted to elicit MEPs with a peak-to-peak amplitude of on average 1 mV. next morning (nm). 25 MEPs were recorded every 5 min at 0.06 Baseline MEP amplitudes were close to 1 mV in all experimental conditions. and subjects took DMO 2 h before MEP measurement next morning. The intensity was adjusted to elicit baseline MEPs of. 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.05). no other side effects of tDCS or medication were reported. This means that all subjects completed all 6 sessions of this experiment. Experimental procedures Experiment 2 In this control experiment. 2B). Monte-Silva et al. here the after-effects were evaluated until same evening after tDCS. material. / Brain Stimulation 6 (2013) 424e432 Cambridge. 13e3e13 or 13e20e13). whether the MEP amplitudes after tDCS differed significantly from the pre-DC amplitudes.1  0.426 K. Protocol 13e0e0 13e0e13 13e3e13 13e-20e13 13e3 he13 13e24 he13 MEP (mV) %MSO 0. Afterwards motor-cortical tDCS was performed as follows: (a) continuous application of one or two 13 min anodal tDCS sessions (13e0e0 or 13e0e13).10 45. Slight but significant differences were obtained between the 13e20e13 protocol on the one hand. .07 0. 25 MEPs were recorded after stable MEP amplitudes were obtained at a frequency of 0. They took place in an air-conditioned laboratory with a constant temperature of 20  C to guarantee constant skin temperature throughout the experimental sessions.5  0. Experiment 1 Experiment 1 included 6 experimental conditions which were conducted in a repeated measures. the time point where a trend of peak excitability enhancement was observed in Experiment 1 (Fig. Aftereffects of tDCS on excitability were monitored for 120 min after the end of stimulation.890  10 44. Two hours after intake of FLU. stimuli before the respective after-effect measures. a second baseline (baseline 2) was determined to control for a possible influence of the drug on cortical excitability and adjusted if necessary (baseline 3). mean interval between the 120 min and the se measure 236  88 (standard deviation) min).08 0. For all stimulation protocols.06 0. and 13e24 he13 on the other. Fig. Since we did not expect longer-lasting aftereffects induced by the single 13 min anodal tDCS session. Table 1 Baseline MEP amplitudes and TMS stimulation intensity of experiment 1. Experimental procedures are summarized in Fig. A P value of <0. 1 mV peak-to-peak amplitude and was kept constant for the post-stimulation assessment. Results With the exception of a slight itching sensation under the tDCS electrodes mentioned by some participants during stimulation. Identification of the hot-spot took about 20e30 min in each subject. Data analysis and statistics Experiment 1 MEP amplitude means were calculated for each time bin covering baseline and post-stimulation values. The Mauchly test of sphericity was conducted and GreenhouseGeisser correction was performed when appropriate. P > 0. and the 13e0e13. 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. without FLU or DMO).999  0.7  0. randomized complete crossover design.3  0. Baseline MEP amplitudes and percentage of maximal stimulator output did not show major differences between sessions (Table 1). because it allows obtaining motor cortex excitability enhancements or reductions without the danger of bottom or ceiling effects.10 43. Immediately after the last tDCS session.13 44.951  0. Values are given as means  standard deviation (sd). S1) show no effects of a single 13 min session of tDCS the day after stimulation. This required usually obtaining about five to seven TMS Experiment 3 The protocol of two consecutive tDCS with 20 min interval was chosen. The experimental sessions were separated by at least one week to exclude interference between the stimulation conditions. with head and arm rests. (c) two 13 min long blocks of tDCS with a long interval of 3 or 24 h (after after-effects. UK). and medication (with vs. and with time course. it was regularly checked if the actual coil position in threedimensional space elicited the largest MEPs during the after-effect measures.947  0. on average. and tDCS condition as within-subjects factors.05 was considered significant for all statistical analyses. the procedure was identical to that of 26 min contiguous tDCS with the exception that flunarizine (FLU) was administered 2 h before the plasticity induction procedure. 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 (Fisher’s LSD tests. 13e3 he13 or 13e24 he13). and a control experiment (see Suppl. and then every 30 min up to 2 h after the end of each stimulation. which did however not affect the main analyses.12 44. next afternoon (na).3  0. The post-stimulation MEPs were normalized intra-individually and are given as baseline ratios. and next evening (ne). The TMS recordings were also performed at four additional time points: same day evening (se. and whether the MEP amplitudes of the repeated tDCS conditions differed from the 13e0e0 protocol.07 1. The left motor-cortical representational field of the right abductor digiti minimi muscle (ADM) was identified using TMS (the coil position which produced the largest MEPs in the resting ADM).9  0. All results are given as mean and standard error of the mean (SEM).947  13 45.003  0. Determination of baseline TMS intensity took 5e10 min for each subject. Additionally Fisher’s LSD tests were used to test whether baseline MEP amplitudes differed significantly between the tDCS protocols.25 Hz for baseline determination. We chose this stimulation intensity. 1. The motor cortex tDCS electrode (anode) was fixed on the ADM hot-spot and the other (the cathode) was fixed at the contralateral forehead above the orbit.06 0. To guarantee optimal coil positioning during the course of the experiment. The EMG electrode and coil positions were marked with a water-proof pen to guarantee identical positions during the whole course of the experiments. (b) two 13 min long blocks of tDCS with a short interval of 3 or 20 min (during after-effects. The volunteers were seated in a comfortable chair.25 Hz for half an hour.

