Transcranial direct current stimulation (tDCS) is a non-invasive method for modulating neuronal excitability. It does so by altering the neuronal membrane at rest, and appears to be a promising tool for improving motor skills and initiating neuronal plasticity in the motor cortex (Kidgell et al., 2013), with its strongest effects seen when co-applied with motor activity (Reis and Fritsch, 2011; Saucedo Marquez et al., 2013). In tDCS, a weak and constant current in the range of 0.5–2 milliampere (mA) is applied to the cerebral cortex through the skull (Nitsche and Paulus, 2000; Priori, 2003). This is thought to provoke a polarity-specific subthreshold influence on the resting membrane potential (Nitsche and Paulus, 2000). While anodal stimulation facilitates spontaneous neuronal activity, it is assumed that cathodal tDCS leads to the converse effect and results in inhibition of the stimulated brain area (Stagg and Nitsche, 2011; Dayan et al., 2013). Direct current is conventionally applied using two or more rectangular sponge or rubber electrodes with a size of 25–35 cm² (Ruffini et al., 2014). The so-called high-definition tDCS (HD-tDCS) is a modified tDCS-method that uses smaller (1–5 cm²) and usually round sponge or gel electrodes (AgCl) to stimulate the brain region of interest. Based on physiological and computational studies, it is thought that HD-tDCS applies the current flow more focally, allowing a more target-oriented application of transcranial direct currents (Edwards et al., 2013; Miranda, 2013; Villamar et al., 2013; Nikolin et al., 2015; Alam et al., 2016). The first attempts by our group to target the motor domain using this technique have already indicated successful modulation of motor adaptation (Doppelmayr et al., 2016) and sensorimotor performance (Pixa et al., 2017).

There is growing evidence that tDCS of the motor cortex improves motor performance and motor learning in both healthy individuals as well as patients suffering from diverse neurological diseases (Bastani and Jaberzadeh, 2012; Brunoni et al., 2012). Such positive tDCS effects on motor skills have been reported for single-session applications, as well as for multiple applications over several days of training (Vines et al., 2008; Reis et al., 2009, 2015; Schambra et al., 2011; Saucedo Marquez et al., 2013; Waters-Metenier et al., 2014), with prolonged improvement of up to 3 months (Reis et al., 2009), suggesting an impact on neuroplasticity mediated by synaptic long-term potentiation (LTP) within the motor cortex (Liebetanz et al., 2002; Reis and Fritsch, 2011; Stagg and Nitsche, 2011). Unfortunately, most tDCS studies have focused almost exclusively on unimanual motor performance (for an overview, see Buch et al., 2017).

Since humans spend over half of their time each day grasping and manipulating objects with both of their hands (Kilbreath and Heard, 2005), motor behavior in everyday life—whether in sport or in occupational and recreational activities—most often involves skilled and precise bimanual movements. There is also evidence that bimanual motor training leads to improvements in hand and arm function in the paretic limb, e.g., in stroke patients (Cauraugh et al., 2010). Consequently, more research is needed on how to support bimanual training with specific stimulation protocols. tDCS appears to be a promising tool in this regard, due to evidence of its ability to improve unimanual motor skills, its low cost, its relative ease of use and its noninvasive nature.

