Behavioral entrainment to rhythmic auditory stimulation can be modulated by tACS depending on the electrical stimulation field properties

Reviewer #1 (Public Review):

Summary of what the authors were trying to achieve.

This paper studies the possible effects of tACS on the detection of silence gaps in an FM-modulated noise stimulus. Both FM modulation of the sound and the tACS are at 2Hz, and the phase of the two is varied to determine possible interactions between the auditory and electric stimulation. Additionally, two different electrode montages are used to determine if variation in electric field distribution across the brain may be related to the effects of tACS on behavioral performance in individual subjects.

Major strengths and weaknesses of the methods and results.

The study appears to be well-powered to detect modulation of behavioral performance with N=42 subjects. There is a clear and reproducible modulation of behavioral effects with the phase of the FM sound modulation. The study was also well designed, combining fMRI, current flow modeling, montage optimization targeting, and behavioral analysis. A particular merit of this study is to have repeated the sessions for most subjects in order to test repeat-reliability, which is so often missing in human experiments. The results and methods are generally well-described and well-conceived. The portion of the analysis related to behavior alone is excellent. The analysis of the tACS results is also generally well described, candidly highlighting how variable results are across subjects and sessions. The figures are all of high quality and clear. One weakness of the experimental design is that no effort was made to control for sensation effects. tACS at 2Hz causes prominent skin sensations which could have interacted with auditory perception and thus, detection performance.

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

Unfortunately, the main effects described for tACS are encumbered by a lack of clarity in the analysis. It does appear that the tACS effects reported here could be an artifact of the analysis approach. Without further clarification, the main findings on the tACS effects may not be supported by the data.

Likely impact of the work on the field, and the utility of the methods and data to the community.

The central claim is that tACS modulates behavioral detection performance across the 0.5s cycle of stimulation. However, neither the phase nor the strength of this effect reproduces across subjects or sessions. Some of these individual variations may be explainable by individual current distribution. If these results hold, they could be of interest to investigators in the tACS field.

The additional context you think would help readers interpret or understand the significance of the work.

The following are more detailed comments on specific sections of the paper, including details on the concerns with the statistical analysis of the tACS effects.

The introduction is well-balanced, discussing the promise and limitations of previous results with tACS. The objectives are well-defined.

The analysis surrounding behavioral performance and its dependence on the phase of the FM modulation (Figure 3) is masterfully executed and explained. It appears that it reproduces previous studies and points to a very robust behavioral task that may be of use in other studies.

There is a definition of tACS(+) vs tACS(-) based on the relative phase of tACS that may be problematic for the subsequent analysis of Figures 4 and 5. It seems that phase 0 is adjusted to each subject/session. For argument's sake, let's assume the curves in Fig. 3E are random fluctuations. Then aligning them to best-fitting cosine will trivially generate a FM-amplitude fluctuation with cosine shape as shown in Fig. 4a. Selecting the positive and negative phase of that will trivially be larger and smaller than a sham, respectively, as shown in Fig 4b. If this is correct, and the authors would like to keep this way of showing results, then one would need to demonstrate that this difference is larger than expected by chance. Perhaps one could randomize the 6 phase bins in each subject/session and execute the same process (fit a cosine to curves 3e, realign as in 4a, and summarize as in 4b). That will give a distribution under the Null, which may be used to determine if the contrast currently shown in 4b is indeed statistically significant.

Results of Fig 5a and 5b seem consistent with the concern raised above about the results of Fig. 4. It appears we are looking at an artifact of the realignment procedure, on otherwise random noise. In fact, the drop in "tACS-amplitude" in Fig. 5c is entirely consistent with a random noise effect.

To better understand what factors might be influencing inter-session variability in tACS effects, we estimated multiple linear models ..." this post hoc analysis does not seem to have been corrected for multiple comparisons of these "multiple linear models". It is not clear how many different things were tried. The fact that one of them has a p-value of 0.007 for some factors with amplitude-difference, but these factors did not play a role in the amplitude-phase, suggests again that we are not looking at a lawful behavior in these data.

"So far, our results demonstrate that FM-stimulus driven behavioral modulation of gap detection (FM-amplitude) was significantly affected by the phase lag between the FM-stimulus and the tACS signal (Audio-tACS lag) ..." There appears to be nothing in the preceding section (Figures 4 and 5) to show that the modulation seen in 3e is not just noise. Maybe something can be said about 3b on an individual subject/session basis that makes these results statistically significant on their own. Maybe these modulations are strong and statistically significant, but just not reproducible across subjects and sessions?