Experimental sessions were separated by at least one week from each other. excitability was again significantly enhanced for up to about 30 percent as compared to baseline MEPs. and a significant main effect of tDCS condition (Table 2).25 Hz Single Pulse TMS 0. and the different tDCS protocols were applied in randomized order. and 13e24 he13 (MEP amplitude size 1.25 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 5 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 0 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. an initial excitability enhancement of about 10e20 percent compared to baseline MEP amplitudes was seen only trendwise during the first 30 min after tDCS (non-significant results of the post-hoc tests for contrasts comparing baseline with posttDCS data). Baseline TMS intensity in percentage of maximum stimulator output was identical between conditions (Table 1. 60 and 120 min after tDCS. / Brain Stimulation 6 (2013) 424e432 Current Stimulation Baseline MEPs Stimulation Parameters 427 Single Pulse TMS Post tDCS MEPs Repeated Anodal tDCS 0. the effect of different intervals of 13 min anodal tDCS on motor cortex excitability. Experiment 1 Baseline MEP amplitudes did not differ between the respective tDCS conditions. the single session of anodal tDCS for 13 min resulted in an excitability enhancement of about 20% versus baseline which was significant for up to 60 min after stimulation (significant results of the posthoc tests for contrasts comparing baseline with post-tDCS data from five to 25 min after tDCS. A complete crossover design was performed. with the exception of a significant excitability enhancement immediately after tDCS in the 13e3e13 condition. The ANOVA resulted in a significant interaction of tDCS condition  time course. TMS intensities between conditions did not differ.0 mV). pairwise comparisons between all combinations of conditions). This . and 60 min after tDCS). Monte-Silva et al. a calcium channel blocker. with the exception that there was a slight. and the 13e0e13 (MEP amplitude size 1. Some hours later however. or 90 min (13e20e13 condition) after stimulation. and 26 min continuous stimulation (13e0e13) were compared with two 13 min stimulation protocol separated by short (3 min (13e3e13). As shown by the post-hoc tests. Since the MEP differences were small. on the excitability diminution induced by 26 min continuous tDCS. 20 min (13e20e13)) or long (3 h (13e3 he13). 13 min stimulation only (13e0e0 protocol). excitability was monitored up to the evening after the (second) DC stimulation. and MEP values of the 13e0e0 condition at the same time point (significant results of the post-hoc tests for comparisons of 13e0e0 condition versus 13e3e13. but significant. and 13e20e13 conditions). and the MEP amplitudes of the directly compared (see below) 13e0e0 and the 13e20e13 condition did not differ significantly. at the evening of the stimulation day. as monitored via single pulse TMS. and completely disappeared 30 (13e3e13). If 13 min tDCS was repeated during the after-effects of the first stimulation (13e3e13 or 13e20e13). this should not have a relevant impact on the results of this study. In experiment 2. In experiment 1. Table 1). difference between the 13e20e13 (MEP amplitude size 0. where excitability measures were terminated the evening of the day of tDCS application. was explored. 24 h (13e24 he13)) intervals. significant 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).0 mV) on the other (pairwise comparisons between all combinations of conditions. we explored the effect of flunarizine.K. and 5 min after tDCS. With the exception of the 13e0e0 protocol. 26 min contiguous stimulation-induced a significant reduction of motor cortex excitability of about 20% compared to baseline lasting approximately equally long (significant results of the post-hoc tests for contrasts comparing baseline with post-tDCS data immediately after. 15e25 min. Specifically.9 mV) condition on the one hand.