There is consensus that bimanual movements are not driven by a single brain region; rather, the complex organization of bimanual behavior is based on a motor network within and between both brain hemispheres (Swinnen and Wenderoth, 2004; Grefkes et al., 2008). In this neural network, the primary motor cortices (M1) play an essential role due to their key functions in goal-oriented movement execution (Halsband and Lange, 2006; Hardwick et al., 2013). In addition, M1 is associated with the consolidation of newly learned motor skills (Brashers-Krug et al., 1996; Doyon and Benali, 2005; Dayan and Cohen, 2011; Wessel et al., 2016). Further insights into the cortical activation pattern of bimanual motor behavior come from transcranial magnetic stimulation (TMS) studies. McCombe Waller et al. (2008) demonstrated strong bilateral excitation in left and right M1, when both hands were used simultaneously. Changes in motor cortical excitability due to bimanual training of bilateral wrist movements revealed strong activation of the M1 in both hemispheres (Neva et al., 2012). During synchronous (i.e., in-phase) movements of both wrists, which requires intra- and intercortical interactions, Byblow et al. (2012) reported decreased intracortical inhibition (ICI) as well as interhemispheric inhibition (IHI), with no changes in ICI or IHI during anti-phase movements. In line with that finding are observations of increased bilateral cortical activity in asynchronous compared to synchronous bilateral tasks (Haslinger et al., 2004). Considering these cortical activation patterns during bimanual movements, it seems appropriate to target both M1s as a first step when investigating tDCS effects on bimanual motor performance. This, however, has not been done systematically as of yet. Consequently, reports on the effects of tDCS on bimanual behavior and motor skill learning are scarce. Carter et al. (2015) used tDCS to modulate the excitability of the supplementary motor area (SMA) and to investigate polarity-dependent consequences in bimanual behavior. They reported that only anodal tDCS enhanced the stability and consistency of bimanual anti-phase wrist movements along a delayed transition from anti-phase to in-phase movements (Carter et al., 2015). The same group also showed that anodal tDCS over the SMA facilitates the switch between anti-phase and in-phase when performed intentionally (Carter et al., 2017). Others (Vancleef et al., 2016) applied tDCS to the left M1 (lM1) or left dorsolateral prefrontal cortex (lDLPFC) concurrently over 4 days (one session per day) of learning a complex bimanual task. Bimanual motor skill learning did not differ between the groups, nor were there any neurophysiological changes following tDCS. The lines of research focusing on bimanual tasks have either stimulated the SMA or only one M1. To our knowledge, the extent to which simultaneous stimulation of both M1s impacts bimanual performance has only been investigated by two studies. In the first, Gomes-Osman and Field-Fote (2013) applied anodal tDCS using two anodes, one over the left and one over the right M1, with two bilateral cathodes over the left and right supraorbital cortex. tDCS was applied in between performance of a bimanual typing task for five consecutive days. The authors observed significantly greater task improvements from day 1 to day 5 for the stimulated participants compared to sham-stimulated control group. However, this effect vanished after a retention interval of 1 week, possibly due to the non-concurrent timing of tDCS with motor practice. The second study was recently published by our group and showed improvements in unimanual and bimanual dexterity assessed with the Purdue Pegboard Test (PPT) after HD-atDCS bilateral M1 co-applied with motor training for three consecutive days (Pixa et al., 2017). Here, prolonged improvements were seen after a retention interval of 1 week, indicating increased bimanual motor learning and consolidation.

To summarize, it has been consistently shown that unimanual motor performance can be improved by anodal stimulation of M1, but little is known about the effect on bimanual motor performance. Thus, further exploration of whether tDCS can improve bimanual motor performance is required—a request that has been frequently made, since many daily activities require skillful coordinated use of both hands to interact with the surrounding environment. Such knowledge is relevant beyond a simply theoretical point of view, given that a) rehabilitation of bimanual motor behavior is essential for healthy and independent living in old age or after disease (e.g., for persons with bilateral hand dysfunction) and b) patients benefit from bimanual training, e.g., during recovery after stroke. Therefore, the central purpose of the present study was to investigate the effects of HD-atDCS on performance in a bimanual sequential sensorimotor task. The present approach uses a sport stacking task that requires the subject to build up (stack up) and dismantle (stack down) a formation of objects (cups) in a predefined and fixed formation (objects, actions and order) as quickly and as accurately as possible using both hands. Since sport stacking has already been the object of investigation in earlier studies concerning saccadic eye movements and attention (Foerster et al., 2011, 2012), impact on hand-eye coordination and reaction time (Udermann et al., 2004) and influence on motor and cognitive functions (McKune et al., 2011), it seems perfectly suited to our investigation. In addition to requiring well-established coordination between the upper limbs, sport stacking requires bimanual goal-oriented dexterity to precisely grasp and manipulate the cups at a high speed. Furthermore, it has several advantages, such as novelty, competitive character, task variations, ecological validity and no ceiling effects expected in a short time period (Foerster et al., 2011, 2012).

We hypothesized that sport stacking training conducted over multiple days with co-application of HD-atDCS to both M1s simultaneously would lead to superior performance gains (expressed in shorter movement time) compared to a sham-stimulated control group.

