Transcranial focused ultrasound induces source localizable cortical activation in resting state humans when applied concurrently with transcranial electric stimulation

Domain: biology
Published: 2026-03-10
Editorial outcome: minor_revision

Abstract
The effects of transcranial focused ultrasound (tFUS) on the human brain are poorly understood. Currently, the field is at odds with whether tFUS is sub-threshold modulating the brain’s excitability towards other stimuli, producing suprathreshold neural stimulation on its own, or if it even has a spatially specific non-auditory induced effect. Herein, we investigated the ability of tFUS, transcranial direct current stimulation (tDCS), and a combination of the two (transcranial electro-acoustic stimulation; tEAS) to evoke cortical target-location specific activity in 27 resting state humans with whole-brain electroencephalography recordings. In none of the exogenous event-related potentials did tFUS or tDCS result in location-specific activations. However, the co-modulatory combination of tEAS provided evidence that tFUS has a location-specific subthreshold modulatory effect. We propose a minimally modified Hodgkin-Huxley model that explains our results and provides a unifying framework for the field-wide observed effects (or lack thereof) of tFUS.

Introduction
Therapeutic intervention of the human nervous system dates back centuries. Medical texts from c. 1st century CE Rome1 explicitly prescribe uses of electric fish for pain management, and surviving records from c. 1550 BCE Egypt2 imply the practice may date even earlier. A modern, philosophical descendant of these methods, transcranial direct current stimulation (tDCS), applies subthreshold (meaning it will modulate spontaneous firing, but will not induce stimulation time-locked spikes on its own) current across brain tissue using non-invasive scalp electrodes3–6. The subthreshold current depolarizes (or hyperpolarizes, in the case of inhibitory current) the neurons towards (or away from) their action potential threshold, which will make the neurons more (or less) sensitive to additional stimuli. One of the biggest limitations with this form of neuromodulation has to do with the spatial precision at which it can be applied due to the volume conduction effect7. Non-invasive electrical stimulation has spatial precision on the order of centimeters8, which can contain tens of millions of neurons9, and unintentionally modulate off-target brain regions as well3,10.

A recently introduced technique, transcranial focused ultrasound (tFUS), promises to overcome that limitation with a spatial precision on the order of millimeters11,12. tFUS’ mechanism of action continues to be under investigation, but one prevalent theory is that the ultrasound pressure waves open mechanosensitive ion channels13,14. This theory would be consistent with behavioral outcomes dependent on the specific brain region targeted with tFUS, which have been reported in a range of human studies11,15–20.

However, there is a critical lack of understanding when it comes to tFUS’ effect on the human brain. The bulk of human tFUS studies pair it with a secondary stimulation (such as tactile vibrations11,15, transcranial magnetic stimulation (TMS) pulses21, or visual stimuli18), which point toward, but do not confirm, a subthreshold modulation-based mechanism of action. Other studies directly looked into tFUS’ ability to elicit percepts as the sole stimulus, but their results pointed towards, at best, an inconsistent ability to do so—a study investigating S1 activation only elicited percepts in 54% of trials16, and a study investigating V1 stimulation reported visual percepts in only 11 of 19 participants17. Beyond even the question of suprathreshold stimulation vs. subthreshold modulation, some studies report that the effects of tFUS may not actually be due to the pressure wave, but rather auditory conduction. tFUS, typically, is not applied as one continuous wave, but instead pulsed at some lower frequency (pulse repetition frequency; PRF; Fig. 1D). This PRF makes a beeping noise, which is associated with activating auditory pathways22,23, and some studies could not detect an effect beyond auditory activation24,25. In humans, it was reported that actively playing a tone over the duration of tFUS sonication led to no significant differences in EEG between sham and active conditions26.

These studies raise critical questions about whether tFUS merely activates auditory pathways or if it evokes location-specific neuronal responses—and, if it does have a location-specific effect, whether that effect is subthreshold or suprathreshold. Resolving these questions requires a comprehensive analysis of whole-brain responses. While functional magnetic resonance imaging (fMRI) studies have investigated these questions and reported activations at the focal site of tFUS sonication27,28, their results may be confounded by multiple mechanisms. fMRI detects changes in blood-oxygen-level-dependent (BOLD) signal as a correlate for neural firing, but blood flow can be changed through a variety of methods not necessarily dictated by neuronal firing, such as vasodilation, which is directly inducible by ultrasound29. Further, unless tFUS is applied strictly parallel to the MRI’s magnetic field, physical interaction between the ultrasound pressure waves and the MRI’s magnetic fields will generate localized electric fields30, potentially creating artifactual neuronal activations absent in non-MRI environments. Consequently, fMRI measurements of tFUS’ neural activation ability are not definitive. Electroencephalography (EEG) based recordings, which directly measure electrical activity and do not require a magnetic resonance environment, may offer more insight into the true effects of tFUS as a sole stimulus.

Herein, we apply excitatory (e) and inhibitory (i) tDCS, tFUS (500 kHz fundamental frequency), and a combination of the modalities (transcranial electro-acoustic stimulation; tEAS) to the cortex of resting-state humans (Fig. 1). In follow-up studies, we broaden our tFUS parameter scope. For all tFUS experiments, we record and analyze simultaneous whole-scalp EEG recordings and image their corresponding cortical sources to characterize targeted-location specific, or lack thereof, responses. We also present a first-principles verification of our findings using a minimally modified Hodgkin-Huxley model.

Results
Transcranial electro-acoustic stimulation produces a hemisphere-specific exogenous response
Butterfly plots of each condition’s preprocessed EEG are shown in Fig. 2A (dosing information is available in Supplementary Table T1). Statistical differences between EEG signals of the region of interest (ROI; electrodes C1, C3, FC1, and FC3) and their contralateral homologous counterparts (C2, C4, FC2, and FC4) were assessed using spatio-temporal permutation cluster tests within the 0–200 ms exogenous (stimulus-evoked) response window. The tests identified significant differences (p < 0.05) in the itEAS (30 Hz PRF tFUS with overlapping cathodal tDCS) and etEAS (3 kHz PRF tFUS with overlapping anodal tDCS) conditions, but none for itFUS (30 Hz PRF), etFUS (3 kHz PRF), itDCS (cathodal tDCS with spatial-control 30 Hz PRF tFUS) or etDCS (anodal tDCS with spatial-control 3 kHz PRF tFUS) conditions (Fig. 2B). Both significant clusters encompassed electrode C3 and the 10–63 ms time window. We used the shared components across both tEAS conditions’ significant clusters as spatial and temporal windows for further analysis.

Inhibitory vs. excitatory tEAS produces significant changes to EEG polarity
EEG data were averaged over the permutation cluster test-identified time frame of 10–63 ms. Resultant topographic maps present with visually apparent ROI activations for tEAS conditions and primarily hemisphere-symmetric topographies for the other conditions (Fig. 2C). Effects on electrode C3 (selected based on its presence in permutation cluster test results) polarity for this time average were assessed with McNemar’s test and False Discovery Rate (FDR) multiple comparison correction (Fig. 2D). Inhibitory tEAS led to a significant change (p_adj < 0.001, χ2=12.1) in ROI polarity compared to etEAS. Neither e/itFUS nor e/itDCS conditions induced significant polarity changes (p_adj > 0.05).

tEAS induces significantly localizable responses to the targeted ROI
EEG data were back-projected to the subjects’ cortices using the dynamic statistical parametric mapping (dSPM) source localization method, and the resultant power of source waveforms was averaged over the 10–63 ms window. Source-morphing to a common brain model and averaging the response across subjects showed strong concordance with the targeted ROI, defined by the subspace contained within electrodes C1, C3, FC1, and FC3 orthogonally projected to the cortex (Fig. 3A). Statistical analysis to determine if ipsilateral (IL) ROI power was significantly higher than the homologous contralateral (CL) power was conducted with permutation t-tests with FDR multiple comparison correction. Both tEAS conditions had significantly increased IL ROI source power (etEAS_IL = 2.59 ± 1.18 z_dSPM, CL = 1.62 ± 0.63 z_dSPM, p_adj < 0.001, t = 4.10, d = 1.03; itEAS_IL = 2.40 ± 1.08 z_dSPM, CL = 1.22 ± 0.45 z_dSPM, p_adj < 0.001, t = 4.78, d = 1.42). None of the other conditions had significantly increased IL source power (p_adj > 0.05; Fig. 3B).

Pairwise comparisons using each subject’s condition average were made between IL source power, as well as precision, recall, and F1 score of source localization with respect to the ROI (Supplementary Fig. S1). For all metrics, tEAS conditions were found to have significantly higher outcomes. Cohen’s d analysis yielded that tEAS conditions had at least moderate effect sizes (d > 0.5) for all comparisons with other conditions, and often large ones (d > 0.8). Pairwise FDR-adjusted p-values and Cohen’s d values are provided within the relevant sub-figures.

Transcranial focused ultrasound results in significant auditory activations
Despite the presence of three distinct exogenous time window event-related potentials (ERPs) induced from 3 kHz tFUS in the topographic maps (30, 90, and 170 ms peaks; Fig. 2A), spatio-temporal permutation cluster tests did not return any significant differences for left hemisphere ROI electrodes compared to their right hemisphere counterparts. This points to a hemisphere-symmetric response, despite tFUS only being applied to one side. Topographic map visualizations of each peak (20–40 ms, 80–100 ms, and 160–180 ms averages; Fig. 4A) do not provide any evidence of a target-location specific effect. Source imaging metrics of these peak windows continue to support a lack of ROI response (20–40 ms precision w.r.t. ROI: 0.013 ± 0.014; 80–100 ms: 0.015 ± 0.019; 160–180 ms: 0.022 ± 0.020).

In 21 of the 22 subjects with etFUS, we also tested a 3 kHz PRF right prefrontal cortex (RH PFC) spatial control condition. The resultant waveforms and ERP spatial topographies (Fig. 4B.i) share a striking resemblance to those that arose from applying tFUS to the left motor cortex area (LH M1). Spatio-temporal permutation cluster testing did not yield any significantly different clusters between LH M1 and RH PFC conditions (Fig. 4B.ii). Further, when we directly quantified statistical similarity between the LH ROI electrodes for the two conditions using Bayes Factor (BF) analysis, the evidence that the 10–63, 20–40, and 80–100 ms responses of the two conditions were the same was robust (log10 BF < 0 for all electrodes/scaling factors; Fig. 4B.iii). For the 160–180 ms window, BF analysis was inconclusive for small-medium effect sizes (scaling factors < 0.707; 0 < log10 BF < 0.5), though it continued to provide robust evidence in favor of the null for medium-large effect sizes (scaling factor > 0.707). For comparison, BF analysis of etEAS compared to etFUS yielded evidence that certain ROI electrode responses were significantly different (log10 BF > 0.5; Supplementary Fig. S2).

Granger causality analysis of both itFUS and etFUS conditions showed significant increases in information flow from the primary auditory cortex to the deep brain, and no changes in net information flow from the targeted ROI to the deep brain (Fig. 4C). In a follow-up study with 11 subjects, it was found that even an inaudible PRF (30 kHz) continued to induce significant auditory to deep brain Granger causality (Supplementary Fig. S3).

tEAS conditions were also found to induce significant increases in auditory to deep brain connectivity and have no effect on the net flow of information from the targeted ROI to deep brain (Supplementary Fig. S4).

itFUS, but not tEAS, induces endogenous dissociations between the ROI and deep brain
Despite not inducing a significant effect on net Granger causality, itFUS was found to have significant dissociation effects on undirected endogenous (>200 ms) spectral imaginary coherence (ImCoh; Supplementary Fig. S5). None of these dissociations were present in the tEAS conditions.

Increased tFUS parameter searches continued to yield no evidence of ROI suprathreshold stimulation
A follow-up call-back study was conducted in 10 of the 22 original subjects (Supplementary Table T2), in which additional tFUS parameters were tested. These parameters included 1,500 and 40 Hz PRFs with a 30% duty cycle (DC), and, initially, 10% and 60% DCs with both 30 and 3000 Hz PRFs. All four of the first four subjects experienced thermal discomfort with 60% DC, and so we did not test it further. From the fourth subject onward, we tested 0.6% DC instead. Source imaging results continued to point towards deep brain and temporal lobe activations (Fig. 5A) and ROI analysis continued to find no increases in ipsilateral source power for any of the new tested conditions (Fig. 5B). Analysis of the effects of holding the DC or PRF constant and changing the other parameter yielded no significant differences (permutation one-way ANOVA; all p > 0.05) and Bayes Factors provide robust evidence that the resting-state tFUS responses are statistically similar across the parameter spaces.

In an additional follow-up study with 11 subjects, the effects of higher pressure were investigated by normalizing the in situ pressure to 300 kPa with Sim4Life simulations (original study in situ mean pressure was 231 kPa). The sonication duration was reduced from 500 ms to 100 ms to compensate for this increase. Even still, 2 of the 11 subjects (18.2%) experienced scalp thermal discomfort and their experiments were aborted. Analysis of the other 9 subjects’ data continued to yield no significant increases in IL compared to CL ROI source power (Supplementary Fig. S6; p > 0.05).

tEAS is more than a linear sum of its parts
We took the summation of the source-space tFUS and tDCS conditions to produce a condition representing the sum of independent electric and acoustic stimulation. The summation did not result in increased concordance with the ROI (Supplementary Fig. S7A), nor to significant increases in ipsilateral over contralateral ROI source power (Supplementary Fig. S7B; permutation t-test p > 0.05). We also modeled the tEAS ROI response as function of tDCS scalp current, cortical current, and tFUS intensity and found none of these components to be meaningfully predictive (Supplementary Fig. S7C–E).

tEAS response is driven by tDCS polarity, not tFUS PRF
Anodal tDCS was combined with 30 and 3000 Hz PRF tFUS and analyzed over the 10–63 ms window (Supplementary Fig. S8). Both conditions led to brain sources concordant with the ROI. Comparisons of the ROI source power yielded no significant differences across conditions (permutation t-test p > 0.05), and Bayes Factor analysis provided evidence that the response powers were statistically similar (log10 BF < 0).

A minimally modified Hodgkin-Huxley model provides support for an electro-acoustic co-modulatory effect
The Hodgkin-Huxley model was modified (Supplementary Methods) to have an additional parallel pathway representing piezo-electric ion channels (Supplementary Fig. 9A). Simulations revealed that subthreshold ultrasound-based opening of piezo-electric channels (Supplementary Fig. S9B) and subthreshold direct current, representing tDCS (Supplementary Fig. S9C), could combine to form a suprathreshold stimulus (Supplementary Fig. S9D-F).

Methods
MRI and MRI segmentation
Subjects underwent a T1-weighted 3T Magnetic Resonance Imaging (MRI) structural scan of their brain at the CMU-Pitt BRIDGE Center (RRID:SCR_023356). The brain images were then segmented in FreeSurfer (version 7)69,70 using the Desikan-Killiany atlas for post-hoc analysis. One subject was not MRI eligible due to an implanted device, so the FreeSurfer FSAverage brain was used71.

For EEG source imaging cortical parcellation, the primary auditory cortices and deep brain regions were defined by the left and right transverse temporal and medial wall parcellations, respectively. The IL/CL M1 ROI was defined by projecting the four IL/CL ROI electrodes orthogonally to the brain model’s inflated cortex and creating a square slice whose corners were defined by the electrodes.

Freewater ultrasound calibration
This experiment used a 500 kHz f0 (fundamental frequency) single-element ultrasound transducer (1.5 inch focal length, 1 inch acoustic aperture diameter, 0.75 inch radius of curvature; Blatek Industries Inc., Boalsburg, PA, USA). To characterize the beam shape and determine the needed input voltage for the desired free water pressure, the transducer was calibrated in a water tank. The water tank was filled with deionized water. Pressure measurements were recorded with an HNR-0500 hydrophone (Onda Corp., Sunnyvale, CA, USA).

In silico transcranial focused ultrasound simulations
In the initial subject cohort, tFUS pressure was normalized with respect to the energy delivered on the scalp (0.98 MPa). To calculate the resulting on-cortical target ultrasound metrics (denoted with .T) across subjects, Sim4Life (Version 7.0, Zurich Med Tech; Zurich, Switzerland)72 computer simulations of the single-element tFUS transducer were conducted. Structural MRI files were converted to pseudo computed tomography (pCT) for use with the software73,74. The pCTs were manually segmented using iSeg (v3.10) into air (1.16 kg/m3 density, 343 m/s speed of sound, 0.000399 MRayl acoustic impedance), bone (1910 kg/m3 density, 3510 m/w speed of sound, 6.71 MRayl acoustic impedance), and soft tissue (1050 kg/m3 density, 1550 m/s speed of sound, 1.61 MRayl acoustic impedance). Sim4Life solved the acoustic simulations with the Linear Acoustic Pressure Wave Equation and the automatic discretization settings (0.45 mm resolution, 0.3 mm maximum step, and a time step of 1.0/(speed of sound * sqrt(1/dx2 + 1/dy2 + 1/dz2))). For the simulations, the transducer was positioned over the subject’s left motor cortex, and the resultant pressure loss from brain tissue and skull bone attenuation was used to calculate spatial-peak temporal-average intensity (ISPTA.T; subject range = 48.6–372 mW/cm2), derated spatial-peak pulse-average intensity (ISPPA.T; 0.16–1.24 W/cm2), and Mechanical Index (MI.T; 0.10–0.28). The values for each subject are listed in Supplementary Table T1. In the case of two subjects, a pCT could not be generated (one subject did not have an MRI, and the other’s MRI had quality issues). All metrics were within the Food and Drug Administration’s guidelines for ultrasound (MI < 1.9, ISPPA < 190 W/cm2, and ISPTA < 720 mW/cm2)75.

For later subject cohorts, Sim4Life simulations were used to normalize in situ tFUS pressure to 250 kPa and 300 kPa. The equivalent free water pressures are provided in the relevant Supplementary data.

Neuromodulation-EEG experiment
Once the MRI segmentation was completed, subjects were asked to return for the neuromodulation-EEG portion of the study, which took place in an acoustic and electromagnetically shielded booth (IAC Acoustics; Naperville, Illinois, USA). Subjects were fit with a 64-channel EEG cap (BrainCap TMS, Brain Products, Gilching, Germany). EEG caps were aligned with subject landmarks following standard convention (centering electrode Cz between both the inion and nasion, as well as the right pre-auricular and left pre-auricular). EEG electrode impedances were lowered to be less than 10 kΩ using ABRALYT HiCl electrode gel.

The 4×1 high-definition tDCS (Soterix Medical Inc., Woodbridge, NJ, USA) electrodes were placed in the following locations: 1 peripheral electrode between AFz (ground), Fz, F1, and AF3, 1 peripheral electrode between CP1, CPz, Pz, and P1, 1 peripheral electrode between FC3, C3, C5, and FC5, 1 peripheral electrode between FC2, FC4, C2, and C4, and the center electrode between FCz (reference), Cz, C1, and FC1 (Fig. 1B). The electrical dosage for each subject was based on their own personal threshold for feeling scalp sensations. To determine the dosage, current was slowly ramped up from 0 until the subjects reported sensations. The dosage was set at 0.1 mA below their perception threshold, up to a maximum of 2 mA (Supplementary Table T1). The same amperage was used across all conditions involving tDCS (etEAS, itEAS, etDCS, and itDCS). Excitatory tDCS consisted of center-anode current, while center-cathode current was defined as inhibitory tDCS current. tDCS remained on throughout for the entirety of the 200 trials per condition.

For on-target ultrasound conditions (etEAS, itEAS, etFUS, and itFUS), the transducer was attached between EEG electrodes C1, C3, FC1, and FC3. Using an image-guided neuronavigation system (MagVenture Localite TMS Navigator; Alpharetta, Georgia, USA), the ultrasound target was visually confirmed to align with the subject’s left motor cortex. For off-target ultrasound spatial control conditions (etDCS, itDCS, and an additional 3 kHz tFUS), the ultrasound was targeted to the subject’s right prefrontal cortex area between electrodes AF4, AF8, F4, and F6. Based on our lab’s prior work, excitatory ultrasound was defined by a PRF of 3 kHz, and inhibitory ultrasound by a PRF of 30 Hz39 (Fig. 1B). The duty cycle (30%) and sonication duration (500 ms) were held constant.

For each condition, subjects were asked to assume a resting state for 200 trials (Fig. 1D). Specifically, they were instructed to close their eyes, remain as still as possible, and to relax their thoughts in a meditative-like state. The inter-trial interval was 2 seconds with up to a 20% (0.4 s) random jitter. A five-minute break was taken between each condition to allow the subjects to stretch and let any cumulative effects of neuromodulation dissipate. Experimental condition order was randomized across subjects, though data for e/itDCS control conditions were not collected in the initial subjects (Fig. 2D).

One follow-up study was conducted to investigate additional duty cycles (0.6%, 10%, and 60%) for 30 Hz and 3 kHz PRFs, and additional pulse repetition frequencies (1500 Hz and 40 Hz) with 30% DC. Changing tEAS parameters was also done in this follow-up to determine if the tEAS response differed as a function of PRF, but these conditions were always tested after the resting-state tFUS ones in order to minimize possible long-term contamination effects of tDCS. Another follow-up session was done to investigate the effects of normalizing in situ brain pressure. Other than these parameter changes, the protocol remained the same.

EEG analysis
EEG preprocessing. EEG data were band pass filtered between 1 and 40 Hz with a zero-phase infinite impulse response filter. Data were epoched from 500 ms before the trial onset time to 1.5 seconds after. Bad channels were detected by averaging all the trials and identifying double median absolute deviation (double MAD; threshold = 3) outlier pre-stimulus variance or total trial peak-to-peak amplitude. On the unaveraged trials, independent component analysis eyeblink and muscle artifacts were removed using MNE Python’s76 automatic artifact detection with a z-threshold of 2.5 (Python version 3.9, MNE Python version 1.8). The data were common average referenced, and the previously identified bad channels were interpolated back into the signal using MNE Python’s interpolate_bads function. The cleaned data were averaged over all the trials to get the evoked waveform and z-scored with respect to the −500 to −100 ms pre-stimulus window. In a few instances, active tDCS created noticeable EEG artifacts. These data were excluded from the analysis (Fig. 2D).

EEG sensor domain analysis. Preprocessed EEG waveforms were averaged across subjects for each condition and used to produce butterfly plots (Fig. 2A). Statistical analysis was conducted using non-parametric spatio-temporal two-tailed cluster tests (5000 permutations, α=0.05; Fig. 2B). Paired t-tests were conducted comparing ipsilateral ROI electrodes C1, C3, FC1, and FC3 against their homologous contralateral counterparts C2, C4, FC2, and FC4 within the exogenous (0–200 ms37) brain response window. Significantly different adjacent data were clustered together in time and space. Data were randomly shuffled, and the largest significant cluster from each permutation was used to construct a cluster-size histogram. The cluster-forming threshold was set at a significance level of 0.02. Clusters from the original data with sizes greater than the α percentile (corresponding to p < 0.05) were marked as significant.

ROI polarity analysis for the largest overlapping significant cluster components of tEAS was determined by taking the sign of electrode C3’s average over the response window (10–63 ms). McNemar’s test, a statistical procedure for assessing paired binary data, was used to investigate the effect of excitatory and inhibitory modulation parameters on ROI polarity for each condition. False discovery rate (FDR) multiple comparison correction was applied.

EEG source imaging. EEG source imaging (ESI) was solved by minimizing the difference between cortical current density produced by scalp EEG and recorded EEG7 using MNE Python’s implementation of dSPM, which normalizes the minimum norm estimation relative to a noise covariance matrix77. The noise covariance used for dSPM was calculated from each condition’s baseline window (−500 to −100 ms). EEG electrodes were anatomically aligned to the subject’s specific MRI model using the standard montage setup. The dSPM source maps were averaged over the 10–63 ms response window for ROI activity and localization analysis. Subject source maps were morphed from their individual head models to FreeSurfer’s FSAverage71 brain model for group-level visualizations (taking the average of each subject’s max-min scaled data) and connectivity analysis. For grand average ERP analysis (Supplementary Fig. S11), the subjects’ EEG data were averaged prior to EEG source imaging with the FSAverage brain model.

For each subject, the mean activity, precision (Eq. 1), recall (Eq. 2), and F1 score (Eq. 3) were calculated for the source-imaged activity with respect to the ipsilateral ROI. The ROI was defined by all the cortical space within a bounding box formed by projecting electrodes C1, C3, FC1, and FC3 (the electrodes between which tFUS was targeted) orthogonally from the scalp to the cortex. For precision, recall, and F1 score, true or false positive were defined as active brain sources that were inside, or outside, respectively, the region of interest. False negative were defined as brain voxels within the region of interest that were not estimated as active sources. The threshold for what constituted a brain source, compared to noise, was defined using Otsu’s method56,57. Pairwise statistical comparisons between the mean activity, precision, recall, and F1 score were conducted with linear mixed effect modeling (see Linear Mixed Effect Modeling for more details).

Precision = True Positives / (True Positives + False Positives) (1)

Recall = True Positives / (True Positives + False Negatives) (2)

F1 Score = 2 * Precision * Recall / (Precision + Recall) (3)

The contralateral ROI was defined by the cortical space contained within the projection of electrodes C2, C4, FC2, and FC4. Comparisons between ipsilateral and contralateral mean activity were conducted with 5000 permutation one-tailed paired t-tests and FDR multiple comparison correction (H1: μIL > μCL). A permutation t-test is a non-parametric alternative to the parametric t-test, which relies on no underlying assumptions about data distributions. Instead, it shuffles the data between conditions an N_permutations number of times (in this case 5000) and calculates the T-statistic for each instance. This generates a histogram of T-statistics, against which the unshuffled data’s T-statistic is compared against to determine its significance. The p-value represents the proportion of permuted T-statistics that are at least as extreme as the unpermuted T-statistic. In some instances, comparisons across the ipsilateral activity for multiple conditions were assessed with permutation ANOVA tests. The permutation ANOVA test follows the same logic as the permutation t-test, except it is based on the F-statistic. Bayes Factor ANOVA testing was performed in R (version 4.3.3) with the BayesFactor package.

