Brain Polarization Enhances the Formation and Retention of Motor Memories

Joseph M. Galea and Pablo Celnik

J Neurophysiol 102:294-301, 2009. First published 22 April 2009; doi: 10.1152/jn.00184.2009 You might find this additional info useful... This article cites 42 articles, 21 of which you can access for free at: http://jn.physiology.org/content/102/1/294.full#ref-list-1 This article has been cited by 16 other HighWire-hosted articles: http://jn.physiology.org/content/102/1/294#cited-by Updated information and services including high resolution figures, can be found at: http://jn.physiology.org/content/102/1/294.full Additional material and information about Journal of Neurophysiology can be found at: http://www.the-aps.org/publications/jn This information is current as of September 14, 2012.
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Journal of Neurophysiology publishes original articles on the function of the nervous system. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2009 the American Physiological Society. ISSN: 0022-3077, ESSN: 1522-1598. Visit our website at http://www.the-aps.org/.

J Neurophysiol 102: 294 301, 2009. First published April 22, 2009; doi:10.1152/jn.00184.2009.

Brain Polarization Enhances the Formation and Retention of Motor Memories

Joseph M. Galea and Pablo Celnik
Department of Physical Medicine and Rehabilitation, Johns Hopkins Medical Institution, Baltimore, Maryland
Submitted 2 March 2009; accepted in nal form 13 April 2009

Galea JM, Celnik P. Brain polarization enhances the formation and retention of motor memories. J Neurophysiol 102: 294 301, 2009. First published April 22, 2009; doi:10.1152/jn.00184.2009. One of the rst steps in the acquisition of a new motor skill is the formation of motor memories. Here we tested the capacity of transcranial DC stimulation (tDCS) applied over the motor cortex during motor practice to increase motor memory formation and retention. Nine healthy individuals underwent a crossover transcranial magnetic stimulation (TMS) study designed to test motor memory formation resulting from training. Anodal tDCS elicited an increase in the magnitude and duration of motor memories in a polarity-specic manner, as reected by changes in the kinematic characteristics of TMS-evoked movements after anodal, but not cathodal or sham stimulation. This effect was present only when training and stimulation were associated and mediated by a differential modulation of corticomotor excitability of the involved muscles. These results indicate that anodal brain polarization can enhance the initial formation and retention of a new motor memory resulting from training. These processes may be the underlying mechanisms by which tDCS enhances motor learning.
INTRODUCTION

The ability to learn an unlimited repertoire of motor skills is one of the main characteristics of human beings. Recent studies have suggested that the primary motor cortex (M1) is crucially involved in this ability by forming and retaining short-term memory representations of recently practiced movements (Classen et al. 1998; Hadipour-Niktarash et al. 2007; MolinaLuna et al. 2008; Muellbacher et al. 2002). In particular, it has been shown that although disruption of M1 activity can interfere with retention of practiced motor tasks (HadipourNiktarash et al. 2007; Muellbacher et al. 2002), enhancing M1 excitability with transcranial DC stimulation can improve it (Reis et al. 2009). However, it is not clear whether this benecial effect on retention is mediated by a larger amount of motor memory formation or changes in retention mechanisms. One of the initial steps in the acquisition of new complex motor skills is the formation of motor memories (Classen et al. 1998; Muellbacher et al. 2002). Classen et al. (1998) used transcranial magnetic stimulation (TMS) over M1 to ascertain how simple motor memories are formed. In this paradigm, the preferred direction of TMS-evoked thumb movements is measured before and after performance of motor training. In this manner, it is possible to observe that training results in changes in the direction of TMS-evoked movements, indicative of formation of new motor memories containing the kinematic details of the practiced motions retrieved by the magnetic stimulation. This phenomenon is mediated by N-methyl-Daspartate (NMDA), muscarinic, adrenergic and GABAergic
Address for reprint requests and other correspondence: P. Celnik, Department of Physical Medicine and Rehabilitation, Johns Hopkins Medical Institution, 98 North Broadway, Baltimore, MD 21231 (E-mail: pcelnik @jhmi.edu). 294

