Abstract
Cortical excitability, the tendency of neurons to respond to various stimuli, is impaired in most neuropsychiatric conditions. Non-invasive brain stimulation can exert therapeutic effects by modulating the cortical excitability. Transcranial direct current stimulation (tDCS) and transcranial alternating current stimulation (tACS) have shown promise in various neuropsychiatric disorders, including improving cognitive abilities and motor function following stroke. Oscillatory transcranial direct current stimulation (otDCS), as a novel stimulation paradigm, combines tDCS and tACS to simultaneously regulate neuronal membrane potentials and oscillatory rhythms. This combination may produce more significant effects on neurons. To investigate this, participants received the following stimuli for 20 min on different days: (i) 2 mA 40 Hz otDCS, (ii) 2 mA 40 Hz tACS, (iii) 2 mA tDCS, and (iv) sham stimulation. Motor evoked potentials (MEPs) and transcranial magnetic stimulation combined with electroencephalography (TMS-EEG) were assessed both before and after stimulation. The increase in MEPs amplitudes was most pronounced under otDCS conditions compared with tACS and tDCS. Furthermore, analysis of TMS-EEG data revealed that changes in time-varying brain network patterns were most pronounced after otDCS, manifesting as enhanced brain-wide information connectivity. Our results indicate that gamma otDCS has significant potential for regulating cortical excitability and activating brain networks.
Introduction
Non-invasive brain stimulation (NIBS) is a non-invasive neural regulating technology that regulates the activities of brain neurons by acting on the cerebral cortex through physical means (electricity, magnetism, light, ultrasound, etc.) (Cambiaghi et al. 2023; Davidson et al. 2024; Toth et al. 2024). Transcranial electrical stimulation (tES), a typical NIBS method, is widely used to treat various types of neuronal lesions, such as cognitive impairment, Parkinson's disease, and stroke (Elsner et al. 2020; Chen et al. 2022; Ni et al. 2022).
Transcranial direct current stimulation (tDCS) and transcranial alternating current stimulation (tACS) are two types of tES commonly used in clinical practice. TDCS can polarize or depolarize neuronal membrane potentials, increasing or decreasing cortical excitability in a polarity-specific manner (Nitsche and Paulus 2000). TACS alters the phase of sinusoidal currents by changing the frequency of the stimulating current, thus affecting neural oscillation rhythms and regulating neural activity to achieve therapeutic effects (Antal et al. 2008; Kanai et al. 2008). TDCS and tACS have demonstrated efficacy in treating numerous neuropsychiatric disorders, such as improving cognitive ability and motor dysfunction after stroke (Veldema and Gharabaghi 2022; Grover et al. 2023). However, recent studies have indicated that their clinical therapeutic response is not significant (Marquez et al. 2013; Dissanayaka et al. 2017; Lafleur et al. 2021). Therefore, we urgently need to explore new strategies to regulate cortical excitability to a greater extent, aiming to achieve significant improvement and broader amelioration of nervous system diseases. Oscillatory transcranial direct current stimulation (otDCS), a novel tES technology, combines tDCS with tACS to deliver frequency-modulated currents without reversing polarity. The current oscillates in a sinusoidal manner at a specific frequency within either positive (e.g. between +1 and + 2 mA) or negative polarity. OtDCS simultaneously modulates neuronal membrane potential and oscillatory rhythms (Živanović et al. 2022). This combination may result in a more pronounced effect on neural stimulation, potentially leading to more significant improvements in cognitive functions such as memory and attention, motor functions like coordination and balance, as well as other aspects of brain function. It may have greater advantages in the treatment and rehabilitation of neurological diseases.
Gamma oscillations are very important in the human brain. The application of gamma tACS in healthy individuals has been associated with improved cognitive performance (Santarnecchi et al. 2013; Santarnecchi et al. 2016; Lee et al. 2023). In this experiment, we investigated the changes in cortical excitability before and after otDCS stimulation at gamma frequency (40 Hz) in healthy participants, and compared these changes with those observed under tACS or tDCS conditions at the same current intensity. We observed alterations in corticospinal excitability of the motor cortex by measuring motor evoked potential (MEP). Furthermore, we evaluated the changes in brain network connectivity before and after stimulation using transcranial magnetic stimulation combined with electroencephalography (TMS-EEG). MEP is used to evaluate NIBS effects on cerebral cortex excitability. TMS-EEG, a multimodal imaging technique, has emerged as a powerful tool to non-invasively probe brain circuits in humans, allowing for the assessment of several cortical properties such as excitability and connectivity (Kimiskidis 2016; Tremblay et al. 2019). By comparing the changes in MEP and TMS-EEG before and after applying three different electrical stimulations with the same current intensity, we aimed to explore the significance of otDCS at gamma frequency in modulating cortical excitability and brain network connectivity.
