Experimental set-up

EEG acquisition equipment

The Enobio system (Neuroelectrics) is used to acquire EEG from 32 channels (P7, P4, CZ, PZ, P3, P8, O1, O2, C2, F8, C4, F4, FP2, FZ, C3, F3, FP1, C1, F7, OZ, PO4, FC6, FC2, AF4, CP6, CP2, CP1, CP5, FC1, FC5, AF3, and PO3) according to the international 10/10 system at a sampling rate of 500 Hz.

tDCS supply

Two or three stimulation channels of the Starstim system (Neuroelectrics) are used to supply anodal tDCS through electrodes with 1 cm radius, depending on the evaluated tDCS modality. These electrodes are denoted as electrodes 1, 2 and 3. Electrodes 1 and 2 represent the anode position for two possible versions of the montage approach that are used to provide different stimulation modalities in which either the right-hand or feet motor area is stimulated, while electrode 3 is the cathode in both montage versions. Electrode 2 is placed on the midpoint of Cz and FC1 in order to stimulate the SMA and feet motor cortex. In contrast, electrode 1 is located in front of C3, after an imaginary line that is traced from C1 to FC5. This electrode is aimed to stimulate right hand motor and premotor areas, while keeping the distance to C3 similar to the one found between electrode 2 and Cz. Electrode 3 is positioned at the inion level, but displaced approximately 3 cm to the left hemisphere. This location was selected based on [47], which states that various studies of cerebellar tDCS place the stimulating electrode 3 cm lateral to the inion, which is close to cerebellum. These stimulation electrodes are shown in Fig. 1.

Fig. 1 tDCS montage. Scheme of tDCS electrodes position in reference to EEG electrodes and inion (left), and placement of tDCS electrodes on the EEG cap (right). Electrodes 1,2 and 3 are highlighted in red, green and blue, respectively Full size image

This way, the proposed montage version that targets feet motor area, attempts to increase the excitability of the SMA and feet M1, which is expected to increase feet motor performance and improve the initiation of motor activity, regardless of the kind of movement due to the broad stimulation of SMA. In contrast, the montage version that targets right-hand motor area is expected to improve right-hand motor performance by exciting right-hand M1 and PM. Both montage versions would inhibit left cerebellum, considering that the cathode is placed over it. Since the position of electrode 3 (displaced 3 cm lateral to the inion) is also used in [33] for decreasing ipsilateral motor adaptation, we hypothesized that task-adaptation would be decreased for the left limbs. However, it could be expected also the possible facilitation of the right motor cortex (associated to the left limb) due to CBI suppression.

tDCS simulation

SimNIBS free platform [48] is employed in order to produce two simulations of the electric field that is produced by the proposed tDCS montage on a standardized healthy brain: one including electrodes 1 and 3, and another with electrodes 2 and 3. Current values represented approximately the maximum current that is used on the experiments, i.e. 188 μA. In particular, the current in the simulations is configured as 180 μA. Electrodes are modeled with radius of 1 cm, thickness of 3 mm and a space for conductive gel of 4 mm, based on measures of the electrodes that are used in the experiments.

Subjects

Five volunteers (two male and three female between 27 and 35 years old) without metallic implants or known neurological disorders participated in this study. Subjects were labeled as S1, S2, …, S5.

Experimental procedure

Each subject went through twelve experimental sessions performed in different days, leaving between sessions at least two days to allow recovery from tDCS effects. This recovery interval was selected taking into account that an intersession interval of 48 hours to one week is recommended for protocols with long-lasting effects of more than one hour [6]. Since aftereffects duration has not been evaluated yet for the proposed montage, it was assumed that the duration would be similar to the one obtained with another cephalic montage for stimulating the motor cortex [49]. With such montage, the effects of stimulating with a current intensity of 1 mA and an electrode size of 35 cm2 (an approximate current density of 0.03mA/cm2) for nine and eleven minutes provided significant aftereffects for up to thirty and fifty minutes, respectively.