Statistical analysis of sport stacking performance (fST) using three-way ANOVA with repeated measures showed a significant main effect for TIME (F_(2,60) = 221.27, p < 0.001, ωp2 = 0.874, 90% CI (0.827, 0.905)) and a significant TIME × GROUP interaction (F_(2,60) = 4.66, p < 0.05, ωp2 = 0.104, 90% CI (0.017, 0.253)). Neither the interaction of FORMATION × GROUP (F_(2,60) = 0.53, p > 0.05, ωp2 = 0.104, 90% CI (0.017, 0.253)) nor the three-way interaction among TIME × GROUP × FORMATION was significant. Finally, no significant main effect for GROUP was found (p > 0.05). To analyze the sport stacking performance for the 3-6-3 stack and the 1-10-1 stack, separate two-way ANOVAs with repeated measures were calculated. The two-way ANOVA with repeated measures for the 3-6-3 stack revealed a significant main effect of TIME (F_(2,60) = 239.87, p < 0.001, ωp2 = 0.883, 90% CI (0.840, 0.912)) and a significant TIME × GROUP interaction (F_(2,60) = 11.21, p < 0.001, ωp2 = 0.245, 90% CI (0.109, 0.396)), with improved performance in the STIM-M1 group. No significant main effect for GROUP was found (p > 0.05; Figure 4A). Bonferroni-corrected post hoc t-tests revealed no significant differences between both groups at any time point (p > 0.05). An ANCOVA for the 3-6-3 stack (fST) revealed that the covariate (pretest score 3-6-3) was significantly related to performance in the posttest (F_(1,29) = 26.81, p < 0.001, ωp2 = 0.454, 90% CI (0.243, 0.621)). The significant effect of the stimulation on posttest performance remained after controlling for the effect of the pretest score (F_(1,29) = 13.36, p < 0.001, ωp2 = 0.285, 90% CI (0.095, 0.489)). The pretest score was also significantly related to performance in the follow-up test (F_(1,29) = 8.73, p < 0.05, ωp2 = 0.200, 90% CI (0.042, 0.416)) and the stimulation had still a significant effect on follow-up test performance (F_(1,29) = 4.80, p < 0.05, ωp2 = 0.109, 90% CI (0.005, 0.327)). Analysis of performance improvements for the 3-6-3 stack by Bonferroni-corrected t-tests revealed significant differences between groups in both performance gain scores: (1) Pre-Post Score (t₍₃₀₎ = −4.25, p < 0.05, d = −1.503, 95% CI (−2.282, −0.703)), with an achieved power of 1-β = 0.99. (2) Pre-Follow-up Score: (t₍₃₀₎ = −3.14, p < 0.05, d = −1.110, 95% CI (−1.849, −0.355)), with an achieved power of 1-β = 0.92. Both performance gain scores indicate stronger improvements for the STIM-M1 group (Figure 4B).

Analysis of sport stacking performance (fST) for the 1-10-1 stack using two-way ANOVA with repeated measures (pre-, post- and follow-up test) showed a significant main effect of TIME (F_(2,60) = 142.21, p < 0.001, ωp2 = 0.818, 90% CI (0.751, 0.862)). However, no significant TIME × GROUP interaction emerged (p > 0.05). Again, no significant main effect for GROUP was found (p > 0.05; Figure 4C) and Bonferroni-corrected post hoc t-tests showed no significant differences (p > 0.05). The ANCOVA for the 1-10-1 stack (fST) revealed that the covariate (pretest score 1-10-1) was significantly related to performance in the posttest (F_(1,29) = 11.41, p < 0.05, ωp2 = 0.251, 90% CI (0.072, 0.461)). The significant effect of stimulation on posttest performance remained after controlling for the effect of the pretest score (F_(1,29) = 4.77, p < 0.05, ωp2 = 0.108, 90% CI (0.046, 0.327)). The pretest score was also significantly related to performance in the follow-up test (F_(1,29) = 14.80, p < 0.001, ωp2 = 0.308, 90% CI (0.112, 0.508)) but no significant stimulation effect was found on follow-up test performance (F_(1,29) = 2.80, p = 0.105, ωp2 = 0.055, 90% CI (0.000, 0.265)). For the 1-10-1 stack, the performance improvements showed no significant differences between groups (p > 0.05; Figure 4D).