Materials and methods
Participants
Sixteen healthy people (eight males and eight females; Mean Age 23.5 ± 0.67 years) participated in the experiment. All participants were free from medication and counter indications to TMS and tES. According to the Edinburgh Handedness Inventory (Oldfield 1971), all participants were right-handed. Prior to the experiment participants gave their written informed consent. This study was approved by the Ethics Committee of the First Hospital of Hebei Medical University (ID: Research Review No.111[2024]), and following the Declaration of Helsinki.
Transcranial magnetic stimulation and MEPs recording
MagVenture MagPro X100 (MagVenture A/S, Farum, Denmark) provided single pulse transcranial magnetic stimulation (sTMS). The transcranial magnetic stimulation (TMS) coil was held tangentially to the skull over the left M1 with the handle pointing posterolaterally at a 45° angle to the sagittal plane. The optimal coil positioning on the hotspot of the M1 was identified for inducing the largest MEP amplitude first dorsal interosseous (FDI) muscle. Mark the hotspots with a red skin marker to ensure that the coil remains in the correct position throughout the entire experiment. Resting motor threshold (RMT) was determined before and immediately after stimulation session. The RMT, defined as the intensity required to elicit MEPs of at least 50 μV peak-to-peak amplitude in five out of 10 consecutive trials, was adjusted if necessary after stimulation. Participants were instructed to maintain a relaxed hand position throughout the testing procedure.
MEP amplitudes of the right FDI muscle were recorded at 120% RMT before (baseline) and immediately, 10, 20, and 30 min after each stimulation session: otDCS, tACS, tDCS, and sham stimulation. Fifteen peak-to-peak MEP amplitudes at 5 s intervals (varied randomly by ±1 s) were measured at each time point. The values were subsequently averaged for each of five time points. MEP modulation (ΔMEP%, baseline corrected) was expressed along with the following equation:
(A stands for post-stimulus amplitude, and B stands for pre-stimulus amplitude).
Measurement of TMS-EEG
TMS-EEG data (8 min) were acquired using a magnetic field-compatible EEG amplifier (Yunshen Ltd, Beijing, China). The cap (Greentek Ltd, Wuhan, China) with 32 TMS-compatible electrodes were positioned according to the 10–20 montage. The sampling rate was 1024 Hz. Electrode impedance was maintained below 5 kΩ. The AFz channel served as the reference, and the nasion electrode was used as the ground. sTMS was stimulated in the C3 position in the 10/20 system at 90% RMT. Each sTMS was applied at an interval of 4 s to avoid previous TMS effect. Synchronous EEG recordings were made. Participants wore earplugs to shield environmental noise and coil discharge noise.
We conducted pre-processing and time-varying network analysis using the MATLAB platform (R2019b from MathWorks, USA). The pre-processing of EEG data included a bandpass filter (3–30 Hz) and data segmentation. We used single TMS disturbance as the stimulus labels. For every disturbance event label, the time point corresponding to the peak of the label was set as time “0”. Data corresponding to 0.5 s before (as baseline) and 2 s after “0” were extracted. Next, we applied the time-varying multivariate adaptive autoregressive (TV-MVAAR) model from previous research (Song et al. 2019a; Song et al. 2022). Using a two-tailed t-test, we analyzed the time-varying EEG networks before and after each stimulation, obtaining data on the alterations in the brain network post-stimulation compared to the pre-stimulation state. Last, we mapped the brain networks as previously described (Song et al. 2019b; Song et al. 2020).