Connectivity analysis. Changes in Granger causality were calculated on source waveforms78 for the primary auditory cortex to deep brain and the targeted ROI to deep brain. Granger causality was calculated using the Toda and Yamamoto procedure79. The lag for each pathway was selected using the Akaike Information Criterion with possible choices between 10 and 15 ms based on reported values for transmission delays80. Net Granger causality (GC_Net) was calculated by subtracting the reverse Y→X Granger causality Wald statistic from the forward X→Y Granger causality Wald statistic. GC_Net was calculated for each 200 ms time bin from stimulus onset to one second after, as well as for the −0.5 to −0.1 s pre-stimulus baseline period. Permutation paired two-tailed t-tests were used to compare each time bin’s GC_Net to the pre-stimulus period. FDR multiple comparison correction was applied locally for each series.

Spectral connectivity was assessed with imaginary coherence (Eq. 4), a metric used for quantifying non-zero time-lagged undirected connectivity81. The power spectral density (PSD) for each series was calculated using Welch’s method with a 0.2 s window in 0.2 s bins over the 0 to 1 second post-stimulus period and mean-normalized against the −0.5 to −0.1 s baseline period. Imaginary coherence was calculated for alpha (10–13 Hz), beta (13–30 Hz), and gamma (30–40 Hz) frequency bands. Comparison between the ipsilateral and contralateral changes in ImCoh for each time bin was assessed with 5000 permutation t-tests. FDR multiple comparison correction was applied locally for each condition/frequency band level.

ImCoh = |Imag(PSD_XY)| / sqrt(PSD_XX * PSD_YY) (4)

Linear mixed effect modeling. The partially-paired data (Fig. 2D) made linear mixed-effect models excellent candidates for analyzing statistical differences across conditions. Linear mixed effect models account for both fixed effects (experimental conditions) and random effects (subject-to-subject differences) and estimate the true mean of the predictors on the outcome variables. For all our analyses, we modeled the relationship as Eq. 5. The outcome variable name is a stand-in for mean ROI activity, precision, recall, or F1 score. The outcome_j,k variable for condition j and subject k is a function of some fixed offset for all subjects and all conditions, β0, the estimated true mean effect of experimental condition j, β1,j, and a subject-specific random effect for subject k, bk. The difference between the model’s prediction and the observed outcome is the error term ϵ_j,k. Since our independent variables are categorical, β1 is a (1xJ) vector of the estimated true means of the effects, and Condition is a (Jx1) one-hot vector. Modeling was done in R (version 4.3.3) with the lme4 and multcomp packages.

Outcome_j,k = β0 + β1,j * Condition_j + bk + ϵ_j,k (5)

Pairwise comparisons were made using R’s glht function, which calculates the mean difference of the estimated true effect across conditions and their pooled standard error. These values are used in z-tests to statistically compare condition means with FDR multiple comparison correction. The pooled standard error, used in calculating Cohen’s d (Eq. 6), was found by multiplying the pooled standard error by the square root of the subject number.

Cohen’s d = |μ1 - μ2| / σ_pooled (6)

The primary assumptions of a linear mixed effect model are: (1) the response variable is linearly related to the predictor variable(s), (2) the model errors are independent, (3) the error variances are homogeneous, and (4) the errors are normally distributed. Since the independent variables are categorical (i.e., experimental condition), the model is calculating group means and not fitting a line, so assumption 1 is automatically satisfied. Non-violation of assumption 2 was tested by plotting the autocorrelation of the model residuals and identifying no significant systematic patterns. Assumptions 3 and 4 were assessed with Levene and Shapiro-Wilks tests, respectively. To meet the requirements of assumptions 3 and 4, ROI activity (strictly positive) was natural log transformed, and classification metric data (precision, recall, and F1; inclusively bound between 0 and 1) were square-root transformed.