neurotransmission (Butesch et al. 2000; Sawaki et al. 2002b, 2003) and can be enhanced by dopaminergic (Floel et al. 2005a,b) and adrenergic agents (Butesch et al. 2002; Sawaki et al. 2002a), D-amphetamine, congruent action observation (Celnik et al. 2006; Stefan et al. 2008), and hebbian-type noninvasive stimulation (Butesch et al. 2004). Transcranial DC stimulation (tDCS) is a noninvasive, painless cortical stimulation technique (Nitsche and Paulus 2000, 2001; Nitsche et al. 2003a,b, 2005). Whereas anodal tDCS results in increased cortical excitability without direct neuronal depolarization, cathodal tDCS decreases excitability (Nitsche and Paulus 2000; Nitsche et al. 2003; Purpura and McMurtry 1965). Interestingly, anodal tDCS appears to have a facilitatory effect on different forms of learning in healthy individuals (Antal et al. 2004; Floel et al. 2008; Kincses et al. 2004; Nitsche et al. 2003c; Reis et al. 2009) and in stroke (Hummel et al. 2005) and Parkinson patients (Boggio et al. 2006; for a review see Wu et al. 2007). However, it is not clearly understood what specic process mediates the enhanced learning when tDCS is applied during training. For instance, it is possible that the performance improvement described after learning a motor task under the inuence of tDCS is mediated by increased memory formation, stronger retention of the formed memories, or better recall. Antal et al. (2004) and Nitsche et al. (2003c) previously showed performance improvements during early learning, which suggests that tDCS may inuence memory formation. In this study, we used a well-established paradigm that assesses the formation and retention of a motor memory without engaging voluntary recall to test the hypothesis that anodal tDCS over M1 enhances these processes relative to sham stimulation. In addition, in three separate controls we determine 1) the polarity specicity of the effects by testing cathodal stimulation, 2) the longevity of the effects by periodically reassessing the effects of stimulation and training, and 3) the association between motor practice and stimulation by testing the effects of anodal tDCS without training.

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METHODS

Subjects
Nine right-handed (self-assessed) healthy individuals with no history of neurological or psychiatric conditions (mean SD: 30 9 yr old, six females) participated in the study. All subjects denied the use of acute or chronic CNS-acting medication, drinking alcohol in the previous 24 h, and reported to have had 5 h of sleep the previous night. All subjects signed informed consent approved by Johns Hopkins Medical Institution Institutional Review Board and in accordance with the Declaration of Helsinki.
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Experimental design
All subjects participated in two randomly ordered sessions separated by 5 days and designed to evaluate the effect of anodal tDCS on the encoding of motor memories (Fig. 1) (Classen et al. 1998).
RECORDINGS AND STIMULATION PROCEDURES.

Subjects sat comfortably in a chair while the right forearm was restrained in a semipronated position with a molded cast with the four ngers in a slightly extended position and the thumb left entirely free. Using a two-dimensional accelerometer (Kistler Instruments, Amherst, NY) placed on the proximal phalanx of the thumb, movement directions were determined and calculated from the rst peak acceleration vector in both the vertical axis (extension/exion) and the horizontal axis (adduction/adduction) (Fig. 1) (Classen et al. 1998). Electromyographic (EMG) activity was recorded through pairs of disposable electrodes placed over the right extensor pollicis brevis and right exor pollicis brevis muscles. EMG signals were recorded, amplied, and ltered (bandwidth 5 Hz to 1 kHz) with a Viking IVP (Nicolet, Viasys Healthcare). All signals were sampled at 2 kHz, visually displayed on-line, and stored for off-line analysis using a custom LabVIEW program (National Instruments, Austin, TX).

Rogue Research, Montreal, Quebec, Canada) we coregistered the subjects heads to a standard magnetic resonance image and marked this hot spot. We decided to use BrainSight because this is an accurate way to ensure the TMS coil remains in the same position within the X, Y, and Z directions during and between each session. Resting motor threshold for the muscle agonist to the training (see following text) was determined at this position and dened as the minimum TMS intensity that evoked motor-evoked potentials (MEPs) of 50 V in 5 of 10 trials (Rossini et al. 1994). Muscle relaxation was monitored by visual feedback of the EMG recording. Transcranial DC stimulation (tDCS) was delivered through two sponge electrodes (surface area: 25 cm2) embedded in a saline-soaked solution. Depending on the session, the anode or cathode was positioned on the marked motor hot spot and the other electrode on the skin overlying the contralateral supraorbital region. During training, 1-mA anodal tDCS was delivered for about 30 min in the corresponding session and 30 s in the sham session using a Phoresor Auto (Model PM850; IOMED, Salt Lake City, UT). At the onset and offset of tDCS, the current was increased or decreased in a ramplike fashion, a method shown to achieve good blinding level (Gandiga 2006).
TRANSCRANIAL DC STIMULATION. EXPERIMENTAL PROCEDURE. The formation of a motor memory was assessed using a previously described protocol (Butesch et al. 2000; Celnik et al. 2006; Classen et al. 1998; Stefan et al. 2008). At the beginning of each session, 65 TMS stimuli were delivered at 0.1 Hz over the hot spot to determine the baseline TMS-evoked thumb movement direction (Pre; Fig. 1). Subjects subsequently performed