Transcranial electrical stimulation
TES was delivered by a 4 × 1 multichannel stimulation device (Model XPNS208-B, Xpnos Medical Instrument Co., Ltd, Suzhou, China). The left M1 was targeted by placing the central (anode) electrode at C3 position in the 10/20 system (Fig. 1). We set four return (cathode) electrodes in a radius of ~7 cm from the anode (corresponding to Cz, F3, T3, and P3 in the left hemisphere). During tES, the electrodes were fixed in place using a brain-positioning cap that employs an internationally standardized 10–20 system for electrode placement. The otDCS (sinusoidal waveform with an amplitude between 0 and 2 mA), tACS (sinusoidal waveform with an amplitude between −1 and 1 mA), and tDCS (2 mA) are applied. The stimulus frequency was set at 40 Hz for both otDCS and tACS, with a duration of 20 min. For the sham condition, no constant current was delivered, except for the first and last 15 s.
The diagram of the tES electrodes on the scalp. Central (anode) electrode, C3. Return (cathode) electrodes, F3, T3, Cz, C3. tES, transcranial electrical stimulation.
Experimental procedures
This was a double-blind, sham-controlled, randomized study. Participants underwent four separate stimulation sessions (tDCS, tACS, otDCS, and sham) on different days, with the order of sessions randomized. To minimize the impact of circadian rhythm, all sessions were conducted at the same time of day. A minimum of five days was allowed between sessions to avoid residual effects from previous stimulation. During each stimulation session (Fig. 2), we first determined the correct location of the TMS stimulation point, and then measured the RMT, baseline MEP, and baseline TMS-EEG. Next, one of the stimulation conditions was applied for 20 min. MEPs were measured immediately following stimulation (labeled as 0 min) and at 10, 20, and 30 min post-stimulation, after which TMS-EEG was performed. MEPs and TMS-EEG measurements were performed by professional researchers who were blinded to the type of stimulation used. During stimulation, participants were asked about adverse reactions, including tingling, dizziness, pain. We also examined them for any skin redness or skin burns.
Schematic representation of the experimental design. TMS, transcranial magnetic stimulation; RMT, resting motor threshold; MEP, motor evoked potential; TMS-EEG, transcranial magnetic stimulation combined with electroencephalography.
We included two subjects for the pre-experiment and calculated the sample size based on the results of the pre-experiment. We used the “Repeated Measures Analysis” module of the PASS 2023 software to calculate the sample size. The parameters are set as follows: Based on Pillai-Bartlett Corrected F Test; Tests: Pillai-Bartlett Corrected F Test; power = 0.9, a = 0.05; 1 between factor (group) and 1 within factor (time); Mean Input Type is “Means, Interaction Effects”; Between factor has four levels, the list of means is “69.34 42.59 30.93 4.04”; Within factor has four levels, the list of means is “39.15 38.42 36.32 33.02”; Effect Multipliers (K) is set as “0.5 1 1.5”; Equal subjects are allocated to groups during the search for sample size. The average group sample size was calculated as 6 (K = 0.5 or 1.0) and 5 (K = 1.5). Considering the Dropout Rate (20%), the sample size was 8. In this study, we enrolled 16 subjects.
Statistical analysis
Statistical analyses were performed using SPSS software version 26.0 (IBM Corporation, Armonk, NY, USA). The ΔMEP% was analyzed using two-way repeated measures ANOVA with type of stimulation and time as within-subject factors (type of stimulation×time). Post-hoc comparisons with Bonferroni correction were conducted to identify significant differences in ΔMEP% among stimulation conditions at each time point. Paired samples t-tests were used to compare the lines (enhanced connections) in TMS-EEG pre- and post-stimulation. The statistical significance level was set at P < 0.05.
Results
All 16 participants exhibited a good tolerance to tES and completed the study. None reported any adverse effects during or after the experiment.
Changes of RMT before and after stimulation
No significant change occurred in RMT before and after stimulation [F(3, 1) = 0.004, P = 1.000] (Table 1).
RMT before and immediately after stimulation in four conditions.
| otDCS | tACS | tDCS | Sham | |
|---|---|---|---|---|
| Pre-stimulation | 43.94 ± 10.85 | 44.00 ± 11.08 | 44.13 ± 11.31 | 43.69 ± 10.96 |
| Post-stimulation | 43.56 ± 10.61 | 43.75 ± 11.23 | 43.94 ± 11.23 | 43.56 ± 11.01 |
| otDCS | tACS | tDCS | Sham | |
|---|---|---|---|---|
| Pre-stimulation | 43.94 ± 10.85 | 44.00 ± 11.08 | 44.13 ± 11.31 | 43.69 ± 10.96 |
| Post-stimulation | 43.56 ± 10.61 | 43.75 ± 11.23 | 43.94 ± 11.23 | 43.56 ± 11.01 |
Data are presented as mean ± standard deviation.