and error bars standard deviation of the mean (SEM). na ¼ next afternoon. and for the 5 min time point in the 13e24 he13 condition. however. nm ¼ next morning. interval duration determines the effects of repeated anodal tDCS on motor cortex excitability. excitability enhancement remained significant 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.05). Instead. 20. no significant excitability enhancement was seen for the whole course of the experiments. se ¼ same evening. Inter-stimulation interval determines the effects of anodal tDCS on motor cortex excitability. and thus for more than 24 h. 30. significant results of the post-hoc test for contrasts comparing the 13e0e0 with the 13e3 he13 condition for time points 5. 13e24 he13 protocols) however abolishes the excitability enhancement. (A) A single session of 13 min anodal tDCS (13e0e0) enhances excitability up to 60 min after DC stimulation. motor cortex excitability is again enhanced significantly. As can be seen from the results of the study. An inter tDCS-interval of a few minutes (13e3e13. which however is present trendwise up to about 90 min after tDCS. and next afternoon and evening for the 13e20e13 condition). Filled symbols indicate significant differences from baseline MEPs. and 60 min after tDCS. This late excitability enhancement is more prominent for the 20 min interval condition. ne ¼ next evening. but significant reduction of excitability (about 10% as compared to baseline values) took place after the second stimulation (significant 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. asterisks significant differences of the effects of 13 min only tDCS on MEP amplitudes at identical time points (P  0. (C) Prolonging the inter tDCS-intervals for 3 or 24 h (13e3 he13. From the evening of the stimulation day on for up to the next evening. For the long interval conditions (second stimulation after the after-effects of the first stimulation). / Brain Stimulation 6 (2013) 424e432 A B C D Figure 2. and for the .428 K. Monte-Silva et al. (B) Prolonging stimulation duration for 26 min (13e0e13) however converts the after-effects into excitability diminution. 13e20e13 protocols) primarily diminishes the efficacy of tDCS to enhance excitability. afternoon and evening for the 13e3e13 condition. and turns it slightly into inhibition. a slight.

239 1.233). As can be seen by the results. A late-onset.e.3 none 1. as revealed by the respective post-hoc contrasts. asterisks significant differences between the effects of FLU on MEP amplitudes at identical time points (P  0.335 0. significant results for contrasts comparing no medication and DMO for the respective time points next morning and next noon) (Fig. and baseline MEPs were identical between the experimental sessions with and without FLU (baseline 3 with FLU 1. which was not present under calcium channel block by FLU (no significant results in the respective post-hoc tests comparing baseline with post-tDCS MEP values).002a 0. 20.008 mV  0.634 1.2 1.913). and asterisks represent significant differences between the conditions with and without DMO (P  0. baseline 2 1. Filled symbols indicate significant differences between baseline and post-tDCS MEPs.182 0.058. The NMDA receptor antagonist DMO abolishes the late-onset excitability enhancement induced by repeated tDCS with a short interval.5 0 5 10 15 20 25 30 60 90 120 Time course (min) Figure 3.4 1. next morning.002 mV  0.5 FLU 1. DMO abolished the long-lasting excitability enhancement.114 0. Time course (min) Figure 4. In experiment 3. and was significantly enhanced the same evening until next day noon without medication (significant results in the respective posthoc tests comparing baseline with post-tDCS MEP values for the time points same evening.6 0. These excitability alterations obtained by spaced tDCS cannot be caused by total stimulation duration alone.301 0.6 MEP amplitude normalized by pre-tDCS baseline Experiment 1 429 3.8 0.4 tDCS condition 13-20-13 Medication DMO None * * 0 5 10 15 20 25 30 60 90 120 se nm na ne 0.9 The ANOVA showed a significant interaction of time course  medication.331 3. no significant results for contrasts comparing no medication and DMO for the respective time points). and placebo medication conditions for the time points 5.001a In experiment 1. Dextromethorphan (DMO) blocked this l-LTP-like excitability enhancement when administered 2 h before the surge of MEP next morning.005a 0.105 sd.K. The ANOVA reveals a significant main effect of medication in experiment 2. A similar effect of this tDCS protocol was observed in the DMO condition before the drug was applied (no significant results in the respective post-hoc tests comparing baseline with post-tDCS MEP values with the exception of the time points 25. F-value P-value 11 5 11 10 1 10 14 1 14 1. Filled symbols indicate significant differences from baseline MEPs. 3. error bars: SEM). 26 min continuous anodal tDCS was performed under the calcium channel blocker flunarizine (FLU). se ¼ same evening.2 2.143 sd. FLU did not affect MEP baseline values (baseline 1 1.0 mV  0.704 30.007a 0. the combined after-effects of the two blocks were present for more than 24 h after tDCS. the ANOVA shows a significant main effect of time course and a significant interaction of medication  time course.4 2.0 2. l-LTP-like excitability enhancements of the primary motor cortex. In contrast.8 2.1 1. 1. FLU abolishes the excitability diminution induced by the prolonged stimulation. the ANOVA resulted in a significant main effect of time course. This effect critically depends on the duration of the interval between tDCS applications. The reduction of cortical excitability induced by 26 min anodal tDCS depends on calcium channel activity. 