The aim of the present study was to examine whether bipolar tDCS, with the anode over the right AG and the cathode over the contralateral supraorbital region, modulates the BOLD signal within any of the canonical RSN. Using methods previously validated for analysing RSN [66], [86]–[90], [95], we examined eleven RSN immediately before and after tDCS. Significant differences between the two fMRI sessions were found in seven RSN (summarized in Table 1). Following tDCS, the BOLD signal at rest was decreased in bilateral primary and secondary visual areas, and in the right putamen. Increased BOLD signal at rest following tDCS was found in several parts of the brain, including thalamic, frontal, parietal, and occipital clusters of activity.

We could induce widespread changes in several RSN following bipolar tDCS with the anode over the right AG and the cathode over the contralateral supraorbital region. In accordance with previous studies [37], [38], [40], [41], [44], [77], [96]–[98], we thus demonstrated tDCS-induced changes in distinct RSN beyond the RSN comprising the stimulation site. Concerning functional connectivity during rest, tDCS seems to induce extensive changes, not limited to the stimulation site but distributed across the whole brain. We provide both a replication and an extension of previous findings with the present study, demonstrating that also bipolar tDCS with the anode over the right AG and the cathode over the contralateral supraorbital region induces large-scale modulations of multiple RSN. Detailed, specific comparisons with previous studies are complicated by several crucial experimental parameter variations across studies concerning site, duration, intensity, and polarity of stimulation as well as analysis techniques. Nevertheless, the present results corroborate previous findings [37], [38], [40], [41], [44], [77], [96]–[98] in showing that a single session of tDCS induces complex network modulations, including but not limited to the RSN comprising the stimulation site itself, changing resting-state activity within anatomically and functionally connected brain areas. We discuss the present results more specifically in the following paragraphs.

In the context of the right frontoparietal RSN, the putamen was also less active following tDCS. It is well known that the putamen, together with other basal ganglia structures such as the caudate nucleus, is involved in motor functions, including selection, preparation, and execution of movements [102] , [103] . Moreover, the putamen and other basal ganglia regions have been shown to play an important role in cognitive and emotional processing [104] – [106] . An explanation for decreased activity in the putamen might be related to connectivity between the AG and the putamen, as it was previously shown that both functional connectivity during rest and anatomical connectivity exist between the AG and the basal ganglia [107] – [109] . To some extent our findings replicate this connection between the AG and the putamen during rest and extend previous findings, by showing that stimulation of the AG also results in activity changes in the putamen during resting-state.

We found decreased activity after tDCS for the DMN and the right frontoparietal RSN. For both networks, differences in activity were localized in the occipital cortex, covering primary and secondary visual areas. A possible explanation for the differences found in these visual areas might be that these areas are relatively close to the stimulation site, because we stimulated the right AG with a sponge electrode of 35 cm 2 . Thus, not only the AG but probably also the surrounding brain areas (e.g. secondary visual areas) were affected by tDCS, albeit to a lesser extent. Additionally, especially the right AG has been shown to exert strong functional connectivity with occipital areas [99] . Via the middle longitudinal fasciculus and the posterior part of the arcuate fasciculus, the AG is anatomically connected to several ventral temporal regions which are located in close proximity to the visual clusters of activity detected here [76] , [100] , [101] . Thus, changes in activity in visual areas following tDCS might be related to the fact that the AG is connected to these areas both anatomically and functionally. Regarding the fact that brain activity in occipital regions was decreased in response to anodal tDCS, it was previously shown that the application of anodal tDCS can also lead to decreased BOLD signal in areas close to the stimulation site [36] , [39] during task paradigms. Future studies will have to address which stimulation parameters (localization of electrodes, intensity, duration, polarity) influence the effect of anodal tDCS on the BOLD signal, both during task paradigms and during rest.

4.2 Increased resting-state activity following tDCS

Following tDCS, increased BOLD signal was found in the bilateral thalamus and the caudate nucleus. Because the AG is connected to these structures [107]–[109], increased activity might be caused by the excitability increasing stimulation induced at the AG. Although current density is highest directly underneath the stimulating electrodes, it has to be acknowledged that areas in the path of the current from the anode to the cathode will also be affected by stimulation, because the electrical field induced by tDCS is distributed across the whole brain [110]–[115]. Because electrical current will inevitably take the fastest route through the brain, possibly running along direct anatomical connections (e.g. from the AG to the thalamus), increased activity in this part of the brain may be explained by the current passing through the thalamus and the caudate nucleus on its way to the cathodal electrode. However, we can only infer the real current flow. Accurate simulations of current flow are beyond the scope of this exploratory study, but will be an interesting direction for future studies.