Ethics
27 healthy human subjects volunteered to participate in the study. Subjects took part in a pre-experiment session where they were safety-screened for magnetic resonance imaging (MRI) and tFUS eligibility. Each participant gave informed written consent. Every experiment involving human participants has been carried out following a protocol approved by an ethical commission (Advarra Institutional Review Board). Subject recruitment and experiments were conducted in cohorts of 22 (subjects 101–122; 14F/8M; mean ± std age: 24.5 ± 4.0 years old), 11 (subjects 201-211; 7F/4M; mean ages = 23.22 ± 1.10 years old), 11 (subjects 301-311; 5F/6M; mean age = 24.2 ± 2.3 years old), 6 (subjects 401–406; 4F/2M; mean ages = 23.6 ± 2.6 years old), and 8 (subjects 501–508; 5F/3M; mean ages = 23.6 ± 2.3 years old) participants. Some subjects participated in multiple cohorts of experimentation. Subjects were compensated at a rate of $20 per hour. We did not obtain consent to publish subject-identifying information.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors combine transcranial focused ultrasound with whole brain electroencephalography in resting state humans. The study demonstrates that tFUS or tDCS do not induce source localizable cortical activity, while tEAS (i.e., combined tFUS and tDCS) does result in activity in the targeted cortical region of interest. The paper is interesting, of high quality, and well written, and gives an answer to the important question whether tFUS results in sub- or suprathreshold neuromodulation in the human cortex. I have some comments and suggestions that I believe could further improve the impact of the manuscript.\nIn the Discussion section, the authors state that the data provides evidence that tFUS induces a location-specific subthreshold effect. However, the authors also demonstrate that (e/i)tFUS waveforms result in significant auditory activation (e.g., Granger causality and dSPM ESI analysis). tEAS also does not induce significant M1 to deep brain Granger causality. This raises the question if the observed source localizable response in tEAS stimulation could in part be explained by non-linear summation of tFUS-induced indirect subthreshold activation of M1 through an auditory pathway (non localizable) and tDCS subthreshold depolarization (localizable). In theory, only one of the stimulation modalities would need to be focal, for tEAS to result in a suprathreshold localizable response. For example, also considering that the tEAS localization scores (e.g., averaged subject precision of reconstructed sources) are relatively low. It would be interesting if the authors could discuss these points in the Discussion section.\nIn the Discussion section, it is mentioned that the auditory evoked response might mask a subthreshold localizable tFUS response. In my opinion, it would be valuable to include a small study with participants that are insonicated with ramped ultrasonic waveforms, designed to minimize auditory confounding effects. This could potentially allow the authors to evaluate the small subthreshold effects of tFUS with reduced auditory indirect effects.\nI would suggest to omit the computational section on the minimally modified Hodgkin-Huxley model or to move this section to the supplementary information. As mentioned by the authors, the modelling results are conceptual, because of the approximations made in the computational model. E.g., a membrane model of the squid giant axon is used and ultrasound is included by a single mechanosensitive Piezo1 channel. I believe there are also some limitations in the modelling of Piezo1, that could be included in the Discussion. In particular, the authors model the open probability of Piezo1 as proportional to the instantaneous pressure (eq. 7), based on reference [38] (Methods section “Hodgkin-Huxley modeling”). However, it is good to note that [38] does not use ultrasound to activate Piezo1 channels. In my opinion, it is not expected that the Piezo1 open state will follow the instantaneous acoustic pressure as in Fig. 6(e), because the opening time constant of Piezo1 will be higher than the ultrasonic period. Another consideration is that Piezo1 channels are activated by membrane tension instead of by the harmonic pressure: the relationship between membrane tension and pressure is nonlinear and not instantaneous. For example, increase in membrane tension is likely caused by radiation force induced tissue displacement, instead of directly by the harmonic pressure. This also would mean that the Piezo1 open probability would be non-oscillating, because of the steady (non-oscillating) driving radiation force. For these reasons, I would recommend to move this section to the supplementary information, because of these limitations and because the conceptual results in this section have been shown before in the computational literature (i.e., that multiple input stimuli will summate non-linearly in neuron models).\nMinor comments:\n- P. 4, “[...] and some studies go so far as to propose 100% of tFUS modulation is due to auditory activation [21,22]”. Perhaps this sentence can be nuanced, because [21,22] mention in their Discussion sections, that their results do not imply that tFUS has no direct neuromodulatory effects. Although these studies indeed demonstrate the importance of controlling for auditory confounds in tFUS studies.\n- Fig. 1(c): I would recommend to zoom out on the right subplot, to visualize also the FWHM in the axial direction. Fig. 1(d): I recommend to adjust the tDCS stimulation plot, because this now seems to imply that alternating tDCS currents similar to ultrasound are applied.\n- Results, p. 14, “[...] with both 30 and 3,000 Hz PRFs” -> with both 40 and 3,000 Hz PRFs.\n- Methods, hydrophone measurements and transducer: please include details on the hydrophone measurements (Fig. 1(c)) and on the transducer (aperture, radius of curvature).\n- Methods, In silico transcranial focused ultrasound simulations: it would be good to also include details on the pressure field simulation (e.g., tissue parameters, mapping from Hounsfield units (pCT) to acoustic parameters, used solver (discretisation step, Courant number, etc.)).\nIn this section, the notation could be confusing. E.g., the notation Isppa.3 is normally used to denote the derated SPPA intensity with homogeneous attenuation of 0.3 dB/(cm-MHz). However, in this section more realistic heterogeneous attenuation is used by Sim4life simulations of the skull and brain tissue. For this reason, the simulated attenuation is likely not equal to 0.3 dB/(cm-MHz), as indicated by the subscript.\n- Methods, p. 28: “Excitatory tDCS consisted of center-cathode current, while center-anode current was defined as inhibitory tDCS current”. I am expecting this is the other way around.\n- Minor textual comments: (abstract) affect -> effect. (Fig. 2(b), caption): [...] right subplot [...] left subplot -> other way around. (Fig. S1, caption) Ostu -> Otsu. (Fig. 4(b.ii), caption): PRF -> PFC.\n- Figures: in some figures (e.g., Fig 6d) the readability could be improved by increasing the font size of the labels and ticks. Fig. 1(c): please include a colorbar for this subplot.", "reviewer_2": "A main methodological issue is the dose titration of tDCS. The dosage was set at 0.1 mA below skin perception threshold, up to a maximum of 2 mA (0.7-2 mA). This is an absolutely unusual, currents finally entering the brain vary almost 3 fold. They should of course correlate with intensity but the individual skin perception with individual current flow not. Current flow calculations e.g. with SIMNibs will be appreciated.\nFor each condition the inter-trial interval was 2 seconds with up to a 20% (0.4 s) random jitter, so each condition lasting 6.6-8min. A five-minute break was taken between each condition to allow for the subjects to stretch and let any cumulative effects of neuromodulation dissipate. This is a rather short interval neglecting results in the literature suggesting e.g that 3 min or 20 min breaks boost plasticity and influence hereby succeeding stimulation results. The authors are of course welcome to come up with references that this is not of relevance here.\n3 kHz PRF tFUS under the right prefrontal cortex share a striking resemblance to tFUS applied under left motor cortex area (Left M1) with 10 – 63, 20 – 40, and 80 – 100 ms responses. Both cause a hemisphere-symmetric response, despite tFUS only being applied to one side. This needs explanation since seemingly this does not support evidence of a target-location specific effect. Also, Granger causality analysis of both itFUS and etFUS conditions shows significant increases in information flow from primary auditory cortex to the deep brain, and no changes in net information flow from the targeted ROI to the deep brain.\nEven increased tFUS parameter (including duty cycle; higher pressure; sonication duration) continued to yield no changes in net information flow from the targeted ROI to the deep brain. Transcranial electro-acoustic stimulation（tEAS）under the left M1 produces a hemisphere specific exogenous ERPs（10-63ms），which is claimed to be driven by tDCS polarity, not the excitatory or inhibitory parameters of tFUS (but see tDCS comments above).\nAnodal tDCS combined with 30Hz PRF tFUS (inhibitory) and 3,000 Hz (excitatory) PRF tFUS both led stronger ipsilateral than contralateral responses. But again, here tEAS conditions were also found to induce significant increases in auditory to deep brain connectivity without an effect on the net flow of information from the targeted ROI to deep brain.\nWhy does tDCS applied under the left M1 synchronize the tFUS under the right frontal without causing local changes; instead, it still results in a hemisphere-symmetric response.\nIs the claim that tEAS is more than a linear sum of its parts really supported by the summation of the source-space tFUS and tDCS conditions which in turn did not result in increased concordance with the ROI, nor to significant increases in ipsilateral under contralateral ROI source power.\nSo, in summary two main concerns regarding the tDCS intensity and intervals between different types of stimulation are worrying.\nMinor:\n„subthreshold (meaning it will not induce neural firing on its own)“ better „modulating sponteanous firing without stimulation time locked additional spikes“\n„but one prevalent theory is the ultrasound pressure waves open mechanosensitive ion channels“\nInstead „but one prevalent theory is that the ultrasound pressure waves open mechanosensitive ion channels“\n„Electroencephalogram (EEG) based recordings, which directly measure electrical activity without the need for magnetic fields, may offer more insight into the true effects of tFUS as a sole stimulus.„ unclear needs to be broken down to tangential MEG sensitivity.\n„In follow-up studies, we broaden our tFUS parameter scope. We record and analyze simultaneous whole-scalp EEG recordings and image their corresponding cortical sources to characterize targeted-location specific, or lack-thereof, responses.“ Here an immediate switch from stimulation to recording occurs with a logical brake“\nSentences like „While the effect of tFUS is commonly considered to be based on the targeted brain region, there is a lack of agreement for whether tFUS is modulating neural excitability or stimulating the neurons on its own.“ are not particularly logic. The first half refers to topography the second to neurophysiology. So not mutually exclusive. This occurs throughout the manuscript, see e.g. „Our data provide strong evidence that, while there are prominent auditory effects, the applied tFUS has location-specific subthreshold modulatory effects on the human cortex.“ Sentences like „While the clusters from permutation cluster tests are not guaranteed to be significant for their entire extent, we did consider the overlapping windows (10 – 63 ms) for significant itEAS and etEAS clusters to be interesting enough due to their shared presence to warrant use in our further analysis.“ ambiguous.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We express our sincere gratitude to the editors and reviewers for their constructive comments. In response, we have undertaken substantial efforts, including additional human experiments, computer simulations, and data analyses to address the reviewers’ comments. Specifically, we conducted two more human experiments–one with a seven-day wash-out period between sessions and another with a ramping ultrasound waveform–and compared the resultant sources to those of our main study. We also investigated the effects of varied tDCS dose, both at the scalp level and cortical level using simulations. We believe these efforts have significantly enhanced and improved the work.\nBelow, you will find our detailed responses to each reviewer comment in BLUE, with the original reviewer comments in BLACK. Figures in the main manuscript are denoted as Fig. x, while figures in the supplemental information are denoted as Fig. Sx. Revised or added text in the manuscript is highlighted in RED.\nIn addition to the responses to reviewers below, there are a couple of small errors we noticed in the original manuscript that we have corrected in the latest version. They are listed below.\n1. There was a bug in our pairwise ESI metric comparisons in which two subjects were incorrectly being excluded. This has led to slight numerical differences in the reported mean and standard deviations, as well as associated p-values and Cohen’s d values in Supplemental Figure 1C. No comparisons changed from significant to not significant; in some cases, results became more significant (the tEAS IL and CL comparison changed from p < 0.01 to p < 0.001).\n2. There was a different bug that was causing multiple comparison p-value correction not to apply to the etFUS imaginary coherence computations (Supplemental Figure S5). After properly implementing FDR correction, etFUS ImCoh is no longer statistically significant at any frequency band. However, itFUS continues to have significant differences. We have updated all the relevant results and discussion points in the manuscript to reflect this.\nReviewer #1 (Remarks to the Author):\nIn this study, the authors combine transcranial focused ultrasound with whole brain electroencephalography in resting state humans. The study demonstrates that tFUS or tDCS do not induce source localizable cortical activity, while tEAS (i.e., combined tFUS and tDCS) does result in activity in the targeted cortical region of interest. The paper is interesting, of high quality, and well written, and gives an answer to the important question whether tFUS results in sub- or suprathreshold neuromodulation in the human cortex. I have some comments and suggestions that I believe could further improve the impact of the manuscript.\nWe thank the reviewer for their overall favorable assessment of the work. We also thank the reviewer for their constructive comments and suggestions for further improvement, which we have addressed below.\nIn the Discussion section, the authors state that the data provides evidence that tFUS induces a location-specific subthreshold effect. However, the authors also demonstrate that (e/i)tFUS waveforms result in significant auditory activation (e.g., Granger causality and dSPM ESI analysis). tEAS also does not induce significant M1 to deep brain Granger causality. This raises the question if the observed source localizable response in tEAS stimulation could in part be explained by non-linear summation of tFUS-induced indirect subthreshold activation of M1 through an auditory pathway (non localizable) and tDCS subthreshold depolarization (localizable). In theory, only one of the stimulation modalities would need to be focal, for tEAS to result in a suprathreshold localizable response. For example, also considering that the tEAS localization scores (e.g., averaged subject precision of reconstructed sources) are relatively low. It would be interesting if the authors could discuss these points in the Discussion section.\nWe thank the reviewer for raising this excellent point. Our study design actually takes this into account – for the tDCS conditions, a spatial sham ultrasound (ultrasound to right prefrontal cortex) is also applied. If the localizable tDCS with a non-localizable tFUS summation is what caused the focal response, then we would also have seen the focal response on tDCS conditions.\nWe have added the following statement to our Discussion to clarify this point to future readers (page 15):\n“tDCS driving tEAS polarity also raises the question on whether the focality of tEAS’ response could be due to tDCS locally subthreshold modulating whole brain induced tFUS auditory response. However, this is already accounted for in our experimental set up. For our tDCS conditions, we implemented a spatial tFUS control (the set up is shown in Figure 1B). If the source response of tEAS was the result of tDCS interacting with the whole brain auditory response, the tDCS conditions would have resulted in focal activations as well.”\nIn the Discussion section, it is mentioned that the auditory evoked response might mask a subthreshold localizable tFUS response. In my opinion, it would be valuable to include a small study with participants that are insonicated with ramped ultrasonic waveforms, designed to minimize auditory confounding effects. This could potentially allow the authors to evaluate the small subthreshold effects of tFUS with reduced auditory indirect effects.\nWe thank the reviewer for this suggestion. We conducted a follow up study with 6 human subjects in which we tested a ramped waveform. The results continued to show a lack of ROI response from tFUS-only, and a significantly increased ROI response from the smoothed tEAS condition.\nWe have added the following analysis / discussion to our manuscript (page 17):\n“We further investigated whether a smoothed ultrasound waveform could potentially ‘unmask’ an ROI response. We tested a smoothed waveform (pulse duration: 3.25 ms, 1 ms tukey ramping, 250 Hz pulse-repetition frequency)1 in six human subjects (Supplemental Figure S10). All six subjects reported that they could not hear the ultrasound. However, the smoothed tFUS by itself continued to lack increased ipsilateral ROI source power, whereas the smoothed tEAS waveform did. We also note that the smoothed tEAS waveform does appear overall less focal than the one that resulted from our rectangular pulses, with additional strong activations in the ipsilateral frontal/premotor regions. While the results of this study indicate that ultrasound PRF may not play a substantial role in the outcome of tEAS (Supplemental Figure S8), it may be that the ultrasound pulse shape does. Future tEAS parameter optimization studies are warranted.”\nSupplemental Figure S10: A follow up study was conducted with 6 subjects to investigate whether a smoothed ultrasound waveform (pulse duration: 3.25 ms, 1 ms tukey ramping, 250 Hz pulse-repetition frequency) could reduce auditory activations and potentially ‘unmask’ target region specific source activity.\na) The subject average source maps are presented for the smoothed tFUS and tEAS conditions. b) Smoothed ultrasound continued to result in no increased ipsilateral source power, while smoothed tEAS did. ns p > 0.05; * p < 0.05\nI would suggest to omit the computational section on the minimally modified Hodgkin-Huxley model or to move this section to the supplementary information. As mentioned by the authors, the modelling results are conceptual, because of the approximations made in the computational model. E.g., a membrane model of the squid giant axon is used and ultrasound is included by a single mechanosensitive Piezo1 channel. I believe there are also some limitations in the modelling of Piezo1, that could be included in the Discussion. In particular, the authors model the open probability of Piezo1 as proportional to the instantaneous pressure (eq. 7), based on reference [38] (Methods section “Hodgkin-Huxley modeling”). However, it is good to note that [38] does not use ultrasound to activate Piezo1 channels. In my opinion, it is not expected that the Piezo1 open state will follow the instantaneous acoustic pressure as in Fig. 6(e), because the opening time constant of Piezo1 will be higher than the ultrasonic period. Another consideration is that Piezo1 channels are activated by membrane tension instead of by the harmonic pressure: the relationship between membrane tension and pressure is nonlinear and not instantaneous. For example, increase in membrane tension is likely caused by radiation force induced tissue displacement, instead of directly by the harmonic pressure. This also would mean that the Piezo1 open probability would be non-oscillating, because of the steady (non-oscillating) driving radiation force. For these reasons, I would recommend to move this section to the supplementary information, because of these limitations and because the conceptual results in this section have been shown before in the computational literature (i.e., that multiple input stimuli will summate non-linearly in neuron models).\nWe thank the reviewer for their input and have moved the HH modeling to the supplementary information.\nMinor comments:\n- P. 4, “[...] and some studies go so far as to propose 100% of tFUS modulation is due to auditory activation [21,22]”. Perhaps this sentence can be nuanced, because [21,22] mention in their Discussion sections, that their results do not imply that tFUS has no direct neuromodulatory effects. Although these studies indeed demonstrate the importance of controlling for auditory confounds in tFUS studies.\nWe thank the reviewer for their suggestion. We have reworded the sentence:\n“[…] and some studies could not detect an effect beyond auditory activation.”\n- Fig. 1(c): I would recommend to zoom out on the right subplot, to visualize also the FWHM in the axial direction. Fig. 1(d): I recommend to adjust the tDCS stimulation plot, because this now seems to imply that alternating tDCS currents similar to ultrasound are applied.\nThank you for your suggestions. Please see our updated Figure 1 with a new tDCS representation and new tFUS scan showing the full FWHM below.\nFigure 1\n- Results, p. 14, “[...] with both 30 and 3,000 Hz PRFs” -> with both 40 and 3,000 Hz PRFs.\nThank you for your attention to detail. 30 Hz PRF is actually correct for 10% and 60% DC conditions, but we had a typo earlier in the sentence where 30 should have been 40. The corrected statement is below:\n“These parameters included 1,500 and 40 Hz PRFs with 30% duty cycle (DC), and, initially, 10% and 60% DC with both 30 and 3,000 Hz PRFs.”\n- Methods, hydrophone measurements and transducer: please include details on the hydrophone measurements (Fig. 1(c)) and on the transducer (aperture, radius of curvature).\nWe’ve added in details for the hydrophone and transducer to the methods section:\n“Free water ultrasound calibration\nThis experiment used a 500 kHz f (fundamental frequency) single-element\n0\nultrasound transducer (1.5 inch focal length, 1 inch acoustic aperture diameter, 0.75 inch radius of curvature; Blatek Industries Inc., Boalsburg, PA, USA). To characterize the beam shape and determine needed input voltage for desired free water pressure, the transducer was calibrated in a water tank. The water tank was filled with deionized water. Pressure measurements were recorded with an HNR-0500 hydrophone (Onda Corp., Sunnyvale, CA, USA).”\n- Methods, In silico transcranial focused ultrasound simulations: it would be good to also include details on the pressure field simulation (e.g., tissue parameters, mapping from Hounsfield units (pCT) to acoustic parameters, used solver (discretisation step, Courant number, etc.)).\nWe thank the reviewer for their suggestion. We have added the following information to our methods:\n“The pCTs were manually segmented using iSeg (v3.10) into air (1.16 kg/m3 density, 343 m/s speed of sound, 0.000399 MRayl acoustic impedance), bone (1910 kg/m3 density, 3510 m/w speed of sound, 6.71 MRayl acoustic impedance), and soft tissue (1050 kg/m3 density, 1550 m/s speed of sound, 1.61 MRayl acoustic impedance). Sim4Life solved the acoustic simulations with the Linear Acoustic Pressure Wave Equation and the ‘automatic’ discretization settings (0.45 mm resolution, 0.3 mm maximum step, and a time step of 1.0 / (speed of sound * !1/dx² + 1/dy² + 1/dz² ).”\nmax\nIn this section, the notation could be confusing. E.g., the notation Isppa.3 is normally used to denote the derated SPPA intensity with homogeneous attenuation of 0.3 dB/(cm-MHz). However, in this section more realistic heterogeneous attenuation is used by Sim4life simulations of the skull and brain tissue. For this reason, the simulated attenuation is likely not equal to 0.3 dB/(cm-MHz), as indicated by the subscript.\nThank you for raising this possible source of confusion. We have replaced “the .3” notation with “.T” for “target” throughout the manuscript. For example, Isppa.3 is now Isppa.T.\n- Methods, p. 28: “Excitatory tDCS consisted of center-cathode current, while center-anode current was defined as inhibitory tDCS current”. I am expecting this is the other way around.\nThank you for your comment. We have fixed this typo.\n- Minor textual comments: (abstract) affect -> effect. (Fig. 2(b), caption): [...] right subplot [...] left subplot -> other way around. (Fig. S1, caption) Ostu -> Otsu. (Fig. 4(b.ii), caption): PRF -> PFC.\nThank you for your comments. We have fixed these typos.\n- Figures: in some figures (e.g., Fig 6d) the readability could be improved by increasing the font size of the labels and ticks. Fig. 1(c): please include a colorbar for this subplot.\nThank you for your suggestions. We have remade our figures for increased readability. Please find the updated versions in the manuscript and supplemental files. For Figure 1C, a color bar is included to the right.\nReviewer #2 (Remarks to the Author):\nA main methodological issue is the dose titration of tDCS. The dosage was set at 0.1 mA below skin perception threshold, up to a maximum of 2 mA (0.7-2 mA). This is an absolutely unusual, currents finally entering the brain vary almost 3 fold. They should of course correlate with intensity but the individual skin perception with individual current flow not. Current flow calculations e.g. with SIMNibs will be appreciated.\nWe thank the reviewer for this comment. We respectfully point towards other examples of highly varied transcranial electric stimulation titration. Pilloni et al used between 1.5 or 2.5 mA based on subject tolerability2. Simpson et al delivered between 1.0 – 1.5 mA based on subject tolerability3. Lafon et al delivered currents varying between 0.3 and 2 mA4. We also note there have also been numerous reports of no meaningful differences in outcomes from using different tDCS amperages5–7.\nPerhaps more important, our study design is a cross-over experimental design, which looks at changes in the brain response within subjects. For each subject, the tDCS dose was held constant. Our results show, despite varying levels of initial scalp current, there is a significant increase in brain response for the tEAS conditions compared to others.\nNevertheless, we have run the requested simulations to estimate cortical current density. They are provided in Supplemental Table T1. Analysis of both scalp-level current and cortical current density do not show current intensity to be a meaningful (linear) predictor of the subjects’ tEAS response levels (all R2 < 0.05, all p > 0.05), which is consistent with the past studies reporting little-to-no differences from currents in our range5–7.\nSupplemental Figure S7d and S7e: Mean tEAS-induced ROI activity is not well predicted by tDCS intensity. The tEAS cortical responses were fit with an affine function of tDCS scalp current and the cortical current density (estimated with SimNIBS). Neither of these hold meaningful predictive power for the tEAS responses (both p > 0.05, both R2 < 0.05).\nWe also performed a follow-up experiment with a 7-day washout out between etFUS and etEAS conditions. As part of this study, we used a consistent 0.7 mA of tDCS in the tEAS conditions for all subjects. The results were consistent with the main study, in which tEAS led to strong ROI activity while tFUS alone did not (Supplemental Figure S12).\nWe have added discussion the following manuscript (page 20):\n“We note the varied level of scalp current applied to subjects during our experiments. There are, broadly, two approaches to transcranial electric stimulation dose determination for human experiments. One is to normalize the dose for all subjects (either at the scalp- or the cortical-level using simulations). The other is to use a varied dose across subjects2–4. We opted for the latter approach in our study for two reasons. The first reason is that we wanted to prioritize subject comfort and safety. tDCS is generally considered safe, and the scalp sensations of tingling, itching, and/or burning are often reported as transient8. However, it is possible for tDCS to induce skin lesions after only a single session9. Since our participants were healthy human subjects, we wanted to avoid this possibility. The second reason is that skin perceptions are the result of activated sensory neurons10, which can contaminate the resting-state EEG recordings, both from the firing of the scalp (i.e., non-cortical) neurons, but also from the activated pain circuits within the brain itself. We wished to avoid such possible contamination.\nSince we used the same current density for each condition within a subject, the varied current density across subjects is not a major concern. This is because our cross-over study design looks at the changes in brain response across conditions for subjects.\nOur results show a significant increase in ROI activity for tEAS conditions, regardless of initial tDCS dosage. We did conduct further analysis to determine if scalp or cortical (estimated with SIMNibs simulations11) tDCS dose (Supplemental Table T1) was a meaningful predictor of ROI response. Our results indicate tDCS dose, at the between-subjects level, is not a meaningful predictor of tEAS activation (Supplemental Figure S7; R2 < 0.05, p < 0.05). This is consistent with other studies that have reported little-to-no differences in the outcomes of different tDCS doses5–7, and further supports that our subject-specific dosage should not be viewed as a study limitation. In a follow-up study, we used a constant 0.7 mA tDCS for all subjects and found a consistent activation pattern across subjects for the tEAS condition (Supplemental Figure S12).”\nSupplemental Figure S12: Results of a 7-day washout experiment and constant 0.7 mA scalp current are consistent with the main study. We repeated our etEAS and etFUS resting state conditions in 8 subjects, this time with a 7-day washout period between the two conditions. For etEAS, we used a consistent 0.7 mA of current across all subjects. We also tested each condition twice; once before and once after a 5 minute break. a) The ROI activity for etFUS was not significantly altered pre/post break (two-tailed permutation t-test; p > 0.05), and Bayes Factor analysis reveals the activities to be robustly statistically similar for a range of scaling factors. b) The ROI activity for the etEAS condition was also statistically similar before and after the 5 minute break period. c) The induced sources are consistent with those from the main study.\nFor each condition the inter-trial interval was 2 seconds with up to a 20% (0.4 s) random jitter, so each condition lasting 6.6-8min. A five-minute break was taken between each condition to allow for the subjects to stretch and let any cumulative effects of neuromodulation dissipate. This is a rather short interval neglecting results in the literature suggesting e.g that 3 min or 20 min breaks boost plasticity and influence hereby succeeding stimulation results. The authors are of course welcome to come up with references that this is not of relevance here.\nWe thank the reviewer for the comment. We removed the comment about letting neuromodulation effects dissipate from our methods:\n“For each condition the inter-trial interval was 2 seconds with up to a 20% (0.4 s) random jitter, so each condition lasting 6.6-8min. A five-minute break was taken between each condition to allow for the subjects to stretch.”