The twelve sessions were done in three blocks of four sessions where it was evaluated only one of the following stimulation modalities:

Supply of tDCS on the brain region related to right-hand movement before EEG recording : First, anodal tDCS is applied for 10 min with 3-seconds ramps at the beginning and the end of stimulation. Electrode 1 works as anode, while electrode 3 as cathode. Then, EEG is recorded while the subject stares at a screen that shows sequences of instructions. If the screen is cleared (4 to 4.5 s), the user has to remain at rest. On the other hand, if an arrow pointing to the right or down appears (5 s), the subject has to imagine to move the right hand or feet, respectively. Every session consists of three runs of fifteen sequences or trials of each kind of motor imagery (right hand or feet) plus a corresponding period of rest, i.e., each run includes the random presentation of fifteen trials of right hand motor imagery and fifteen trials of feet motor imagery. Figure 2 shows the temporal sequence of tDCS and a run for this modality, in which a total of thirty trials (motor imagery plus rest) are recorded. Between runs there are given breaks of 3 min. Fig. 2 Temporal sequence when tDCS is provided before EEG recording. Series of events in time for stimulation ( top ) and a run ( bottom ) when tDCS is provided before EEG recording Full size image

Supply of tDCS on the brain region related to feet movement before EEG recording : The procedure for this modality is similar to the previous one, with the only difference of using electrode 2 as anode and that the feet motor region of the brain is stimulated instead of the hand area.

Supply of tDCS on the brain region related to right-hand or feet movement during EEG recording: In this tDCS scheme, the subject looks at a screen that can show three possible visual cues: an arrow, a blank screen (both with the same meaning as in the two previous modalities) or the word “Stimulation”. When the latter appears, tDCS is applied over the brain motor area that is associated with the next visual cue, which is one of MI. This means that before displaying an arrow directed toward the right or the bottom, tDCS is administered using electrode 3 as cathode and electrode 1 or 2 as anode, respectively. The visual cue of stimulation lasts 16 s, although the tDCS pulse actually has a duration of 4 s with a ramp of 3 s at the beginning and the end of stimulation, as can be observed in Fig. 3. This is done because the activation and deactivation of the Starstim device have a delay of variable duration that introduces noise in the EEG, so these time lapses have to be discarded. Moreover, the rest cue lasts 4 s more in comparison to other modalities as an effort to dissipate the tDCS effects during the first seconds of the cue. This extra time was selected considering that tDCS of 4 s and current density of 0.029 mA/cm2 with another cephalic montage [3] needs just a break of 10 s to avoid tDCS effects. Even though the duration of the effect depends on the montage, this was the only reference for the unknown value of the persistence of the effect. Fig. 3 Temporal sequence when tDCS is provided during EEG recording. Series of events in time for stimulation (top) and a run (bottom) when tDCS is provided simultaneously to EEG recording Full size image In this modality, every session consisted of nine runs of five trials of both kinds of MI, with their associated rest time. There were breaks of one minute between the runs. Note that the short stimulation duration from this tDCS modality is not considered to induce long-lasting aftereffects. Then, it is unlikely that it produces neuroplasticity due to cumulative effects, but maybe only for strong current intensities that extend their effects to overlap with the next stimulation pulse. The main interest in implementing this stimulation modality is to evaluate the changes in classification when the tDCS is applied only to increase excitability during MI and the EEG band power changes are compared to the rest state without any excitability effect, which would allow short-time comparisons in band power. This approach could have application in just enhancing MI detection during training so EEG changes are better detected. However, this is not expected to reduce the required training sessions to induce plasticity in neurorehabilitation, which would make the use of tDCS inefficient. On the other hand, MI detection enhancement could be useful for improving the detection of individualized EEG features for proper calibration of systems for brain entrainment before training is started.

In every block of tDCS modality, each of the four sessions had a different current density value that was assigned in random order: 0 (or sham), 0.02, 0.04 or 0.06 mA/cm2. Also, the order of evaluation of tDCS modalities was counterbalanced between users. Sham stimulation was used only when tDCS was applied before EEG adquisition. In contrast, when tDCS pulse lasted 4 s the current intensity analog to sham was just configured as 0 mA/cm2 for practical reasons. In addition, the lowest current density value was set as 0.02 mA/cm2 in order to have a reference that was close to 0.028 mA/cm2, which is the recommended maximum limit of current density [50]. Despite this recommendation, most tDCS studies evaluate current densities between 0.029 and 0.08 mA/cm2 [6]. Hence, maximum current density was defined as 0.06 mA/cm2. Figure 4 shows the experimental setup that was used in this study.