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TRANSCRANIAL MAGNETIC STIMULATION. Transcranial magnetic stimulation (TMS) was delivered using a 70-mm loop-diameter gure-of-eight coil (Magstim BiStim2, Whitland, South West Wales, UK) placed over the left M1 to optimally activate the corticospinal tract and elicit focal isolated and directionally consistent thumb movements. Using a frameless neuronavigation system (BrainSight,

B
anodal/sham tDCS

accelerometer

pre 65 trials

motor training 30 mins Extension

p1 65 trials

rest 10 mins

p2 65 trials

Abduction

Adduction

Flexion
1. Schematic representation of the main experiment setup. A: pre. Transcranial magnetic stimulation (TMS) evoked movement directions were derived from the rst-peak acceleration in the 2 major axes of the movement (extension/exion and abduction/adduction) measured by an accelerometer mounted on the proximal phalanx of the thumb. Black arrows indicate the direction of individual TMS-evoked thumb movements (in this case extension and abduction). B: motor training. Pre was followed in 2 separate sessions randomly ordered by 30 min of motor training with either sham or anodal transcranial DC stimulation (tDCS) being applied over the left primary motor cortex (M1). Voluntary thumb movements were performed in a direction opposite to the baseline TMS-evoked movement direction (in this case: exion and adduction). C: post 1 (p1). The direction of TMS-evoked thumb movements was determined as previously measured in pre. D: post 2 (p2). The subjects rested for 10 min and the direction of TMS-evoked thumb movements was again determined. J Neurophysiol VOL
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FIG.

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motor training, consisting of voluntary thumb brisk movements paced by an audio metronome at 1 Hz for 30 min in a direction opposite to the baseline TMS-evoked direction. For example, if the principal baseline direction was extension and abduction, then subjects were instructed to perform repetitive exion and adduction movements (Figs. 1 and 2). The participants were instructed to relax and let the thumb return to its original position after each motion. This was ensured by monitoring on-line EMG and acceleration signals and providing verbal feedback when needed. Consistency and accuracy of training movements were quantied off-line by calculating the angular variance and compound acceleration of the rst peak acceleration vector (Table 1) (Stefan et al. 2008). In the main experiment, while subjects performed the motor training, one of two interventions were delivered in separate sessions: 1) anodal tDCS applied over the active M1 for a total of 30 min and 2) sham tDCS applied in an identical manner to the anodal session except that stimulation lasted for only 30 s at the beginning of training. At the end of each intervention, 65 TMS-evoked thumb movement directions were determined again as done at baseline for about 11 min [post 1 (p1)]. Then, subjects rested for 10 min and another 65 TMS pulses were applied [post 2 (p2); Fig. 1]. At the end of each session, subjects reported their alertness, attention, and perceived pain of tDCS using a self-scored visual analog scale (VAS), where 1 represents poorest attention, maximal fatigue, and maximal pain and 7 represents maximal attention, least fatigue, and least pain (Table 1) (Stefan et al. 2005).

training was performed under the inuence of cathodal tDCS applied over the active M1 as done in the anodal session.
COMBINATION OF TRAINING AND TDCS. To test whether stimulation alone could elicit changes in TMS-evoked movement directions, three additional subjects (two females, mean age 24 3 yr) were tested using a similar experimental protocol, although they received anodal tDCS for 30 min without performance of motor training.

