Transcranial direct current stimulation modulates primate brain dynamics across states of consciousness

Reviewer #1 (Public review):

Summary:

In this work, the authors apply TDCS to awake and anesthetized macaques to determine the effect of this modality on dynamic connectivity measured by fMRI. The question is to understand the extent to which TDCS can influence conscious or unconscious states. Their target was the PFC. During the conscious states, the animals were executing a fixation task. Unconsciousness was achieved by administering a constant infusion of propofol and a continuous infusion of the muscle relaxant cisatracurium. They observed the animals while awake receiving anodal or cathodal hd-TDCS applied to the PFC. During the cathodal stimulation, they found disruption of functional connectivity patterns, enhanced structure-function correlations, a decrease in Shannon entropy, and a transition towards patterns that were more commonly anatomically based. In contrast under propofol anesthesia anodal hd-TDCS stimulation appreciably altered the brain connectivity patterns and decreased the correlation between structure and function. The PFC stimulations altered patterns associated with consciousness as well as those associated with unconsciousness.

Strengths:

The authors carefully executed a set of very challenging experiments that involved applying tDCS in awake and anesthetized non-human primates while conducting functional imaging.

Weaknesses:

The authors show that tDCS can alter functional connectivity measured by fMRI but they do not make clear what their studies teach the reader about the effects of tDCS on the brain during different states of consciousness. No important finding is stated contrary to what is stated in the abstract. It is also not clear what the work teaches us about how tDCS works nor is it clear what are the "clinical implications for disorders of consciousness." The deep anesthesia is akin to being in a state of coma. This was not discussed.

While the authors have executed a set of technically challenging experiments, it is not clear what they teach us about how tDCS works, normal brain neurophysiology, or brain pathological states such as disorders of consciousness.

Reviewer #2 (Public review):

General comments:

The authors investigated the effects of tDCS on brain dynamics in awake and anesthetized monkeys using functional MRI. They claim that cathodal tDCS disrupts the functional connectivity pattern in awake monkeys while anodal tDCS alters brain patterns in anesthetized monkeys. This study offers valuable insight into how brain states can influence the outcomes of noninvasive brain stimulation. However, there are several aspects of the methods and results sections that should be improved to clarify the findings.

Major comments

(1) For the anesthetized monkeys, the anode location differs between subjects, with the electrode positioned to stimulate the left DLFPC in monkey R and the right DLPFC in monkey N. The authors mention that this discrepancy does not result in significant differences in the electric field due to the monkeys' small head size. However, this is not correct, as placing the anode on the left hemisphere would result in much lower EF in the right DLPFC compared to placing the anode on the right side. Running an electric field simulation would confirm this. Additionally, the small electrode size suggested by the Easy cap configuration for NHP appears sufficient to focally stimulate the targeted regions. If this interpretation is correct, the authors should provide additional evidence to support their claim, such as a computational simulation of the EF distribution.

(2) For the anesthetized monkeys, the authors applied 1 mA tDCS first, followed by 2 mA tDCS. A 20-minute stimulation duration of 1 mA tDCS is strong enough to produce after-effects that could influence the brain state during the 2 mA tDCS. This raises some concerns. Previous studies have shown that 1 mA tDCS can generate EF of over 1 V/m in the brain, and the effects of stimulation are sensitive to brain state (e.g., eye closed vs. eye open). How do the authors ensure that there are no after-effects from the 1 mA tDCS? This issue makes it challenging to directly compare the effects of 1 mA and 2 mA stimulation.

(3) The occurrence rate of a specific structural-functional coupling pattern among random brain regions shows significant effects of tDCS. However, these results seem counterintuitive. It is generally understood that noninvasive brain stimulation tends to modulate functional connectivity rather than structural or structural-functional connectivity. How does the occurrence rate of structural-functional coupling patterns provide a more suitable measure of the effectiveness of tDCS than functional connectivity alone? I would recommend that the authors present the results based on functional connectivity itself. If there is no change in functional connectivity, the relevance of changes in structural-functional coupling might not translate into a meaningful alteration in brain function, making it unclear how significant this finding is without corresponding functional evidence.

(4) The authors recorded data from only two monkeys, which may limit the investigation of the group effects of tDCS. As the number of scans for the second monkey in each consciousness condition is lower than that in the first monkey, there is a concern that the main effects might primarily reflect the data from a single monkey. I suggest that the authors should analyze the data for each monkey individually to determine if similar trends are observed in both subjects.

(5) Anodal tDCS was only applied to anesthetized monkeys, which limits the conclusion that the authors are aiming for. It raises questions about the conclusion regarding brain state dependency. To address this, it would be better to include the cathodal tDCS session for anesthetized monkeys. If cathodal tDCS changes the connectivity during anesthesia, it becomes difficult to argue that the effects of cathodal tDCS varies depending on the state of consciousness as discussed in this paper. On the other hand, if cathodal tDCS would not produce any changes, the conclusion would then focus on the relationship between the polarity of tDCS and consciousness. In that case, the authors could maintain their conclusion but might need to refine it to reflect this specific relationship more accurately.