\nWe have added the following discussion to our paper to indicate why our experimental design helps to mitigate confounding sequential effects. In our new 7-day washout follow up study, we also tested each condition twice before/after a 5 minute break. Our results found ROI activity pre and post 5-minute break to be statistically similar from each other, further supporting that our results were not confounded from these breaks (page 21).\nWe randomized the order of our experiments across subjects, which is also known as counterbalancing. While it is possible that conditions tested within a single session may experience a carry-over or sequence-based effect, counterbalancing, at the group-level, helps ensure that these effects were randomly distributed across conditions and should not confound the main results12. To further validate our main findings, we conducted an additional follow-up study, in which we repeated our etEAS and etFUS experiments with a 7-day wash out period (8 participants; split 50/50 for which condition was first). On the day the condition was tested, we tested it twice: once ‘pre’ 5-minute break and once ‘post’ break. The ROI activity for each condition was statistically similar before and after the break (Supplemental Figure S12a-b), suggesting that, even without the counterbalancing of the main study, the results may not have suffered from any sequential-based confounds. The source localizability of the experimental conditions were also consistent with the main study, where etEAS yielded strong targeting-location specific activity, and etFUS did not (Supplemental Figure S12c).\nSupplemental Figure S12: Results of a 7-day washout experiment and constant 0.7 mA scalp current are consistent with the main study. We repeated our etEAS and etFUS resting state conditions in 8 subjects, this time with a 7-day washout period between the two conditions. For etEAS, we used a consistent 0.7 mA of current across all subjects. We also tested each condition twice; once before and once after a 5 minute break. a) The ROI activity for etFUS was not significantly altered pre/post break (two-tailed permutation t-test; p > 0.05), and Bayes Factor analysis reveals the activities to be robustly statistically similar for a range of scaling factors. b) The ROI activity for the etEAS condition was also statistically similar before and after the 5 minute break period. c) The induced sources are consistent with those from the main study.\n3 kHz PRF tFUS under the right prefrontal cortex share a striking resemblance to tFUS applied under left motor cortex area (Left M1) with 10 – 63, 20 – 40, and 80 – 100 ms responses. Both cause a hemisphere-symmetric response, despite tFUS only being applied to one side. This needs explanation since seemingly this does not support evidence of a target-location specific effect. Also, Granger causality analysis of both itFUS and etFUS conditions shows significant increases in information flow from primary auditory cortex to the deep brain, and no changes in net information flow from the targeted ROI to the deep brain.\nThank you for your comment. We apologize that we were not clearer in our explanation of this phenomenon; this is critical evidence of tFUS’ subthreshold location-specific effect. In other words, when tFUS is applied by itself, we only see the whole brain auditory-based response, regardless of where tFUS is targeted. We have reworded our discussion to make this more clear.\nPlease see our revised paragraph below (bold added for emphasis) (page 14):\n“Excitatory (3 kHz PRF) tFUS targeted to the left motor region, as part of etFUS, etEAS, and etDCS (which included a tFUS spatial control), induced distinct ERPs at approximately 30, 90, and 170 ms post stimulation onset (Figure 2a, Figure 4a). This is consistent with a previous study that identified V1-targeted tFUS induced EEG peaks, though their reported peak times were labeled as 55 ms, 100 ms, and 150 ms post sonication. However, when we compared these ERPs to those induced by applying tFUS to an entirely different brain region (right hemisphere prefrontal cortex), the resultant waveforms did not just lack statistical differences from each other—they were found to be statistically similar through Bayes Factor analysis (Figure 4b). These results provide critical evidence that location-specific effects of tFUS are subthreshold; by itself, tFUS produces the same responses regardless of where it is targeted.\nOf course, these shared responses are time-locked event related potentials.\nTo put it another way, tFUS does induce a suprathreshold response. However, since the topology of these ERPs do not differ across targeting locations, they can be attributed to a whole brain auditory response. Indeed, the 3 kHz PRF EEG topographic spatial distributions of the 90 and 170 ms ERPs do share visual resemblance to the previously reported auditory induced N1 and P2 ERPs. Granger causality investigations, which revealed significantly increased A1 to deep brain information flow for both 30 Hz and 3 kHz tFUS conditions (Figure 4c), provide additional support that the ERPs are auditory related. Nevertheless, the present results that combining tFUS with tDCS led to spatially specific responses, when neither could do so individually (Figure 3), as well as itFUS dissociating the spectral connectivity between the ROI and the deep brain (Supplemental Figure S5), indicate that tFUS does have targeted-location specific effects.”\nEven increased tFUS parameter (including duty cycle; higher pressure; sonication duration) continued to yield no changes in net information flow from the targeted ROI to the deep brain. Transcranial electro-acoustic stimulation（tEAS）under the left M1 produces a hemisphere specific exogenous ERPs（10-63ms，which is claimed to be driven by tDCS polarity, not the excitatory or inhibitory parameters of tFUS (but see tDCS comments above).\nThank you for your comments. The continued lack of differences between the tFUS-only conditions are all in support of a subthreshold modulatory effect. It is only when tFUS is combined with tDCS that we start to see location-specific information. We have added the following clarification to our discussion section (page 12):\n“Analysis of the other parameters continued to show a lack of sonication-location specific suprathreshold responses, providing further support that tFUS’ location-specific effect is subthreshold (Figure 5).”\nWe have also added to our discussion on tDCS based on the above tDCS comments / analysis (bold added for emphasis) (page 12):\n“Not only did itEAS and etEAS induce significantly higher source localization metrics and source power than the other tested conditions, but they also led to a significant change in ROI polarity (Figure 2d). While this polarity change is evidence of differing electrophysiological effects, a follow-up study indicates that this change in response is driven from tDCS polarity and not tFUS PRF (Supplemental Figure S7). However, further analysis shows no linear relationship between the applied tDCS dose and tEAS response magnitude (Supplemental Figure S7d-e), nor between tFUS intensity and tEAS response (Supplemental Figure S7c). This points to a non-linear response from a combined transcranial electro-acoustic effect.”\nAnodal tDCS combined with 30Hz PRF tFUS (inhibitory) and 3,000 Hz (excitatory) PRF tFUS both led stronger ipsilateral than contralateral responses. But again, here tEAS conditions were also found to induce significant increases in auditory to deep brain connectivity without an effect on the net flow of information from the targeted ROI to deep brain.\nThank you for your comment. We have added the following statement to our discussion regarding tEAS and Granger causality (page 14-15):\n“We note that these auditory effects persist within the tEAS conditions (Supplemental Figure S4). This is unsurprising, as we would have no reason to think the electro-acoustic effect at M1 would cancel out auditory pathways. However, what is surprising is the continued lack of ROI to deep brain change in net flow of information. M1 is connected to deep brain structures13, so on one hand it would be expected that increased M1 activity would lead to increased deep brain activity, and this would be reflected as an increased net flow of information. However, this was not our finding. One possible explanation for this phenomena is that M1 neuron connections with the deep brain are dependent on cell type13. Prior work has demonstrated tFUS’ parameter choices can alter cell-type selectivity14. Our tFUS parameters may not have been optimal for eliciting M1 to deep brain connections. Future research on tEAS connectivity changes (or lack thereof) are warranted.”\nWhy does tDCS applied under the left M1 synchronize the tFUS under the right frontal without causing local changes; instead, it still results in a hemisphere-symmetric response.\nOur tDCS conditions are combined with a spatial-sham tFUS (Figure 1b). These hemisphere-symmetric responses are the continued results of auditory based effects from tFUS. We have clarified this point in the discussion (bold added for emphasis):\n“Excitatory (3 kHz PRF) tFUS targeted to the left motor region, as part of etFUS, etEAS, and etDCS (which included a tFUS spatial control), induced distinct ERPs at approximately 30, 90, and 170 ms post stimulation onset (Figure 2a, Figure 4a).”\nIs the claim that tEAS is more than a linear sum of its parts really supported by the summation of the source-space tFUS and tDCS conditions which in turn did not result in increased concordance with the ROI, nor to significant increases in ipsilateral under contralateral ROI source power.\nThank you for your comments. We have included linear modeling of tEAS source-space response as a function of tDCS / tFUS intensities (Supplemental Figure S7), which provide additional evidence of a non-linear response. We added the following line to the results section (page 10):\n“We also modeled the tEAS ROI response as function of tDCS scalp current, cortical current, and tFUS intensity and found none of these component to be meaningfully predictive (Supplemental Figure S7c-e).”\nSupplemental Figure S7: The transcranial electro-acoustic effect is more than just the summation of independent electric and acoustic modulation. a) Source-imaging data for itFUS and itDCS, as well as etFUS and tDCS, were added together for each subject, and then averaged across subjects. Visualizations continue to show strong off-targeted sources. b) The average ipsilateral ROI activity of the added sources continues to be not significantly greater than the contralateral ROI for both the summed excitatory and summed inhibitory conditions (p > 0.05). c – e) Mean tEAS-induced ROI activity is not well predicted by tFUS or tDCS intensity. The tEAS cortical responses were fit with an affine function of c) cortical tFUS intensity, d) current, and e) the cortical current density (estimated with SimNIBS). None of the individual components hold meaningful predictive power for the tEAS responses (all p > 0.05).\nSo, in summary two main concerns regarding the tDCS intensity and intervals between different types of stimulation are worrying.\nThank you for your feedback. We have addressed these concerns with our updated analysis, discussion points, and follow-up study.\nMinor:\n„subthreshold (meaning it will not induce neural firing on its own)“ better „modulating sponteanous firing without stimulation time locked additional spikes“\nThank you for your suggestion. We have reworded the statement (bold added for emphasis):\n“A modern, philosophical descendant of these methods, transcranial direct current stimulation (tDCS), applies subthreshold (meaning it will modulate spontaneous firing, but will not induce stimulation time-locked spikes on its own) current across brain tissue using non-invasive scalp electrodes.”\n„but one prevalent theory is the ultrasound pressure waves open mechanosensitive ion channels“\nInstead „but one prevalent theory is that the ultrasound pressure waves open mechanosensitive ion channels“\nThank you for your suggestion. We have implemented this change.\n„Electroencephalogram (EEG) based recordings, which directly measure electrical activity without the need for magnetic fields, may offer more insight into the true effects of tFUS as a sole stimulus.„ unclear needs to be broken down to tangential MEG sensitivity.\nThank you for your comment. We have reworded this sentence to clarify we are mentioning the magnetic field as contrast to fMRI, not MEG (bold added for emphasis).\n“Electroencephalogram (EEG) based recordings, which directly measure electrical activity and do not require a magnetic resonance environment, may offer more insight into the true effects of tFUS as a sole stimulus.”\n„In follow-up studies, we broaden our tFUS parameter scope. We record and analyze simultaneous whole-scalp EEG recordings and image their corresponding cortical sources to characterize targeted-location specific, or lack-thereof, responses.“ Here an immediate switch from stimulation to recording occurs with a logical brake“\nThank you for your comment. We have added a clause to make the transition smoother (bold added for emphasis).\n“In follow-up studies, we broaden our tFUS parameter scope. For all tFUS experiments, we record and analyze simultaneous whole-scalp EEG recordings and image their corresponding cortical sources to characterize targeted-location specific, or lack-thereof, responses.”\nSentences like „While the effect of tFUS is commonly considered to be based on the targeted brain region, there is a lack of agreement for whether tFUS is modulating neural excitability or stimulating the neurons on its own.“ are not particularly logic. The first half refers to topography the second to neurophysiology. So not mutually exclusive. This occurs throughout the manuscript, see e.g. „Our data provide strong evidence that, while there are prominent auditory effects, the applied tFUS has location-specific subthreshold modulatory effects on the human cortex.“\nSentences like „While the clusters from permutation cluster tests are not guaranteed to be significant for their entire extent, we did consider the overlapping windows (10 – 63 ms) for significant itEAS and etEAS clusters to be interesting enough due to their shared presence to warrant use in our further analysis.“ ambiguous.\nThank you for your comments. We have revised the sentences in the following ways (bold added for emphasis):\n“While the effect of tFUS is commonly considered to be based on the targeted brain region, there is a lack of agreement on the magnitude of effect. Specifically, there are unanswered questions on whether tFUS is modulating neural excitability or stimulating the neurons on its own.”\n“Our data provide strong evidence that, while tFUS produces prominent whole brain auditory related activations, it also induces location-specific subthreshold modulatory effects.”\n“While the clusters from permutation cluster tests are not guaranteed to be significant for their entire extent, we did consider shared clusters from both itEAS and etEAS to warrant further analysis. Thus, the remainder of our work will focus on the shared window of 10 – 63 ms.”\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank reviewer #3 for their effort and time co-reviewing our manuscript.\nReference:\n1. Johnstone, A., Nandi, T., Martin, E., Bestmann, S., Stagg, C. & Treeby, B. A range of pulses commonly used for human transcranial ultrasound stimulation are clearly audible. Brain Stimul 14, 1353–1355 (2021).\n2. Pilloni, G., Vogel-Eyny, A., Lustberg, M., Best, P., Malik, M., Walton-Masters, L., George, A., Mirza, I., Zhovtis, L., Datta, A., Bikson, M., Krupp, L. & Charvet, L. Tolerability and feasibility of at-home remotely supervised transcranial direct current stimulation (RS-tDCS): Single-center evidence from 6,779 sessions. Brain Stimulation: Basic, Translational, and Clinical Research in Neuromodulation 15, 707–716 (2022).\n3. Simpson, E. A., Saiote, C., Sutter, E., Lench, D. H., Ikonomidou, C., Villegas, M. A. & Gillick, B. T. Remotely monitored transcranial direct current stimulation in pediatric cerebral palsy: open label trial protocol. BMC Pediatrics 22, 566 (2022).\n4. Lafon, B., Henin, S., Huang, Y., Friedman, D., Melloni, L., Thesen, T., Doyle, W., Buzsáki, G., Devinsky, O., Parra, L. C. & A. Liu, A. Low frequency transcranial electrical stimulation does not entrain sleep rhythms measured by human intracranial recordings. Nat Commun 8, 1199 (2017).\n5. Agboada, D., Mosayebi Samani, M., Jamil, A., Kuo, M.-F. & Nitsche, M. A. Expanding the parameter space of anodal transcranial direct current stimulation of the primary motor cortex. Sci Rep 9, 18185 (2019).\n6. Kidgell, D. J., Daly, R. M., Young, K., Lum, J., Tooley, G., Jaberzadeh, S., Zoghi, M. & Pearce, A. J. Different Current Intensities of Anodal Transcranial Direct Current Stimulation Do Not Differentially Modulate Motor Cortex Plasticity. Neural Plasticity 2013, 603502 (2013).\n7. Jamil, A., Batsikadze, G., Kuo, H.-I., Labruna, L., Hasan, A., Paulus, W. & Nitsche, M. A. Systematic evaluation of the impact of stimulation intensity on neuroplastic after-effects induced by transcranial direct current stimulation. The Journal of Physiology 595, 1273–1288 (2017).\n8. Poreisz, C., Boros, K., Antal, A. & Paulus, W. Safety aspects of transcranial direct current stimulation concerning healthy subjects and patients. Brain Research Bulletin 72, 208–214 (2007).\n9. Wang, J., Wei, Y., Wen, J. & Li, X. Skin Burn After Single Session of Transcranial Direct Current Stimulation (tDCS). Brain Stimulation 8, 165–166 (2015).\n10. van Boekholdt, L., Kerstens, S., Khatoun, A., Asamoah, B. & Mc Laughlin, M. tDCS peripheral nerve stimulation: a neglected mode of action? Mol Psychiatry 26, 456–461 (2021).\n11. Thielscher, A., Antunes, A. & Saturnino, G. B. Field modeling for transcranial magnetic stimulation: A useful tool to understand the physiological effects of TMS? in 2015 37th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) 222–225 (2015). doi:10.1109/EMBC.2015.7318340.\n12. Corriero, E. F. Counterbalancing. in The SAGE Encyclopedia of Communication Research Methods 278–281 (SAGE Publications, Inc, 2017). doi:10.4135/9781483381411.\n13. McColgan, P., Joubert, J., Tabrizi, S. J. & Rees, G. The human motor cortex microcircuit: insights for neurodegenerative disease. Nat Rev Neurosci 21, 401–415 (2020).\n14. Yu, K., Niu, X., Krook-Magnuson, E. & He, B. Intrinsic functional neuron-type selectivity of transcranial focused ultrasound neuromodulation. Nat Commun 12, 2519 (2021)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have significantly improved the manuscript. In particular, the revised manuscript now includes two additional human experiments, addressing possible carry-over effects (7 day wash-out experiment) and auditory confounds (ramped protocols). I would like to thank the authors for their work.\nI have one remaining minor comment. The authors have moved the Hodgkin-Huxley modelling results to the supplementary information. Here, it is mentioned: “Outside of pressure ranges where the channel has a 0 or 1 probability of being open, the open probability (Popen) scales approximately linearly with applied pressure [38]. As a result, Popen was modeled directly as the ultrasound waveform (clipped at 0 pressure to avoid negative probabilities), scaled to have some peak probability less than 1.”\nHowever, in my opinion, this cannot be concluded from [38], because in this publication ultrasound was not used. In particular, Piezo1 opening is mediated by membrane tension instead of directly by ultrasonic pressure. The relation between membrane tension and ultrasonic pressure is nonlinear and non-instantaneous, and is also determined by acoustic radiation force. For this reason, I would recommend to nuance or clarify this statement in the Supplementary Information. This is a minor comment, because the Hodgkin-Huxley modelling is intended by the authors to be conceptual.", "reviewer_2": "I have now reread the whole manuscript with all reviewers comments and feel increasingly uncomfortable. As I understand the main conclusion is based on the results of Fig. 2D representing a highly digested and compressed summary. The contact to original data is lost here.\nThe justification of the paper in the abstract reads as follows: “In none of the\nexogenous event related potentials did tFUS or tDCS result in location-specific activations“. This may indeed by true for special methodology used here, does however not apply for general literature.\nOthers issues now concern me. I did not find anything about earplugging or even double ear-plugging with noise cancellation in order to prevent any influence of acoustic evoked potentials by ultrasound. Did not understand what finally was done by Simbnibs? Taking bone densities into account?\nComments to some references used:\nThe Pilloni et al article summarized only the safety and tolerability of multiple applications of different intensities of tDCS at home. No cortical stimulation effects were included. A pure safety study. The Simpson et al study is no study but a study protocol for a future study. Thus, this sentence “Simpson et al delivered between 1.0 – 1.5 mA based on subject tolerability” is more than irritating in the present context for a study which have not been performed. The results in the Lafon study were essentially negative. Those being reported were local field potentials which can of course vary but cannot be cited as being supportive in the present context. Reference 5 is cherry picking. The literature is quite clear in arguing of intensity and duration sensitive effects, see e.g. doi: 10.1016/j.brs.2022.01.011. and doi: 10.1016/j.brs.2021.03.013. from the same group.\nReference 6 varied intensity in a much smaller range between 0.8, 1.0, and 1.2 mA\nReference 7 can indeed be quoted in the present context but only for anodal not for cathodal stimulation.\nSuppl fig 7 seems to be in line now with intensity dependent changes however does not reflect any reversal from cathodal inhibition to excitation when moving from 1 mA to 2 mA.\nIn this context the paragraph “we note the varied level…” can be omitted. As I said it is not justified to use skin perception as a reason at all to come up with different doses. The Simnibs approach is appreciated however I did not find any hints what was done finally here.\nI do not quite understand the meaning of this sentence: “Our results show, despite varying levels of initial scalp current, there is a significant increase in brain response for the tEAS conditions compared to others.”\nWhat is meant be “regardless of initial tDCS dosage.“ Do not understand the initial here.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "RESPONSE TO REVIEWER COMMENTS\nWe express our sincere gratitude to the editors and reviewers for their constructive comments. In\nresponse, we have undertaken substantial efforts, including additional human experiments,\ncomputer simulations, and data analyses to address the reviewers’ comments. Specifically, we\nconducted two more human experiments–one with a seven-day wash-out period between\nsessions and another with a ramping ultrasound waveform–and compared the resultant sources\nto those of our main study. We also investigated the effects of varied tDCS dose, both at the scalp\nlevel and cortical level using simulations. We believe these efforts have significantly enhanced\nand improved the work.\nBelow, you will find our detailed responses to each reviewer comment in BLUE, with the original\nreviewer comments in BLACK. Figures in the main manuscript are denoted as Fig. x, while figures\nin the supplemental information are denoted as Fig. Sx. Revised or added text in the manuscript\nis highlighted in RED.\nIn addition to the responses to reviewers below, there are a couple of small errors we noticed in\nthe original manuscript that we have corrected in the latest version. They are listed below.\n1. There was a bug in our pairwise ESI metric comparisons in which two subjects were\nincorrectly being excluded. This has led to slight numerical differences in the reported\nmean and standard deviations, as well as associated p-values and Cohen’s d values in\nSupplemental Figure 1C. No comparisons changed from significant to not significant; in\nsome cases, results became more significant (the tEAS IL and CL comparison changed\nfrom p < 0.01 to p < 0.001).\n2. There was a different bug that was causing multiple comparison p-value correction not to\napply to the etFUS imaginary coherence computations (Supplemental Figure S5). After\nproperly implementing FDR correction, etFUS ImCoh is no longer statistically significant\nat any frequency band. However, itFUS continues to have significant differences. We have\nupdated all the relevant results and discussion points in the manuscript to reflect this.\nReviewer #1 (Remarks to the Author):\nIn this study, the authors combine transcranial focused ultrasound with whole brain\nelectroencephalography in resting state humans. The study demonstrates that tFUS or tDCS do\nnot induce source localizable cortical activity, while tEAS (i.e., combined tFUS and tDCS) does\nresult in activity in the targeted cortical region of interest. The paper is interesting, of high quality,\nand well written, and gives an answer to the important question whether tFUS results in sub- or\nsuprathreshold neuromodulation in the human cortex. I have some comments and suggestions\nthat I believe could further improve the impact of the manuscript.\nWe thank the reviewer for their overall favorable assessment of the work. We also thank the\nreviewer for their constructive comments and suggestions for further improvement, which we have\naddressed below.\nIn the Discussion section, the authors state that the data provides evidence that tFUS induces a\nlocation-specific subthreshold effect. However, the authors also demonstrate that (e/i)tFUS\nwaveforms result in significant auditory activation (e.g., Granger causality and dSPM ESI\nanalysis). tEAS also does not induce significant M1 to deep brain Granger causality. This raises\nthe question if the observed source localizable response in tEAS stimulation could in part be\n1\nexplained by non-linear summation of tFUS-induced indirect subthreshold activation of M1\nthrough an auditory pathway (non localizable) and tDCS subthreshold depolarization (localizable).\nIn theory, only one of the stimulation modalities would need to be focal, for tEAS to result in a\nsuprathreshold localizable response. For example, also considering that the tEAS localization\nscores (e.g., averaged subject precision of reconstructed sources) are relatively low. It would be\ninteresting if the authors could discuss these points in the Discussion section.\nWe thank the reviewer for raising this excellent point. Our study design actually takes this into\naccount – for the tDCS conditions, a spatial sham ultrasound (ultrasound to right prefrontal cortex)\nis also applied. If the localizable tDCS with a non-localizable tFUS summation is what caused the\nfocal response, then we would also have seen the focal response on tDCS conditions.\nWe have added the following statement to our Discussion to clarify this point to future readers\n(page 15):\n“tDCS driving tEAS polarity also raises the question on whether the focality of tEAS’\nresponse could be due to tDCS locally subthreshold modulating whole brain induced tFUS\nauditory response. However, this is already accounted for in our experimental set up. For\nour tDCS conditions, we implemented a spatial tFUS control (the set up is shown in Figure\n1B). If the source response of tEAS was the result of tDCS interacting with the whole brain\nauditory response, the tDCS conditions would have resulted in focal activations as well.”\nIn the Discussion section, it is mentioned that the auditory evoked response might mask a\nsubthreshold localizable tFUS response. In my opinion, it would be valuable to include a small\nstudy with participants that are insonicated with ramped ultrasonic waveforms, designed to\nminimize auditory confounding effects. This could potentially allow the authors to evaluate the\nsmall subthreshold effects of tFUS with reduced auditory indirect effects.\nWe thank the reviewer for this suggestion. We conducted a follow up study with 6 human subjects\nin which we tested a ramped waveform. The results continued to show a lack of ROI response\nfrom tFUS-only, and a significantly increased ROI response from the smoothed tEAS condition.\nWe have added the following analysis / discussion to our manuscript (page 17):\n“We further investigated whether a smoothed ultrasound waveform could potentially\n‘unmask’ an ROI response. We tested a smoothed waveform (pulse duration: 3.25 ms, 1\nms tukey ramping, 250 Hz pulse-repetition frequency)1 in six human subjects\n(Supplemental Figure S10). All six subjects reported that they could not hear the\nultrasound. However, the smoothed tFUS by itself continued to lack increased ipsilateral\nROI source power, whereas the smoothed tEAS waveform did. We also note that the\nsmoothed tEAS waveform does appear overall less focal than the one that resulted from\nour rectangular pulses, with additional strong activations in the ipsilateral frontal/premotor\nregions. While the results of this study indicate that ultrasound PRF may not play a\nsubstantial role in the outcome of tEAS (Supplemental Figure S8), it may be that the\nultrasound pulse shape does. Future tEAS parameter optimization studies are warranted.”\nsmoothed ultrasound waveform (pulse duration: 3.25 ms, 1 ms tukey ramping, 250 Hz pulse-repetition\nfrequency) could reduce auditory activations and potentially ‘unmask’ target region specific source activity.\na) The subject average source maps are presented for the smoothed tFUS and tEAS conditions. b)\nSmoothed ultrasound continued to result in no increased ipsilateral source power, while smoothed tEAS\ndid. ns p > 0.05; * p < 0.05\nI would suggest to omit the computational section on the minimally modified Hodgkin-Huxley\nmodel or to move this section to the supplementary information. As mentioned by the authors, the\nmodelling results are conceptual, because of the approximations made in the computational\nmodel. E.g., a membrane model of the squid giant axon is used and ultrasound is included by a\nsingle mechanosensitive Piezo1 channel. I believe there are also some limitations in the modelling\nof Piezo1, that could be included in the Discussion. In particular, the authors model the open\nprobability of Piezo1 as proportional to the instantaneous pressure (eq. 7), based on reference\n[38] (Methods section “Hodgkin-Huxley modeling”). However, it is good to note that [38] does not\nuse ultrasound to activate Piezo1 channels. In my opinion, it is not expected that the Piezo1 open\nstate will follow the instantaneous acoustic pressure as in Fig. 6(e), because the opening time\nconstant of Piezo1 will be higher than the ultrasonic period. Another consideration is that Piezo1\nchannels are activated by membrane tension instead of by the harmonic pressure: the relationship\nbetween membrane tension and pressure is nonlinear and not instantaneous. For example,\nincrease in membrane tension is likely caused by radiation force induced tissue displacement,\ninstead of directly by the harmonic pressure. This also would mean that the Piezo1 open\nprobability would be non-oscillating, because of the steady (non-oscillating) driving radiation force.\nFor these reasons, I would recommend to move this section to the supplementary information,\nbecause of these limitations and because the conceptual results in this section have been shown\n3\nbefore in the computational literature (i.e., that multiple input stimuli will summate non-linearly in\nneuron models).\nWe thank the reviewer for their input and have moved the HH modeling to the supplementary\ninformation.\nMinor comments:\n- P. 4, “[...] and some studies go so far as to propose 100% of tFUS modulation is due to auditory\nactivation [21,22]”. Perhaps this sentence can be nuanced, because [21,22] mention in their\nDiscussion sections, that their results do not imply that tFUS has no direct neuromodulatory\neffects. Although these studies indeed demonstrate the importance of controlling for auditory\nconfounds in tFUS studies.