Auditory confounds can drive online effects of transcranial ultrasonic stimulation in humans

Reviewer #1 (Public Review):

Summary: The authors have used transcranial magnetic stimulation (TMS) and motor evoked potentials (MEPs) to determine whether the peripheral auditory confound arising from TUS can drive motor inhibition on its own. They gathered data from three international centers in four experiments testing:
In Experiment 1 (n = 11), two different TUS durations and intensities under sound masking or without.
Experiment 2 (n = 27) replicates Exp 1 with different intensities and a fixed TUS duration of 500ms.
Experiment 3 ( n = 16) studied the effect of various auditory stimuli testing different duration and pitches while applying TUS in an active site, on-target or no TUS.
Experiment 4 (n = 12) used an inactive control site to reproduce the sound without effective neuromodulation, while manipulating the volume of the auditory confound at different TUS intensities with and without continuous sound masking.

Strengths: This study comes from three very strong groups in noninvasive brain stimulation with long experience in neuromodulation, multimodal and electrophysiological recordings. Although complex to understand due to slightly different methodologies across centers, this study provides quantitative evidence alerting on the potential auditory confound of online US. Their results are in line with reductions seen in motor-evoked responses during online 1kHz TUS, and remarkable efforts were made to isolate peripheral confounds from actual neuromodulation factors, highlighting the confounding effect of sound itself.

Weaknesses: However, there are some points that need attention. In my view, the most important are:
1. Despite the main conclusion of the authors stating that there is no dose-response effects of TUS on corticospinal inhibition, the point estimates for change in MEP and Ipssa indicate a more complex picture. The present data and analyses cannot rule out that there is a dose-response function which cannot be fully attributed to difference in sound (since the relationship in inversed, lower intracranial Isppa leads to higher MEP decrease). These results suggest that dose-response function needs to be further studied in future studies.
2. Other methods to test or mask the auditory confound are possible (e.g., smoothed ramped US wave) which could substantially solve part of the sound issue in future studies or experiments in deaf animals etc...

Reviewer #2 (Public Review):

Summary:
This study aims to test auditory confounds during transcranial ultrasound stimulation (TUS) protocols that rely on audible frequencies. In several experiments, the authors show that a commonly observed suppression of motor-evoked potentials (MEP) during TUS can be explained by acoustic stimulation. For instance, not only target TUS, but also stimulation of a control site and acoustic stimulation led to suppressed MEP.

Strengths:
A clear strength of the study is the multitude of control conditions (control sites, acoustic masking, acoustic stimulation etc) that makes results very convincing.
Indeed, I do not have much to criticise. The paper follows a clear structure and is easy to follow, the research question is clearly relevant, and analyses are sound. Figures are of high quality.
Although auditory confounds during TUS have been demonstrated before, the thorough design of the study will lead to a strong impact in the field.

Weaknesses:
I cannot see major weaknesses. A few minor ones are that (1) the overview of previous related work, and how frequent audible TUS protocols are in the field, could be a bit clearer/more detailed; (2) the acoustic control stimulus can be described in more detail; and (3) the finding that remaining motor inhibition is observed during acoustically masked trials deserves further discussion.