Thirty healthy volunteers were subjected to 1 mA tDCS during 20 minutes with a mid-line frontal electrode (anodal n = 10, cathodal n = 10, sham n = 10) and an extracephalic reference electrode, under continuous cardio-respiratory monitoring. No adverse effect was observed. While the RF decreased progressively, the blood pressure increased steadily over time, without significant difference between groups. The HR remained stable during the monitoring period. The HRV parameters reflecting the tones of the sympathetic and vagal autonomic nervous system suggested a progressive shift in the sympathovagal balance favouring the sympathetic tone. Neither anodal nor cathodal tDCS modified the sympathovagal balance when compared to sham tDCS.

tDCS and the brainstem

Lippold and collaborators reported an episode of disturbed speech and apnoea followed by a transient respiratory depression in a normal female subject who received 16 minutes of 3 mA bi-frontal cathodal tDCS, with an extracephalic reference electrode [14, 15]. The authors concluded that the respiratory depression was due to an unwanted modulation of the brainstem respiratory centres by the DC flowing through the brainstem [14, 15]. Recently, tDCS with an extracephalic reference electrode has been safely applied using various montages: inion and neck base [20], M1 and ipsilateral shoulder ([21] and in two patients with Tourette syndrome [22], left fronto-temporal areas or inion and right shoulder [22], bi-frontal and non-dominant arm [23], bilateral dorsolateral prefrontal cortex and right deltoid [34], cerebellum [35]. However, monitoring of the respiratory frequency or exploration of subtle changes in the sympathovagal balance have not been conducted so far, leaving unanswered the issue whether tDCS could modulate the activity of the brainstem autonomic nervous nuclei.

If tDCS with an extracephalic reference electrode could modulate the activity of the brainstem cardio-respiratory and autonomic centres, this would lead to the exciting possibility to directly test and manipulate homeostatic functions such as respiratory frequency, heart rate. Moreover, this could lead to therapeutic perspectives since the autonomic nervous system is an essential target for pharmacological therapies given its key role in hypertension, heart failure, cardiac arrhythmias, sudden death or dysrhythmic breathing [36–39].

Respiratory frequency

In the present experiment, the extracephalic reference electrode was placed on the right leg and a unique active tDCS electrode over Fz, to maximise the likelihood of orienting the DC flow through the brainstem. The RF decreased steadily throughout the 1-hour monitoring. This mild diminution of the RF could be explained by a progressive relaxation while lying supine. When necessary, the healthy volunteers were allowed to speak briefly to stay awake, mostly during the last 30 minutes of monitoring. Although this may partly explain the decrease of the measured RF in the three groups, this trend was present since the onset of the monitoring period. Nevertheless, there was no significant difference in the temporal evolution of the RF between the sham, anodal and cathodal groups. Therefore, 20-minutes of tDCS applied with an extracephalic reference electrode seem not to interfere with the activity of the brainstem respiratory network and thus appear to be safe in healthy volunteers. Of course, longer stimulation periods, higher tDCS intensities, other electrode montages and the inclusion of patients should be performed before this conclusion can be generalised.

Blood pressure

The blood pressure increased mildly throughout the monitoring period from the onset of the experiment, suggesting an increasing nervousness during the monitoring period rather than transient anxiety at the onset of intervention. This hypothesis is supported by the fact that the HR remained stable over time (see below). The blood pressure remained within the normal range for most of the healthy volunteers during the baseline epoch; some of them showed values compatible with previously unknown mild hypertension, mostly by the end of the monitoring period. The variability outside the mean ± 1.96 SD also increased throughout the epochs in all three groups. Anyway, the blood pressure followed a similar temporal evolution in the three groups.

Heart rate and sympathovagal balance

The HR remained stable over time in the three groups, ruling out transient stress at the onset of intervention or a significant effect of tDCS on HR. There was a significant increase of the LF nu-RRI and a mirroring decrease of the HF nu-RRI over time, as well as resulting increases in the LF/HF ratio and PSD-RRI. These progressive changes were similar for the three groups, suggesting a progressive shift of the sympathovagal balance in favour of the sympathetic tone.

Could this shift of in the power of the LF and HF bands have been driven by the decline of RF? Indeed, whereas the HF band of HRV reflects the vagal tone, it is also partly contaminated by the RF, a phenomenon called the respiratory sinus arrhythmia (RSA) [28, 32]. Tasks such as mental arithmetic test or free talking may shift respiration and the HRV balance within the LF band [40]. In the present experiment, whereas the RF decreased mildly over time, the LF nu-RRI increased. However, since the R-R variability is almost abolished after autonomic ganglion blockade of both the sympathetic and parasympathetic nervous systems in healthy volunteers during supine resting condition, it has been suggested that the HRV is predominantly mediated by the autonomic nervous system [31]. Overall, there was a progressive shift of the sympathovagal balance in favour of the sympathetic tone, similar in the three groups, likely reflecting an increasing nervousness. It is of course acknowledged that the concept of sympathovagal balance is a coarse approximation and does not fully reflect the complex interactions of the sympathetic and vagal systems [28, 32, 41]

In addition, the GEE model disclosed subtle differences that were not picked up by the ANOVA RM . The LF nu-RRI was lower (and the HF nu-RRI higher) in the sham group than in both the anodal or cathodal group but, again, there was no epoch by group interaction. Similarly, the percentage of data outside the mean ± 1.96 SD increased for the LF/HF-RRI ratio in the anodal and cathodal groups. In order to explain this difference between the sham group on the one hand and the anodal and cathodal groups on the other hand, one should hypothesize that both anodal and cathodal tDCS induced the same changes in the sympathovagal balance whereas sham intervention had no influence. This would imply that the DC "interfered" with the activity of these brainstem centres independently of its flow direction, resulting in the same net effect. Alternatively, whereas subjects undergoing tDCS are unable to explicitly point out whether the tDCS stimulation is on (active) or off (sham) [42], they still may unconsciously perceive the active stimulation and experience subliminal stress. This would hold true only if this change started after the onset of intervention; however it was already present during the baseline period and built-up steadily.

