Participants

Seventy-five university students participated in the study with an age range between 19 and 30 (47 females and 28 males). Subjects were randomly assigned to one of five experimental groups, with 15 subjects in each group. No significant differences were observed between experimental groups in age or handedness (see Supplementary Table S2). A screening for contra-indications of tDCS and TMS was administered prior to participation. Participants gave their written informed consent and all procedures were approved by the Ethical Committee of the University of Leuven (KU Leuven) and were in accordance with the Declaration of Helsinki (1964).

Design and procedure

A schematic representation of the main procedure is given in Fig. 3. In four daily sessions (Day 1–4), subjects were trained in a bimanual tracking task. For each subject, all the sessions were conducted at a similar time of the day (±2 h). Prior to each training session, a questionnaire assessed the quality and quantity of sleep, caffeine and alcohol intake to evaluate differences in these confounding variables between the experimental groups. In addition, every day a pre-measurement of the bimanual tracking task was applied. After the pre-measurement, tDCS was applied for 20 minutes. After five minutes of tDCS the bimanual tracking training session of 20 minutes was started. The stimulation was continued for 15 minutes during the training. Following the training, a post-measurements of the bimanual tracking task was administered. Furthermore, a tDCS sensation questionnaire was administered to evaluate effectiveness of blinding subjects to the stimulation type (sham versus anodal tDCS). Six to seven days after the last training session (~Day 11), a delayed post-measurement of the bimanual tracking task was completed without any tDCS to assess long-term consolidation effects.

Figure 3 Schematic overview of the protocol. The top panel shows the protocol for the training days or all stimulation groups. The bottom panel shows the protocol for the retention day. All blocks followed immediately after each other as indicated by the arrows. Full size image

We assessed working memory in the groups aDLPFC, sDLPFC and BEHAV to assess earlier findings on the effect of DLPFC stimulation on working memory. Two well-known working memory tasks were administered: a parametric Go/No-Go task and a 3-back task. To assess the short and long term effects of left DLPFC stimulation, subjects were assessed before the first tDCS session (pre-test on Day 1), after the last tDCS session (post-test on Day 4) and during the delayed post-measurement, one week later (Day 11).

For the group who received stimulation over left M1 (sM1 and aM1), we assessed changes in corticospinal excitability using TMS. Corticospinal excitability was examined immediately before and following tDCS (i.e., anodal and sham on Days 1 through 4) by applying single-pulse TMS to the left M1 targeting right flexor carpi radialis (FCR), one of the prime mover muscles for the task we used. Excitability was also assessed on Day 11.

Bimanual tracking task

Subjects performed a complex bimanual tracking task in which they had to track a moving dot by rotating two dials with both hands simultaneously. A general description of the task is given below, additional details can be found in Beets et al., and Sisti et al.17,38. The task combined skilled motor training in addition to cognitive control over coordination of both hands which requires intensive practice17,38. Starting from the centre of the display, a target dot moved along a straight line towards the edge of the display at different slopes (see Fig. 4). The left hand controlled movements of the cursor in the vertical direction, while the right hand controlled horizontal movements. Complexity of the task was increased by varying the relative frequency ratio (FR) of the hand movements. Different frequency ratios corresponded to different slopes of the target trajectory (see Fig. 4). A frequency ratio of 1:3 indicates the right hand (second digit in the ratio 1:3) had to move three times as fast as the left hand (first digit). Each trial consisted of two phases: a planning phase and a tracking phase. In the planning phase, the target trajectory was shown without the target moving. This allowed subjects to prepare their movements. After two seconds, the target dot started moving along the trajectory at a constant speed and subjects were instructed to track the dot (tracking phase, 7 seconds).