RMT before and immediately after stimulation in four conditions.
| otDCS | tACS | tDCS | Sham | |
|---|---|---|---|---|
| Pre-stimulation | 43.94 ± 10.85 | 44.00 ± 11.08 | 44.13 ± 11.31 | 43.69 ± 10.96 |
| Post-stimulation | 43.56 ± 10.61 | 43.75 ± 11.23 | 43.94 ± 11.23 | 43.56 ± 11.01 |
| otDCS | tACS | tDCS | Sham | |
|---|---|---|---|---|
| Pre-stimulation | 43.94 ± 10.85 | 44.00 ± 11.08 | 44.13 ± 11.31 | 43.69 ± 10.96 |
| Post-stimulation | 43.56 ± 10.61 | 43.75 ± 11.23 | 43.94 ± 11.23 | 43.56 ± 11.01 |
Data are presented as mean ± standard deviation.
MEP alterations under four stimulation types
ANOVA revealed significant main effects of time [F(1.933, 115.969) = 13.474, P < 0.001, η2 = 0.183], condition [F(3, 60) = 30.658, P < 0.001, η2 = 0.51], and a significant interaction between time and stimulation condition [F(5.798, 270) = 4.884, P < 0.001, η2 = 0.196] on MEP amplitudes.
MEP amplitudes for each participant are shown in Fig. 3. The MEP amplitudes increased in most participants following otDCS, tACS, and tDCS, but did not change in the sham condition (P > 0.05; Fig. 4). The MEP amplitudes also increased significantly under the tDCS condition compared with sham stimulation immediately after stimulation (P < 0.05). However, no significant difference was observed between tDCS and sham conditions at 10, 20, and 30 min post-stimulation (P > 0.05). MEP amplitudes were significantly higher in the tACS and otDCS condition compared with tDCS and sham stimulation within 30 min post-stimulation (P < 0.05). Furthermore, the increase in MEP amplitude within 20 min of stimulation was significantly greater in the otDCS condition compared with the tACS condition (P < 0.05).
Individual MEP data for each condition. (A) otDCS condition. (B) tACS condition. (C) tDCS condition. (D) Sham condition. MEP, motor evoked potential; otDCS, oscillatory transcranial direct current stimulation; tACS, transcranial alternating current stimulation; tDCS, transcranial direct current stimulation.
(A). MEP at each time point in four stimulation conditions. The horizontal axis denotes each time point, and the vertical axis denotes the MEP modulation normalized to baseline (%). * denotes the difference from sham stimulus (mean ± SEM, P < 0.05); # denotes the difference from tDCS (mean ± SEM, P < 0.05); △ denotes the difference from tACS (mean ± SEM, P < 0.05). (B). One example of MEP at baseline and 0 min, 30 min after stimulation. MEP, motor evoked potential; otDCS, oscillatory transcranial direct current stimulation; tACS, transcranial alternating current stimulation; tDCS, transcranial direct current stimulation.
Differences in time-varying EEG network patterns following four stimulation types
Figure 5 shows the condition differences of the brain connections in the corresponding time-varying EEG network patterns of healthy participants after four stimulation types. The otDCS and tACS conditions demonstrated early enhancement in information connectivity, persisting throughout the observation period. Conversely, almost no enhancement existed in information connectivity in the tDCS and sham conditions.
The time-varying EEG network connections after sTMS of participants under four different stimulation conditions. Time, after single TMS. otDCS, oscillatory transcranial direct current stimulation; tACS, transcranial alternating current stimulation; tDCS, transcranial direct current stimulation. sTMS, single pulse transcranial magnetic stimulation.