\nWe thank the reviewer for their suggestion. We have reworded the sentence:\n“[…] and some studies could not detect an effect beyond auditory activation.”\n- Fig. 1(c): I would recommend to zoom out on the right subplot, to visualize also the FWHM in\nthe axial direction. Fig. 1(d): I recommend to adjust the tDCS stimulation plot, because this now\nseems to imply that alternating tDCS currents similar to ultrasound are applied.\nThank you for your suggestions. Please see our updated Figure 1 with a new tDCS representation\nand new tFUS scan showing the full FWHM below.\nFigure 1\n- Results, p. 14, “[...] with both 30 and 3,000 Hz PRFs” -> with both 40 and 3,000 Hz PRFs.\nconditions, but we had a typo earlier in the sentence where 30 should have been 40. The\ncorrected statement is below:\n“These parameters included 1,500 and 40 Hz PRFs with 30% duty cycle (DC), and,\ninitially, 10% and 60% DC with both 30 and 3,000 Hz PRFs.”\n- Methods, hydrophone measurements and transducer: please include details on the hydrophone\nmeasurements (Fig. 1(c)) and on the transducer (aperture, radius of curvature).\nWe’ve added in details for the hydrophone and transducer to the methods section:\n“Free water ultrasound calibration\nThis experiment used a 500 kHz f (fundamental frequency) single-element\n0\nultrasound transducer (1.5 inch focal length, 1 inch acoustic aperture diameter, 0.75 inch\nradius of curvature; Blatek Industries Inc., Boalsburg, PA, USA). To characterize the beam\nshape and determine needed input voltage for desired free water pressure, the transducer\nwas calibrated in a water tank. The water tank was filled with deionized water. Pressure\nmeasurements were recorded with an HNR-0500 hydrophone (Onda Corp., Sunnyvale,\nCA, USA).”\n- Methods, In silico transcranial focused ultrasound simulations: it would be good to also include\ndetails on the pressure field simulation (e.g., tissue parameters, mapping from Hounsfield units\n(pCT) to acoustic parameters, used solver (discretisation step, Courant number, etc.)).\nWe thank the reviewer for their suggestion. We have added the following information to our\nmethods:\n“The pCTs were manually segmented using iSeg (v3.10) into air (1.16 kg/m3 density, 343\nm/s speed of sound, 0.000399 MRayl acoustic impedance), bone (1910 kg/m3 density,\n3510 m/w speed of sound, 6.71 MRayl acoustic impedance), and soft tissue (1050 kg/m3\ndensity, 1550 m/s speed of sound, 1.61 MRayl acoustic impedance). Sim4Life solved the\nacoustic simulations with the Linear Acoustic Pressure Wave Equation and the ‘automatic’\ndiscretization settings (0.45 mm resolution, 0.3 mm maximum step, and a time step of 1.0\n/ (speed of sound * !1/dx² + 1/dy² + 1/dz² ).”\nmax\nIn this section, the notation could be confusing. E.g., the notation Isppa.3 is normally used to\ndenote the derated SPPA intensity with homogeneous attenuation of 0.3 dB/(cm-MHz). However,\nin this section more realistic heterogeneous attenuation is used by Sim4life simulations of the\nskull and brain tissue. For this reason, the simulated attenuation is likely not equal to 0.3 dB/(cm-\nMHz), as indicated by the subscript.\nThank you for raising this possible source of confusion. We have replaced “the .3” notation with\n“.T” for “target” throughout the manuscript. For example, Isppa.3 is now Isppa.T.\n- Methods, p. 28: “Excitatory tDCS consisted of center-cathode current, while center-anode\ncurrent was defined as inhibitory tDCS current”. I am expecting this is the other way around.\nThank you for your comment. We have fixed this typo.\n5\n- Minor textual comments: (abstract) affect -> effect. (Fig. 2(b), caption): [...] right subplot [...] left\nsubplot -> other way around. (Fig. S1, caption) Ostu -> Otsu. (Fig. 4(b.ii), caption): PRF -> PFC.\nThank you for your comments. We have fixed these typos.\n- Figures: in some figures (e.g., Fig 6d) the readability could be improved by increasing the font\nsize of the labels and ticks. Fig. 1(c): please include a colorbar for this subplot.\nThank you for your suggestions. We have remade our figures for increased readability. Please\nfind the updated versions in the manuscript and supplemental files. For Figure 1C, a color bar is\nincluded to the right.\nReviewer #2 (Remarks to the Author):\nA main methodological issue is the dose titration of tDCS. The dosage was set at 0.1 mA below\nskin perception threshold, up to a maximum of 2 mA (0.7-2 mA). This is an absolutely unusual,\ncurrents finally entering the brain vary almost 3 fold. They should of course correlate with intensity\nbut the individual skin perception with individual current flow not. Current flow calculations e.g.\nwith SIMNibs will be appreciated.\nWe thank the reviewer for this comment. We respectfully point towards other examples of highly\nvaried transcranial electric stimulation titration. Pilloni et al used between 1.5 or 2.5 mA based on\nsubject tolerability2. Simpson et al delivered between 1.0 – 1.5 mA based on subject tolerability3.\nLafon et al delivered currents varying between 0.3 and 2 mA4. We also note there have also been\nnumerous reports of no meaningful differences in outcomes from using different tDCS\namperages5–7.\nPerhaps more important, our study design is a cross-over experimental design, which looks at\nchanges in the brain response within subjects. For each subject, the tDCS dose was held\nconstant. Our results show, despite varying levels of initial scalp current, there is a significant\nincrease in brain response for the tEAS conditions compared to others.\nNevertheless, we have run the requested simulations to estimate cortical current density. They\nare provided in Supplemental Table T1. Analysis of both scalp-level current and cortical current\ndensity do not show current intensity to be a meaningful (linear) predictor of the subjects’ tEAS\nresponse levels (all R2 < 0.05, all p > 0.05), which is consistent with the past studies reporting\nlittle-to-no differences from currents in our range5–7.\nintensity. The tEAS cortical responses were fit with an affine function of tDCS scalp current and the cortical\ncurrent density (estimated with SimNIBS). Neither of these hold meaningful predictive power for the tEAS\nresponses (both p > 0.05, both R2 < 0.05).\nWe also performed a follow-up experiment with a 7-day washout out between etFUS and etEAS\nconditions. As part of this study, we used a consistent 0.7 mA of tDCS in the tEAS conditions for\nall subjects. The results were consistent with the main study, in which tEAS led to strong ROI\nactivity while tFUS alone did not (Supplemental Figure S12).\nWe have added discussion the following manuscript (page 20):\n“We note the varied level of scalp current applied to subjects during our\nexperiments. There are, broadly, two approaches to transcranial electric stimulation dose\ndetermination for human experiments. One is to normalize the dose for all subjects (either\nat the scalp- or the cortical-level using simulations). The other is to use a varied dose\nacross subjects2–4. We opted for the latter approach in our study for two reasons. The first\nreason is that we wanted to prioritize subject comfort and safety. tDCS is generally\nconsidered safe, and the scalp sensations of tingling, itching, and/or burning are often\nreported as transient8. However, it is possible for tDCS to induce skin lesions after only a\nsingle session9. Since our participants were healthy human subjects, we wanted to avoid\nthis possibility. The second reason is that skin perceptions are the result of activated\nsensory neurons10, which can contaminate the resting-state EEG recordings, both from\nthe firing of the scalp (i.e., non-cortical) neurons, but also from the activated pain circuits\nwithin the brain itself. We wished to avoid such possible contamination.\nSince we used the same current density for each condition within a subject, the\nvaried current density across subjects is not a major concern. This is because our cross-\nover study design looks at the changes in brain response across conditions for subjects.\nOur results show a significant increase in ROI activity for tEAS conditions, regardless of\ninitial tDCS dosage. We did conduct further analysis to determine if scalp or cortical\n(estimated with SIMNibs simulations11) tDCS dose (Supplemental Table T1) was a\nmeaningful predictor of ROI response. Our results indicate tDCS dose, at the between-\nsubjects level, is not a meaningful predictor of tEAS activation (Supplemental Figure S7;\nR2 < 0.05, p < 0.05). This is consistent with other studies that have reported little-to-no\ndifferences in the outcomes of different tDCS doses5–7, and further supports that our\nsubject-specific dosage should not be viewed as a study limitation. In a follow-up study,\n7\nwe used a constant 0.7 mA tDCS for all subjects and found a consistent activation pattern\nacross subjects for the tEAS condition (Supplemental Figure S12).”\nSupplemental Figure S12: Results of a 7-day washout experiment and constant 0.7 mA scalp current are\nconsistent with the main study. We repeated our etEAS and etFUS resting state conditions in 8 subjects,\nthis time with a 7-day washout period between the two conditions. For etEAS, we used a consistent 0.7 mA\nof current across all subjects. We also tested each condition twice; once before and once after a 5 minute\nbreak. a) The ROI activity for etFUS was not significantly altered pre/post break (two-tailed permutation t-\ntest; p > 0.05), and Bayes Factor analysis reveals the activities to be robustly statistically similar for a range\nof scaling factors. b) The ROI activity for the etEAS condition was also statistically similar before and after\nthe 5 minute break period. c) The induced sources are consistent with those from the main study.\nFor each condition the inter-trial interval was 2 seconds with up to a 20% (0.4 s) random jitter, so\neach condition lasting 6.6-8min. A five-minute break was taken between each condition to allow\nfor the subjects to stretch and let any cumulative effects of neuromodulation dissipate. This is a\nrather short interval neglecting results in the literature suggesting e.g that 3 min or 20 min breaks\nboost plasticity and influence hereby succeeding stimulation results. The authors are of course\nwelcome to come up with references that this is not of relevance here.\nWe thank the reviewer for the comment. We removed the comment about letting neuromodulation\neffects dissipate from our methods:\n8\n“For each condition the inter-trial interval was 2 seconds with up to a 20% (0.4 s) random\njitter, so each condition lasting 6.6-8min. A five-minute break was taken between each\ncondition to allow for the subjects to stretch.”\nWe have added the following discussion to our paper to indicate why our experimental design\nhelps to mitigate confounding sequential effects. In our new 7-day washout follow up study, we\nalso tested each condition twice before/after a 5 minute break. Our results found ROI activity pre\nand post 5-minute break to be statistically similar from each other, further supporting that our\nresults were not confounded from these breaks (page 21).\nWe randomized the order of our experiments across subjects, which is also known\nas counterbalancing. While it is possible that conditions tested within a single session may\nexperience a carry-over or sequence-based effect, counterbalancing, at the group-level,\nhelps ensure that these effects were randomly distributed across conditions and should\nnot confound the main results12. To further validate our main findings, we conducted an\nadditional follow-up study, in which we repeated our etEAS and etFUS experiments with\na 7-day wash out period (8 participants; split 50/50 for which condition was first). On the\nday the condition was tested, we tested it twice: once ‘pre’ 5-minute break and once ‘post’\nbreak. The ROI activity for each condition was statistically similar before and after the\nbreak (Supplemental Figure S12a-b), suggesting that, even without the counterbalancing\nof the main study, the results may not have suffered from any sequential-based confounds.\nThe source localizability of the experimental conditions were also consistent with the main\nstudy, where etEAS yielded strong targeting-location specific activity, and etFUS did not\n(Supplemental Figure S12c).\nSupplemental Figure S12: Results of a 7-day washout experiment and constant 0.7 mA scalp current are\nconsistent with the main study. We repeated our etEAS and etFUS resting state conditions in 8 subjects,\nthis time with a 7-day washout period between the two conditions. For etEAS, we used a consistent 0.7 mA\nof current across all subjects. We also tested each condition twice; once before and once after a 5 minute\nbreak. a) The ROI activity for etFUS was not significantly altered pre/post break (two-tailed permutation t-\ntest; p > 0.05), and Bayes Factor analysis reveals the activities to be robustly statistically similar for a range\nof scaling factors. b) The ROI activity for the etEAS condition was also statistically similar before and after\nthe 5 minute break period. c) The induced sources are consistent with those from the main study.\n3 kHz PRF tFUS under the right prefrontal cortex share a striking resemblance to tFUS applied\nunder left motor cortex area (Left M1) with 10 – 63, 20 – 40, and 80 – 100 ms responses. Both\ncause a hemisphere-symmetric response, despite tFUS only being applied to one side. This\nneeds explanation since seemingly this does not support evidence of a target-location specific\neffect. Also, Granger causality analysis of both itFUS and etFUS conditions shows significant\nincreases in information flow from primary auditory cortex to the deep brain, and no changes in\nnet information flow from the targeted ROI to the deep brain.\nThank you for your comment. We apologize that we were not clearer in our explanation of this\nphenomenon; this is critical evidence of tFUS’ subthreshold location-specific effect. In other\nwords, when tFUS is applied by itself, we only see the whole brain auditory-based response,\nregardless of where tFUS is targeted. We have reworded our discussion to make this more clear.\nPlease see our revised paragraph below (bold added for emphasis) (page 14):\n“Excitatory (3 kHz PRF) tFUS targeted to the left motor region, as part of etFUS,\netEAS, and etDCS (which included a tFUS spatial control), induced distinct ERPs at\napproximately 30, 90, and 170 ms post stimulation onset (Figure 2a, Figure 4a). This is\nconsistent with a previous study that identified V1-targeted tFUS induced EEG peaks,\nthough their reported peak times were labeled as 55 ms, 100 ms, and 150 ms post\nsonication. However, when we compared these ERPs to those induced by applying tFUS\nto an entirely different brain region (right hemisphere prefrontal cortex), the resultant\nwaveforms did not just lack statistical differences from each other—they were found to be\nstatistically similar through Bayes Factor analysis (Figure 4b). These results provide\ncritical evidence that location-specific effects of tFUS are subthreshold; by itself,\ntFUS produces the same responses regardless of where it is targeted.\nOf course, these shared responses are time-locked event related potentials.\nTo put it another way, tFUS does induce a suprathreshold response. However, since\nthe topology of these ERPs do not differ across targeting locations, they can be\nattributed to a whole brain auditory response. Indeed, the 3 kHz PRF EEG topographic\nspatial distributions of the 90 and 170 ms ERPs do share visual resemblance to the\npreviously reported auditory induced N1 and P2 ERPs. Granger causality investigations,\nwhich revealed significantly increased A1 to deep brain information flow for both 30 Hz\nand 3 kHz tFUS conditions (Figure 4c), provide additional support that the ERPs are\nauditory related. Nevertheless, the present results that combining tFUS with tDCS led to\nspatially specific responses, when neither could do so individually (Figure 3), as well as\nitFUS dissociating the spectral connectivity between the ROI and the deep brain\n(Supplemental Figure S5), indicate that tFUS does have targeted-location specific effects.”\nEven increased tFUS parameter (including duty cycle; higher pressure; sonication duration)\ncontinued to yield no changes in net information flow from the targeted ROI to the deep brain.\nTranscranial electro-acoustic stimulation（tEAS）under the left M1 produces a hemisphere\nspecific exogenous ERPs（10-63ms，which is claimed to be driven by tDCS polarity, not the\nexcitatory or inhibitory parameters of tFUS (but see tDCS comments above).\nconditions are all in support of a subthreshold modulatory effect. It is only when tFUS is combined\nwith tDCS that we start to see location-specific information. We have added the following\nclarification to our discussion section (page 12):\n“Analysis of the other parameters continued to show a lack of sonication-location specific\nsuprathreshold responses, providing further support that tFUS’ location-specific\neffect is subthreshold (Figure 5).”\nWe have also added to our discussion on tDCS based on the above tDCS comments / analysis\n(bold added for emphasis) (page 12):\n“Not only did itEAS and etEAS induce significantly higher source localization metrics and\nsource power than the other tested conditions, but they also led to a significant change in\nROI polarity (Figure 2d). While this polarity change is evidence of differing\nelectrophysiological effects, a follow-up study indicates that this change in response is\ndriven from tDCS polarity and not tFUS PRF (Supplemental Figure S7). However, further\nanalysis shows no linear relationship between the applied tDCS dose and tEAS\nresponse magnitude (Supplemental Figure S7d-e), nor between tFUS intensity and\ntEAS response (Supplemental Figure S7c). This points to a non-linear response\nfrom a combined transcranial electro-acoustic effect.”\nAnodal tDCS combined with 30Hz PRF tFUS (inhibitory) and 3,000 Hz (excitatory) PRF tFUS\nboth led stronger ipsilateral than contralateral responses. But again, here tEAS conditions were\nalso found to induce significant increases in auditory to deep brain connectivity without an effect\non the net flow of information from the targeted ROI to deep brain.\nThank you for your comment. We have added the following statement to our discussion regarding\ntEAS and Granger causality (page 14-15):\n“We note that these auditory effects persist within the tEAS conditions (Supplemental\nFigure S4). This is unsurprising, as we would have no reason to think the electro-acoustic\neffect at M1 would cancel out auditory pathways. However, what is surprising is the\ncontinued lack of ROI to deep brain change in net flow of information. M1 is connected to\ndeep brain structures13, so on one hand it would be expected that increased M1 activity\nwould lead to increased deep brain activity, and this would be reflected as an increased\nnet flow of information. However, this was not our finding. One possible explanation for\nthis phenomena is that M1 neuron connections with the deep brain are dependent on cell\ntype13. Prior work has demonstrated tFUS’ parameter choices can alter cell-type\nselectivity14. Our tFUS parameters may not have been optimal for eliciting M1 to deep\nbrain connections. Future research on tEAS connectivity changes (or lack thereof) are\nwarranted.”\nWhy does tDCS applied under the left M1 synchronize the tFUS under the right frontal without\ncausing local changes; instead, it still results in a hemisphere-symmetric response.\nOur tDCS conditions are combined with a spatial-sham tFUS (Figure 1b). These hemisphere-\nsymmetric responses are the continued results of auditory based effects from tFUS. We have\nclarified this point in the discussion (bold added for emphasis):\n“Excitatory (3 kHz PRF) tFUS targeted to the left motor region, as part of etFUS, etEAS,\nand etDCS (which included a tFUS spatial control), induced distinct ERPs at\napproximately 30, 90, and 170 ms post stimulation onset (Figure 2a, Figure 4a).”\nIs the claim that tEAS is more than a linear sum of its parts really supported by the summation of\nthe source-space tFUS and tDCS conditions which in turn did not result in increased concordance\nwith the ROI, nor to significant increases in ipsilateral under contralateral ROI source power.\nThank you for your comments. We have included linear modeling of tEAS source-space response\nas a function of tDCS / tFUS intensities (Supplemental Figure S7), which provide additional\nevidence of a non-linear response. We added the following line to the results section (page 10):\n“We also modeled the tEAS ROI response as function of tDCS scalp current, cortical\ncurrent, and tFUS intensity and found none of these component to be meaningfully\npredictive (Supplemental Figure S7c-e).”\nSupplemental Figure S7: The transcranial electro-acoustic effect is more than just the summation\nof independent electric and acoustic modulation. a) Source-imaging data for itFUS and itDCS, as well\nas etFUS and tDCS, were added together for each subject, and then averaged across subjects.\nVisualizations continue to show strong off-targeted sources. b) The average ipsilateral ROI activity of the\nadded sources continues to be not significantly greater than the contralateral ROI for both the summed\nexcitatory and summed inhibitory conditions (p > 0.05). c – e) Mean tEAS-induced ROI activity is not well\n12\npredicted by tFUS or tDCS intensity. The tEAS cortical responses were fit with an affine function of c)\ncortical tFUS intensity, d) current, and e) the cortical current density (estimated with SimNIBS). None of the\nindividual components hold meaningful predictive power for the tEAS responses (all p > 0.05).\nSo, in summary two main concerns regarding the tDCS intensity and intervals between different\ntypes of stimulation are worrying.\nThank you for your feedback. We have addressed these concerns with our updated analysis,\ndiscussion points, and follow-up study.\nMinor:\n„subthreshold (meaning it will not induce neural firing on its own)“ better „modulating sponteanous\nfiring without stimulation time locked additional spikes“\nThank you for your suggestion. We have reworded the statement (bold added for emphasis):\n“A modern, philosophical descendant of these methods, transcranial direct current\nstimulation (tDCS), applies subthreshold (meaning it will modulate spontaneous firing,\nbut will not induce stimulation time-locked spikes on its own) current across brain\ntissue using non-invasive scalp electrodes.”\n„but one prevalent theory is the ultrasound pressure waves open mechanosensitive ion channels“\nInstead „but one prevalent theory is that the ultrasound pressure waves open mechanosensitive\nion channels“\nThank you for your suggestion. We have implemented this change.\n„Electroencephalogram (EEG) based recordings, which directly measure electrical activity without\nthe need for magnetic fields, may offer more insight into the true effects of tFUS as a sole\nstimulus.„ unclear needs to be broken down to tangential MEG sensitivity.\nThank you for your comment. We have reworded this sentence to clarify we are mentioning the\nmagnetic field as contrast to fMRI, not MEG (bold added for emphasis).\n“Electroencephalogram (EEG) based recordings, which directly measure electrical\nactivity and do not require a magnetic resonance environment, may offer more\ninsight into the true effects of tFUS as a sole stimulus.”\n„In follow-up studies, we broaden our tFUS parameter scope. We record and analyze\nsimultaneous whole-scalp EEG recordings and image their corresponding cortical sources to\ncharacterize targeted-location specific, or lack-thereof, responses.“ Here an immediate switch\nfrom stimulation to recording occurs with a logical brake“\nThank you for your comment. We have added a clause to make the transition smoother (bold\nadded for emphasis).\n“In follow-up studies, we broaden our tFUS parameter scope. For all tFUS experiments,\nwe record and analyze simultaneous whole-scalp EEG recordings and image their\ncorresponding cortical sources to characterize targeted-location specific, or lack-thereof,\nresponses.”\nbrain region, there is a lack of agreement for whether tFUS is modulating neural excitability or\nstimulating the neurons on its own.“ are not particularly logic. The first half refers to topography\nthe second to neurophysiology. So not mutually exclusive. This occurs throughout the manuscript,\nsee e.g. „Our data provide strong evidence that, while there are prominent auditory effects, the\napplied tFUS has location-specific subthreshold modulatory effects on the human cortex.“\nSentences like „While the clusters from permutation cluster tests are not guaranteed to be\nsignificant for their entire extent, we did consider the overlapping windows (10 – 63 ms) for\nsignificant itEAS and etEAS clusters to be interesting enough due to their shared presence to\nwarrant use in our further analysis.“ ambiguous.\nThank you for your comments. We have revised the sentences in the following ways (bold added\nfor emphasis):\n“While the effect of tFUS is commonly considered to be based on the targeted brain\nregion, there is a lack of agreement on the magnitude of effect. Specifically, there are\nunanswered questions on whether tFUS is modulating neural excitability or stimulating\nthe neurons on its own.”\n“Our data provide strong evidence that, while tFUS produces prominent whole brain\nauditory related activations, it also induces location-specific subthreshold modulatory\neffects.”\n“While the clusters from permutation cluster tests are not guaranteed to be significant for\ntheir entire extent, we did consider shared clusters from both itEAS and etEAS to\nwarrant further analysis. Thus, the remainder of our work will focus on the shared\nwindow of 10 – 63 ms.”\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank reviewer #3 for their effort and time co-reviewing our manuscript.\nReference:\n1. Johnstone, A., Nandi, T., Martin, E., Bestmann, S., Stagg, C. & Treeby, B. A range of pulses\ncommonly used for human transcranial ultrasound stimulation are clearly audible. Brain\nStimul 14, 1353–1355 (2021).\n2. Pilloni, G., Vogel-Eyny, A., Lustberg, M., Best, P., Malik, M., Walton-Masters, L., George, A.,\nMirza, I., Zhovtis, L., Datta, A., Bikson, M., Krupp, L. & Charvet, L. Tolerability and feasibility\nof at-home remotely supervised transcranial direct current stimulation (RS-tDCS): Single-\ncenter evidence from 6,779 sessions. Brain Stimulation: Basic, Translational, and Clinical\nResearch in Neuromodulation 15, 707–716 (2022).\n3. Simpson, E. A., Saiote, C., Sutter, E., Lench, D. H., Ikonomidou, C., Villegas, M. A. & Gillick,\nB. T. Remotely monitored transcranial direct current stimulation in pediatric cerebral palsy:\nopen label trial protocol. BMC Pediatrics 22, 566 (2022).\n4. Lafon, B., Henin, S., Huang, Y., Friedman, D., Melloni, L., Thesen, T., Doyle, W., Buzsáki, G.,\nDevinsky, O., Parra, L. C. & A. Liu, A. Low frequency transcranial electrical stimulation does\nnot entrain sleep rhythms measured by human intracranial recordings. Nat Commun 8, 1199\n(2017).\n5. Agboada, D., Mosayebi Samani, M., Jamil, A., Kuo, M.-F. & Nitsche, M. A. Expanding the\nparameter space of anodal transcranial direct current stimulation of the primary motor cortex.\nSci Rep 9, 18185 (2019).\n6. Kidgell, D. J., Daly, R. M., Young, K., Lum, J., Tooley, G., Jaberzadeh, S., Zoghi, M. & Pearce,\nA. J. Different Current Intensities of Anodal Transcranial Direct Current Stimulation Do Not\nDifferentially Modulate Motor Cortex Plasticity. Neural Plasticity 2013, 603502 (2013).\n7. Jamil, A., Batsikadze, G., Kuo, H.-I., Labruna, L., Hasan, A., Paulus, W. & Nitsche, M. A.\nSystematic evaluation of the impact of stimulation intensity on neuroplastic after-effects\ninduced by transcranial direct current stimulation. The Journal of Physiology 595, 1273–1288\n(2017).\n8. Poreisz, C., Boros, K., Antal, A. & Paulus, W. Safety aspects of transcranial direct current\nstimulation concerning healthy subjects and patients. Brain Research Bulletin 72, 208–214\n(2007).\n9. Wang, J., Wei, Y., Wen, J. & Li, X. Skin Burn After Single Session of Transcranial Direct\nCurrent Stimulation (tDCS). Brain Stimulation 8, 165–166 (2015).\n10. van Boekholdt, L., Kerstens, S., Khatoun, A., Asamoah, B. & Mc Laughlin, M. tDCS peripheral\nnerve stimulation: a neglected mode of action? Mol Psychiatry 26, 456–461 (2021).\n11. Thielscher, A., Antunes, A. & Saturnino, G. B. Field modeling for transcranial magnetic\nstimulation: A useful tool to understand the physiological effects of TMS? in 2015 37th Annual\nInternational Conference of the IEEE Engineering in Medicine and Biology Society (EMBC)\n222–225 (2015). doi:10.1109/EMBC.2015.7318340.\n12. Corriero, E. F. Counterbalancing. in The SAGE Encyclopedia of Communication Research\nMethods 278–281 (SAGE Publications, Inc, 2017). doi:10.4135/9781483381411.\n13. McColgan, P., Joubert, J., Tabrizi, S. J. & Rees, G. The human motor cortex microcircuit:\ninsights for neurodegenerative disease. Nat Rev Neurosci 21, 401–415 (2020).\n14. Yu, K., Niu, X., Krook-Magnuson, E. & He, B. Intrinsic functional neuron-type selectivity of\ntranscranial focused ultrasound neuromodulation. Nat Commun 12, 2519 (2021).\nWe thank the reviewers for their addi1onal comments. We have responded to all of review comments\nraised and revise the manuscript further. Below, you will find our detailed responses to each reviewer\ncomment in BLUE, with the original reviewer comments in BLACK. Revised or added text in the manuscript\n/ supplementary informa1on is highlighted in RED.\nReviewer #1 (Remarks to the Author):\nIn this study, the authors combine transcranial focused ultrasound with whole brain\nelectroencephalography in resting state humans. The study demonstrates that tFUS or tDCS do\nnot induce source localizable cortical activity, while tEAS (i.e., combined tFUS and tDCS) does\nresult in activity in the targeted cortical region of interest. The paper is interesting, of high quality,\nand well written, and gives an answer to the important question whether tFUS results in sub- or\nsuprathreshold neuromodulation in the human cortex. I have some comments and suggestions\nthat I believe could further improve the impact of the manuscript.\nWe thank the reviewer for their overall favorable assessment of the work. We also thank the\nreviewer for their constructive comments and suggestions for further improvement, which we have\naddressed below.\nIn the Discussion section, the authors state that the data provides evidence that tFUS induces a\nlocation-specific subthreshold effect. However, the authors also demonstrate that (e/i)tFUS\nwaveforms result in significant auditory activation (e.g., Granger causality and dSPM ESI\nanalysis). tEAS also does not induce significant M1 to deep brain Granger causality. This raises\nthe question if the observed source localizable response in tEAS stimulation could in part be\n1\nexplained by non-linear summation of tFUS-induced indirect subthreshold activation of M1\nthrough an auditory pathway (non localizable) and tDCS subthreshold depolarization (localizable).\nIn theory, only one of the stimulation modalities would need to be focal, for tEAS to result in a\nsuprathreshold localizable response. For example, also considering that the tEAS localization\nscores (e.g., averaged subject precision of reconstructed sources) are relatively low. It would be\ninteresting if the authors could discuss these points in the Discussion section.\nWe thank the reviewer for raising this excellent point. Our study design actually takes this into\naccount – for the tDCS conditions, a spatial sham ultrasound (ultrasound to right prefrontal cortex)\nis also applied. If the localizable tDCS with a non-localizable tFUS summation is what caused the\nfocal response, then we would also have seen the focal response on tDCS conditions.\nWe have added the following statement to our Discussion to clarify this point to future readers\n(page 15):\n“tDCS driving tEAS polarity also raises the question on whether the focality of tEAS’\nresponse could be due to tDCS locally subthreshold modulating whole brain induced tFUS\nauditory response. However, this is already accounted for in our experimental set up. For\nour tDCS conditions, we implemented a spatial tFUS control (the set up is shown in Figure\n1B). If the source response of tEAS was the result of tDCS interacting with the whole brain\nauditory response, the tDCS conditions would have resulted in focal activations as well.”