Data analysis
TRAINING TARGET ZONE. We dened a training target zone (TTZ) as a window of 20 centered on the mean direction of the performed training movements (Butesch et al. 2000; Celnik et al. 2006; Stefan et al. 2008). The percentage of TMS-evoked thumb movements falling within the TTZ, the primary outcome measure, was calculated for pre, p1, and p2.

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TMS-EVOKED MOVEMENT DIRECTION DISTANCE RELATIVE TO TRAINING DIRECTION. For each session we calculated the mean angular

direction of the training movements. Then, the angular direction of each individual TMS-evoked movement was subtracted from this mean value (Classen et al. 1998). For example, if the mean training direction was 320 and a movement in pre had an angular direction of 150 then the deviation from training would be 170 (Fig. 1). These values were averaged for each subject for pre, p1, and p2.
COMPOUND ACCELERATION VECTOR. The mean magnitude of the rst-peak acceleration in the extension/exion direction during pre, p1, and p2 was calculated (Stefan et al. 2008). Since the principal training direction differed across subjects, we inverted the values so that all subjects had an extension movement within the pre block (Stefan et al. 2008). CORTICOMOTOR EXCITABILITY.

Controls
DURATION OF MOTOR MEMORIES. To assess the longevity of the effects observed with anodal tDCS, three subjects that took part in the main experiment were exposed to an additional block of TMS 50 min after the cessation of anodal tDCS and training (p3). SPECIFICITY OF POLARITY EFFECTS ON MOTOR TRAINING. To determine whether tDCS effects were polarity specic, three subjects who completed the main experiment underwent a third session where
TABLE

We calculated the MEP amplitudes in muscles acting as either agonist or antagonist of the trained movement direction for each subject. Amplitudes were measured as peak-to-peak for each individual MEP and then averaged for pre, p1, and p2. Ratios between post and pre amplitudes for the agonist and antagonist muscles were calculated.

1.

Baseline measures
Sham Anodal Paired t-Test: Sham Anodal

Statistical analysis
Separate repeated-measures ANOVAs (ANOVARM) were used for the percentage of movements falling in TTZ, deviation from the training direction, and compound acceleration with factors block (pre, p1, p2) and session (sham, anodal). ANOVARM was also used for the MEP ratio with factors muscle (agonist, antagonist), block (p1, p2), and session (sham, anodal). Measures of baseline corticomotor excitability (motor threshold, TMS stimulus intensity, and MEPagonist and MEPantagonist amplitudes), attention, fatigue, pain of tDCS, and training movement kinematics (angular variance and compound acceleration) were analyzed with separate paired t-tests. When signicant differences were found, post hoc analysis was performed using paired t-test. Data are expressed as means SE and effects were considered signicant if P 0.05.
RESULTS

Parameter

A. Psychological measures Attention, VAS Fatigue, VAS Pain, VAS Compound acceleration, m/s2 Angular variance, deg 4.7 0.4 2.3 0.6 1.4 0.2 4.6 0.4 2.8 0.7 2.0 0.6 P 0.5, t 0.7 P 0.5, t 0.6 P 0.38, t 0.9

B. Motor training kinematics 1.07 0.13 1.06 0.23 P 0.6, t 0.5 17.2 2.0 21.0 3.0 P 0.26, t 1.2

C. Measures of corticomotor excitability preceding interventions Motor threshold (agonist muscle), % MEPantagonist, mV MEPagonist, mV 45.0 2.0 2.9 0.7 1.3 0.4 43.0 2.0 3.1 0.6 1.4 0.4 P 0.13, t 1.6 P 0.9, t 0.14 P 0.9, t 0.05

Psychological measures. Values (means SE) depict subjects choice in a visual analog scale (VAS), where 1 represents poorest attention, maximal fatigue and pain, and 7 represents maximal attention, least fatigue, and least pain. Motor training kinematics. Units (means SE) are in meters per second squared for compound acceleration and degrees for angular variance. Measures of corticomotor excitability preceding interventions. Units (means SE) are the percentage of stimulators output for motor threshold and millivolts for MEP amplitudes elicited in muscles acting as antagonist and agonist to the trained thumb movements. P and t values originate from paired t-tests performed on the sham and anodal sessions. J Neurophysiol VOL