26 min anodal tDCS-induced a significant excitability diminution without medication (significant results of the post-hoc tests for the contrasts comparing baseline with post-tDCS values from 5 to 90 min after tDCS). / Brain Stimulation 6 (2013) 424e432 Experiment 3 Table 2 Results of the ANOVAs. ne ¼ next evening.7 0.2 1. and lasted until next noon (no significant results in the respective post-hoc tests comparing baseline with post-tDCS MEP values in the DMO condition.f.8 1. long-lasting enhancement of cortical excitability was observed when a second tDCS was applied within the effective time window after the first tDCS (13e20e13 protocol). a Indicates significant effects (P  0. 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. and a significant main effect of time course (Table 2).008 mV  0. and 60 min after tDCS. Experiment 2 MEP amplitude normalized by pretDCS baseline The ANOVA revealed a significant main effect of medication (Table 2). error bars: SEM).. In a subgroup of participants. Post-hoc tests revealed that the blocking effect was significant for the MEP measurement of next morning.638 1.05. If the second stimulation was performed during the after-effects of the first one (as it was the case with an interval of 3 or 20 min). na ¼ next afternoon. and a significant tDCS condition  time course interaction.461 1. contrasts comparing the 13e0e0 with the 13e24 he13 condition for time point 5 min after tDCS) (Fig. When given the morning after tDCS. two-tailed paired Student’s t-test P ¼ 0.392 4.0 0. FLU abolished the induction of inhibitory after-effects by 26 min anodal stimulation. the same total stimulation duration Medication tDCS condition 13-0-13 1. 2AeD).0 * * 0. an interval of 3 or 24 h abolished the after-effects of tDCS.4 0. nm ¼ next morning. because two contiguous blocks of 13 min of anodal tDCS. . and next noon). i.059 0. 2 h after DMO intake. and 25 min. * * 0. baseline without FLU 1. 4).6 1.176 2.8 Discussion This study shows that periodical anodal tDCS is able to induce long-lasting. Cortical excitability was trendwise increased after repetitive anodal tDCS with 20 min break for up to 120 min after tDCS (no significant results in the respective post-hoc tests comparing baseline with post-tDCS MEP values with the exception of the 15 min time point). two-tailed paired Student’s t-test P ¼ 0. As can be seen in Fig. Monte-Silva et al.003a 0. Consequently. MEP values differed significantly between FLU.05).6 2.

Fig. inter tDCS-intervals of hours prevented any plasticity induction by the second DC stimulation. Interestingly. Interestingly. Whereas the early excitability enhancement induced by spaced tDCS is similar to that induced by a single stimulation session.10. a two-phasic response on MEP amplitudes resulted. This might hamper direct comparison between the experimental conditions to some extent. and should be explored more directly in future studies. as compared to the single stimulation session. as suggested also in the present study.39].10]. abolished l-LTPlike plasticity. which is the alteration of the efficacy of a stimulus to induce neuroplasticity dependent on prior activity of the cortical network. S1). Accordingly the conversion of the tDCS-induced excitability diminution was abolished by calcium channel block via FLU. and RNA synthesis [5]. in animal slice studies. a calcium-mediated alteration of glutamatergic receptors [41] is an attractive candidate mechanism. Protein and RNA synthesis. and both phenomena share certain similarities such as NMDA receptor-dependency [9.37]. Since tDCS induces calcium channel. however somewhat smaller. resulted in reduced motor cortex excitability. amongst others. 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. / Brain Stimulation 6 (2013) 424e432 applied continuously without any tDCS-free interval in between. and their NMDA receptor-dependency [6. MEP measures were terminated at the end of the first day. neuron. the activation of hyperpolarizing potassium channels. Experiment 3 only included subjects showing l-LTP-like excitability changes. which depends on intraneuronal calcium concentration [29]. and are induced by repeated stimulation with an inter-stimulation interval of some minutes [5. and not on total stimulation duration. as obtained in animal slice experiments. these include at least some features of l-LTP. the duration of the after-effects for more than 24 h. Modulation of LTP-like plasticity by an inter tDCS block interval of hours In contrast to the effect of short intervals on plasticity. which are not necessarily paralleled by modifications of corticospinal excitability [46. we do not expect a reoccurrence of after-effects more than 1 h after these had . 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.8. since plasticity is known to be consolidated during sleep [36. however the exact mechanism of action awaits further exploration. in the latter experiment (experiment 3). as described in animal slice preparations [2]. and a control experiment (Supplemental material. an NMDA receptor antagonist. and the excitability measure via monitoring of MEP amplitudes does not cover all aspects of LTP. in the 13e0e0 condition. For the later occurring excitability enhancements. might be caused by homeostatic effects (see below). 