\nIn the Discussion section, it is mentioned that the auditory evoked response might mask a\nsubthreshold localizable tFUS response. In my opinion, it would be valuable to include a small\nstudy with participants that are insonicated with ramped ultrasonic waveforms, designed to\nminimize auditory confounding effects. This could potentially allow the authors to evaluate the\nsmall subthreshold effects of tFUS with reduced auditory indirect effects.\nWe thank the reviewer for this suggestion. We conducted a follow up study with 6 human subjects\nin which we tested a ramped waveform. The results continued to show a lack of ROI response\nfrom tFUS-only, and a significantly increased ROI response from the smoothed tEAS condition.\nWe have added the following analysis / discussion to our manuscript (page 17):\n“We further investigated whether a smoothed ultrasound waveform could potentially\n‘unmask’ an ROI response. We tested a smoothed waveform (pulse duration: 3.25 ms, 1\nms tukey ramping, 250 Hz pulse-repetition frequency)1 in six human subjects\n(Supplemental Figure S10). All six subjects reported that they could not hear the\nultrasound. However, the smoothed tFUS by itself continued to lack increased ipsilateral\nROI source power, whereas the smoothed tEAS waveform did. We also note that the\nsmoothed tEAS waveform does appear overall less focal than the one that resulted from\nour rectangular pulses, with additional strong activations in the ipsilateral frontal/premotor\nregions. While the results of this study indicate that ultrasound PRF may not play a\nsubstantial role in the outcome of tEAS (Supplemental Figure S8), it may be that the\nultrasound pulse shape does. Future tEAS parameter optimization studies are warranted.”\nsmoothed ultrasound waveform (pulse duration: 3.25 ms, 1 ms tukey ramping, 250 Hz pulse-repetition\nfrequency) could reduce auditory activations and potentially ‘unmask’ target region specific source activity.\na) The subject average source maps are presented for the smoothed tFUS and tEAS conditions. b)\nSmoothed ultrasound continued to result in no increased ipsilateral source power, while smoothed tEAS\ndid. ns p > 0.05; * p < 0.05\nI would suggest to omit the computational section on the minimally modified Hodgkin-Huxley\nmodel or to move this section to the supplementary information. As mentioned by the authors, the\nmodelling results are conceptual, because of the approximations made in the computational\nmodel. E.g., a membrane model of the squid giant axon is used and ultrasound is included by a\nsingle mechanosensitive Piezo1 channel. I believe there are also some limitations in the modelling\nof Piezo1, that could be included in the Discussion. In particular, the authors model the open\nprobability of Piezo1 as proportional to the instantaneous pressure (eq. 7), based on reference\n[38] (Methods section “Hodgkin-Huxley modeling”). However, it is good to note that [38] does not\nuse ultrasound to activate Piezo1 channels. In my opinion, it is not expected that the Piezo1 open\nstate will follow the instantaneous acoustic pressure as in Fig. 6(e), because the opening time\nconstant of Piezo1 will be higher than the ultrasonic period. Another consideration is that Piezo1\nchannels are activated by membrane tension instead of by the harmonic pressure: the relationship\nbetween membrane tension and pressure is nonlinear and not instantaneous. For example,\nincrease in membrane tension is likely caused by radiation force induced tissue displacement,\ninstead of directly by the harmonic pressure. This also would mean that the Piezo1 open\nprobability would be non-oscillating, because of the steady (non-oscillating) driving radiation force.\nFor these reasons, I would recommend to move this section to the supplementary information,\nbecause of these limitations and because the conceptual results in this section have been shown\n3\nbefore in the computational literature (i.e., that multiple input stimuli will summate non-linearly in\nneuron models).\nWe thank the reviewer for their input and have moved the HH modeling to the supplementary\ninformation.\nMinor comments:\n- P. 4, “[...] and some studies go so far as to propose 100% of tFUS modulation is due to auditory\nactivation [21,22]”. Perhaps this sentence can be nuanced, because [21,22] mention in their\nDiscussion sections, that their results do not imply that tFUS has no direct neuromodulatory\neffects. Although these studies indeed demonstrate the importance of controlling for auditory\nconfounds in tFUS studies.\nWe thank the reviewer for their suggestion. We have reworded the sentence:\n“[…] and some studies could not detect an effect beyond auditory activation.”\n- Fig. 1(c): I would recommend to zoom out on the right subplot, to visualize also the FWHM in\nthe axial direction. Fig. 1(d): I recommend to adjust the tDCS stimulation plot, because this now\nseems to imply that alternating tDCS currents similar to ultrasound are applied.\nThank you for your suggestions. Please see our updated Figure 1 with a new tDCS representation\nand new tFUS scan showing the full FWHM below.\nFigure 1\n- Results, p. 14, “[...] with both 30 and 3,000 Hz PRFs” -> with both 40 and 3,000 Hz PRFs.\nconditions, but we had a typo earlier in the sentence where 30 should have been 40. The\ncorrected statement is below:\n“These parameters included 1,500 and 40 Hz PRFs with 30% duty cycle (DC), and,\ninitially, 10% and 60% DC with both 30 and 3,000 Hz PRFs.”\n- Methods, hydrophone measurements and transducer: please include details on the hydrophone\nmeasurements (Fig. 1(c)) and on the transducer (aperture, radius of curvature).\nWe’ve added in details for the hydrophone and transducer to the methods section:\n“Free water ultrasound calibration\nThis experiment used a 500 kHz f (fundamental frequency) single-element\n0\nultrasound transducer (1.5 inch focal length, 1 inch acoustic aperture diameter, 0.75 inch\nradius of curvature; Blatek Industries Inc., Boalsburg, PA, USA). To characterize the beam\nshape and determine needed input voltage for desired free water pressure, the transducer\nwas calibrated in a water tank. The water tank was filled with deionized water. Pressure\nmeasurements were recorded with an HNR-0500 hydrophone (Onda Corp., Sunnyvale,\nCA, USA).”\n- Methods, In silico transcranial focused ultrasound simulations: it would be good to also include\ndetails on the pressure field simulation (e.g., tissue parameters, mapping from Hounsfield units\n(pCT) to acoustic parameters, used solver (discretisation step, Courant number, etc.)).\nWe thank the reviewer for their suggestion. We have added the following information to our\nmethods:\n“The pCTs were manually segmented using iSeg (v3.10) into air (1.16 kg/m3 density, 343\nm/s speed of sound, 0.000399 MRayl acoustic impedance), bone (1910 kg/m3 density,\n3510 m/w speed of sound, 6.71 MRayl acoustic impedance), and soft tissue (1050 kg/m3\ndensity, 1550 m/s speed of sound, 1.61 MRayl acoustic impedance). Sim4Life solved the\nacoustic simulations with the Linear Acoustic Pressure Wave Equation and the ‘automatic’\ndiscretization settings (0.45 mm resolution, 0.3 mm maximum step, and a time step of 1.0\n/ (speed of sound * !1/dx² + 1/dy² + 1/dz² ).”\nmax\nIn this section, the notation could be confusing. E.g., the notation Isppa.3 is normally used to\ndenote the derated SPPA intensity with homogeneous attenuation of 0.3 dB/(cm-MHz). However,\nin this section more realistic heterogeneous attenuation is used by Sim4life simulations of the\nskull and brain tissue. For this reason, the simulated attenuation is likely not equal to 0.3 dB/(cm-\nMHz), as indicated by the subscript.\nThank you for raising this possible source of confusion. We have replaced “the .3” notation with\n“.T” for “target” throughout the manuscript. For example, Isppa.3 is now Isppa.T.\n- Methods, p. 28: “Excitatory tDCS consisted of center-cathode current, while center-anode\ncurrent was defined as inhibitory tDCS current”. I am expecting this is the other way around.\nThank you for your comment. We have fixed this typo.\n5\n- Minor textual comments: (abstract) affect -> effect. (Fig. 2(b), caption): [...] right subplot [...] left\nsubplot -> other way around. (Fig. S1, caption) Ostu -> Otsu. (Fig. 4(b.ii), caption): PRF -> PFC.\nThank you for your comments. We have fixed these typos.\n- Figures: in some figures (e.g., Fig 6d) the readability could be improved by increasing the font\nsize of the labels and ticks. Fig. 1(c): please include a colorbar for this subplot.\nThank you for your suggestions. We have remade our figures for increased readability. Please\nfind the updated versions in the manuscript and supplemental files. For Figure 1C, a color bar is\nincluded to the right.\nReviewer #2 (Remarks to the Author):\nA main methodological issue is the dose titration of tDCS. The dosage was set at 0.1 mA below\nskin perception threshold, up to a maximum of 2 mA (0.7-2 mA). This is an absolutely unusual,\ncurrents finally entering the brain vary almost 3 fold. They should of course correlate with intensity\nbut the individual skin perception with individual current flow not. Current flow calculations e.g.\nwith SIMNibs will be appreciated.\nWe thank the reviewer for this comment. We respectfully point towards other examples of highly\nvaried transcranial electric stimulation titration. Pilloni et al used between 1.5 or 2.5 mA based on\nsubject tolerability2. Simpson et al delivered between 1.0 – 1.5 mA based on subject tolerability3.\nLafon et al delivered currents varying between 0.3 and 2 mA4. We also note there have also been\nnumerous reports of no meaningful differences in outcomes from using different tDCS\namperages5–7.\nPerhaps more important, our study design is a cross-over experimental design, which looks at\nchanges in the brain response within subjects. For each subject, the tDCS dose was held\nconstant. Our results show, despite varying levels of initial scalp current, there is a significant\nincrease in brain response for the tEAS conditions compared to others.\nNevertheless, we have run the requested simulations to estimate cortical current density. They\nare provided in Supplemental Table T1. Analysis of both scalp-level current and cortical current\ndensity do not show current intensity to be a meaningful (linear) predictor of the subjects’ tEAS\nresponse levels (all R2 < 0.05, all p > 0.05), which is consistent with the past studies reporting\nlittle-to-no differences from currents in our range5–7.\nintensity. The tEAS cortical responses were fit with an affine function of tDCS scalp current and the cortical\ncurrent density (estimated with SimNIBS). Neither of these hold meaningful predictive power for the tEAS\nresponses (both p > 0.05, both R2 < 0.05).\nWe also performed a follow-up experiment with a 7-day washout out between etFUS and etEAS\nconditions. As part of this study, we used a consistent 0.7 mA of tDCS in the tEAS conditions for\nall subjects. The results were consistent with the main study, in which tEAS led to strong ROI\nactivity while tFUS alone did not (Supplemental Figure S12).\nWe have added discussion the following manuscript (page 20):\n“We note the varied level of scalp current applied to subjects during our\nexperiments. There are, broadly, two approaches to transcranial electric stimulation dose\ndetermination for human experiments. One is to normalize the dose for all subjects (either\nat the scalp- or the cortical-level using simulations). The other is to use a varied dose\nacross subjects2–4. We opted for the latter approach in our study for two reasons. The first\nreason is that we wanted to prioritize subject comfort and safety. tDCS is generally\nconsidered safe, and the scalp sensations of tingling, itching, and/or burning are often\nreported as transient8. However, it is possible for tDCS to induce skin lesions after only a\nsingle session9. Since our participants were healthy human subjects, we wanted to avoid\nthis possibility. The second reason is that skin perceptions are the result of activated\nsensory neurons10, which can contaminate the resting-state EEG recordings, both from\nthe firing of the scalp (i.e., non-cortical) neurons, but also from the activated pain circuits\nwithin the brain itself. We wished to avoid such possible contamination.\nSince we used the same current density for each condition within a subject, the\nvaried current density across subjects is not a major concern. This is because our cross-\nover study design looks at the changes in brain response across conditions for subjects.\nOur results show a significant increase in ROI activity for tEAS conditions, regardless of\ninitial tDCS dosage. We did conduct further analysis to determine if scalp or cortical\n(estimated with SIMNibs simulations11) tDCS dose (Supplemental Table T1) was a\nmeaningful predictor of ROI response. Our results indicate tDCS dose, at the between-\nsubjects level, is not a meaningful predictor of tEAS activation (Supplemental Figure S7;\nR2 < 0.05, p < 0.05). This is consistent with other studies that have reported little-to-no\ndifferences in the outcomes of different tDCS doses5–7, and further supports that our\nsubject-specific dosage should not be viewed as a study limitation. In a follow-up study,\n7\nwe used a constant 0.7 mA tDCS for all subjects and found a consistent activation pattern\nacross subjects for the tEAS condition (Supplemental Figure S12).”\nSupplemental Figure S12: Results of a 7-day washout experiment and constant 0.7 mA scalp current are\nconsistent with the main study. We repeated our etEAS and etFUS resting state conditions in 8 subjects,\nthis time with a 7-day washout period between the two conditions. For etEAS, we used a consistent 0.7 mA\nof current across all subjects. We also tested each condition twice; once before and once after a 5 minute\nbreak. a) The ROI activity for etFUS was not significantly altered pre/post break (two-tailed permutation t-\ntest; p > 0.05), and Bayes Factor analysis reveals the activities to be robustly statistically similar for a range\nof scaling factors. b) The ROI activity for the etEAS condition was also statistically similar before and after\nthe 5 minute break period. c) The induced sources are consistent with those from the main study.\nFor each condition the inter-trial interval was 2 seconds with up to a 20% (0.4 s) random jitter, so\neach condition lasting 6.6-8min. A five-minute break was taken between each condition to allow\nfor the subjects to stretch and let any cumulative effects of neuromodulation dissipate. This is a\nrather short interval neglecting results in the literature suggesting e.g that 3 min or 20 min breaks\nboost plasticity and influence hereby succeeding stimulation results. The authors are of course\nwelcome to come up with references that this is not of relevance here.\nWe thank the reviewer for the comment. We removed the comment about letting neuromodulation\neffects dissipate from our methods:\n8\n“For each condition the inter-trial interval was 2 seconds with up to a 20% (0.4 s) random\njitter, so each condition lasting 6.6-8min. A five-minute break was taken between each\ncondition to allow for the subjects to stretch.”\nWe have added the following discussion to our paper to indicate why our experimental design\nhelps to mitigate confounding sequential effects. In our new 7-day washout follow up study, we\nalso tested each condition twice before/after a 5 minute break. Our results found ROI activity pre\nand post 5-minute break to be statistically similar from each other, further supporting that our\nresults were not confounded from these breaks (page 21).\nWe randomized the order of our experiments across subjects, which is also known\nas counterbalancing. While it is possible that conditions tested within a single session may\nexperience a carry-over or sequence-based effect, counterbalancing, at the group-level,\nhelps ensure that these effects were randomly distributed across conditions and should\nnot confound the main results12. To further validate our main findings, we conducted an\nadditional follow-up study, in which we repeated our etEAS and etFUS experiments with\na 7-day wash out period (8 participants; split 50/50 for which condition was first). On the\nday the condition was tested, we tested it twice: once ‘pre’ 5-minute break and once ‘post’\nbreak. The ROI activity for each condition was statistically similar before and after the\nbreak (Supplemental Figure S12a-b), suggesting that, even without the counterbalancing\nof the main study, the results may not have suffered from any sequential-based confounds.\nThe source localizability of the experimental conditions were also consistent with the main\nstudy, where etEAS yielded strong targeting-location specific activity, and etFUS did not\n(Supplemental Figure S12c).\nSupplemental Figure S12: Results of a 7-day washout experiment and constant 0.7 mA scalp current are\nconsistent with the main study. We repeated our etEAS and etFUS resting state conditions in 8 subjects,\nthis time with a 7-day washout period between the two conditions. For etEAS, we used a consistent 0.7 mA\nof current across all subjects. We also tested each condition twice; once before and once after a 5 minute\nbreak. a) The ROI activity for etFUS was not significantly altered pre/post break (two-tailed permutation t-\ntest; p > 0.05), and Bayes Factor analysis reveals the activities to be robustly statistically similar for a range\nof scaling factors. b) The ROI activity for the etEAS condition was also statistically similar before and after\nthe 5 minute break period. c) The induced sources are consistent with those from the main study.\n3 kHz PRF tFUS under the right prefrontal cortex share a striking resemblance to tFUS applied\nunder left motor cortex area (Left M1) with 10 – 63, 20 – 40, and 80 – 100 ms responses. Both\ncause a hemisphere-symmetric response, despite tFUS only being applied to one side. This\nneeds explanation since seemingly this does not support evidence of a target-location specific\neffect. Also, Granger causality analysis of both itFUS and etFUS conditions shows significant\nincreases in information flow from primary auditory cortex to the deep brain, and no changes in\nnet information flow from the targeted ROI to the deep brain.\nThank you for your comment. We apologize that we were not clearer in our explanation of this\nphenomenon; this is critical evidence of tFUS’ subthreshold location-specific effect. In other\nwords, when tFUS is applied by itself, we only see the whole brain auditory-based response,\nregardless of where tFUS is targeted. We have reworded our discussion to make this more clear.\nPlease see our revised paragraph below (bold added for emphasis) (page 14):\n“Excitatory (3 kHz PRF) tFUS targeted to the left motor region, as part of etFUS,\netEAS, and etDCS (which included a tFUS spatial control), induced distinct ERPs at\napproximately 30, 90, and 170 ms post stimulation onset (Figure 2a, Figure 4a). This is\nconsistent with a previous study that identified V1-targeted tFUS induced EEG peaks,\nthough their reported peak times were labeled as 55 ms, 100 ms, and 150 ms post\nsonication. However, when we compared these ERPs to those induced by applying tFUS\nto an entirely different brain region (right hemisphere prefrontal cortex), the resultant\nwaveforms did not just lack statistical differences from each other—they were found to be\nstatistically similar through Bayes Factor analysis (Figure 4b). These results provide\ncritical evidence that location-specific effects of tFUS are subthreshold; by itself,\ntFUS produces the same responses regardless of where it is targeted.\nOf course, these shared responses are time-locked event related potentials.\nTo put it another way, tFUS does induce a suprathreshold response. However, since\nthe topology of these ERPs do not differ across targeting locations, they can be\nattributed to a whole brain auditory response. Indeed, the 3 kHz PRF EEG topographic\nspatial distributions of the 90 and 170 ms ERPs do share visual resemblance to the\npreviously reported auditory induced N1 and P2 ERPs. Granger causality investigations,\nwhich revealed significantly increased A1 to deep brain information flow for both 30 Hz\nand 3 kHz tFUS conditions (Figure 4c), provide additional support that the ERPs are\nauditory related. Nevertheless, the present results that combining tFUS with tDCS led to\nspatially specific responses, when neither could do so individually (Figure 3), as well as\nitFUS dissociating the spectral connectivity between the ROI and the deep brain\n(Supplemental Figure S5), indicate that tFUS does have targeted-location specific effects.”\nEven increased tFUS parameter (including duty cycle; higher pressure; sonication duration)\ncontinued to yield no changes in net information flow from the targeted ROI to the deep brain.\nTranscranial electro-acoustic stimulation（tEAS）under the left M1 produces a hemisphere\nspecific exogenous ERPs（10-63ms，which is claimed to be driven by tDCS polarity, not the\nexcitatory or inhibitory parameters of tFUS (but see tDCS comments above).\nconditions are all in support of a subthreshold modulatory effect. It is only when tFUS is combined\nwith tDCS that we start to see location-specific information. We have added the following\nclarification to our discussion section (page 12):\n“Analysis of the other parameters continued to show a lack of sonication-location specific\nsuprathreshold responses, providing further support that tFUS’ location-specific\neffect is subthreshold (Figure 5).”\nWe have also added to our discussion on tDCS based on the above tDCS comments / analysis\n(bold added for emphasis) (page 12):\n“Not only did itEAS and etEAS induce significantly higher source localization metrics and\nsource power than the other tested conditions, but they also led to a significant change in\nROI polarity (Figure 2d). While this polarity change is evidence of differing\nelectrophysiological effects, a follow-up study indicates that this change in response is\ndriven from tDCS polarity and not tFUS PRF (Supplemental Figure S7). However, further\nanalysis shows no linear relationship between the applied tDCS dose and tEAS\nresponse magnitude (Supplemental Figure S7d-e), nor between tFUS intensity and\ntEAS response (Supplemental Figure S7c). This points to a non-linear response\nfrom a combined transcranial electro-acoustic effect.”\nAnodal tDCS combined with 30Hz PRF tFUS (inhibitory) and 3,000 Hz (excitatory) PRF tFUS\nboth led stronger ipsilateral than contralateral responses. But again, here tEAS conditions were\nalso found to induce significant increases in auditory to deep brain connectivity without an effect\non the net flow of information from the targeted ROI to deep brain.\nThank you for your comment. We have added the following statement to our discussion regarding\ntEAS and Granger causality (page 14-15):\n“We note that these auditory effects persist within the tEAS conditions (Supplemental\nFigure S4). This is unsurprising, as we would have no reason to think the electro-acoustic\neffect at M1 would cancel out auditory pathways. However, what is surprising is the\ncontinued lack of ROI to deep brain change in net flow of information. M1 is connected to\ndeep brain structures13, so on one hand it would be expected that increased M1 activity\nwould lead to increased deep brain activity, and this would be reflected as an increased\nnet flow of information. However, this was not our finding. One possible explanation for\nthis phenomena is that M1 neuron connections with the deep brain are dependent on cell\ntype13. Prior work has demonstrated tFUS’ parameter choices can alter cell-type\nselectivity14. Our tFUS parameters may not have been optimal for eliciting M1 to deep\nbrain connections. Future research on tEAS connectivity changes (or lack thereof) are\nwarranted.”\nWhy does tDCS applied under the left M1 synchronize the tFUS under the right frontal without\ncausing local changes; instead, it still results in a hemisphere-symmetric response.\nOur tDCS conditions are combined with a spatial-sham tFUS (Figure 1b). These hemisphere-\nsymmetric responses are the continued results of auditory based effects from tFUS. We have\nclarified this point in the discussion (bold added for emphasis):\n“Excitatory (3 kHz PRF) tFUS targeted to the left motor region, as part of etFUS, etEAS,\nand etDCS (which included a tFUS spatial control), induced distinct ERPs at\napproximately 30, 90, and 170 ms post stimulation onset (Figure 2a, Figure 4a).”\nIs the claim that tEAS is more than a linear sum of its parts really supported by the summation of\nthe source-space tFUS and tDCS conditions which in turn did not result in increased concordance\nwith the ROI, nor to significant increases in ipsilateral under contralateral ROI source power.\nThank you for your comments. We have included linear modeling of tEAS source-space response\nas a function of tDCS / tFUS intensities (Supplemental Figure S7), which provide additional\nevidence of a non-linear response. We added the following line to the results section (page 10):\n“We also modeled the tEAS ROI response as function of tDCS scalp current, cortical\ncurrent, and tFUS intensity and found none of these component to be meaningfully\npredictive (Supplemental Figure S7c-e).”\nSupplemental Figure S7: The transcranial electro-acoustic effect is more than just the summation\nof independent electric and acoustic modulation. a) Source-imaging data for itFUS and itDCS, as well\nas etFUS and tDCS, were added together for each subject, and then averaged across subjects.\nVisualizations continue to show strong off-targeted sources. b) The average ipsilateral ROI activity of the\nadded sources continues to be not significantly greater than the contralateral ROI for both the summed\nexcitatory and summed inhibitory conditions (p > 0.05). c – e) Mean tEAS-induced ROI activity is not well\n12\npredicted by tFUS or tDCS intensity. The tEAS cortical responses were fit with an affine function of c)\ncortical tFUS intensity, d) current, and e) the cortical current density (estimated with SimNIBS). None of the\nindividual components hold meaningful predictive power for the tEAS responses (all p > 0.05).\nSo, in summary two main concerns regarding the tDCS intensity and intervals between different\ntypes of stimulation are worrying.\nThank you for your feedback. We have addressed these concerns with our updated analysis,\ndiscussion points, and follow-up study.\nMinor:\n„subthreshold (meaning it will not induce neural firing on its own)“ better „modulating sponteanous\nfiring without stimulation time locked additional spikes“\nThank you for your suggestion. We have reworded the statement (bold added for emphasis):\n“A modern, philosophical descendant of these methods, transcranial direct current\nstimulation (tDCS), applies subthreshold (meaning it will modulate spontaneous firing,\nbut will not induce stimulation time-locked spikes on its own) current across brain\ntissue using non-invasive scalp electrodes.”\n„but one prevalent theory is the ultrasound pressure waves open mechanosensitive ion channels“\nInstead „but one prevalent theory is that the ultrasound pressure waves open mechanosensitive\nion channels“\nThank you for your suggestion. We have implemented this change.\n„Electroencephalogram (EEG) based recordings, which directly measure electrical activity without\nthe need for magnetic fields, may offer more insight into the true effects of tFUS as a sole\nstimulus.„ unclear needs to be broken down to tangential MEG sensitivity.\nThank you for your comment. We have reworded this sentence to clarify we are mentioning the\nmagnetic field as contrast to fMRI, not MEG (bold added for emphasis).\n“Electroencephalogram (EEG) based recordings, which directly measure electrical\nactivity and do not require a magnetic resonance environment, may offer more\ninsight into the true effects of tFUS as a sole stimulus.”\n„In follow-up studies, we broaden our tFUS parameter scope. We record and analyze\nsimultaneous whole-scalp EEG recordings and image their corresponding cortical sources to\ncharacterize targeted-location specific, or lack-thereof, responses.“ Here an immediate switch\nfrom stimulation to recording occurs with a logical brake“\nThank you for your comment. We have added a clause to make the transition smoother (bold\nadded for emphasis).\n“In follow-up studies, we broaden our tFUS parameter scope. For all tFUS experiments,\nwe record and analyze simultaneous whole-scalp EEG recordings and image their\ncorresponding cortical sources to characterize targeted-location specific, or lack-thereof,\nresponses.”\nbrain region, there is a lack of agreement for whether tFUS is modulating neural excitability or\nstimulating the neurons on its own.“ are not particularly logic. The first half refers to topography\nthe second to neurophysiology. So not mutually exclusive. This occurs throughout the manuscript,\nsee e.g. „Our data provide strong evidence that, while there are prominent auditory effects, the\napplied tFUS has location-specific subthreshold modulatory effects on the human cortex.“\nSentences like „While the clusters from permutation cluster tests are not guaranteed to be\nsignificant for their entire extent, we did consider the overlapping windows (10 – 63 ms) for\nsignificant itEAS and etEAS clusters to be interesting enough due to their shared presence to\nwarrant use in our further analysis.“ ambiguous.\nThank you for your comments. We have revised the sentences in the following ways (bold added\nfor emphasis):\n“While the effect of tFUS is commonly considered to be based on the targeted brain\nregion, there is a lack of agreement on the magnitude of effect. Specifically, there are\nunanswered questions on whether tFUS is modulating neural excitability or stimulating\nthe neurons on its own.”\n“Our data provide strong evidence that, while tFUS produces prominent whole brain\nauditory related activations, it also induces location-specific subthreshold modulatory\neffects.”\n“While the clusters from permutation cluster tests are not guaranteed to be significant for\ntheir entire extent, we did consider shared clusters from both itEAS and etEAS to\nwarrant further analysis. Thus, the remainder of our work will focus on the shared\nwindow of 10 – 63 ms.”\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank reviewer #3 for their effort and time co-reviewing our manuscript.\nReference:\n1. Johnstone, A., Nandi, T., Martin, E., Bestmann, S., Stagg, C. & Treeby, B. A range of pulses\ncommonly used for human transcranial ultrasound stimulation are clearly audible. Brain\nStimul 14, 1353–1355 (2021).\n2. Pilloni, G., Vogel-Eyny, A., Lustberg, M., Best, P., Malik, M., Walton-Masters, L., George, A.,\nMirza, I., Zhovtis, L., Datta, A., Bikson, M., Krupp, L. & Charvet, L. Tolerability and feasibility\nof at-home remotely supervised transcranial direct current stimulation (RS-tDCS): Single-\ncenter evidence from 6,779 sessions. Brain Stimulation: Basic, Translational, and Clinical\nResearch in Neuromodulation 15, 707–716 (2022).\n3. Simpson, E. A., Saiote, C., Sutter, E., Lench, D. H., Ikonomidou, C., Villegas, M. A. & Gillick,\nB. T. Remotely monitored transcranial direct current stimulation in pediatric cerebral palsy:\nopen label trial protocol. BMC Pediatrics 22, 566 (2022).\n4. Lafon, B., Henin, S., Huang, Y., Friedman, D., Melloni, L., Thesen, T., Doyle, W., Buzsáki, G.,\nDevinsky, O., Parra, L. C. & A. Liu, A. Low frequency transcranial electrical stimulation does\nnot entrain sleep rhythms measured by human intracranial recordings. Nat Commun 8, 1199\n(2017).\n5. Agboada, D., Mosayebi Samani, M., Jamil, A., Kuo, M.-F. & Nitsche, M. A. Expanding the\nparameter space of anodal transcranial direct current stimulation of the primary motor cortex.\nSci Rep 9, 18185 (2019).\n6. Kidgell, D. J., Daly, R. M., Young, K., Lum, J., Tooley, G., Jaberzadeh, S., Zoghi, M. & Pearce,\nA. J. Different Current Intensities of Anodal Transcranial Direct Current Stimulation Do Not\nDifferentially Modulate Motor Cortex Plasticity. Neural Plasticity 2013, 603502 (2013).\n7. Jamil, A., Batsikadze, G., Kuo, H.-I., Labruna, L., Hasan, A., Paulus, W. & Nitsche, M. A.