Our results indicate that during the initial phase (200 ms) of sTMS stimulation, changes in brain network connectivity were similar across all four groups. In the otDCS condition, enhanced information connectivity originated in the right prefrontal lobe (Fp2). During the early stage (599 ms–935 ms), Fp2 exhibited enhanced connectivity with temporal (T3, T4, T5, T6), parietal (P3, P4, Pz), and occipital (O1, O2) regions. These changes tended to stabilize during the mid-to-late stage (1216 ms–1716 ms). In the tACS condition, increased brain network connections also originated from Fp2, initially showing enhanced connectivity with temporal (T3, T4, T5, T6), parietal (P3, P4, Pz), and occipital regions (O1, O2, Oz), accompanied by weakened connectivity between other brain nodes. Subsequently, connections gradually increased from prefrontal regions (Fp2, Fpz, Fp1) to other nodes. Connections from the left occipital region (O1) to the temporal (T3, T4, T5, T6) and left frontal (Fp1, Fpz) regions also gradually increased. These connections stabilized during the mid-to-late stage (1216 ms–1716 ms). In the tDCS condition, the early stage (599 ms–935 ms) was characterized by weakened connectivity between various nodes of the whole brain. During the mid-to-late stage (1216 ms–1716 ms), a decrease in these weakened connections was observed, primarily involving the prefrontal cortex region (Fp1, Fp2). Subsequently, enhanced connectivity between the frontal lobe regions (Fp1, F8) and other nodes emerged. The sham condition group consistently exhibited weakened brain network connections throughout the entire observation period.
We performed further statistical analyses on main nodes (Fp1, Fp2, F3, C3, O1, O2) in the TMS-EEG results. First, we analyzed the time-varying brain network maps of each participant under different stimulation conditions, pre- and post-stimulation. The lines (enhanced connections) at each node (Fp1, Fp2, F3, C3, O1, and O2) were counted at six time points (200, 599, 935, 1216, 1466, 1716 ms) pre- and post-stimulation. For each node, the number of lines (enhanced connections) at the six time points pre- and post-stimulation was summed, respectively. For each node (Fp1, Fp2, F3, C3, O1, and O2), we compared the total number of lines (enhanced connections) pre- and post-stimulation. The results are shown in Fig. 6. Compared with pre-stimulation, under otDCS condition, the total number of lines (enhanced connections) significantly increased (P < 0.05) at all nodes post-stimulation. Under tACS condition, the total number of lines (enhanced connections) significantly increased (P < 0.05) at three nodes (Fp1, Fp2, and O1). Under tDCS condition, only the total number of lines (enhanced connections) at Fp1 and Fp2 significantly increased (P < 0.05). Notably, in the sham group, no significant changes were observed in the total number of lines (enhanced connections) at any of the six nodes when comparing the pre- and post-stimulation values.
Differences in the total number of lines (enhanced connections) between pre- and post-treatment for each of the four groups separately. otDCS, oscillatory transcranial direct current stimulation; tACS, transcranial alternating current stimulation; tDCS, transcranial direct current stimulation. TMS, transcranial magnetic stimulation. *P < 0.05.
Discussion
To the best of our knowledge, this study is the first to reveal that 40 Hz otDCS stimulation of the motor cortex can increase corticospinal excitability. Our results revealed several important findings. The 40 Hz otDCS applied to the primary motor cortex of healthy individuals enhanced corticospinal excitability, occurring immediately following stimulation, and lasting for at least 30 min. Furthermore, the degree of MEP amplitude increase was higher than that of the tACS and tDCS conditions under the same frequency and current intensity. Under the four stimulation conditions, alterations in the time-varying brain network patterns following otDCS were the most conspicuous, manifesting as enhanced brain-wide information connectivity. This corroborated the MEP changes elicited by otDCS, indicating that otDCS is the most stable stimulation method for increasing cortical excitability compared with tACS and tDCS.