Summary At baseline, TMS-evoked movements were consistent across sessions. Subjects were then trained in the opposite direction for 30 min (Fig. 2). Both, anodal and sham sessions showed training-induced effects where the proportion of TMS-evoked movements reected the practiced movement direction. However, this effect was more prominent in the anodal session
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pre TMS-evoked mvts p1 TMS-evoked mvts p2 TMS-evoked mvts

297

motor training

Extension

anodal tDCS
Adduction Abduction

Flexion

B
sham tDCS

FIG. 2. Intervention-dependent changes in TMSevoked thumb movement directions in a representative subject. Each line represents the rst-peak acceleration direction vector of a single thumb movement. The pre block is characterized by predominantly extension/adduction movement directions in both sessions. A: after motor training with anodal tDCS the direction of TMS-evoked thumb movements (p1) changed to a direction similar to training (exion/abduction). This was partially maintained in p2. B: after training with sham tDCS the direction of TMS-evoked thumb movements within p1 did not have such a dramatic change with only a small proportion of movements moving in a direction similar to motor training. For p2 all movements were in a direction similar to the pre block.

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2 m/s2

immediately after the training (p1) and lasted longer (p2) relative to sham (Fig. 2). Training parameters Subjects ratings of attention, fatigue, and pain were comparable across sessions [separate paired t-tests: t(8) 0.9, P 0.38; Table 1]. For the sham session ve of nine subjects believed they had received real stimulation and seven of nine subjects felt that way in the anodal session. Motor training kinematics measured as compound acceleration and angular variance did not differ across sessions [separate paired t-test: t(8) 1.2, P 0.13; Table 1]. Preceding interventions, motor thresholds, and both MEPantagonist and MEPagonist amplitudes were comparable across sessions [separate paired t-test: t(8) 1.6, P 0.13; Table 1]. Effects of tDCS on motor memory formation ANOVARM revealed a signicant main effect for block [F(2,16) 21.7, P 0.001], session [F(1,8) 16, P 0.004], and block session interaction [F(2,16) 3.6, P 0.05] for the percentage of movements falling within TTZ, the primary outcome measure. Paired t-tests showed that in both sham and anodal sessions there was a signicant increase in the percentage of movements falling in the TTZ from pre to p1 [from 2 1 to 12 4% in sham, t(8) 2.9, P 0.02; and from 2 1 to 17 4% in anodal, t(8) 7.4, P 0.001; Fig. 3A]. However, only in the anodal session was this effect signicantly maintained at p2 [11 4%, t(8) 2.9, P 0.002; Fig. 3A]. In addition, compared with the sham the anodal session had signicantly more movements falling in the TTZ within p1 [t(8) 2.6, P 0.015] and p2 [t(8) 3.6, P 0.004]. TMS-evoked movement direction distance relative to training direction ANOVARM revealed a signicant main effect for block [F(2,16) 23.6, P 0.001], session [F(1,8) 16.8, P 0.003],
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and block session interaction [F(2,16) 5.4, P 0.016]. Post hoc paired t-tests showed that both sham and anodal sessions had a signicant decrease in the relative angular distance from pre to p1 and p2 [sham: pre (146 4) to p1 (112 9) and p2 (128 10), t(8) 4, P 0.002, t(8) 2.3, P 0.03; and anodal: pre (150 4) to p1 (83 10) and p2 (98 14), t(8) 8.4, P 0.001, t(8) 4.6, P 0.001; Fig. 4A]. However, the TMS-evoked movement directions in the anodal intervention were signicantly closer to the training direction than sham during p1 [t(8) 3.4, P 0.005] and p2 [t(8) 2.7, P 0.02; Fig. 4A]. Compound acceleration vector ANOVARM revealed a signicant main effect for block [F(2,16) 10, P 0.001], session [F(1,8) 5.4, P 0.05], and block session interaction [F(2,16) 6, P 0.01]. Paired t-tests revealed that in both sham and anodal sessions there was a signicant change in direction of compound acceleration from pre to p1 [from pre (0.36 0.14 m/s2) to p1 (0.06 0.05 m/s2) in sham, t(8) 2.5, P 0.02; and from pre (0.42 0.11 m/s2) to p1 (0.19 0.1 m/s2) in anodal, t(8) 3.6, P 0.003; Fig. 4B]. However, only in the anodal session was this effect signicantly maintained at p2 [0.12 0.08 m/s2, t(8) 3, P 0.009; Fig. 4B]. In addition, there was a signicantly greater change in compound acceleration vector in the anodal session for p1 and p2 relative to sham [t(8) 2.4, P 0.02, t(8) 2.7, P 0.02, respectively; Fig. 4B]. Corticomuscular excitability ANOVARM revealed a signicant main effect of session [F(1,8) 7.1, P 0.03], block [F(1,8) 20, P 0.002], and muscle [F(1,8) 41, P 0.001] in assessment of MEP amplitudes. In addition, there were signicant interactions between session and muscle [F(1,8) 10.2, P 0.01] and block and muscle [F(1,8) 7, P 0.03]. Paired t-tests showed that within the sham session there was a signicant difference
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B
20