A similar explanation as given above may apply to the activity differences found in the left SFG and SMG, which were detected in the context of the DMN. Because the cathodal electrode was placed over the left supraorbital area, the current may have reached the left superior frontal brain regions on its way to the reference electrode. The change in activity may also be a direct effect of the cathodal electrode placed over the left supraorbital area, as it was previously shown that cathodal tDCS can lead to increased BOLD activity [34].

In the context of the TPN, we found changes of activity at the stimulation site itself. The right AG, which was directly stimulated with anodal tDCS, was found to be more active in the second fMRI session. We therefore cautiously suggest that the excitability increasing stimulation induced by anodal tDCS also increased brain activity at the stimulated area, as shown previously [35], [116]. Thus, both previous and the present results demonstrate that neuroimaging measures, such as the BOLD signal, can be increased at the stimulation site following anodal tDCS. Importantly, we also need to consider the possibility that the increased AG activity detected during resting-state might be related to a spill-over effect from the cognitive task, which was performed before the second resting-state measurement. But there was a clear difference between the pattern of activity changes detected during the task and those detected during resting-state measurements: whereas tDCS-induced changes assessed during cognitive task performance were rather focused and limited to the AG, SMA, and the retrosplenial cortex [35], we found widespread changes in many different brain areas during resting-state. Thus, we suggest that spill-over effects – if present at all – most likely affected primarily task-related activity and not the task-irrelevant resting-state activity studied here. Nevertheless, some participants may have still processed cognitive or emotional aspects related to the previously performed task during the second resting-state measurement. Participants may have for example been re-calculating or still thinking about some of the trials they could not solve, or they may have been emotionally aroused in a positive or negative manner in response to their self-perceived task performance. Such internal thought processes can distract participants away from a strict resting-state and activate brain regions related to cognitive and emotional processing. In this context, it has to be considered that such brain regions may generally be activated during a conscious resting-state, because participants might be engaged in conceptual processes involving semantic knowledge retrieval, self-awareness, and directed knowledge manipulation for organisational purposes. Thus, some of the brain regions (e.g. AG, SFG, SMG, thalamus) showing increased resting-state activity during the second measurement, which one may attribute to the modulatory effects of tDCS, might have also shown greater resting-state activity because they were involved in the aforementioned cognitive and emotional processes. Such an involvement in turn might have been influenced by the previously performed cognitive task. Thus, the arithmetic task itself might have also influenced changes in RSN. This alternative explanation for some of the present findings should be explicitly tested in future studies aimed at examining the influence of arithmetic tasks on resting-state activity, because a detailed examination of task effects on resting-state activity goes beyond the scope of the present study.