Fig. 4 Experimental setup. The experimental setup consists in the user wearing an EEG cap, the Enobio system and the Starstim device, while watching visual cues on a screen. A laptop that records EEG is able to control cues and activate Starstim in other computer Full size image

Considering that current density is estimated as the ratio of current intensity and the electrode area [6], the current values required to produce the current densities of 0.02, 0.04 or 0.06 mA/cm2 are 63, 126 and 188 μA, respectively. This estimation of the current density can be inaccurate for small electrodes [51]. However, [52] reported that the reduction of ten times of the electrode size produced no significant difference in the elicited effect of tDCS if the current density was kept constant in a cephalic montage. In particular, the effects obtained with electrodes of 35 cm2 and 1000 μA were similar to the case when 100 μA were used with electrodes of 3.5 cm2, which is an electrode area close to the one of the present study (π cm2). Therefore, the ratio of current intensity and electrode size was used in the study to calculate the current density.

EEG processing and analysis

On each session, the percentage of correct classifications (i.e., accuracy) for each kind of MI and rest is obtained. The changes on event-related syncronization (ERS) for μ and β bands, which is the increment on power related to a task, is also calculated for those tDCS modalities that show an improvement in accuracy when stimulation is applied. All the EEG processing required to obtain accuracy and ERS, as well as their further analysis was performed using MATLAB software [53].

In order to obtain the accuracy and power in one session, the EEG signals are first preprocessed with the aim of getting EEG signals that are clean from artifacts for further analysis. The initial part of preprocessing consists in filtering EEG signals with a fourth-order bandpass Butterworth with cut-off frequencies of 5 and 45 Hz. Then, each trial undergoes independent component analysis (ICA) with EEGLAB toolbox [54] in order to detect visually the presence of blinking artifacts. When an artifact is found in a ICA component, the latter is processed with an adaptive Wiener filter with the objective of estimating the artifact component in the ICA segment. The estimated contamination is subtracted from the ICA component in question and EEG signals are reconstructed. This method allows removing artifacts from EEG with small distortion and loss of physiological information [55]. Finally, reconstructed EEG of each trial is inspected visually to verify its quality and noisy trials are excluded from the analysis.

Once preprocessing is performed, the EEG of every trial is processed with a spatial filter that subtracts from C3, Cz and C4 the mean of their four neighbouring electrodes. The electrodes considered for C3 are FC5, FC1, CP5, and CP1, while for Cz are FC1, FC2, CP1, and CP2, and the ones for C4 are FC2, FC6, CP2, and CP6. The output of these three channels is divided in a MI epoch and its corresponding rest EEG epoch and also labeled with the kind of MI that was performed at the trial, so EEG epochs of MI and its rest period are separated for right hand and feet MI. In the case of the stimulation modality in which tDCS is applied during EEG acquisition, the first 4 s of the rest state are omitted. After obtaining the signal epochs, spectra after the second 2 for every epoch of MI and rest is calculated. Then, the same procedure is carried out for each kind of MI (hand and feet), in which the epochs corresponding to the EEG information at Cz, C3 and C4 is used to calculate the accuracy of classification between rest and MI events, as well as the ERS changes associated to the performance of MI at each session. These three channels were selected for the analysis because they are located on the neighborhood of the motor areas that may present a higher impact after tDCS, considering that C3 is located over the right hand motor cortex area and Cz is over the feet motor cortex region [56]. In addition, there is evidence of EEG connectivity between C3 and C4 at some frequencies and it has also been reported an increasing ERS surrounding a region of EEG desynchronization during motor imagery for some subjects [57]. The relevance of these channels is supported by the observation of ERS changes at their location during right or left hand and foot motor imagery [58], as well as their use in motor imagery analysis [59, 60]. The process performed for estimating the accuracy and ERS is described next.