Figure 4 Bimanual tracking task. The left panel shows the task set-up. Subjects were instructed to rotate two dials with both hands simultaneously in order to track the target dot that moves along the blue path on the screen. The trial shown here included delayed feedback (red line that showed the tracking trajectory at the end of the trial). Hands were hidden from view by a table-top bench. On the right panel, a schematic illustration of the frequency ratios with corresponding target blue paths is presented. The semi-circled arrows indicate the direction of movement for the left (LH) and the right (RH) hand, while the ratios indicate the required movement speed of the hands, i.e., the antecedent represents the speed of the left hand, while the consequent indicates the speed of the right hand. Full size image

On every training day (Day 1–4), subjects underwent a pre-measurement, training, and a post-measurement. Pre- and post-measurements consisted of 2 trials per condition (5 FR × 2 quadrants × 2 trials = 20 trials) that were presented in one block and without feedback (only the target trajectory was visible, not the tracking trajectory). The training phase consisted of 10 trials per condition (5 FR × 2 quadrants × 10 trials = 100 trials). During training, conditions were presented in random order, a training scheme that has shown to be advantageous over blocked learning37,60. To optimize learning, we opted for fading feedback over training days. Participants either received concurrent feedback (showing the last second of the actual tracking trajectory along with the target trajectory), delayed feedback (static representation of the tracking trajectory was shown along with the target trajectory after the trial and not during tracking) or no feedback during the training trials. The number of trials with concurrent feedback was decreased during training and the number of trials without feedback was increased61,62. To keep participants motivated, a constant number of trials with delayed feedback was included in each training block. At retention (Day 11), a post-measurement of 4 trials per condition without feedback was completed (5 FR × 2 quadrants × 4 trials = 40 trials). Our outcome measure was the mean target deviation or the average Euclidian distance between target and position of the cursor at any point in time.

Working memory tasks

To confirm previous findings on the beneficial effect of tDCS over DLPFC on working memory, we included two tasks in our protocol that measured different components of working memory in groups aDLPFC, sDLPFC, and BEHAV. First, to measure sustained attention, response inhibition and set-shifting, we applied a parametric Go/No-Go task63. Second, a traditional n-back working memory task was included in the protocol. Following previous studies27,45 and a pilot experiment, we have opted for a 3-back task to avoid ceiling and floor effects that might obscure subtle performance differences following tDCS. Detailed descriptions of the tasks are given as Supplementary Material.

Questionnaires

At the start of each session, subjects reported the number of hours of sleep and the quality of sleep in the night proceeding the session on a 10-point scale. In addition, we asked for the number of units of alcohol and caffeine intake in the last 12 hours before the session.

After the BTT training with tDCS, the stimulation groups (aDLPFC, sDLPFC, aM1, and sM1) received a questionnaire about their perceived sensations during the stimulation. We asked to report the presence and severity of feelings of itchiness, tingling, headache, neck pain, scalp pain, burning, warmth/heat, pinching, iron taste, fatigue, concentration difficulties and acute mood changes. In addition, the onset and duration of these sensations was questioned and subjects were asked if the sensations influenced their performance.

tDCS

tDCS was delivered by a battery-driven constant current stimulation (HDCStim class IIa; Model: HDCelEN-05, Newronika s.r.l., Milano, Italy). The common safety guidelines were followed64,65,66. The current was transferred by an anodal conductive-rubber electrode of 5 by 5 cm (current density: 0.08 mA/cm2) and a cathodal electrode of 6 by 8.5 cm (current density: 0.04 mA/cm2), which were placed in saline-soaked sponges. The choice for a bigger cathode was guided by the observation that increasing the size of the electrode turns the cathode in an effectively inactive electrode55. The centre of anodal electrode was placed over left M1 or left DLPFC. M1 was localized using a TMS hot spotting procedure. The left DLPFC, which corresponds to the F3 position in the international 10–20 system of EEG electrode placement, was determined by the method described in Beam, Borckardt, Reeves, and George67. In this method, the F3 position is calculated based on circumference, nasion-inion distance and the distance between the left and right pre-aurical point. The cathodal electrode was placed over the right supraorbital area at a minimal distance of 6 cm from the anodal electrode to decrease the probability of shunting of current through the scalp68. Electrodes were fixed to the head with a tubular elastic net bandage. In active conditions (aDLPFC and aM1), the stimulation lasted for 20 minutes at an intensity of 2 mA with a ramping up and down over 12 seconds. In the sham conditions (sDLPFC and sM1), the current turned off after 36 seconds in order to produce the same sensations as in the active conditions without the long-lasting effects of stimulation69. Subjects were blind to stimulation condition. Impedance was monitored by the stimulator.