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. the later one differs relevantly. The presence of the respective excitability alterations the day after stimulation might be furthermore supported by sleep-dependent processes. which show no alteration of cortical excitability versus baseline after 13 min anodal tDCS the day after plasticity induction). or synapse. Moreover. and thus for over 24 h. and thus share similarities with glutamatergic early LTP. although it has been proposed that l-LTP is the physiological basis of long term memory formation and consolidation. direct evidence in humans is missing at present. NMDA receptors are involved in the induction and maintenance of l-LTP [10].48]. Since the effects of tDCS on cortical excitability are calcium-dependent. Furthermore. these were usually obtained by an inter-stimulation interval of some minutes. but was enhanced again in the later excitability measures. The abolishment of facilitatory plasticity might be due to homeostatic mechanisms. which prevents over-excitation. Moreover. 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. and the excitability enhancement accomplished by these mechanisms might only partially overlap with the early ones.35]. For experiment one. They last considerably longer than 3 h. these occur hours after the overt effects of tDCS on the MEP amplitude have vanished.27.16]. This late excitability enhancement. to stabilize enhancements of synaptic connectivity. which are essential features of l-LTP in animal slice experiments. the reduction of motor cortex excitability between both MEP enhancements is compatible with discernable mechanisms. the after-effects of tDCS on MEP amplitudes show not the reduction of excitability present in experiment 1 (13e20e13 condition) 120 min after stimulation. Taking into account the proposed relevance of l-LTP for long term memory formation. One likely mechanism of action of the conversion of the after-effects of tDCS is a neuronal counter-regulation. the induction procedures in humans and animal slices differ for some aspects. Whereas homeostatic mechanisms have been identified in humans before by combined application of different non-invasive brain stimulation techniques [13. and thus preserves the stability of the network.42e45]. General remarks Some limiting aspects of the present study should be taken into account. Thus the relationship between both phenomena should be explored to a larger extent in future studies. In contrast with e-LTP.and NMDA receptordependent plasticity.430 K. start with a delay of up to 4 h after plasticity induction [6].47]. They are in good accordance with the observation that spaced learning results in better effects than massed learning [22. l-LTP requires protein kinase A activity. The slight reduction of their magnitude. These effects depend on NMDA receptors and calcium channel activity [15. including adaption and consolidation [21e27]. whereas the respective condition in experiment 1 included subjects with and without l-LTP-like after-effects. Monte-Silva et al. was stable up to the evening after the day of tDCS.9. However.38. In accordance with the results of previous experiments [28]. An initial excitability enhancement was present for up to 90 min after stimulation. Multiple cellular mechanisms of homeostatic effects have been identified in animal and slice experimentation [40]. Motor cortex excitability was at baseline level 2 h after stimulation. protein synthesis. Future studies should explore if these are due to intracortical excitability changes. Although these long-lasting alterations of synaptic efficacy share some physiological similarities with late phase LTP. is a candidate mechanism. Here much longer intervals induce homeostatic effects. such as the relevance of a specific time windows of some minutes for the induction of the after-effects. which helps to avoid “runaway” excitation. where DMO. and not continued to the next day. which might be larger for the 20 min interval condition. it might be that the biphasic excitability enhancement is caused by different mechanisms of action. The early excitability enhancements might follow similar mechanisms as those induced by a single tDCS session.

Sossin WS. Neurosci Res 2006. because most of the subjects included in these experiments had already participated in six tDCS sessions before (experiment 1). Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons. the results might help to improve the efficacy of tDCS as a therapeutic tool for the treatment of neuropsychiatric diseases accompanied by pathological alterations of brain activity. Abbas AK. if periodical stimulation protocols. Rodriguez J. or clinical symptoms. Since the pharmacological control studies were planned and conducted after completion of experiment one. Donoghue J. as proposed by the current study. due to additional physiological differences between the subjects participating in the present experiment. References [1] Feldman DE. . Science 1993. Klann E.260(5114):1661e4. i-wave facilitation [47]. [9] Dozmorov M.55(2):182e8. Future experiments should explore the specific mechanisms of action to a larger extent. [4] Rioult-Pedotti M. might be limited. Neuropharmacology 2007.2012. ‘synaptic tagging’. [7] Valero-Cabre A. the number of subjects is relatively small.brs. This might mean that also for clinical application. assuming that l-LTP is one important physiological underlying mechanism of long term memory formation. the efficacy of tDCS to improve motor functions after stroke. might contribute. For the pharmacological control experiments. and thus even prolonged stimulation might not suffice to enhance intracellular calcium to a level inducing LTP-like effects. and plasticity duration.011. These results are compatible with similar mechanisms of action for both. 60(1):1e5. Brazil. Supplementary material Supplementary data related to this article can be found online at doi:10. although to a somewhat minor extent. Huang YY. as obtained in animal experiments. from the results of the present study it cannot be concluded that these are identical to late LTP.04. 