p3 after anodal tDCS

Percentage of movements falling in TTZ (pTTZ)

25

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* * *

pTTZ

pre

p1

p2

p3

20

C sham vs. cathodal

Sham Anodal 10 pTTZ Sham Cathodal

15

10

5 0 0 pre p1 p2 pre p1 p2

FIG. 3. Percentage of movements falling in the training target zone (pTTZ). A: with anodal tDCS applied during training there was a signicant increase in movements falling within the TTZ during p1 and p2. Asterisks denote P 0.02. B: 50 min after the cessation of anodal tDCS (p3) TMS-evoked movement directions returned to premotor training values (n 3). C: TMS-evoked movement directions were similar between sham and cathodal sessions at p1 and p2 times (n 3). D: application of anodal tDCS for 30 min without motor training did not elicit changes in pTTZ. Data are means SE.

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D anodal tDCS without motor training

10 pTTZ 0

pre

p1

between the increase of the MEPagonist ratio (post/pre) (1.4 0.15) compared with the MEPantagonist ratio (1.18 0.07) for p1 only [t(8) 2.4, P 0.02; Fig. 5B], whereas in the anodal

session there was a signicant difference at p1 [MEPagonist (1.8 0.2), MEPantagonist (1.14 0.16), t(8) 5.5, P 0.002] and p2 measures] MEPagonist (1.38 0.17), MEPantagonist

A
170

* *

* * * *

TMS-evoked movement direction angles relative to training (degrees)

150 130 110 90 70

Sham Anodal
FIG. 4. Angular distance and compound acceleration of TMS-evoked movements. A: angular distance of TMS-evoked movements relative to training during pre, p1, and p2. In comparison to sham, anodal tDCS led to a greater reduction in the angular distance between training movements and those evoked by TMS at p1 and p2 times. Please note that at pre, both sessions have similar angular difference from the trained direction. B: average compound acceleration during pre, p1, and p2. Anodal tDCS led to a greater change in direction of compound acceleration during p1 and p2 relative to sham. Asterisks, P 0.03. Data are means SE.

Training direction 0

B
0.6
Compound acceleration (m/s2)

pre

p1

p2

* *

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0.2

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0 Sham Anodal

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A
5 MEP amplitude (mV) 4 3 2 1 0 pre p1 Sham p2 pre p1 Anodal p2
FIG. 5. Corticomotor excitability, measured by the motorevoked potential (MEP) amplitude for the agonist and antagonist muscles involved in motor training. A: changes in the MEP amplitude recorded from the training antagonist (closed squares) and agonist (open diamonds) muscles. MEP amplitudes increased with both sham and anodal tDCS. B: MEP amplitude ratio (post/pre) signicantly increased in the MEPagonist muscle compared with the MEPantagonist during p1 for both sham and anodal tDCS. For anodal tDCS this difference was maintained during p2. For both p1 and p2, anodal tDCS resulted in a greater increase in the MEPagonist ratio. Asterisks, P 0.05. Data are means SE.

antagonist agonist

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* *
2 MEP ratio (post/pre)