"Inter-individual variability in the simulated E-field predicts tACS effects" Authors here are attempting to predict a property of the subjects that was just shown to not be a reliable property of the subject. Authors are picking 9 possible features for this, testing 33 possible models with N=34 data points. With these circumstances, it is not hard to find something that correlates by chance. And some of the models tested had interaction terms, possibly further increasing the number of comparisons. The results reported in this section do not seem to be robust, unless all this was corrected for multiple comparisons, and it was not made clear?

"Can we reduce inter-individual variability in tACS effects ..." This section seems even more speculative and with mixed results.

Given the concerns with the statistical analysis above, there are concerns about the following statements in the summary of the Discussion:

"2) does modulate the amplitude of the FM-stimulus induced behavioral modulation (FM-amplitude)"
This seems to be based on Figure 4, which leaves one with significant concerns.

"4) individual variability in tACS effect size was partially explained by two interactions: between the normal component of the E-field and the field focality, and between the normal component of the E-field and the distance between the peak of the electric field and the functional target ROIs."
The complexity of this statement alone may be a good indication that this could be the result of false discovery due to multiple comparisons.

For the same reasons as stated above, the following statements in the Abstract do not appear to have adequate support in the data:
"We observed that tACS modulated the strength of behavioral entrainment to the FM sound in a phase-lag specific manner. ... Inter-individual variability of tACS effects was best explained by the strength of the inward electric field, depending on the field focality and proximity to the target brain region. Spatially optimizing the electrode montage reduced inter-individual variability compared to a standard montage group."
In particular, the evidence in support of the last sentence is unclear. The only finding that seems related is that "the variance test was significant only for tACS(-) in session 2". This is a very narrow result to be able to make such a general statement in the Abstract. But perhaps this can be made more clear.

Reviewer #2 (Public Review):

In "Behavioral entrainment to rhythmic auditory stimulation can be modulated by tACS depending on the electrical stimulation field properties" Cabral-Calderin and collaborators aimed to document 1) the possible advantages of personalized tACS montage over standard montage on modulating behavior; 2) the inter-individual and inter-session reliability of tACS effects on behavioral entrainment and, 3) the importance of the induced electric field properties on the inter-individual variability of tACS.

To do so, in two different sessions, they investigated how the detection of silent gaps occurring at random phases of a 2Hz- amplitude modulated sound could be enhanced with 2Hz tACS, delivered at different phase lags. In addition, they evaluated the advantage of using spatially optimized tACS montages (information-based procedure - using anatomy and functional MRI to define the target ROI and simulation to compare to a standard montage applied to all participants) on behavioral entrainment. They first show that the optimized and the standard montages have similar spatial overlap to the target ROI. While the optimized montage induced a more focal field compared to the standard montage, the latter induced the strongest electric field. Second, they show that tACS does not modify the optimal phase for gap detection (phase of the frequency-modulated sound) but modulates the strength of behavioral entrainment to the frequency-modulated sound in a phase-lag specific manner. However, and surprisingly, they report that the optimal tACS lag, and the magnitude of the phasic tACS effect were highly variable across sessions. Finally, they report that the inter-individual variability of tACS effects can be explained by the strength of the inward electric field as a function of the field focality and on how well it reached the target ROI.

The article is interesting and well-written, and the methods and approaches are state-of-the-art.

Strengths:
- The information-based approach used by the authors is very strong, notably with the definition of subject-specific targets using a fMRI localizer and the simulation of electric field strength using 3 different tACS montages (only 2 montages used for the behavioral experiment).
- The inter-session and inter-individual variability are well documented and discussed. This article will probably guide future studies in the field.

Weaknesses:
- The addition of simultaneous EEG recording would have been beneficial to understand the relationship between tACS entrainment and the entrainment to rhythmic auditory stimulation.
- It would have been interesting to develop the fact that tACS did not "overwrite" neural entrainment to the auditory stimulus. The authors try to explain this effect by mentioning that "tACS is most effective at modulating oscillatory activity at the intended frequency when its power is not too high" or "tACS imposes its own rhythm on spiking activity when tACS strength is stronger than the endogenous oscillations but it decreases rhythmic spiking when tACS strength is weaker than the endogenous oscillations". However, it is relevant to note that the oscillations in their study are by definition "not endogenous" and one can interpret their results as a clear superiority of sensory entrainment over tACS entrainment. This potential superiority should be discussed, documented, and developed.
- The authors propose that "by applying tACS at the right lag relative to auditory rhythms, we can aid how the brain synchronizes to the sounds and in turn modulate behavior." This should be developed as the authors showed that the tACS lags are highly variable across sessions. According to their results, the optimal lag will vary for each tACS session and subtle changes in the montage could affect the effects.
- In a related vein, it would be very useful to show the data presented in Figure 3 (panels b,d,e) for all participants to allow the reader to evaluate the quality of the data (this can be added as a supplementary figure).