27(3):765e74. Translational control of long-lasting synaptic plasticity and memory. et al. [10] Lynch MA. [8] Frey U. The results of the current study show brain stimulation-induced l-LTP-like effects in the human motor cortex.g. Proc Natl Acad Sci U S A 2004. Hess G. Similarly. [2] Malenka R. Bear M. Annu Rev Neurosci 2009. Strengthening of horizontal cortical connections following skill learning. However. Future experiments should explore these parameters. The other authors declare no conflict of interest.55]. Pittenger C.52(1):24e40. double-blinded design.101(3):859e64. Li R. DMO is not a pure NMDA receptor antagonist. which might also have an impact on the results. 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. Neuroscience 1994. or depressive symptoms [51e53]. learning-related synaptic plasticity in the hippocampus induced by heterosynaptic low-frequency pairing. Huang FS. 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. prolongation of stimulation duration might not result in higher efficacy in each case.K. inhibition. Rushmore RJ. [5] Huang YY. The transferability of the results to stimulation protocols aiming to modify behavior. and patients suffering from neuropsychiatric diseases. 44(1):5e21. whereas in most clinical studies tDCS was repeated on five consecutive days or more. as compared to the results of the present study.32:33e55. in that study conduction of the second stimulation protocol during the after-effects of the first one enhanced efficacy of stimulation. Increase in synaptic vesicle proteins accompanies long-term potentiation in the dentate gyrus. and the Rose Foundation. Monte-Silva et al. Neuron 2009. or other molecular or cellular alterations. Furthermore. are applied instead of stimulation once patients is hypothetical. Sonenberg N. because effects of tDCS on other parameters not explored in the present experiments. This project was further supported by the German Ministry for Education and Research. the GABAergic system [56]. however.1(3):230e4. A relevant effect of DMO on ion channels is however improbable at the present dosage [17]. Synaptic mechanisms for plasticity in neocortex. phases. Science 2000.290(5491):533e6. Bernstein-Center for Computational Neuroscience. Financial disclosures MAN is member of the advisory board of Neuroelectrics. LTP and LTD: an embarrassment of riches. the transferability of these results e especially with regard to specific stimulation parameters . but resulted in a prolongation. Voss KL.1016/j. Nat Neurosci 1998. Moreover. Eur J Neurosci 2008. the occurrence of expectancy effects is improbable in our opinion. Moreover. because it has been shown recently that MEP amplitudes elicited by single pulse TMS are stable for at least 24 h [49]. We only explored global corticospinal excitability alterations induced by tDCS. anodal and cathodal. Friedman D. and their importance for the respective functional alterations. which is currently applied most often in clinical studies. Neuron 2004. Acknowledgments KM-S was supported by CAPES. [11] Costa-Mattioli M. on intracortical facilitation. As subjects were not aware of specific hypotheses. the results of the 13e0e13 tDCS sessions hints for a non-linear connection between stimulation duration. doubling the stimulation duration of cathodal tDCS without a break did not convert the after-effects. The late maintenance of hippocampal LTP: requirements. in which the effects of repeated excitability-diminishing cathodal tDCS were explored [50]. Kandel ER. in our healthy volunteers group. but is improbable. Specifically. the results look reasonable clear and stable. Finally. tDCS with regard to the effects of repeated stimulation on prolongation of after-effects. Hellberg F. Farre C. Contribution of AMPA and NMDA receptors to early and late phases of LTP in hippocampal slices. as far as volunteers already involved in experiment one were tested. We did not perform a separate experimental session without stimulation to control for a “drift” of MEP amplitudes. to a larger extent. They demonstrate that this kind of plasticity is induced by periodical stimulation. and should be explored directly in future studies. but has additional ion channel-blocking effects. and the respective control experiments were conducted months after experiment 1 in most of the subjects. and be not in complete accordance with the global excitability measures. However. However. / Brain Stimulation 6 (2013) 424e432 vanished. a one-to-one transferability of the present results obtained on plasticity to behavioral and clinical effects is not self-evident. e. It might also be speculated that optimal timing of plasticity induction is suited to improve learning in humans.61(1):10e26. The reason for this dissociation might be that cathodal tDCS at the present intensity is expected to result in much lower calcium influx than anodal tDCS. Bliss TV. Cumulative sessions of repetitive transcranial magnetic stimulation (rTMS) build up facilitation to subsequent TMS-mediated behavioural disruptions. whereas a break duration of hours diminished the effects of tDCS. Donoghue J. in the present study we accomplished only the 431 combination of two tDCS sessions. Goettingen. might be enhanced. ‘late-associativity’ and implications. [3] Rioult-Pedotti M. Learning-induced LTP in neocortex. and that a specific time window is critical for its induction. Finally. Kandel ER. [6] Reymann KG. An order effect for these experiments cannot be ruled out completely. The results show similarities with another study of our group. once daily stimulation has been shown to result in e in some studies cumulative e effects on clinical symptoms [54. Frey JU. A form of long-lasting. Indeed. and direction. it was not possible to conduct them in a randomized. Pascual-Leone A. Friedman D.