* *

*
1

0 ago antag p1 ago antag p2 ago antag p1 ago antag p2

Sham

Anodal

(0.93 0.11), t(8) 2.8, P 0.04; Fig. 5B]. In addition, relative to sham the anodal session showed a signicantly greater increase in the MEPagonist ratio for p1 [t(8) 2, P 0.05] and p2 [t(8) 2, P 0.05]. Control results Only the anodal group showed a clear training effect during p2, suggesting that the motor memory had been retained for a longer period (30 min). To test whether this effect was still present 50 min after the cessation of training, three subjects were given an additional block of TMS (p3, Fig. 3B). The percentage of movements falling in the TTZ were not signicantly different between pre and p3 [2.2 2 and 3 1%, respectively; paired t-test: t(2) 0.6, P 0.6]. Similarly, at p3 there were no signicant differences in MEPagonist and MEPantagonist ratios [1.1 0.03 and 0.9 0.08 mV, respectively; paired t-test: t(2) 2.1, P 0.16], indicating a return to similar excitability to the pre measure. To determine whether the previously described anodal tDCS effects were polarity specic, three subjects participated in a third session in which cathodal stimulation was applied over M1 during training. Here, the percentage of movements falling in the TTZ were not signicantly different from sham stimulation in either p1 or p2 [cathodal: 10.4 0.6 and 2.1 0.6%, respectively, ANOVARM: session: F(1,2) 0.3, P 0.3; block:
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F(1,2) 53, P 0.001; session block F(2,4) 0.6 P 0.6; Fig. 3C]. MEP ratios were also statistically similar across sham and cathodal sessions for p1 [MEPagonist (1.39 0.03), MEPantagonist (1.1 0.09)] and p2 measures [MEPagonist (1.0 0.04), MEPantagonist (1.0 0.1); ANOVARM: session: F(1,2) 0.3, P 0.66; block: F(1,2) 23, P 0.04; muscle: F(1,2) 50, P 0.019; all interactions: F(1,2) 5.6, P 0.14]. Finally, to determine whether anodal tDCS without training could elicit changes in TMS-evoked movement directions we studied an additional group of three subjects. In this group there was no signicant difference in the percentage of movements falling in the TTZ between pre (1 0.7%) and p1 [1 0.5%, paired t-test: t(2) 0.5, P 0.7; Fig. 3D]. Similarly, there was no signicant difference between MEPagonist and MEPantagonist muscle ratios [1.09 0.02 and 1.1 0.07, respectively; paired t-test: t(2) 0.3, P 0.8].
DISCUSSION

The main nding of the present study was that anodal transcranial DC stimulation (tDCS) over the primary motor cortex, engaged in generating the training movements, enhanced the encoding and retention of motor memories. This effect was reected by 1) changes in all kinematic measures (movements falling in the training zone, angular differences, and comwww.jn.org