\nSystematic evaluation of the impact of stimulation intensity on neuroplastic after-effects\ninduced by transcranial direct current stimulation. The Journal of Physiology 595, 1273–1288\n(2017).\n8. Poreisz, C., Boros, K., Antal, A. & Paulus, W. Safety aspects of transcranial direct current\nstimulation concerning healthy subjects and patients. Brain Research Bulletin 72, 208–214\n(2007).\n9. Wang, J., Wei, Y., Wen, J. & Li, X. Skin Burn After Single Session of Transcranial Direct\nCurrent Stimulation (tDCS). Brain Stimulation 8, 165–166 (2015).\n10. van Boekholdt, L., Kerstens, S., Khatoun, A., Asamoah, B. & Mc Laughlin, M. tDCS peripheral\nnerve stimulation: a neglected mode of action? Mol Psychiatry 26, 456–461 (2021).\n11. Thielscher, A., Antunes, A. & Saturnino, G. B. Field modeling for transcranial magnetic\nstimulation: A useful tool to understand the physiological effects of TMS? in 2015 37th Annual\nInternational Conference of the IEEE Engineering in Medicine and Biology Society (EMBC)\n222–225 (2015). doi:10.1109/EMBC.2015.7318340.\n12. Corriero, E. F. Counterbalancing. in The SAGE Encyclopedia of Communication Research\nMethods 278–281 (SAGE Publications, Inc, 2017). doi:10.4135/9781483381411.\n13. McColgan, P., Joubert, J., Tabrizi, S. J. & Rees, G. The human motor cortex microcircuit:\ninsights for neurodegenerative disease. Nat Rev Neurosci 21, 401–415 (2020).\n14. Yu, K., Niu, X., Krook-Magnuson, E. & He, B. Intrinsic functional neuron-type selectivity of\ntranscranial focused ultrasound neuromodulation. Nat Commun 12, 2519 (2021).\nWe thank the reviewers for their addi1onal comments. We have responded to all of review comments\nraised and revise the manuscript further. Below, you will find our detailed responses to each reviewer\ncomment in BLUE, with the original reviewer comments in BLACK. Revised or added text in the manuscript\n/ supplementary informa1on is highlighted in RED.\nReviewer #1 (Remarks to the Author):\nIn this study, the authors combine transcranial focused ultrasound with whole brain\nelectroencephalography in resting state humans. The study demonstrates that tFUS or tDCS do\nnot induce source localizable cortical activity, while tEAS (i.e., combined tFUS and tDCS) does\nresult in activity in the targeted cortical region of interest. The paper is interesting, of high quality,\nand well written, and gives an answer to the important question whether tFUS results in sub- or\nsuprathreshold neuromodulation in the human cortex. I have some comments and suggestions\nthat I believe could further improve the impact of the manuscript.\nWe thank the reviewer for their overall favorable assessment of the work. We also thank the\nreviewer for their constructive comments and suggestions for further improvement, which we have\naddressed below.\nIn the Discussion section, the authors state that the data provides evidence that tFUS induces a\nlocation-specific subthreshold effect. However, the authors also demonstrate that (e/i)tFUS\nwaveforms result in significant auditory activation (e.g., Granger causality and dSPM ESI\nanalysis). tEAS also does not induce significant M1 to deep brain Granger causality. This raises\nthe question if the observed source localizable response in tEAS stimulation could in part be\n1\nexplained by non-linear summation of tFUS-induced indirect subthreshold activation of M1\nthrough an auditory pathway (non localizable) and tDCS subthreshold depolarization (localizable).\nIn theory, only one of the stimulation modalities would need to be focal, for tEAS to result in a\nsuprathreshold localizable response. For example, also considering that the tEAS localization\nscores (e.g., averaged subject precision of reconstructed sources) are relatively low. It would be\ninteresting if the authors could discuss these points in the Discussion section.\nWe thank the reviewer for raising this excellent point. Our study design actually takes this into\naccount – for the tDCS conditions, a spatial sham ultrasound (ultrasound to right prefrontal cortex)\nis also applied. If the localizable tDCS with a non-localizable tFUS summation is what caused the\nfocal response, then we would also have seen the focal response on tDCS conditions.\nWe have added the following statement to our Discussion to clarify this point to future readers\n(page 15):\n“tDCS driving tEAS polarity also raises the question on whether the focality of tEAS’\nresponse could be due to tDCS locally subthreshold modulating whole brain induced tFUS\nauditory response. However, this is already accounted for in our experimental set up. For\nour tDCS conditions, we implemented a spatial tFUS control (the set up is shown in Figure\n1B). If the source response of tEAS was the result of tDCS interacting with the whole brain\nauditory response, the tDCS conditions would have resulted in focal activations as well.”\nIn the Discussion section, it is mentioned that the auditory evoked response might mask a\nsubthreshold localizable tFUS response. In my opinion, it would be valuable to include a small\nstudy with participants that are insonicated with ramped ultrasonic waveforms, designed to\nminimize auditory confounding effects. This could potentially allow the authors to evaluate the\nsmall subthreshold effects of tFUS with reduced auditory indirect effects.\nWe thank the reviewer for this suggestion. We conducted a follow up study with 6 human subjects\nin which we tested a ramped waveform. The results continued to show a lack of ROI response\nfrom tFUS-only, and a significantly increased ROI response from the smoothed tEAS condition.\nWe have added the following analysis / discussion to our manuscript (page 17):\n“We further investigated whether a smoothed ultrasound waveform could potentially\n‘unmask’ an ROI response. We tested a smoothed waveform (pulse duration: 3.25 ms, 1\nms tukey ramping, 250 Hz pulse-repetition frequency)1 in six human subjects\n(Supplemental Figure S10). All six subjects reported that they could not hear the\nultrasound. However, the smoothed tFUS by itself continued to lack increased ipsilateral\nROI source power, whereas the smoothed tEAS waveform did. We also note that the\nsmoothed tEAS waveform does appear overall less focal than the one that resulted from\nour rectangular pulses, with additional strong activations in the ipsilateral frontal/premotor\nregions. While the results of this study indicate that ultrasound PRF may not play a\nsubstantial role in the outcome of tEAS (Supplemental Figure S8), it may be that the\nultrasound pulse shape does. Future tEAS parameter optimization studies are warranted.”\nsmoothed ultrasound waveform (pulse duration: 3.25 ms, 1 ms tukey ramping, 250 Hz pulse-repetition\nfrequency) could reduce auditory activations and potentially ‘unmask’ target region specific source activity.\na) The subject average source maps are presented for the smoothed tFUS and tEAS conditions. b)\nSmoothed ultrasound continued to result in no increased ipsilateral source power, while smoothed tEAS\ndid. ns p > 0.05; * p < 0.05\nI would suggest to omit the computational section on the minimally modified Hodgkin-Huxley\nmodel or to move this section to the supplementary information. As mentioned by the authors, the\nmodelling results are conceptual, because of the approximations made in the computational\nmodel. E.g., a membrane model of the squid giant axon is used and ultrasound is included by a\nsingle mechanosensitive Piezo1 channel. I believe there are also some limitations in the modelling\nof Piezo1, that could be included in the Discussion. In particular, the authors model the open\nprobability of Piezo1 as proportional to the instantaneous pressure (eq. 7), based on reference\n[38] (Methods section “Hodgkin-Huxley modeling”). However, it is good to note that [38] does not\nuse ultrasound to activate Piezo1 channels. In my opinion, it is not expected that the Piezo1 open\nstate will follow the instantaneous acoustic pressure as in Fig. 6(e), because the opening time\nconstant of Piezo1 will be higher than the ultrasonic period. Another consideration is that Piezo1\nchannels are activated by membrane tension instead of by the harmonic pressure: the relationship\nbetween membrane tension and pressure is nonlinear and not instantaneous. For example,\nincrease in membrane tension is likely caused by radiation force induced tissue displacement,\ninstead of directly by the harmonic pressure. This also would mean that the Piezo1 open\nprobability would be non-oscillating, because of the steady (non-oscillating) driving radiation force.\nFor these reasons, I would recommend to move this section to the supplementary information,\nbecause of these limitations and because the conceptual results in this section have been shown\n3\nbefore in the computational literature (i.e., that multiple input stimuli will summate non-linearly in\nneuron models).\nWe thank the reviewer for their input and have moved the HH modeling to the supplementary\ninformation.\nMinor comments:\n- P. 4, “[...] and some studies go so far as to propose 100% of tFUS modulation is due to auditory\nactivation [21,22]”. Perhaps this sentence can be nuanced, because [21,22] mention in their\nDiscussion sections, that their results do not imply that tFUS has no direct neuromodulatory\neffects. Although these studies indeed demonstrate the importance of controlling for auditory\nconfounds in tFUS studies.\nWe thank the reviewer for their suggestion. We have reworded the sentence:\n“[…] and some studies could not detect an effect beyond auditory activation.”\n- Fig. 1(c): I would recommend to zoom out on the right subplot, to visualize also the FWHM in\nthe axial direction. Fig. 1(d): I recommend to adjust the tDCS stimulation plot, because this now\nseems to imply that alternating tDCS currents similar to ultrasound are applied.\nThank you for your suggestions. Please see our updated Figure 1 with a new tDCS representation\nand new tFUS scan showing the full FWHM below.\nFigure 1\n- Results, p. 14, “[...] with both 30 and 3,000 Hz PRFs” -> with both 40 and 3,000 Hz PRFs.\nconditions, but we had a typo earlier in the sentence where 30 should have been 40. The\ncorrected statement is below:\n“These parameters included 1,500 and 40 Hz PRFs with 30% duty cycle (DC), and,\ninitially, 10% and 60% DC with both 30 and 3,000 Hz PRFs.”\n- Methods, hydrophone measurements and transducer: please include details on the hydrophone\nmeasurements (Fig. 1(c)) and on the transducer (aperture, radius of curvature).\nWe’ve added in details for the hydrophone and transducer to the methods section:\n“Free water ultrasound calibration\nThis experiment used a 500 kHz f (fundamental frequency) single-element\n0\nultrasound transducer (1.5 inch focal length, 1 inch acoustic aperture diameter, 0.75 inch\nradius of curvature; Blatek Industries Inc., Boalsburg, PA, USA). To characterize the beam\nshape and determine needed input voltage for desired free water pressure, the transducer\nwas calibrated in a water tank. The water tank was filled with deionized water. Pressure\nmeasurements were recorded with an HNR-0500 hydrophone (Onda Corp., Sunnyvale,\nCA, USA).”\n- Methods, In silico transcranial focused ultrasound simulations: it would be good to also include\ndetails on the pressure field simulation (e.g., tissue parameters, mapping from Hounsfield units\n(pCT) to acoustic parameters, used solver (discretisation step, Courant number, etc.)).\nWe thank the reviewer for their suggestion. We have added the following information to our\nmethods:\n“The pCTs were manually segmented using iSeg (v3.10) into air (1.16 kg/m3 density, 343\nm/s speed of sound, 0.000399 MRayl acoustic impedance), bone (1910 kg/m3 density,\n3510 m/w speed of sound, 6.71 MRayl acoustic impedance), and soft tissue (1050 kg/m3\ndensity, 1550 m/s speed of sound, 1.61 MRayl acoustic impedance). Sim4Life solved the\nacoustic simulations with the Linear Acoustic Pressure Wave Equation and the ‘automatic’\ndiscretization settings (0.45 mm resolution, 0.3 mm maximum step, and a time step of 1.0\n/ (speed of sound * !1/dx² + 1/dy² + 1/dz² ).”\nmax\nIn this section, the notation could be confusing. E.g., the notation Isppa.3 is normally used to\ndenote the derated SPPA intensity with homogeneous attenuation of 0.3 dB/(cm-MHz). However,\nin this section more realistic heterogeneous attenuation is used by Sim4life simulations of the\nskull and brain tissue. For this reason, the simulated attenuation is likely not equal to 0.3 dB/(cm-\nMHz), as indicated by the subscript.\nThank you for raising this possible source of confusion. We have replaced “the .3” notation with\n“.T” for “target” throughout the manuscript. For example, Isppa.3 is now Isppa.T.\n- Methods, p. 28: “Excitatory tDCS consisted of center-cathode current, while center-anode\ncurrent was defined as inhibitory tDCS current”. I am expecting this is the other way around.\nThank you for your comment. We have fixed this typo.\n5\n- Minor textual comments: (abstract) affect -> effect. (Fig. 2(b), caption): [...] right subplot [...] left\nsubplot -> other way around. (Fig. S1, caption) Ostu -> Otsu. (Fig. 4(b.ii), caption): PRF -> PFC.\nThank you for your comments. We have fixed these typos.\n- Figures: in some figures (e.g., Fig 6d) the readability could be improved by increasing the font\nsize of the labels and ticks. Fig. 1(c): please include a colorbar for this subplot.\nThank you for your suggestions. We have remade our figures for increased readability. Please\nfind the updated versions in the manuscript and supplemental files. For Figure 1C, a color bar is\nincluded to the right.\nReviewer #2 (Remarks to the Author):\nA main methodological issue is the dose titration of tDCS. The dosage was set at 0.1 mA below\nskin perception threshold, up to a maximum of 2 mA (0.7-2 mA). This is an absolutely unusual,\ncurrents finally entering the brain vary almost 3 fold. They should of course correlate with intensity\nbut the individual skin perception with individual current flow not. Current flow calculations e.g.\nwith SIMNibs will be appreciated.\nWe thank the reviewer for this comment. We respectfully point towards other examples of highly\nvaried transcranial electric stimulation titration. Pilloni et al used between 1.5 or 2.5 mA based on\nsubject tolerability2. Simpson et al delivered between 1.0 – 1.5 mA based on subject tolerability3.\nLafon et al delivered currents varying between 0.3 and 2 mA4. We also note there have also been\nnumerous reports of no meaningful differences in outcomes from using different tDCS\namperages5–7.\nPerhaps more important, our study design is a cross-over experimental design, which looks at\nchanges in the brain response within subjects. For each subject, the tDCS dose was held\nconstant. Our results show, despite varying levels of initial scalp current, there is a significant\nincrease in brain response for the tEAS conditions compared to others.\nNevertheless, we have run the requested simulations to estimate cortical current density. They\nare provided in Supplemental Table T1. Analysis of both scalp-level current and cortical current\ndensity do not show current intensity to be a meaningful (linear) predictor of the subjects’ tEAS\nresponse levels (all R2 < 0.05, all p > 0.05), which is consistent with the past studies reporting\nlittle-to-no differences from currents in our range5–7.\nintensity. The tEAS cortical responses were fit with an affine function of tDCS scalp current and the cortical\ncurrent density (estimated with SimNIBS). Neither of these hold meaningful predictive power for the tEAS\nresponses (both p > 0.05, both R2 < 0.05).\nWe also performed a follow-up experiment with a 7-day washout out between etFUS and etEAS\nconditions. As part of this study, we used a consistent 0.7 mA of tDCS in the tEAS conditions for\nall subjects. The results were consistent with the main study, in which tEAS led to strong ROI\nactivity while tFUS alone did not (Supplemental Figure S12).\nWe have added discussion the following manuscript (page 20):\n“We note the varied level of scalp current applied to subjects during our\nexperiments. There are, broadly, two approaches to transcranial electric stimulation dose\ndetermination for human experiments. One is to normalize the dose for all subjects (either\nat the scalp- or the cortical-level using simulations). The other is to use a varied dose\nacross subjects2–4. We opted for the latter approach in our study for two reasons. The first\nreason is that we wanted to prioritize subject comfort and safety. tDCS is generally\nconsidered safe, and the scalp sensations of tingling, itching, and/or burning are often\nreported as transient8. However, it is possible for tDCS to induce skin lesions after only a\nsingle session9. Since our participants were healthy human subjects, we wanted to avoid\nthis possibility. The second reason is that skin perceptions are the result of activated\nsensory neurons10, which can contaminate the resting-state EEG recordings, both from\nthe firing of the scalp (i.e., non-cortical) neurons, but also from the activated pain circuits\nwithin the brain itself. We wished to avoid such possible contamination.\nSince we used the same current density for each condition within a subject, the\nvaried current density across subjects is not a major concern. This is because our cross-\nover study design looks at the changes in brain response across conditions for subjects.\nOur results show a significant increase in ROI activity for tEAS conditions, regardless of\ninitial tDCS dosage. We did conduct further analysis to determine if scalp or cortical\n(estimated with SIMNibs simulations11) tDCS dose (Supplemental Table T1) was a\nmeaningful predictor of ROI response. Our results indicate tDCS dose, at the between-\nsubjects level, is not a meaningful predictor of tEAS activation (Supplemental Figure S7;\nR2 < 0.05, p < 0.05). This is consistent with other studies that have reported little-to-no\ndifferences in the outcomes of different tDCS doses5–7, and further supports that our\nsubject-specific dosage should not be viewed as a study limitation. In a follow-up study,\n7\nwe used a constant 0.7 mA tDCS for all subjects and found a consistent activation pattern\nacross subjects for the tEAS condition (Supplemental Figure S12).”\nSupplemental Figure S12: Results of a 7-day washout experiment and constant 0.7 mA scalp current are\nconsistent with the main study. We repeated our etEAS and etFUS resting state conditions in 8 subjects,\nthis time with a 7-day washout period between the two conditions. For etEAS, we used a consistent 0.7 mA\nof current across all subjects. We also tested each condition twice; once before and once after a 5 minute\nbreak. a) The ROI activity for etFUS was not significantly altered pre/post break (two-tailed permutation t-\ntest; p > 0.05), and Bayes Factor analysis reveals the activities to be robustly statistically similar for a range\nof scaling factors. b) The ROI activity for the etEAS condition was also statistically similar before and after\nthe 5 minute break period. c) The induced sources are consistent with those from the main study.\nFor each condition the inter-trial interval was 2 seconds with up to a 20% (0.4 s) random jitter, so\neach condition lasting 6.6-8min. A five-minute break was taken between each condition to allow\nfor the subjects to stretch and let any cumulative effects of neuromodulation dissipate. This is a\nrather short interval neglecting results in the literature suggesting e.g that 3 min or 20 min breaks\nboost plasticity and influence hereby succeeding stimulation results. The authors are of course\nwelcome to come up with references that this is not of relevance here.\nWe thank the reviewer for the comment. We removed the comment about letting neuromodulation\neffects dissipate from our methods:\n8\n“For each condition the inter-trial interval was 2 seconds with up to a 20% (0.4 s) random\njitter, so each condition lasting 6.6-8min. A five-minute break was taken between each\ncondition to allow for the subjects to stretch.”\nWe have added the following discussion to our paper to indicate why our experimental design\nhelps to mitigate confounding sequential effects. In our new 7-day washout follow up study, we\nalso tested each condition twice before/after a 5 minute break. Our results found ROI activity pre\nand post 5-minute break to be statistically similar from each other, further supporting that our\nresults were not confounded from these breaks (page 21).\nWe randomized the order of our experiments across subjects, which is also known\nas counterbalancing. While it is possible that conditions tested within a single session may\nexperience a carry-over or sequence-based effect, counterbalancing, at the group-level,\nhelps ensure that these effects were randomly distributed across conditions and should\nnot confound the main results12. To further validate our main findings, we conducted an\nadditional follow-up study, in which we repeated our etEAS and etFUS experiments with\na 7-day wash out period (8 participants; split 50/50 for which condition was first). On the\nday the condition was tested, we tested it twice: once ‘pre’ 5-minute break and once ‘post’\nbreak. The ROI activity for each condition was statistically similar before and after the\nbreak (Supplemental Figure S12a-b), suggesting that, even without the counterbalancing\nof the main study, the results may not have suffered from any sequential-based confounds.\nThe source localizability of the experimental conditions were also consistent with the main\nstudy, where etEAS yielded strong targeting-location specific activity, and etFUS did not\n(Supplemental Figure S12c).\nSupplemental Figure S12: Results of a 7-day washout experiment and constant 0.7 mA scalp current are\nconsistent with the main study. We repeated our etEAS and etFUS resting state conditions in 8 subjects,\nthis time with a 7-day washout period between the two conditions. For etEAS, we used a consistent 0.7 mA\nof current across all subjects. We also tested each condition twice; once before and once after a 5 minute\nbreak. a) The ROI activity for etFUS was not significantly altered pre/post break (two-tailed permutation t-\ntest; p > 0.05), and Bayes Factor analysis reveals the activities to be robustly statistically similar for a range\nof scaling factors. b) The ROI activity for the etEAS condition was also statistically similar before and after\nthe 5 minute break period. c) The induced sources are consistent with those from the main study.\n3 kHz PRF tFUS under the right prefrontal cortex share a striking resemblance to tFUS applied\nunder left motor cortex area (Left M1) with 10 – 63, 20 – 40, and 80 – 100 ms responses. Both\ncause a hemisphere-symmetric response, despite tFUS only being applied to one side. This\nneeds explanation since seemingly this does not support evidence of a target-location specific\neffect. Also, Granger causality analysis of both itFUS and etFUS conditions shows significant\nincreases in information flow from primary auditory cortex to the deep brain, and no changes in\nnet information flow from the targeted ROI to the deep brain.\nThank you for your comment. We apologize that we were not clearer in our explanation of this\nphenomenon; this is critical evidence of tFUS’ subthreshold location-specific effect. In other\nwords, when tFUS is applied by itself, we only see the whole brain auditory-based response,\nregardless of where tFUS is targeted. We have reworded our discussion to make this more clear.\nPlease see our revised paragraph below (bold added for emphasis) (page 14):\n“Excitatory (3 kHz PRF) tFUS targeted to the left motor region, as part of etFUS,\netEAS, and etDCS (which included a tFUS spatial control), induced distinct ERPs at\napproximately 30, 90, and 170 ms post stimulation onset (Figure 2a, Figure 4a). This is\nconsistent with a previous study that identified V1-targeted tFUS induced EEG peaks,\nthough their reported peak times were labeled as 55 ms, 100 ms, and 150 ms post\nsonication. However, when we compared these ERPs to those induced by applying tFUS\nto an entirely different brain region (right hemisphere prefrontal cortex), the resultant\nwaveforms did not just lack statistical differences from each other—they were found to be\nstatistically similar through Bayes Factor analysis (Figure 4b). These results provide\ncritical evidence that location-specific effects of tFUS are subthreshold; by itself,\ntFUS produces the same responses regardless of where it is targeted.\nOf course, these shared responses are time-locked event related potentials.\nTo put it another way, tFUS does induce a suprathreshold response. However, since\nthe topology of these ERPs do not differ across targeting locations, they can be\nattributed to a whole brain auditory response. Indeed, the 3 kHz PRF EEG topographic\nspatial distributions of the 90 and 170 ms ERPs do share visual resemblance to the\npreviously reported auditory induced N1 and P2 ERPs. Granger causality investigations,\nwhich revealed significantly increased A1 to deep brain information flow for both 30 Hz\nand 3 kHz tFUS conditions (Figure 4c), provide additional support that the ERPs are\nauditory related. Nevertheless, the present results that combining tFUS with tDCS led to\nspatially specific responses, when neither could do so individually (Figure 3), as well as\nitFUS dissociating the spectral connectivity between the ROI and the deep brain\n(Supplemental Figure S5), indicate that tFUS does have targeted-location specific effects.”\nEven increased tFUS parameter (including duty cycle; higher pressure; sonication duration)\ncontinued to yield no changes in net information flow from the targeted ROI to the deep brain.\nTranscranial electro-acoustic stimulation（tEAS）under the left M1 produces a hemisphere\nspecific exogenous ERPs（10-63ms，which is claimed to be driven by tDCS polarity, not the\nexcitatory or inhibitory parameters of tFUS (but see tDCS comments above).\nconditions are all in support of a subthreshold modulatory effect. It is only when tFUS is combined\nwith tDCS that we start to see location-specific information. We have added the following\nclarification to our discussion section (page 12):\n“Analysis of the other parameters continued to show a lack of sonication-location specific\nsuprathreshold responses, providing further support that tFUS’ location-specific\neffect is subthreshold (Figure 5).”\nWe have also added to our discussion on tDCS based on the above tDCS comments / analysis\n(bold added for emphasis) (page 12):\n“Not only did itEAS and etEAS induce significantly higher source localization metrics and\nsource power than the other tested conditions, but they also led to a significant change in\nROI polarity (Figure 2d). While this polarity change is evidence of differing\nelectrophysiological effects, a follow-up study indicates that this change in response is\ndriven from tDCS polarity and not tFUS PRF (Supplemental Figure S7). However, further\nanalysis shows no linear relationship between the applied tDCS dose and tEAS\nresponse magnitude (Supplemental Figure S7d-e), nor between tFUS intensity and\ntEAS response (Supplemental Figure S7c). This points to a non-linear response\nfrom a combined transcranial electro-acoustic effect.”\nAnodal tDCS combined with 30Hz PRF tFUS (inhibitory) and 3,000 Hz (excitatory) PRF tFUS\nboth led stronger ipsilateral than contralateral responses. But again, here tEAS conditions were\nalso found to induce significant increases in auditory to deep brain connectivity without an effect\non the net flow of information from the targeted ROI to deep brain.\nThank you for your comment. We have added the following statement to our discussion regarding\ntEAS and Granger causality (page 14-15):\n“We note that these auditory effects persist within the tEAS conditions (Supplemental\nFigure S4). This is unsurprising, as we would have no reason to think the electro-acoustic\neffect at M1 would cancel out auditory pathways. However, what is surprising is the\ncontinued lack of ROI to deep brain change in net flow of information. M1 is connected to\ndeep brain structures13, so on one hand it would be expected that increased M1 activity\nwould lead to increased deep brain activity, and this would be reflected as an increased\nnet flow of information. However, this was not our finding. One possible explanation for\nthis phenomena is that M1 neuron connections with the deep brain are dependent on cell\ntype13. Prior work has demonstrated tFUS’ parameter choices can alter cell-type\nselectivity14. Our tFUS parameters may not have been optimal for eliciting M1 to deep\nbrain connections. Future research on tEAS connectivity changes (or lack thereof) are\nwarranted.”\nWhy does tDCS applied under the left M1 synchronize the tFUS under the right frontal without\ncausing local changes; instead, it still results in a hemisphere-symmetric response.\nOur tDCS conditions are combined with a spatial-sham tFUS (Figure 1b). These hemisphere-\nsymmetric responses are the continued results of auditory based effects from tFUS. We have\nclarified this point in the discussion (bold added for emphasis):\n“Excitatory (3 kHz PRF) tFUS targeted to the left motor region, as part of etFUS, etEAS,\nand etDCS (which included a tFUS spatial control), induced distinct ERPs at\napproximately 30, 90, and 170 ms post stimulation onset (Figure 2a, Figure 4a).”\nIs the claim that tEAS is more than a linear sum of its parts really supported by the summation of\nthe source-space tFUS and tDCS conditions which in turn did not result in increased concordance\nwith the ROI, nor to significant increases in ipsilateral under contralateral ROI source power.\nThank you for your comments. We have included linear modeling of tEAS source-space response\nas a function of tDCS / tFUS intensities (Supplemental Figure S7), which provide additional\nevidence of a non-linear response. We added the following line to the results section (page 10):\n“We also modeled the tEAS ROI response as function of tDCS scalp current, cortical\ncurrent, and tFUS intensity and found none of these component to be meaningfully\npredictive (Supplemental Figure S7c-e).”\nSupplemental Figure S7: The transcranial electro-acoustic effect is more than just the summation\nof independent electric and acoustic modulation. a) Source-imaging data for itFUS and itDCS, as well\nas etFUS and tDCS, were added together for each subject, and then averaged across subjects.\nVisualizations continue to show strong off-targeted sources. b) The average ipsilateral ROI activity of the\nadded sources continues to be not significantly greater than the contralateral ROI for both the summed\nexcitatory and summed inhibitory conditions (p > 0.05). c – e) Mean tEAS-induced ROI activity is not well\n12\npredicted by tFUS or tDCS intensity. The tEAS cortical responses were fit with an affine function of c)\ncortical tFUS intensity, d) current, and e) the cortical current density (estimated with SimNIBS). None of the\nindividual components hold meaningful predictive power for the tEAS responses (all p > 0.05).\nSo, in summary two main concerns regarding the tDCS intensity and intervals between different\ntypes of stimulation are worrying.\nThank you for your feedback. We have addressed these concerns with our updated analysis,\ndiscussion points, and follow-up study.\nMinor:\n„subthreshold (meaning it will not induce neural firing on its own)“ better „modulating sponteanous\nfiring without stimulation time locked additional spikes“\nThank you for your suggestion. We have reworded the statement (bold added for emphasis):\n“A modern, philosophical descendant of these methods, transcranial direct current\nstimulation (tDCS), applies subthreshold (meaning it will modulate spontaneous firing,\nbut will not induce stimulation time-locked spikes on its own) current across brain\ntissue using non-invasive scalp electrodes.”\n„but one prevalent theory is the ultrasound pressure waves open mechanosensitive ion channels“\nInstead „but one prevalent theory is that the ultrasound pressure waves open mechanosensitive\nion channels“\nThank you for your suggestion. We have implemented this change.\n„Electroencephalogram (EEG) based recordings, which directly measure electrical activity without\nthe need for magnetic fields, may offer more insight into the true effects of tFUS as a sole\nstimulus.