The central purpose of this study was to examine the effects of HD-atDCS applied over the left and right M1 on the performance of the sequential sensorimotor task of sport stacking compared to sham stimulation. Overall, the results demonstrate that the application of HD-atDCS concurrently with motor training over multiple days significantly improved cumulative performance in sport stacking (fST) in the stimulation group (STIM-M1). This effect remained after a 1-week retention interval, reflected by superior performance gain scores in the follow-up test in STIM-M1. This finding fits well with our hypothesis that anodal stimulation of both M1s leads to performance gains on a bimanual task compared to sham stimulation, and is in line with previous reports on cumulative improvements (Reis et al., 2009, 2015; Gomes-Osman and Field-Fote, 2013; Saucedo Marquez et al., 2013; Pixa et al., 2017). In a differentiated analysis, however, a clear and significant positive effect with sufficient effect sizes was only found for the 3-6-3 task, and not for the 1-10-1 task, although a similar pattern was observed that did not reach statistical significance. This result is only partially in line with our hypothesis. In spite of the randomization of group assignment, the pretest values for the 3-6-3 stack varied slightly between the groups, which might have had an impact on the outcome score and may have biased the treatment effects of HD-atDCS. However, the ANCOVAs that treated pretest scores as covariates showed significant correlations to posttest scores. There was still a significant and strong effect of the stimulation, which means that the significant interaction was not only attributable to baseline performance differences but also considerably to the stimulation itself. Generally, our results support previous studies reporting positive tDCS effects on unimanual motor behavior (for a review, see Buch et al., 2017) and extend these findings to bimanual motor performance (Gomes-Osman and Field-Fote, 2013; Pixa et al., 2017).

Given the fact that M1 is a major source of motor output and is substantially involved in motor consolidation, our data reveal that training with concomitant stimulation influences the motor processes of bimanual movements. In our analysis, both groups showed improved performance from pre- to posttest, demonstrating that training alone, as well as training combined with HD-atDCS, resulted in cumulative learning from day 1 to day 5. However, the performance gain in the posttest was significantly higher for STIM-M1 than for SHAM; the STIM-M1 group had a lower performance level at pretest and ended with a higher performance level in the posttest. This higher magnitude performance gain for STIM-M1 may be attributable to increased excitability in bilateral M1 (Reis and Fritsch, 2011; Stagg et al., 2011) and indicates enhanced motor learning due to neuroplastic changes in the motor system associated with LTP (Reis et al., 2009; Dayan and Cohen, 2011; Reis and Fritsch, 2011). However, it must be noted that only the performance gain on the 3-6-3 stack was significantly higher for STIM-M1 (not the 1-10-1 stack).

The interpretation that increased excitability in M1 led to superior performance gains in the STIM-M1 group is supported by studies that have reported increased bilateral activation in M1 during bimanual movements (McCombe Waller et al., 2008; Byblow et al., 2012; Neva et al., 2012). Thus, bilateral HD-atDCS combined with bimanual training may have boosted the cortical excitability in both M1 compared to bimanual training alone. Furthermore, sport stacking requires the alternate (asynchronous) use of both hands, and, as was briefly reviewed in the introduction, asynchronous bimanual movements elicit stronger bilateral activation in both M1 compared to synchronous bimanual movements (Haslinger et al., 2004). This may also have contributed to superior performance when HD-atDCS was co-applied with the asynchronous task of sport staking. It remains to be seen whether these effects vanish when participants perform sport stacking in a synchronized pattern, with synchronous movements of both hands. Although the comparison of synchronous vs. asynchronous bimanual movements would be an interesting question, a) this was out of scope of the present approach, and b) parallel stacking (i.e., synchronous movements) is expressly forbidden (according to the regulations of the WSSA).

One may argue that the interaction in absolute performance originated in higher baseline values (i.e., lower task performance) in the STIM-M1 group. Nevertheless, superior performance gains for STIM-M1 were preserved at the follow-up test after a 1-week retention interval. Consequently, and in line with recent findings (Pixa et al., 2017), these results indicate that our HD-atDCS protocol was sufficient to influence motor consolidation of the stacking task, possibly due to the well-described excitability increase induced by anodal tDCS (Nitsche and Paulus, 2000; Nitsche et al., 2005) combined with the excitatory effect of bimanual motor training (McCombe Waller et al., 2008; Neva et al., 2012). This effect might have led to stronger neuroplastic changes (increased LTP) in the motor cortex compared to pure motor training alone or stimulation alone without simultaneous motor activity (Reis and Fritsch, 2011; Stagg et al., 2011). It may also explain why no retention effect was found by Gomes-Osman and Field-Fote (2013). Moreover, the present study used a similar study design and stimulation protocol as was used in a previous study by our group (Pixa et al., 2017), which revealed comparable results with cumulative effects over the training period as well as after a retention interval. In that study, we observed improved dexterity in unimanual as well as in bimanual tasks (PPT) after 3 days of training. This supports our assumption that bimanual skills are sensitive to bilateral HD-atDCS co-applied with motor skill training.