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pound muscle accelerations), 2) longer-lasting effects relative to training alone, 3) the required association of training and stimulation, and 4) the polarity specicity. Previous studies have shown that performing simple motor training results in the formation of motor memories containing kinematic details of the practiced motions (Butesch et al. 2002, 2004; Celnik et al. 2006; Classen et al. 1998; Floel et al. 2005a,b; Stefan et al. 2008). Consistent with these, we found that motor training with sham tDCS increased the probability of TMS-evoked movements falling in the TTZ, decreased the net TMS-evoked movement direction distance relative to the training direction, and changed toward the direction of the practiced movements the net acceleration of all TMS-evoked thumb movements. However, when the same group of participants performed the motor training under the inuence of anodal tDCS they experienced larger gains in all measures in p1 and maintained the effects into p2. These ndings appear to be polarity specic, since training during cathodal tDCS resulted in effects similar to those during training with sham and required the association of physical practice with anodal stimulation because tDCS alone did not result in any kinematic change. Since subjects performing the training combined with anodal tDCS have persistence of the effects 30 min after the completion of training (p2)longer motor memory retentionwe decided to investigate the longevity of the motor memory formed. With this aim, we retested a subgroup of participants 50 min after the cessation of training and stimulation (p3). Across all measures, performance returned to pretraining values, indicating that any changes within the motor cortex representation had reverted to baseline. This type of motor memory encoding enhancement has been previously observed with D-amphetamine (Butesch et al. 2001; Sawaki et al. 2002a), hebbian-type TMS stimulation during training (Butesch et al. 2004), and congruent action observation (Celnik et al. 2006; Stefan et al. 2008). Of note, because ratings of attention, fatigue, and pain of the tDCS were similar across sessions, it is unlikely that these results could directly be explained by unspecic psychological differences across sessions. Similar to previous studies, we found that training resulted in a specic increase in the excitability of the training muscles (Butesch et al. 2004; Celnik et al. 2006; Classen et al. 1998). Interestingly, anodal tDCS increased the excitability of the muscle agonist to the training without a similar change in the antagonist muscle, suggesting that tDCS had a training-specic effect rather than a global increase in corticomotor excitability. These results, together with the ndings that the no-training plus tDCS control group had minimal changes in the excitability of either muscle, suggest that the effect of tDCS on cortical excitability is more prominent on neuronal populations engaged in motor actions. Cathodal tDCS, although known to reduce the excitability of the motor cortex at rest (Nitsche et al. 2003a; Purpura and McMurtry 1965), in the present study did not inuence corticomotor excitability relative to sham. It is possible that this lack of effect was due to the performance of motor training, which elicits increased excitability (Butesch et al. 2002, 2004; Celnik et al. 2006; Classen et al. 1998; Floel et al. 2005a,b;
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Stefan et al. 2008), thus overiding or masking the effect of cathodal tDCS. The mechanism behind the current enhancement of motor memory encoding through anodal tDCS is unknown. However, this form of motor memory encoding is inuenced by NMDAreceptor function (Butesch et al. 2000) and it has been indirectly shown that within humans the mechanisms underlying the effect of tDCS involves the modulation of NMDA receptors (Nitsche et al. 2003d). Therefore it is plausible that the observed results are due to anodal tDCS increasing the efcacy of NMDA-receptor function, possibly through an increase in intracellular calcium concentration (Bennett 2000). A previous study by Rosenkranz et al. (2000) explored the effect of tDCS during a paradigm identical to that of the present study by applying tDCS only during the last 10 min of training. The authors found that both anodal and cathodal tDCS had a negative effect on the formation and retention of a motor memory. We believe that the stark difference in results may be due to the differing tDCS protocols. Work by Iezzi et al. (2008) and Siebner et al. (2004) have shown that a TMS protocol can be either facilitatory or inhibitory, depending on the prior state of the system. It is possible that applying tDCS after 20 min of training has an effect different from that of tDCS at the onset of training due to the preconditioning of the motor cortex resulting from training. Alternatively, it is possible that the lack of ndings in the Rosenkranz investigation was due to a shorter stimulation period. Noninvasive cortical stimulation with tDCS elicits behavioral gains during implicit motor learning (Nitsche et al. 2003d), visuomotor processing (Antal et al. 2004; Reis et al. 2009), and probabilistic learning (Kincses et al. 2004) in healthy volunteers. In addition, it has been shown to improve motor performance in stroke (Hummel et al. 2005) and Parkinson disease patients (Boggio et al. 2006). More recently, Reis et al. (2009) reported that tDCS in association with training enhances learning of a novel skill by modulation of short-term retention. The present study supports these observations and suggests that anodal tDCS enhances both the process of encoding and retention of simple motor memories. In addition, we can rule out changes in memoryrecall mechanisms since the behavioral measures obtained with the TMS paradigm used here do not require voluntary engagement of recall processes. One of the initial steps in the development of more complex skills is the encoding of motor memories (Classen et al. 1998; Molina-Luna et al. 2008). It has been suggested that the motor cortex may encode the memory trace of a skill (Monls et al. 2005) because changes in corticomotor organization occur during training (Karni et al. 1995; Kleim et al. 2004; Nudo et al. 1996a,b,c) and skill learning is associated with an enlargement of representations of trained body parts (PascualLeone et al. 1995). Therefore this study, showing that anodal tDCS over M1 enhances the motor memory formed after simple repetitive practice, may provide evidence as to how this form of stimulation results in the behavioral gains observed in previous studies (Antal et al. 2004; Kincses et al. 2004; Nitsche et al. 2003d; Reis et al. 2009). In summary, we showed that anodal tDCS enhances the effects of simple repetitive training on the formation and retention of motor memories. This effect was polarity specic, relied on the interaction between motor training and tDCS, and lasted longer than performance of training without stimulation. The observed changes in the motor cortex representation may
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play an important role in the acquisition of more complex skills. The current results provide a rationale for the possible mechanism that may underlie the behavioral gains observed with the application of tDCS in previous studies. It also supports the view that such noninvasive cortical stimulation can be a useful tool in the rehabilitation of patients with brain damage.
GRANTS

This work was supported by National Institute of Child Health and Human Development Grants R01 HD-053793 and R21 HD-060169.
REFERENCES

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