The increase in MEP amplitude following 40 Hz otDCS was the greatest compared with tACS and tDCS in healthy individuals. The increase in MEP amplitude is believed to reflect enhanced synaptic plasticity at the motor cortex level, and the increase in cortical excitability is correlated with the enhancement of synaptic plasticity (Hallett 2000). TES applies microcurrents to the scalp, with a portion of this current entering the brain, causing changes in the neuronal membrane potential, thereby affecting the cerebral cortex (Nitsche and Paulus 2000). Cortical excitability shifts, induced during tDCS in humans, depend on membrane polarization, thus modulating the conductance of sodium and calcium channels. These changes may depend on N-methyl-D-aspartic acid (NMDA) receptor (Liebetanz 2002; Nitsche et al. 2003; Monai et al. 2016). TACS exerts its function primarily by regulating neuronal oscillations in the cerebral cortex (Helfrich Randolph et al. 2014). Some studies reported that endogenous theta rhythms and TMS-induced changes in theta oscillations are mediated by NMDA receptor activation (Leung and Desborough 1988; Barr et al. 1995; Labedi et al. 2014). The abnormal rhythm of the gamma band is related to NMDA receptor function decline (Roopun et al. 2008; Jadi et al. 2016). Therefore, we speculate that otDCS in this study may have enhanced NMDA receptors efficacy, thereby regulating changes in neuronal plasticity and increasing corticospinal motor cortex excitability. OtDCS combines the mechanisms of tDCS and tACS, and this combined mechanism may explain why the increase in MEP amplitude following otDCS treatment was more significant than that after tDCS or tACS treatment. The complexity and non-linearity of the neurophysiological system make it difficult to determine whether the effects of otDCS are simply a superposition of tACS and tDCS effects or if synergistic interactions are involved. In subsequent study, we will further explore the relevant mechanisms.
In this study, the increase in MEP amplitude following otDCS or tACS persisted for at least 30 min after stimulation. Previous studies revealed that the online and offline effects of tACS are attributed to two phenomena respectively: entrainment and neuroplasticity (Vosskuhl et al. 2018). Entrainment and plasticity are not mutually exclusive, and may rely on each other. These findings suggest that a combination of neural entrainment and synaptic plasticity may underlie the observed aftereffects. Successful entrainment of neural oscillations during stimulation may be a prerequisite for inducing lasting synaptic plasticity (Vossen et al. 2015). This may explain why MEP increase, following otDCS or tACS, lasts for at least 30 min, whereas the duration of MEP increase following tDCS is less than 10 min, probably due to the successful entrainment arising from brain oscillation modulations by otDCS or tACS. Previous studies have indicated that tDCS has no significant effect on the MEP amplitude at the group level (Horvath et al. 2016). In our study, the increase in MEP amplitude following tDCS stimulation was low, and the duration was less than 10 min, suggesting that the effect of a single tDCS session on cortical excitability may not be significant.
Following otDCS, most patients exhibited varying degrees of enhancement in their MEPs; however, a minority of participants did not experience significant MEP changes. In response to this variation, we proposed the following possible explanations: First, variation exists in the thickness of the skull across individuals. When an electrical current of the same intensity is applied, thicker skulls encounter greater impedance in current conduction, causing a decrease in the electric-field density at the stimulation site (Eichelbaum et al. 2014; Farahani et al. 2024). This reduced electric field density may fail to elicit cortical excitability changes. Second, for a few participants in this experiment, a 20-min stimulation duration was insufficient to elicit changes in synaptic plasticity (Song et al. 2021). Therefore, future in-depth exploration of individualized response mechanisms may provide clearer and more profound insights into the efficacy and reliability of non-invasive neurostimulation techniques (Siebner 2010).
In the TMS-EEG results, compared with the sham condition, the time-varying brain network patterns of participants following 40 Hz otDCS/tACS reversed the weakening trend of whole-brain network connectivity, which was significantly enhanced. After 40 Hz tACS, the brain network pattern mainly exhibited activation in the frontal, temporal, and occipital lobes. This result aligns with previous studies that 40 Hz tACS stimulation enhances auditory perception (Rufener et al. 2016; Meier et al. 2019), visual perception (Gonzalez-Perez et al. 2019), and memory (Hoy et al. 2015). Previous studies have shown that 40 Hz rTMS modulates gamma-band oscillations in the left posterior parietal lobe, strengthens information flow from the left posterior parietal area to the frontal area, and enhances the dynamic connection between the anterior and posterior brain regions. Modulating gamma-band oscillations effectively improves cognitive function in patients with Alzheimer’s disease by promoting local, remote, and dynamic connections in the brain (Liu et al. 2022). In our study, 40 Hz tACS also promoted robust information output from the parieto-occipital region to the frontal region strengthening the dynamic connections between the anterior and posterior brain regions. Furthermore, compared with the sham condition, tDCS improved the weakened trend of brain network connectivity in the anterior frontal lobe. However, the overall improvement was not significant, consistent with the insignificant change in MEP amplitude following tDCS. Our findings indicate that during the initial stage of sTMS stimulation (200 ms), changes in brain network connectivity were similar across all groups. However, in the early stage (599 ms–935 ms), the brain network connectivity in the otDCS, tACS, and tDCS groups exhibited distinct patterns of alteration. These changes tended to stabilize during the mid-to-late stage (1216 ms–1716 ms), suggesting that these three types of tES primarily produce stable alterations in brain network connectivity during the mid-to-late stage following sTMS induction. Both otDCS and tDCS involve continuous anodal current stimulation. The otDCS group showed significantly enhanced connectivity changes across the whole brain network, whereas the tDCS group exhibited notable changes only in the enhancement of brain network connectivity in the frontal regions (Fp1, Fp2). This may be attributed to the characteristic of otDCS in modulating neuronal oscillations, enabling it to have a broader regulatory scope over the brain network.