Another important aspect of the present study was that we had chosen to present participants a cognitive task during tDCS. We included this task during tDCS because we wanted to reduce the variability of cognitive processing during tDCS as much as possible. However, previous investigations have shown that on-going task activity during non-invasive brain stimulation can significantly alter stimulation-induced neuroplastic changes [117]–[120]. In 2008, Silvanto and colleagues [121] demonstrated the importance of state dependency in the context of non-invasive brain stimulation. They concluded that brain stimulation studies often do not take the current activation state of the brain into account, although the brain's response to any external stimulation (including tDCS) will be partly determined by this current activation state [121]. Thus, the brain does not react passively to external stimulation, and the effect of tDCS does not only depend on the stimulation parameters but also on the activation state of the stimulated area during the application of tDCS [122]. In accordance with these conclusions, Antal and colleagues [118] revealed that a cognitive task performed during tDCS substantially altered stimulation-induced plasticity. Interestingly, the typical pattern of increased cortical excitability following anodal tDCS and decreased cortical excitability following cathodal tDCS was reversed when tDCS over M1 was applied in combination with a cognitive task. In another study, Teo and colleagues [119] found that improvement of working memory performance was only achieved during tDCS, when stimulation and task were performed simultaneously. The authors suggested that the increased cortical excitability needed to perform the working memory task during tDCS might have resulted in a cumulative effect, leading to significant behavioural improvements only when stimulation and task were performed simultaneously. Corroborating the hypothesis of such a cumulative effect, working memory performance assessed after tDCS was not significantly improved. Furthermore, Andrews and colleagues [117] demonstrated that tDCS applied during a working memory task resulted in greater improvement of another working memory task, as compared to tDCS alone and sham tDCS applied during the working memory task. Regarding possible explanations for these findings, different authors have put forward the aforementioned hypothesis, stating that cumulative effects will be achieved if tDCS is combined with a cognitive task. Thus, task performance and simultaneous application of tDCS might lead to a greater increase in excitability than tDCS alone, which might than result in greater behavioural effects of tDCS. If task-relevant neuronal populations are already activated, they may be closer to the threshold for inducing neuroplasticity and thus are more likely to reach this threshold, if further stimulated with tDCS. Such additional task effects might induce strong synaptic activation leading to persistent strengthening of synaptic transmission, which might thus further enhance the effects of tDCS. Combining a cognitive task with tDCS could thus lead to greater neuroplastic changes, specifically in task relevant brain regions. This might also be relevant for the present study, as the AG was shown to be specifically activated by the cognitive task performed during tDCS [35]. Thus, changes in resting-state activity in the AG observed here might have been significantly enhanced by the excitability changes in this area induced by the cognitive task performed during tDCS. On the other hand, stimulation-induced neuroplastic changes might also be reversed or reduced if stimulation is combined with a cognitive task. Task-irrelevant brain regions might be deactivated, and this deactivation process might interfere with the neurophysiological processes underlying stimulation-induced neuroplastic changes [118]. Jacobson and colleagues [123] proposed that tDCS-induced neuroplastic changes can only be fully expressed if they appear in a low-competition environment, thus if participants are at rest. The authors hypothesize that if stimulated brain regions are already activated by a task, and thus in a high-competition environment, it might be more difficult to promote even further changes by introducing external stimulation. Over all, we think that the influence of cognitive task activity during tDCS represents a rather complex issue and deserves further scientific attention. For the present study, we conclude that – although we examined changes in resting-state activity only – the differences between the first and second resting-state measurement, especially in task-relevant areas such as the AG, were influenced not only by tDCS but also by the cognitive task performed during tDCS.

Because bipolar tDCS, with the anode over the right AG and the cathode over the contralateral supraorbital region, led to increased activity within several frontal and parietal areas, we suggest that the stimulation somehow increased the alert state of the brain. Consequently, this effect was best visible in areas of the brain sub-serving alertness and other attention dependent cognitive functions. For both the parietal and the aforementioned left frontal cluster, there is broad evidence that these brain areas are involved in various attention demanding tasks [74], [124]–[126]. However, the AG sub-serves other functions as well, such as verbally mediated mathematical processing [35], [75] and semantic processing [73]. Nevertheless, we cautiously conclude that the modulatory effect seen in frontal and parietal areas is due to modulation of the attentional state of the brain induced by our stimulation paradigm. This interpretation is in agreement with a previous study examining RSN in response to tDCS [37]. A similar explanation might also apply to changes found in the executive control RSN. Here, we found differences in BA 8 and BA 9 at the location of the DLPFC. The DLPFC has been linked to attentional processing numerous times and is also involved in attentional networks together with the AG [127]–[129]. Previous studies demonstrated that the DLPFC is involved in various components of attentional processing, such as reorienting, predictive coding and generation of spatially selective responses [126], [130]–[132]. We suggest that a change of the attentional state of the brain during rest, induced by bipolar tDCS, with the anode over the right AG and the cathode over the contralateral supraorbital region, led to the activity changes in this DLPFC cluster.

Overall, we found that changes in RSN were distributed across the whole brain and not limited to the stimulation sites at the right AG and the contralateral supraorbital region. This pattern of changes generally confirms previous findings [37], [38], [40], [41], [44], [77], [96]–[98], showing that connectivity between distant brain areas can be modulated using tDCS. Both the present and previous results indicate that tDCS can modulate resting-state activity in brain areas directly underneath the stimulating electrode and also in networks of functionally connected brain areas. One might thus speculate that tDCS-induced cognitive and behavioural changes reported in previous studies are not purely the result of modulated activity in a single brain region, but rather stem from a reconfiguration of different functional networks. Such a complex reconfiguration might involve changes in functional network connectivity expressed at multiple brain regions, underscoring the dynamic interplay and interactions underpinning functional networks in the human brain. The stimulation of a single brain region might thus have widely distributed effects concerning activity and connectivity of areas which are functionally and/or anatomically connected to the stimulated brain area. Such conclusions might also be important for clinical settings, in which stimulation of an appropriate cortical area might help to normalize brain activity in large-scale functional networks. However, future studies are needed to comprehensively clarify these issues and uncover the functional and behavioural relevance of such tDCS-induced changes in large-scale brain networks.