The separate GEE models disclosed a decrease in both the LF nu-RRI and HF nu-RRI variability exclusively in the cathodal group; this was reflected in the fact that the analysis of the percentage of variance outside the mean ± 1.96 SD also showed a difference for the PSD-RRI for the cathodal group only. This isolated result is difficult to interpret since the baseline difference that prompted to compute separate GEE models concerned the sham group versus the anodal and cathodal group.

Asymmetry of the DC flow

Could the 1964 episode of respiratory depression [14, 15] result from an asymmetrical distribution of the DC within a specific part of the brainstem or on its right aspect (the extracephalic electrode was on the right leg)? Lateralised tDCS with an extracephalic electrode could theoretically impact on cardio-respiratory homeostasis through three additional mechanisms. First, when applied over the lateral aspect of the head, the DC could potentially modulate the activity of the cortical areas involved in the control of autonomic nervous functions such as the insula and amygdala [43, 44]. Whereas the debate about the hemispheric lateralisation of autonomic control in human is still open [45], the insula and parietal cortices may be particularly important in the control of heart rate and have been involved in sudden cardiac death and cardiac arrhythmia after stroke [39, 46, 47].

Second, since DC can modulate the excitability of peripheral nerves [48], the DC flowing preferentially through the lateral aspect of the neck could theoretically interfere with the vagus nerve excitability. It is worth noting that the right vagus nerve seems conveying a predominant outflow toward the heart [49]. Recently, two out of three patients receiving right vagus nerve stimulation for refractory epilepsy suffered from respiratory events suggestive of bronchoconstriction [50]. Therefore, it cannot be ruled out that episode of respiratory depression under tDCS with a right extracephalic electrode [14, 15] was due to a modulation of the right vagus nerve excitability, resulting in breathing difficulties.

Third, the phrenic nerve might also be influenced by DC, maybe leading to a modulation of the RF and respiratory depression. However, whereas unilateral paralysis of the phrenic nerve may result in hemidiaphragmatic paralysis leading to severe respiratory complications [51, 52], a stunning of the phrenic nerve is speculative.

Cephalic and extracephalic tDCS reference electrode

The modern safety guidelines for tDCS [53] recommend using bi-cephalic montages, which have the obvious advantage of avoiding any stimulation of the brainstem but introduce an ambiguity: are the observed effects exclusively due to modulation of the target cortical area activity or to the combination of the modulation of the target area and of the contralateral cortex located under the so-called "reference" electrode? It is only recently that this issue was partly resolved by using a large "reference" electrode placed over the contralateral orbita [54]. Since this large electrode is theoretically neutral (the current under the large electrode is so dispersed that it should be ineffective), the effects observed could be attributed solely to the modulation of the target M1 by a small "active" electrode.

Nevertheless, using an extracephalic reference electrode could lead to two opposite but equally interesting conclusions. On the one hand, if the direction of the DC flow is a key factor determining the (after-)effects of tDCS as suggested by recent modelling studies [7–13], then using an extracephalic reference electrode may potentially expand the variety of potential interventions with tDCS.

On the other hand, the impact of tDCS on a given cortical target may be insensitive to the DC flow direction, whether a cephalic or an extracephalic reference electrode is used. Therefore, using an extracephalic electrode as a genuine neutral reference would help to substantiate the conclusions of several recently published tDCS studies with bi-cephalic montages regarding the spatial location of the observed tDCS effects. Whereas the measures of the cortical excitability of M1 after tDCS are not influenced by a "reference" electrode placed over the controlateral frontopolar cortex, the issue is much more debatable for cognitive studies or studies using behavioural outcome measure. As long as behavioural studies are concerned, the definition of a cortical area as "functionally inert for the relevant task" for a control experiment may be questioned. These control experiments are of course of paramount importance but their interpretation may not be as straightforward as commonly accepted.

Limitations of the study

Several issues should be taken in account when evaluating the outcomes of the present experiment. Firstly, whereas bi-frontal electrodes were used by Lippold and collaborators [14, 15], in this experiment, a single cephalic electrode was placed on Fz in order to maximise the chance of directing the DC flow towards the brainstem. Moreover, during pilot experiences, positioning the cephalic electrode more anteriorly (i.e. over FPz) induced a typical metallic taste in the mouth in most subjects, which raised concerns for the double-blind character of the experiment.

Secondly, as already mentioned, the intensity of the DC was much larger in the study of Lippold et al [14, 15] (16 minutes of 3 mA bi-frontal cathodal tDCS); this may explain the lack of effect observed in the present experiment. However, we deliberately decided to apply 20 minutes of 1 mA tDCS because 1) these parameters were used in the majority of the modern tDCS studies, 2) the blinding of healthy subjects is questionable when using high tDCS intensities such as 3 mA (tingling and itching cutaneous sensations), and 3) this study was designed as a first step towards other experiments exploring different parameters (larger cohorts of healthy volunteers, higher tDCS intensities, different locations of the cephalic electrode, inclusion of patients).