Accuracy: Fisher criterion ( F ) [61] of the spectra of MI and rest states in C3, C4 and Cz at 9-30 Hz is calculated as follows: $$\begin{array}{@{}rcl@{}} F=\frac{\left(\mu_{1f}-\mu_{2f}\right)^{2}}{\delta_{1f}^{2}+\delta_{2f}^{2}}, \end{array} $$ (1) where μ 1 f and μ 2 f denote the mean power at frequency f for conditions 1 (MI) and 2 (rest), respectively, while δ 1 f and δ 2 f correspond to the standard deviation of power at frequency f for conditions 1 and 2, respectively. Then, the maximum value of F for the evaluated frequency range is determined for C3, Cz and C4, so a characteristic frequency is obtained for each of the three channels in every session. This characteristic feature represents the frequency in which the MI and rest states show a greater difference in their means and lower variance, which means both conditions are more discernible. Then, 100 iterations are conducted in which 30 random epochs are selected from the total epochs of both MI and rest and then they are labeled as either MI or rest with a linear discriminant analysis (LDA) classifier. This classifier is trained with the spectral power of C3, Cz and C4 of the rest of the epochs of the session at the characteristic frequency of these channels. Once the classification is performed, the percentage of correct classification of the 30 epochs is calculated per iteration. After completing the iterations, a distribution of the accuracy with a size of 100 samples is obtained for the session. It should be mentioned that the methodology that was described previously is focused on obtaining individual features at each session. Subject-specific features were calculated based on the approach of BCI technology personalization due to high inter-subject variability [62]. In addition, features were obtained for each session in order to take into account intra-subject variability of the sensorimotor rhythm modulation, which is the main reason for which MI-based BCIs include multiple training sessions [63].

ERS: ERS on C3, Cz and C4 is obtained for each trial as the difference of the natural logarithms of the mean spectral power during MI and the mean spectral power during rest of μ or β band. The logarithm is used in this case to reduce skewness of ERS distribution. Considering that S 1 (f) and S 2 (f) represent the spectral power of conditions 1 (MI) and 2 (rest), respectively, at frequency f, which is within μ or β band, ERS is calculated as:

$$\begin{array}{@{}rcl@{}} ERS_{\mu}=\ln{\sum\limits^{f=12}_{9}\frac{S_{1}(f)}{4}}-\ln{\sum\limits^{f=12}_{9} \frac{S_{2}(f)}{4}} \end{array} $$ (2)

$$\begin{array}{@{}rcl@{}} ERS_{\beta}=\ln{\sum\limits^{f=30}_{13}\frac{S_{1}(f)}{18}}-\ln{\sum\limits^{f=30}_{13} \frac{S_{2}(f)}{18}}, \end{array} $$ (3)

After the values of accuracy and ERS are obtained for all sessions, statistical comparisons can be made between current densities. In the case of ERS, possible outliers are excluded from data of each session for further statistical analysis. This is done by discarding data that is out of the 95% confidence interval, according to the method described in [56] to determine significant spectral changes. Also, for band power analysis, mean ERS of sham sessions are subtracted from all sessions of the same tDCS modality in order to set sham session as a reference of ERS with zero value. Then, Barlett tests (p>0.05) were performed to confirm that about 70% of the sessions from all users can be assumed to have homogeneous variance if the variances of the sessions of the same user and stimulation scheme are compared. In the case of accuracy, analysis of variance (ANOVA) tests (p<0.001) are performed in order to identify significant statistical differences in accuracy between sessions of the same tDCS modality for each subject. When significant differences are detected, multiple comparisons are made using Tukey-Kramer’s method (p<0.001). In the case of ERS, t-tests (p<0.05) are performed only for the stimulation schemes when significant improvements are found for accuracy to compare just sham session with results from other current densities. Significance threshold is set as 0.001 for accuracy to assure that the probability of detecting improvements on classification is lower, as we consider the use of tDCS to have a high cost. ERS analysis is used for providing a greater scope about the differences in EEG that could enhance good classification.