To date, relatively few studies have investigated the effects of otDCS, and most have focused on beta and theta frequency oscillations. One study showed that otDCS/tACS at the theta frequency, can improve associative memory in healthy individuals, with otDCS demonstrating superior improvement effects (Bjekić et al. 2022). Another study indicated that beta-frequency otDCS had a more significant effect on enhancing cortical-muscular coherence and corticospinal excitability in healthy individuals (Kudo et al. 2022). We innovatively validated that otDCS at gamma frequency can effectively enhance cortical excitability and time-varying brain networks. Currently, 40 Hz tACS, as a potential method for restoring brain connectivity and potentially alleviating cognitive impairment disorders, has been widely applied in clinical practice (De Paolis et al. 2024). However, this study suggests that otDCS may be a more effective tES method owing to its greater ability to modulate cortical excitability and enhance whole-brain network connectivity compared with tACS and tDCS.
Notably, tDCS, tACS, and otDCS had no effect on RMT aligning with the finding that RMT is not changed by either anodal tDCS or 5-Hz repetitive TMS (Lang et al. 2004). In another study, anodal tDCS and transcranial near-infrared light stimulation(tNIRS) did not result in a change in RMT (Song et al. 2021). Given that RMT serves as an indicator of changes in ion channel conductivity, reflecting membrane excitability (Ziemann et al. 2004), the unchanged RMT in our study suggests that the observed effects of otDCS may primarily be mediated by synaptic plasticity rather than changes in membrane excitability.
Limitations
First, this study was conducted in a sample of healthy young adults, limiting the generalizability of the findings to older adults, individuals with cognitive impairment, and patients with stroke. Further research is needed to determine whether age or disease status affect these outcomes. Second, the study utilized a single stimulation duration (20 min). Future studies should investigate the effects of different stimulation durations to determine the optimal stimulation parameters. Third, the study focused on short-term effects, with MEP amplitude measured within 30 min of stimulation. Longitudinal studies are needed to assess the long-term effects of otDCS on cortical excitability and cognitive function. Finally, this study primarily assessed changes in cortical excitability as measured by MEP amplitude. Future studies should incorporate neuropsychological assessments to investigate the functional implications of these changes on specific cognitive functions.
Conclusions
Our findings demonstrate that 40 Hz otDCS exerts a stronger effect on modulating cortical excitability compared with tACS and tDCS in healthy individuals. This suggests that the application of otDCS holds promise as an effective therapeutic approach for treating neurological diseases.
Acknowledgments
The authors thank all the participants.
Author contributions
Ziyu Guo (Methodology, Writing—original draft, Writing—review & editing), Huiqing Qiu (Investigation, Project administration, Supervision, Writing—original draft), Yang Li (Formal analysis, Resources), Shuaixiang Wang (Formal analysis, Software), Yan Gao (Methodology, Supervision, Validation), Mengwei Yuan (Formal analysis, Investigation), Sha He (Formal analysis, Resources), Fangyuan Yan (Funding acquisition, Validation), Yuping Wang (Conceptualization, Supervision), Xiaowei Ma (Methodology, Project administration, Supervision, Validation).
Funding
This work was supported by the Science and Technology Innovation 2030- “Brain Science and Brain-like Project” major Project (grant numbers: 2021ZD0201800) and the 2025 Government-funded Clinical Medicine Outstanding Talent Cultivation Project (grant numbers: ZF2025067).
Conflict of interest statement. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
Author notes
Ziyu Guo and Huiqing Qiu contributed equally to this work.