Unexpectedly, the acquisition of tasks were slightly differentially affected by stimulation, since the two-way ANOVAs showed a significant interaction for 3-6-3 but not for 1-10-1. Performance outcomes of the ANCOVAs, however, still revealed a significant effect of the stimulation after controlling for pretest values, with the exception of the follow-up test performance for the 1-10-1 stack. These findings indicate that pretest performance might be an important factor (He et al., 2016) to prospectively take into account for training and stimulation protocols (Shmuelof and Krakauer, 2014). Besides the higher ANCOVA effect sizes for the 3-6-3 compared to the 1-10-1 stack, the differential results might be explained by different demands on fine motor skills (e.g., dexterity) and cognitive workload between the tasks: both stacks differ not only in terms of the required motor sequence (3-6-3 vs. 1-10-1), the amount of repetition (the 6-stack must also be built within the 10-stack, and as such, the number of repetitions is doubled), and the refined fine motor skills needed to handle 3, 6 and 3 cups with both hands (maximum of 3 cups in the right hand and 2 cups in the left) as compared to 1, 10 and 1 cups (maximum of 5 cups in the right hand and 4 cups in the left), but also in terms of the cognitive work load of planning the 1-10-1 stack, which is higher or more complex than for the 3-6-3 stack. Thus, the 1-10-1 stack may require higher involvement of fine dexterity when handling more cups in both hands compared to the 3-6-3. Besides these motor aspects, handling more cups at the same time might also require additional cognitive resources to plan and build the formation: In 1-10-1, the 10-stack consists of ten cups built on four levels, with four cups forming the base, followed by three cups, two cups and one cup on top. In contrast, in the 3-6-3 stack, fewer cups are handled at the same time to build the formation of six cups on three levels, where three cups form the base, followed by two cups, and one cup on top (see Figure 2). Whether the stacking formations differ in terms of cognitive demand was not tested in the present study and requires further exploration. That slight differences in task complexity may had led to different demands and different performance outcomes is speculative, although it has already been reported that small task variations can lead to different performance outcomes—an effect that has been linked to the involvement of different learning processes, e.g., implicit learning (Nitsche et al., 2003; Saucedo Marquez et al., 2013; Coffman et al., 2014). Thus, whether or not the higher demand for precise fine-motor skills and cognitive processes in the 1-10-1 task requires a longer training period and/or a varied stimulation protocol warrants further investigation.

Alternatively, our method of alternating training might have caused interference effects known to occur when two explicit motor tasks are trained in an alternating fashion (Brashers-Krug et al., 1996; Krakauer et al., 2005; Krakauer and Shadmehr, 2006). However, whether possible interference effects differentially influenced the sensitivity to neural excitation induced by HD-atDCS is highly speculative and appears to be unlikely, since both tasks yielded a similar performance pattern. Therefore, differences in the fine-motor skills required, repetition and complexity are more likely to account for our findings and should be explored in future studies in order to develop appropriate training and stimulation protocols for clinical and rehabilitative use.

Following on previous studies that stimulated M1 by tDCS and showed successful modulation of unimanual motor performance, our findings indicate that HD-atDCS bilaterally applied to both M1s elicits improved bimanual motor performance. Although significant effects were clearly found for one of our tasks (3-6-3 stack) and only partially for the other (1-10-1 stack), strong effect sizes (ωp2) and high statistical power (1-β) indicate that the present findings represent a valid starting point to fill a relevant gap in the tDCS literature concerning stimulation effects on complex bimanual movements. The findings are important for basic research as well as for different populations, such as patients suffering from neurological diseases and their associated bilateral motor impairments, and may help in the development of treatments to enhance motor recovery of the upper limbs.

NHP contributed to the conceptualization and realization of the study, performed the data acquisition, analysis and interpretation, wrote the manuscript and acted as corresponding author. FS substantially contributed to the data analysis and interpretation and wrote parts of the manuscript. MD contributed to the conceptualization and design of the study, and was involved in the data analysis and interpretation. He critically revised the manuscript and approved the final version and its content.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.