TMS

In aM1 and sM1, muscle activity was measured via EMG surface electrodes (Ag/AgCl) that were placed in belly-tendon montage over right FCR. A reference electrode was placed over the lateral epicondyle of the right arm. Raw signals were amplified with a gain of 1000 band-passed filtered (10–500 Hz) and sampled at 2000 Hz using a 16-bit analog to digital system (CED Limited, Cambridge, UK).

Single-pulse TMS was delivered with a standard figure of eight coil (70 mm loop diameter) connected to a Magstim 200 stimulator (Magstim Company, Dyfed, UK). The coil was positioned tangentially over scalp with the handle holding backward and laterally at 45° away from the mid-sagittal line. To maintain constant coil-positioning over the M1 hotspot of right FCR throughout the subsequent days of testing, the positioning and angles of the coil were monitored by a neuronavigation system (ANT, Enschede, The Netherlands). For navigation, a standard 3D anatomical MRI was used which was co-registered with the positions of the subject’s nasion, left ear, right ear and heap shape. After localization of the hotspot of the right FCR muscle in left M1, the position was marked with a semi-permanent marker for tDCS electrode placement. Subsequently, we measured resting motor threshold (rMT) on each day, prior to training and tDCS. The rMT is defined as the lowest intensity that elicits a motor evoked potential (MEP) of minimum peak-to-peak amplitude of 50 microvolts in 3 out of 5 consecutive trials. Before and after tDCS, we elicited MEPs by delivering 20 pulses at 130% of the individual rMT with an inter-stimulus interval of 5 seconds.

Data analyses

BTT data were pre-processed in Labview (National Instruments) and MATLAB (Mathworks). A cluster analysis was performed in SPSS (IBM). All other statistical analyses were performed in R70. In case of violation of the sphericity assumption in ANOVA, a Greenhouse-Geisser correction was applied to the degrees of freedom and the p-value. Bonferonni correction for multiple comparisons was applied when necessary. The level of significance was set at p < 0.05. The data were also examined by estimating a Bayes factor using JZS Bayes factor repeated measures ANOVA with default prior scales, comparing the likelihood of the data under the null hypothesis and the alternative hypothesis41. For main effects, we have reported Bayes factors comparing the model including the relevant main effect and an effect of subjects (to account for the repeated measures) versus a null model with only an effect of subjects. For the interactions effects, the comparison between a model with the relevant interaction and an effect of subjects versus a null model with only an effect of subjects is meaningless if there is no evidence for including the main effects in the model. Therefore, Bayes factors for interaction effects are only reported when evidence for all main effects was observed. In that case, the reported Bayes factor compares a model with interaction, both main effects and an effect of subjects versus a null model that includes the main effects, the subject’s effect, but no interaction effect.

In the TMS data, trials in which root mean square (RMS) EMG exceeded 15 μV71 during the 40 ms immediately preceding the TMS pulse were discarded. Across subjects, 5.24% of the trials were excluded from analyses. In addition, we checked the stability of the baseline measures (pre-measurement on Day 1) by performing an unpaired t-test on the first and second half of the baseline data for each subject. After Bonferroni correction, no indication of an unstable baseline in any of the subjects was found. Subsequently, data were inspected for deviations from normality and averaged over all trials of a session. Next, data were normalized to an individual baseline by dividing the average MEP amplitude of each session by the average MEP amplitude of the first session (pre-measurement on Day 1).