Siebner HR. / Brain Stimulation 6 (2013) 424e432 [12] Sossin WS. et al. J Neurosci 2009. Brain Stimul 2008. Monte-Silva K. Cephalalgia 1982. Neuroreport 2005.92(1):66e72. Reiber H. Lang N. Klein C. Molecular memory traces. J Neurosci 2004. Brain 2002. et al. Eur J Neurosci 2008. Leung V. Nitsche M. et al. Regulation of ion channel localization and phosphorylation by neuronal activity. Nature 1995. [23] Overduin SA. Mohapatra DP. [21] Karni A. Neurology 2001. Pharmacokinetics of dextromethorphan and dextrorphan: a single dose comparison of three preparations in human volunteers. Siebner HR. Transcranial direct current stimulation of the unaffected hemisphere in stroke patients. and ERK/MAPK activation in the dorsal hippocampus.20(12):3453e62. [32] Stoica E.224(1):57e60. Wylezinska M. Kincses ZT.169:3e25. et al. Int J Neuropsychopharmacol 2008. Shaping the optimal repetition interval for cathodal transcranial direct current stimulation (tDCS). Mejia-Perez C. [15] Nitsche M. Nitsche MA. Temporal sensitivity of protein kinase a activation in late-phase long term potentiation. Mei L.1(1):60e6. Zhen D. Karttunen P.24(13):3379e85. Nat Neurosci 2004. Ferreira MJ. PascualLeone A. et al. Tukiainen H. Large CH. Intermittent practice facilitates stable motor memories. Modulation of associative human motor cortical plasticity by attention. Turrigiano GG. et al. Functional stages in the formation of human long-term motor memory. Nitsche MA. [17] Liebetanz D. [28] Nitsche M. 118(5):1028e32. Pharmacological modulation of cortical excitability shifts induced by transcranial direct current stimulation in humans. J Neurophysiol 2004. Scote-Blachon C. Nitsche MA. Comparison of flunarizine (Sibelium) and pizotifen (Sandomigran) in migraine treatment: a double-blind study. Romcy-Pereira R. Adams M.27(14):3807e12. Giraux P.25(2):498e503. Boggio PS. Tormos JM. Misonou K. Lemon R. Ferreira MJ. Premotor transcranial direct current stimulation (tDCS) affects primary motor excitability in humans. Classen J.553(Pt 1): 293e301. [27] Xue G. Pozo K. Int J Clin Pharmacol Ther Toxicol 1987. Long-lasting forms of synaptic potentiation in the mammalian hippocampus. Learn Mem 2001.20(1):550e60.111(5):800e5. Kim M. Neuropsychopharmacology 2004. Nitsche MA. J Neurosci 2007. Comte JC. Pharmacological approach to the mechanisms of transcranial DC-stimulation-induced after-effects of human motor cortex excitability. Effects of non-invasive cortical stimulation on skilled motor function in chronic stroke.21(10): 2299e306. Abel T. [31] Louis P. Modifying motor learning through gating and homeostatic metaplasticity. glutamate receptor protein levels. [35] Huang YY. Preconditioning of low-frequency repetitive transcranial magnetic stimulation with transcranial direct current stimulation: evidence for homeostatic plasticity in the human motor cortex. Nguyen PV. Neurosci Lett 1997. [19] Baumer T. Chen C. Unraveling mechanisms of homeostatic synaptic plasticity. et al. Distinct modulatory effects of sleep on the maintenance of hippocampal and medial prefrontal cortex LTP. Boggio PS. Holcomb H. Stagg CJ. Best JG. Lu ZL. Liebetanz D. Chen RS.32(2):227e40. Modulation of corticospinal excitability by repetitive transcranial magnetic stimulation.27(1):6e44. Nitsche M.219(1):14e9. Pascual-Leone A. Poldrack R. [14] Stefan K. Siebner H. Wagner T. The influence of amitriptyline and flunarizine on catecholamine response to light in patients with migraine. Press DZ. Science 1997. J Neurosci 1997. Timingdependent modulation of associative plasticity by general network excitability in the human motor cortex. Rigonatti SP. Abel T. Nitsche M. Fregni F. The self-tuning neuron: synaptic scaling of excitatory synapses.11(2):249e54. Blackwell KT. Boggio PS. Paulus W. Mansur CG. Münchau A. A sham-controlled. et al. [30] Holmes B. Park EW. Mussa-Ivaldi FA.31(1):11e9. Meyer G.6(2): e1000691. [25] Shadmehr R. phase II trial of transcranial direct current stimulation for the treatment of central pain in traumatic spinal cord injury. Goda Y. Weiller C. Lima MC. Kokkonen P. Kuo M-F. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic use. Paulus W.432 K. Hasan A. Cereb Cortex 2011. [24] Shadmehr R.7(7):711e8. Liepert J. Brashers-Krug T. Eur J Neurosci 2007. Lang N. J Neurosci 2006. [22] Kornell N.17(1):409e19. Rizzo V.125(Pt 10):2238e47. Boros K. Eich TS. Kandel ER. Paulus W.2(4):197e203. et al. Pavlides C. State of the art: pharmacologic effects on cortical excitability measures tested by transcranial magnetic stimulation. Neuron 2010.8(1): 20e5.29(16):5202e6. et al. Ernst D. Homeostatic plasticity in human motor cortex demonstrated by two consecutive sessions of paired associative stimulation. Rigonatti SP. [20] Maeda F. Turner R. Flunarizine. Lima MC. [16] Nitsche M.128(Pt 3):490e9. Heel RC. Jaussi W. Functional MRI evidence for adult motor cortex plasticity during motor skill learning. Drugs 1984.27(5):1292e300. Seeber A. Nykanen S. Pachoud B. Lemon R. Brain Stimul 2008. Spacing as the friend of both memory and induction in young and older adults. Exp Neurol 2009. Speight TM. Effects of pharmacologically induced changes in NMDAreceptor activity on long-term memory in humans. Treatment of depression with transcranial direct current stimulation (tDCS): a review.26(46):11888e92. Siebner H. Schugens MM.1(3):151e63. Müller JFM. Liebetanz D.16(14):1551e5. Pain 2006. Gerloff C. Psychol Aging 2010.14(5 Pt 2):3208e24. A randomized. Frommann K. Enulescu O. Monte-Silva et al. Floel A. Poreisz C. et al. Modulating parameters of excitability during and after transcranial direct current stimulation of the human motor cortex. Brogden RN. et al. Wagner T. Liu Y. [33] Silvasti M. Liebetanz D. Spaced learning enhances subsequent recognition memory by reducing neural repetition suppression. Nitsche MA. Nitsche M.29(8):1573e8.377(6545):155e8. Di Lazzaro V. 122(1e2):197e209. Dong Q. Tergau F.3(2e3):74e85. double-blind clinical trial on the efficacy of cortical direct current stimulation for the treatment of major depression. [13] Ziemann U. Huang T. Kuo M-F. Myczkowski ML. Paulus W. Rochford C. et al. Lang N. Bizzi E. PLoS Comput Biol 2010. [26] Shadmehr R. Cell 2008. Clin Neurophysiol 2000. Sleep 2009. Ungerleider L. Castel AD. Richardson AG. Hummel F. Neural correlates of motor memory consolidation. Learn Mem 1996.57(10):1899e901. . Classen J. Cohen LG. Nitsche M. The after-effect of human theta burst stimulation is NMDA receptor dependent. Gay N. Rom J Neurol Psychiatry 1993.135(3):422e35. Jezzard P. Prog Brain Res 2008. Consolidation of human motor cortical neuroplasticity by D-cycloserine. Sueske E. Henschke U. Celnik P. [36] Ravassard P. Wu W. Tergau F.66(3):337e51. Loschmann PA. Repeated premotor rTMS leads to cumulative plastic changes of motor cortex excitability in humans. [34] Paulus W. Schlitterlau A. Hanninen U. Paradoxical (REM) sleep deprivation causes a large and rapidly reversible [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] decrease in long-term potentiation. Neuroimage 2003. Rothkegel H.277(5327):821e5. Preconditioning with transcranial direct current stimulation sensitizes the motor cortex to rapid-rate transcranial magnetic stimulation and controls the direction of after-effects. Lang N. et al. Schepelmann K. Roth A. Polarity-sensitive modulation of cortical neurotransmitters by transcranial stimulation. Eur J Neurosci 2004. Fregni F. Daum I. Ribeiro RB. Wycislo M. Paulus W. Lang N. Brain 2005. Nitsche M. Paulus W. J Physiol. Rammsayer TH. Pascual-Leone A. et al. Ziemann U. Sustained excitability elevations induced by transcranial DC motor cortex stimulation in humans. [29] Misonou H. O’Shea J.25(11):3461e8. The NMDA antagonist memantine impairs classical eyeblink conditioning in humans. Paulus W.23(7):1624e33. Klockgether T. Clin Neurophysiol 2007. 2005. Stephenson MC. Fischer AK. Lang N. [18] Huang YZ. Fricke K. Keenan JP. Avery GS. Egerter R. J Cogn Neurosci 2011. Boggio PS.25(9):493e7. Wen HY. Circadian modulation of GABA-mediated cortical inhibition.103(4):1735e40. Liebetanz D. J Neurosci 1994. Lange R. Lane CE. synaptic transmission. Rothwell JC.56(9):634e9. Orekhov Y. Bjork RA. Tergau F. Rothwell JC. Biol Psychiatry 2004. Fregni F. Topka H. J Physiol 2003. Adaptive representation of dynamics during learning of a motor task. J Neurophysiol 2010. Spierings EL.