Reviewer #3 (Public review):

Summary:

This study used transcranial direct current stimulation administered using small 'high-definition' electrodes to modulate neural activity within the non-human primate prefrontal cortex during both wakefulness and anaesthesia. Functional magnetic resonance imaging (fMRI) was used to assess the neuromodulatory effects of stimulation. The authors report on the modification of brain dynamics during and following anodal and cathodal stimulation during wakefulness and following anodal stimulation at two intensities (1 mA, 2 mA) during anaesthesia. This study provides some possible support that prefrontal direct current stimulation can alter neural activity patterns across wakefulness and sedation in monkeys. However, the reported findings need to be considered carefully against several important methodological limitations.

Strengths:

A key strength of this work is the use of fMRI-based methods to track changes in brain activity with good spatial precision. Another strength is the exploration of stimulation effects across wakefulness and sedation, which has the potential to provide novel information on the impact of electrical stimulation across states of consciousness.

Weaknesses:

The lack of a sham stimulation condition is a significant limitation, for instance, how can the authors be sure that results were not affected by drowsiness or fatigue as a result of the experimental procedure?

In the anaesthesia condition, the authors investigated the effects of two intensities of stimulation (1 mA and 2 mA). However, a potential confound here relates to the possibility that the initial 1 mA stimulation block might have caused plasticity-related changes in neural activity that could have interfered with the following 2 mA block due to the lack of a sufficient wash-out period. Hence, I am not sure any findings from the 2 mA block can really be interpreted as completely separate from the initial 1 mA stimulation period, given that they were administered consecutively. Several previous studies have shown that same-day repeated tDCS stimulation blocks can influence the effects of neuromodulation (e.g., Bastani and Jaberzadeh, 2014, Clin Neurophysiol; Monte-Silva et al., J. Neurophysiology).

The different electrode placement for the two anaesthetised monkeys (i.e., Monkey R: F3/O2 montage, Monkey N: F4/O1 montage) is problematic, as it is likely to have resulted in stimulation over different brain regions. The authors state that "Because of the small size of the monkey's head, we expected that tDCS stimulation with these two symmetrical montages would result in nearly equivalent electric fields across the monkey's head and produce roughly similar effects on brain activity"; however, I am not totally convinced of this, and it really would need E-field models to confirm. It is also more likely that there would in fact be notable differences in the brain regions stimulated as the authors used HD-tDCS electrodes, which are generally more focal.

Given the very small sample size, I think it is also important to consider the possibility that some results might also be impacted by individual differences in response to stimulation. For instance, in the discussion (page 9, paragraph 2) the authors contrast findings observed in awake animals versus anaesthetised animals. However, different monkeys were examined for these two conditions, and there were only two monkeys in each group (monkeys J and Y for awake experiments [both male], and monkeys R and N [male and female] for the anaesthesia condition). From the human literature, it is well known that there is a considerable amount of inter-individual variability in response to stimulation (e.g., Lopez-Alonso et al., 2014, Brain Stimulation; Chew et al., 2015, Brain Stimulation), therefore I wonder if some of these differences could also possibly result from differences in responsiveness to stimulation between the different monkeys? At the end of the paragraph, the authors also state "Our findings also support the use of tDCS to promote rapid recovery from general anesthesia in humans...and suggest that a single anodal prefrontal stimulation at the end of the anesthesia protocol may be effective." However, I'm not sure if this statement is really backed-up by the results, which failed to report "any behavioural signs of awakening in the animals" (page 7)?

Author response:

We thank the reviewers for their thoughtful and critical comments. We will revise and improve the manuscript according to the public reviews. In particular, we will:

(1) provide a broader perspective on the potential clinical implications of our experiments regarding the mechanisms and the treatment of coma and disorders of consciousness. In particular, we will address how the reported increase in dynamical features associated with consciousness, even without behavioral signs, might be relevant to characterize patients with a motor-cognitive dissociation.

(2) use the term "tDCS" to qualify the technique we used in the paper instead of "HD-tDCS" to avoid any potential confusion. We understand that "HD-tDCS", which we used in our paper to refer to high-density tDCS (small size electrodes), may cause some confusion with high-definition tDCS, which is more commonly used in the literature to design a 4x1 tDCS montage with smaller high-definition electrodes. We will also provide the full characteristics of the carbon electrodes we used for stimulation.

(3) clarify the location sites of stimulation and provide structural MRI images with the accurate localization of the stimulating electrodes.

(4) clarify the fMRI data analyses we performed and provide a schematic illustration of the analysis process.