„ unclear needs to be broken down to tangential MEG sensitivity.\nThank you for your comment. We have reworded this sentence to clarify we are mentioning the\nmagnetic field as contrast to fMRI, not MEG (bold added for emphasis).\n“Electroencephalogram (EEG) based recordings, which directly measure electrical\nactivity and do not require a magnetic resonance environment, may offer more\ninsight into the true effects of tFUS as a sole stimulus.”\n„In follow-up studies, we broaden our tFUS parameter scope. We record and analyze\nsimultaneous whole-scalp EEG recordings and image their corresponding cortical sources to\ncharacterize targeted-location specific, or lack-thereof, responses.“ Here an immediate switch\nfrom stimulation to recording occurs with a logical brake“\nThank you for your comment. We have added a clause to make the transition smoother (bold\nadded for emphasis).\n“In follow-up studies, we broaden our tFUS parameter scope. For all tFUS experiments,\nwe record and analyze simultaneous whole-scalp EEG recordings and image their\ncorresponding cortical sources to characterize targeted-location specific, or lack-thereof,\nresponses.”\nbrain region, there is a lack of agreement for whether tFUS is modulating neural excitability or\nstimulating the neurons on its own.“ are not particularly logic. The first half refers to topography\nthe second to neurophysiology. So not mutually exclusive. This occurs throughout the manuscript,\nsee e.g. „Our data provide strong evidence that, while there are prominent auditory effects, the\napplied tFUS has location-specific subthreshold modulatory effects on the human cortex.“\nSentences like „While the clusters from permutation cluster tests are not guaranteed to be\nsignificant for their entire extent, we did consider the overlapping windows (10 – 63 ms) for\nsignificant itEAS and etEAS clusters to be interesting enough due to their shared presence to\nwarrant use in our further analysis.“ ambiguous.\nThank you for your comments. We have revised the sentences in the following ways (bold added\nfor emphasis):\n“While the effect of tFUS is commonly considered to be based on the targeted brain\nregion, there is a lack of agreement on the magnitude of effect. Specifically, there are\nunanswered questions on whether tFUS is modulating neural excitability or stimulating\nthe neurons on its own.”\n“Our data provide strong evidence that, while tFUS produces prominent whole brain\nauditory related activations, it also induces location-specific subthreshold modulatory\neffects.”\n“While the clusters from permutation cluster tests are not guaranteed to be significant for\ntheir entire extent, we did consider shared clusters from both itEAS and etEAS to\nwarrant further analysis. Thus, the remainder of our work will focus on the shared\nwindow of 10 – 63 ms.”\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank reviewer #3 for their effort and time co-reviewing our manuscript.\nReference:\n1. Johnstone, A., Nandi, T., Martin, E., Bestmann, S., Stagg, C. & Treeby, B. A range of pulses\ncommonly used for human transcranial ultrasound stimulation are clearly audible. Brain\nStimul 14, 1353–1355 (2021).\n2. Pilloni, G., Vogel-Eyny, A., Lustberg, M., Best, P., Malik, M., Walton-Masters, L., George, A.,\nMirza, I., Zhovtis, L., Datta, A., Bikson, M., Krupp, L. & Charvet, L. Tolerability and feasibility\nof at-home remotely supervised transcranial direct current stimulation (RS-tDCS): Single-\ncenter evidence from 6,779 sessions. Brain Stimulation: Basic, Translational, and Clinical\nResearch in Neuromodulation 15, 707–716 (2022).\n3. Simpson, E. A., Saiote, C., Sutter, E., Lench, D. H., Ikonomidou, C., Villegas, M. A. & Gillick,\nB. T. Remotely monitored transcranial direct current stimulation in pediatric cerebral palsy:\nopen label trial protocol. BMC Pediatrics 22, 566 (2022).\n4. Lafon, B., Henin, S., Huang, Y., Friedman, D., Melloni, L., Thesen, T., Doyle, W., Buzsáki, G.,\nDevinsky, O., Parra, L. C. & A. Liu, A. Low frequency transcranial electrical stimulation does\nnot entrain sleep rhythms measured by human intracranial recordings. Nat Commun 8, 1199\n(2017).\n5. Agboada, D., Mosayebi Samani, M., Jamil, A., Kuo, M.-F. & Nitsche, M. A. Expanding the\nparameter space of anodal transcranial direct current stimulation of the primary motor cortex.\nSci Rep 9, 18185 (2019).\n6. Kidgell, D. J., Daly, R. M., Young, K., Lum, J., Tooley, G., Jaberzadeh, S., Zoghi, M. & Pearce,\nA. J. Different Current Intensities of Anodal Transcranial Direct Current Stimulation Do Not\nDifferentially Modulate Motor Cortex Plasticity. Neural Plasticity 2013, 603502 (2013).\n7. Jamil, A., Batsikadze, G., Kuo, H.-I., Labruna, L., Hasan, A., Paulus, W. & Nitsche, M. A.\nSystematic evaluation of the impact of stimulation intensity on neuroplastic after-effects\ninduced by transcranial direct current stimulation. The Journal of Physiology 595, 1273–1288\n(2017).\n8. Poreisz, C., Boros, K., Antal, A. & Paulus, W. Safety aspects of transcranial direct current\nstimulation concerning healthy subjects and patients. Brain Research Bulletin 72, 208–214\n(2007).\n9. Wang, J., Wei, Y., Wen, J. & Li, X. Skin Burn After Single Session of Transcranial Direct\nCurrent Stimulation (tDCS). Brain Stimulation 8, 165–166 (2015).\n10. van Boekholdt, L., Kerstens, S., Khatoun, A., Asamoah, B. & Mc Laughlin, M. tDCS peripheral\nnerve stimulation: a neglected mode of action? Mol Psychiatry 26, 456–461 (2021).\n11. Thielscher, A., Antunes, A. & Saturnino, G. B. Field modeling for transcranial magnetic\nstimulation: A useful tool to understand the physiological effects of TMS? in 2015 37th Annual\nInternational Conference of the IEEE Engineering in Medicine and Biology Society (EMBC)\n222–225 (2015). doi:10.1109/EMBC.2015.7318340.\n12. Corriero, E. F. Counterbalancing. in The SAGE Encyclopedia of Communication Research\nMethods 278–281 (SAGE Publications, Inc, 2017). doi:10.4135/9781483381411.\n13. McColgan, P., Joubert, J., Tabrizi, S. J. & Rees, G. The human motor cortex microcircuit:\ninsights for neurodegenerative disease. Nat Rev Neurosci 21, 401–415 (2020).\n14. Yu, K., Niu, X., Krook-Magnuson, E. & He, B. Intrinsic functional neuron-type selectivity of\ntranscranial focused ultrasound neuromodulation. Nat Commun 12, 2519 (2021).\nWe thank the reviewers for their addi1onal comments. We have responded to all of review comments\nraised and revise the manuscript further. Below, you will find our detailed responses to each reviewer\ncomment in BLUE, with the original reviewer comments in BLACK. Revised or added text in the manuscript\n/ supplementary informa1on is highlighted in RED.\nReviewer #1 (Remarks to the Author):\nThe authors have significantly improved the manuscript. In par1cular, the revised manuscript now includes\ntwo addi1onal human experiments, addressing possible carry-over effects (7 day wash-out experiment)\nand auditory confounds (ramped protocols). I would like to thank the authors for their work.\nWe are very grateful to the reviewer for favorable and construc1ve comments.\nI have one remaining minor comment. The authors have moved the Hodgkin-Huxley modelling results to\nthe supplementary informa1on. Here, it is men1oned: “Outside of pressure ranges where the channel has\na 0 or 1 probability of being open, the open probability (Popen) scales approximately linearly with applied\npressure [38]. As a result, Popen was modeled directly as the ultrasound waveform (clipped at 0 pressure\nto avoid nega1ve probabili1es), scaled to have some peak probability less than 1.”\nHowever, in my opinion, this cannot be concluded from [38], because in this publica1on ultrasound was\nnot used. In par1cular, Piezo1 opening is mediated by membrane tension instead of directly by ultrasonic\npressure. The rela1on between membrane tension and ultrasonic pressure is nonlinear and non-\ninstantaneous, and is also determined by acous1c radia1on force. For this reason, I would recommend to\nnuance or clarify this statement in the Supplementary Informa1on. This is a minor comment, because the\nHodgkin-Huxley modelling is intended by the authors to be conceptual.\nWe thank the reviewer for their excellent sugges1on and have added nuance into the statement in the\nSupplementary Informa1on:\n“Outside of pressure ranges where the channel has a 0 or 1 probability of being open, the open\nprobability (P ) scales approximately linearly with applied pressure (we note here, though, that\nopen\nreported measurements1 were using membrane tension and not ultrasound pressure. In reality,\nthe two may not correspond to a 1:1 conversion).”\nReviewer #2 (Remarks to the Author):\nI have now reread the whole manuscript with all reviewers comments and feel increasingly uncomfortable.\nAs I understand the main conclusion is based on the results of Fig. 2D represen1ng a highly digested and\ncompressed summary. The contact to original data is lost here.\nWe thank the Reviewer for their 1me and effort rereading the manuscript. However, we think there\nappears to be a misunderstanding as to the main conclusions of the study. The main results flow logically\nfrom the original data. We iden1fy significant permuta1on-cluster test results from the EEG data (Figure\n2B), then analyze those further. Figure 2D is showcasing that the polarity for i/etEAS is significantly\nchanged in those clusters as a result of inhibitory/excitatory parameters, which is important, but not the\nmain conclusion. The main part of the paper, as the 1tle states, is tEAS induces source localizable cor1cal\n1\nac1va1on, but tFUS or tDCS does not. From the permuta1on cluster results of Figure 2B, we perform\nsource imaging (Figure 3), and inves1gate shared poten1als across ROI targeted tFUS and spa1al controls\nto elucidate underlying evoked auditory effects (Figure 4).\nThe jus1fica1on of the paper in the abstract reads as follows: “In none of the\nexogenous event related poten1als did tFUS or tDCS result in loca1on-specific ac1va1ons“. This may\nindeed by true for special methodology used here, does however not apply for general literature.\nWe thank the Reviewer for their comment, though are a bit confused by what is meant by “general\nliterature”. tDCS (/tES) is widely accepted to be a subthreshold modulator. This is supported by a number\nof ac1ve tES labs 2–4.\nFrom San-Juan 20212:\n“Transcranial direct current s1mula1on (tDCS) is a noninvasive method for cor1cal excitability\nmodula1on by subthreshold membrane depolariza1on or hyperpolariza1on”\nFrom Antal et al 20253 :\n“The low intensity tES modulates brain ac1vity primarily through subthreshold neuronal\npolariza1on. tES induces electric fields that shie the membrane poten1als without directly causing\nac1on poten1als”\nFrom Kricheldorf et al 20224:\n“Therefore, tES can only modulate brain ac1vity and is considered subthreshold, i.e., it does not\ndirectly elicit neuronal ac1on poten1als”\nWe have added these addi1onal cita1ons to our Introduc1on.\n“transcranial direct current s1mula1on (tDCS), applies subthreshold (meaning it will modulate\nspontaneous firing, but will not induce s1mula1on 1me-locked spikes on its own) current across\nbrain 1ssue using non-invasive scalp electrodes2–5”\nThat suggests the “general literature” issue is with respect to tFUS. However, there is far from a general\nconsensus on loca1on-specific tFUS ac1va1ons in the field, as we discuss at length in our introduc1on:\n“However, there is a cri1cal lack of understanding when it comes to tFUS’ effect on the human\nbrain. The bulk of human tFUS studies pair it with a secondary s1mula1on (such as tac1le\nvibra1ons6,7, transcranial magne1c s1mula1on (TMS) pulses8, or visual s1muli9), which point\ntoward, but do not confirm, a subthreshold modula1on-based mechanism of ac1on. Other studies\ndirectly looked into tFUS’ ability to elicit percepts as the sole s1mulus, but their results pointed\ntowards, at best, an inconsistent ability to do so—a study inves1ga1ng S1 ac1va1on only elicited\npercepts in 54% of trials10, and a study inves1ga1ng V1 s1mula1on reported visual percepts in\nonly 11 of 19 par1cipants11. Beyond even the ques1on of suprathreshold s1mula1on vs.\nsubthreshold modula1on, some studies report that the effects of tFUS may not actually be due to\nthe pressure wave, but rather auditory conduc1on. tFUS, typically, is not applied as one\ncon1nuous wave, but instead pulsed at some lower frequency (pulse repe11on frequency; PRF;\nFigure 1d). This PRF makes a beeping noise, which is associated with ac1ng auditory\npathways12,13, and some studies could not detect an effect beyond auditory ac1va1on14,15. In\nhumans, it was reported that ac1vely playing a tone over the dura1on of tFUS sonica1on led to\nno significant differences in EEG between sham and ac1ve condi1ons16.\nif it evokes loca1on-specific neuronal responses—and, if it does have a loca1on-specific effect,\nwhether that effect is subthreshold or suprathreshold.”\nThis is the key mo1va1on for our paper.\nWe would like to clarify that the methodology we used for tEAS is indeed “special” and represents a novel\noriginal contribu1on. This study is not, nor does it claim to be, a tradi1onal tDCS study looking at aeer-effects from prolonged s1mula1on. Instead, this study uses the online effects of subthreshold direct\ncurrent to inves1gate possible subthreshold effects of tFUS. This is a special and novel approach to bener\nunderstand tFUS on the human brain. We have added to the manuscript Discussion sec1on to further\nclarify this point.\n“We would also like to emphasize this is not a tradi1onal tDCS study looking at aeer-effects from\nprolonged s1mula1on17–19. Instead, this study uses the online effects of subthreshold direct\ncurrent to inves1gate possible subthreshold effects of tFUS. This approach has not been done\nbefore. Due to the unpredictability of tDCS dose-response rela1onships3,17–19, it is typically\nrecommended to choose dosing levels based on prior research and the intended neurological\neffect3. However, being the first study to inves1gate simultaneous tDCS and tFUS, we did not have\nsuch literature to draw upon. Instead, we priori1zed a dose that would not ac1vate sensory\npathways20 by inducing itching, burning, and 1ngling sensa1ons21. Such skin percep1ons from\ntDCS would contaminate and undermine our inves1ga1ons of tFUS.”\nOthers issues now concern me. I did not find anything about earplugging or even double ear-plugging\nwith noise cancella1on in order to prevent any influence of acous1c evoked poten1als by ultrasound.\nWe thank the Reviewer for acknowledging possible influences of acous1c ar1facts from ultrasound, and\nfor their sugges1on to use earplugs. We would like to clarify that earplugs are not a valid way to control\nfor auditory ar1facts, as the ultrasound noise is transmined through bone conduc1on from scalp contact,\nwhich would not be stopped with earplugs. To account for the influence of acous1c evoked poten1als, we\nused a spa1al sham ultrasound control. The user experiences the same bone-conducted auditory noise,\nbut the pressure wave is focused to a different brain loca1on. The Interna;onal Transcranial Ultrasonic\nS;mula;on Safety and Standards (ITRUSST) consor;um considers the spa;al control (referred to as the\n(in)ac;ve control) as a highly effec;ve control condi;on (their Figure 19)22. Comparisons with these\nspa1al controls are what led to the conclusion of tFUS-evoked whole brain auditory responses.\nWe also inves1gated inaudible ultrasound waves (30 kHz PRF (Supplemental Figure S3)) and smoothed\ntFUS waveforms (Supplemental Figure S10), and have included them in the discussion as well. Using\ninaudible waveforms did not result in an ROI specific response.\nDid not understand what finally was done by Simbnibs? Taking bone densi1es into account?\nWe thank the Reviewer for their comment. We have included addi1onal details about the SimNIBS\nsimula1ons in our Supplemental Methods sec1on:\nSimNIBS Simula;on\nSimNIBS22,23. Subject MRIs were automa1cally segmented using the Complete Head Anatomy\nReconstruc1on Method (CHARM) into different 1ssue types. The simula1ons used the default\nSimNIBS conduc1vity values (e.g., white maner: 0.126 S/m, gray maner: 0.275 S/m, bone: 0.01\nS/m, scalp: 0.465 S/m). Electrodes were modeled as 12 mm diameter cylinders with 1 mm height,\nbased on our HD-tDCS electrodes. The tDCS current intensity was based on the subject’s specific\nexperimental scalp current (Supplemental Table T1). The resultant maximum cor1cal current\ndensity was considered for further analysis (Supplemental Table T1).\nComments to some references used:\nNote: where appropriate, we have put the author name next to the commented reference number, as the\nreference numbering of the R1 response document is not aligned with the reference ordering of this\ndocument.\nThe Pilloni et al ar1cle summarized only the safety and tolerability of mul1ple applica1ons of different\nintensi1es of tDCS at home. No cor1cal s1mula1on effects were included. A pure safety study. The Simpson\net al study is no study but a study protocol for a future study. Thus, this sentence “Simpson et al delivered\nbetween 1.0 – 1.5 mA based on subject tolerability” is more than irrita1ng in the present context for a\nstudy which have not been performed.\nThe Pilloni paper is a paper on safety and tolerability, and they chose subject specific doses. If anything,\nthe fact that their focus is on safety provides support for varied tDCS dosage in our study, where our study\ndesign aims to inves1gate res1ng state brain response to ultrasound s1mula1on without concurrent skin\npercep1on. The Simpson paper, as is pointed out, is a protocol paper. We cited both these papers only in\nsupport of our methods using a varied dose level in response to the Reviewer’s previous comment:\n“A main methodological issue is the dose 1tra1on of tDCS. The dosage was set at 0.1 mA below\nskin percep1on threshold, up to a maximum of 2 mA (0.7-2 mA). This is an absolutely unusual\n[…]”\nWe acknowledge that the tense used should not have been past tense, but rather a future tense indica1ve\nof their proposed methods. We apologize for this oversight. However, in the manuscript itself, we make\nno claims of past/future tense. Instead, we only cite this methods paper in support of subject-specific\ndosage (again, making no men1on of past/future tense). We have bolded this sentence for emphasis. We\nhave also slightly modified the discussion points to acknowledge many studies use fixed doses across\nsubjects, as well as clarifying our ra1onale.\n“We note the varied level of scalp current applied to subjects during our experiments. For tDCS\nexperiments, the dose is most commonly normalized for all subjects (either at the scalp- or the\ncor1cal-level using simula1ons). However, the dose can also be varied across subjects24–26. We\nopted for the laner approach in our study. tDCS is generally considered safe, but can induce\ntransient sensa1ons of 1ngling, itching, and/or burning on the scalp21. The primary goal of our\nstudy was to u1lize tDCS to draw out possible subthreshold effects of tFUS in a res1ng state. Skin\npercep1ons are the result of ac1vated sensory neurons20, which can contaminate the res1ng-state\nEEG recordings, both from the firing of the scalp (i.e., non-cor1cal) neurons, but also from the\nac1vated pain circuits within the brain itself. We wished to avoid such possible contamina1on.\nBeyond this, we also note it is possible for tDCS to induce skin lesions aeer only a single session27.\nSince our par1cipants were healthy human subjects, we wanted to avoid this possibility.”\nwhich can of course vary but cannot be cited as being suppor1ve in the present context.\nThe Lafon study is cited as another example of a research group that used varying levels of transcranial\nelectric s1mula1on dose across subjects in response to the Reviewer’s previous comment:\n“A main methodological issue is the dose 1tra1on of tDCS. The dosage was set at 0.1 mA below\nskin percep1on threshold, up to a maximum of 2 mA (0.7-2 mA). This is an absolutely unusual\n[…]”\nWe cited the Lafon paper only in support of our methodological choice to use a varying dose. The\nreworded statement is below:\n“However, the dose can also be varied across subjects24–26.”\nThe fact that Lafon’s results were “essen1ally nega1ve” does not change the fact they took a subject-\nvarying dose approach. This reference further supports our choice of using varied dose for our use-case,\nas the researchers chose a per-subject maximum dose par1ally based on pa1ent sensa1on since they\nrequired their par1cipants to be asleep for the s1mula1on. Our study used awake par1cipants, but we s1ll\nrequired they be in a res1ng state in order to best capture the brain’s innate response to tFUS s1mula1on.\nReference 5 (Agboada et al) is cherry picking. The literature is quite clear in arguing of intensity and\ndura1on sensi1ve effects, see e.g. doi: 10.1016/j.brs.2022.01.011. and doi: 10.1016/j.brs.2021.03.013.\nfrom the same group.\nWe respectully disagree that the Agboada paper is “cherry picked” just because the same group also\nreported posi1ve results. The existence of one does not nullify the other. If anything, the paper we cited is\nmore applicable to our study, as it was inves1ga1ng effects in young subjects (cited paper’s mean age: 26.5\nyears old; our main study’s mean age: 24.5 years old), while the two other references the Reviewer\nmen1oned both report results in older popula1ons (50 + years old). The heightened relevance to our\noriginal chosen paper is furthered that a key finding of one of provided papers (10.1016/j.brs.2022.01.011)\nis that there is an age-dependency in response to tDCS.\nWe also do not cite Agboada’s paper alone. We cite it with addi1onal papers. We have also now added a\nrecent tES review paper to the suppor1ve cita1ons of a unclear dose-effect rela1onships 3. Wholis1cally,\nthese papers provide evidence of inconsistent dose-effect rela1onships.\n“Our results indicate tDCS dose, at the between-subjects level, is not a meaningful predictor of\ntEAS ac1va1on (Supplemental Figure S7; R2 < 0.05, p > 0.05). This is consistent with other studies\nthat have reported inconsistent outcomes of different tDCS doses3,17–19, and further supports that\nour subject-specific dosage should not be viewed as a study limita1on.”\nReference 6 (Kidgell et al) varied intensity in a much smaller range between 0.8, 1.0, and 1.2 mA\nWe thank the Reviewer for this comment. We wish to state that the fact a smaller range was used (0.8 –\n1.2 mA, which s1ll represents a 1.5 fold increase) does not change this fact they found no meaningful\ndose-response rela1onship.\nReference 7 (Jamil et al) can indeed be quoted in the present context but only for anodal not for cathodal\ns1mula1on.\ninconsistent dose-effect for cathodal s1mula1on (as the authors write, “For both tDCS polari1es, the\nexcitability aeer-effects did not linearly correlate with increasing DC intensity”). We’ve reworded the\nstatement in our manuscript to reflect the inconsistent outcomes as opposed to our previously worded\nli)le-to-no results.\n“Our results indicate tDCS dose, at the between-subjects level, is not a meaningful predictor of\ntEAS ac1va1on (Supplemental Figure S7; R2 < 0.05, p > 0.05). This is consistent with other studies\nthat have inconsistent outcomes of different tDCS doses3,17–19, and further supports that our\nsubject-specific dosage should not be viewed as a study limita1on”\nSuppl fig 7 seems to be in line now with intensity dependent changes however does not reflect any reversal\nfrom cathodal inhibi1on to excita1on when moving from 1 mA to 2 mA.\nWe thank the Reviewer for this comment. It has caused us to realize there was a typo in the previous\nversion, in which we stated in the discussion a p-value was less than 0.05 when it should have been greater\nthan 0.05 (the correct value was reported in Supplemental Fig. S7, but nevertheless the typo existed in the\ndiscussion sec1on).\nOriginal sentence:\nOur results indicate tDCS dose, at the between-subjects level, is not a meaningful predictor of tEAS\nac1va1on (Supplemental Figure S7; R2 < 0.05, p < 0.05).\nWe have fixed this typo:\nOur results indicate tDCS dose, at the between-subjects level, is not a meaningful predictor of tEAS\nac1va1on (Supplemental Figure S7; R2 < 0.05, p > 0.05).\nIn this context the paragraph “we note the varied level…” can be omined. As I said it is not jus1fied to use\nskin percep1on as a reason at all to come up with different doses.\nWe have reworded the paragraph, as follows, to emphasize the jus1fica1ons for our design choice in the\ncontext that, 1) while uncommon, other groups have used, or planned to use, varied dosage, 2) we wanted\nto avoid contamina1ng the res1ng state EEG response, and 3) we priori1zed subject-safety considera1on.\nWe hope the updated paragraph has addressed the concerns raised by the Reviewer.\n“We note the varied level of scalp current applied to subjects during our experiments. For tDCS\nexperiments, the dose is most commonly normalized for all subjects (either at the scalp- or the\ncor1cal-level using simula1ons). However, the dose can also be varied across subjects24–26. We\nopted for the laner approach in our study. tDCS is generally considered safe, but can induce\ntransient sensa1ons of 1ngling, itching, and/or burning on the scalp21. The primary goal of our\nstudy was to u1lize tDCS to draw out possible subthreshold effects of tFUS in a res1ng state. Skin\npercep1ons are the result of ac1vated sensory neurons20, which can contaminate the res1ng-state\nEEG recordings, both from the firing of the scalp (i.e., non-cor1cal) neurons, but also from the\nac1vated pain circuits within the brain itself. We wished to avoid such possible contamina1on.\nBeyond this, we also note it is possible for tDCS to induce skin lesions aeer only a single session27.\nSince our par1cipants were healthy human subjects, we wanted to avoid this possibility.”\nfollow up study in which we used a constant dose for all subjects (0.7 mA). The results con1nued to\nsupport the source localizable effects of tEAS. Further, we would like to re-emphasize our experimental\ndesign always used the same tDCS dosage for tDCS and tEAS condi1ons within a subject and we employed\na cross-over design study.\n“Since we used the same current density for each condi1on within a subject, the varied current\ndensity across subjects is not a major concern. This is because our cross-over study design looks\nat the changes in brain response across condi1ons for subjects.”\nWe would also like to take this opportunity to point to a recent tDCS review paper 3, authored by dozens\nof ac1ve tDCS researchers. In it, they state:\n“Op1mal dosing depends on the intended neuromodulatory effects.”\nOur intended neuromodulatory effect was not to ac1vate sensory pathways in order to maintain a pure\nres1ng state and to bener understand the targeted-loca1on specific effects of tFUS.\nThe same review paper3 also states:\n“At present, in most studies, the dose (including all parameters: intensity, waveform, electrode\nsize, s1mula1on dura1on) is chosen based on previously published data, prior research and\nclinical experience, individual measures like thresholds, computa1onal models, summary metrics\nsuch as the electrode charge density calculated from electrode area, current, and 1me and related\nsafety considera1ons based on human and animal experimental data.”\nWe are the first group to inves5gate transcranial electro-acous5c s5mula5on; there are not previously\npublished works to draw on for our par1cularly use-case. Our usage is novel, and, given the posi1ve\noutcome of the tEAS source localizability, effec1ve. As we stated in the manuscript, we specifically wanted\nnon-percep5ble tDCS in order to avoid ac5va5ng the peripheral sensory pathways, which could\ncontaminate the res5ng state tFUS applica5on / EEG. We used tDCS as a subthreshold tool to inves1gate\nthe underlying effects of tFUS; this is novel and has not been done before.\nWe have added the following to bener clarify this point in the manuscript Discussion:\n“We would also like to emphasize this is not a tradi1onal tDCS study looking at aeer-effects from\nprolonged s1mula1on17–19. Instead, this study uses the online effects of subthreshold direct\ncurrent to inves1gate possible subthreshold effects of tFUS. This approach has not been done\nbefore. Due to the unpredictability of tDCS dose-response rela1onships3,17–19, it is typically\nrecommended to choose dosing levels based on prior research and based on the intended\nneurological effect3. However, being the first study to inves1gate simultaneous tDCS and tFUS, we\ndid not have such literature to draw upon. Instead, we priori1zed a dose that would not ac1vate\nsensory pathways20 by inducing itching, burning, and 1ngling sensa1ons21. Such skin percep1ons\nfrom tDCS would contaminate and undermine our inves1ga1ons of tFUS.”\nThe Simnibs approach is appreciated however I did not find any hints what was done finally here.\nWe thank the Reviewer for their comment. We have added more details regarding SimNIBS to the\nSupplemental Methods:\nSimNIBS Simula;on\nSimNIBS23. Subject MRIs were automa1cally segmented using the Complete Head Anatomy\nReconstruc1on Method (CHARM) into different 1ssue types. The simula1ons used the default\nSimNIBS conduc1vity values (e.g., white maner: 0.126 S/m, gray maner: 0.275 S/m, bone: 0.01\nS/m, scalp: 0.465 S/m). Electrodes were modeled as 12 mm diameter cylinders with 1 mm height,\nbased on our HD-tDCS electrodes. The tDCS current intensity was based on the subject’s specific\nexperimental scalp current (Supplemental Table T1). The resultant maximum cor1cal current\ndensity was considered for further analysis (Supplemental Table T1).\nI do not quite understand the meaning of this sentence: “Our results show, despite varying levels of ini1al\nscalp current, there is a significant increase in brain response for the tEAS condi1ons compared to others.”\nWe thank the Reviewer for their comment. The meaning of the sentence, which is the main point of the\npaper, seems to have gonen lost within the extra discussions on tDCS dosage. We have clarified the\ncomparison is between tEAS, tFUS, and tDCS condi1ons:\n“Our results show a significant increase in ROI ac1vity for tEAS condi1ons, regardless of tDCS\ndosage, compared to tFUS and tDCS condi;ons.”\nWhat is meant be “regardless of ini1al tDCS dosage.” Do not understand the ini1al here.\nWe thank the Reviewer for their comment. We removed the word ‘ini1al.’\nIn summary, we appreciate the efforts of Reviewers #2 and #3 for rereading the R1 manuscript, and for\nvarious comments. We hope our detailed responses above have clarified the reviewer comments.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communica1ons ini1a1ve to facilitate training in peer review and to provide appropriate\nrecogni1on for Early Career Researchers who co-review manuscripts.\nWe thank reviewer #3 for their effort and 1me co-reviewing our manuscript.\nReferences\n1. Lewis, A. H. & Grandl, J. Piezo1 ion channels inherently func=on as independent mechanotransducers. eLife 10,\ne70988 (2021).\n2. San-Juan, D. Cathodal Transcranial Direct Current S=mula=on in Refractory Epilepsy: A Noninvasive\nNeuromodula=on Therapy. J. Clin. Neurophysiol. 38, 503 (2021).\n3. Antal, A. et al. Low intensity transcranial electric s=mula=on: Safety, ethical, legal regulatory and applica=on\nguidelines (2017–2025: An update) – endorsed by the European Society for Brain S=mula=on and by the\nInterna=onal Federa=on for Clinical Neurophysiology. Clin. Neurophysiol. 2111436 (2025)\ndoi:10.1016/j.clinph.2025.2111436.\n4. Kricheldorff, J. et al. Evidence of Neuroplas=c Changes a_er Transcranial Magne=c, Electric, and Deep Brain\nS=mula=on. Brain Sci. 12, 929 (2022).\n5. Nitsche, M. A. et al. Transcranial direct current s=mula=on: State of the art 2008. Brain S8mulat. 1, 206–223\n(2008).\n6. Legon, W. et al. 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Sanguineg, J. L. et al. Transcranial Focused Ultrasound to the Right Prefrontal Cortex Improves Mood and Alters\nFunc=onal Connec=vity in Humans. Front. Hum. Neurosci. 14, (2020).\n13. Niu, X., Yu, K. & He, B. On the Neuromodulatory Pathways of the In Vivo Brain by Means of Transcranial Focused\nUltrasound. Curr. Opin. Biomed. Eng. 8, 61–69 (2018).\n14. Guo, H. et al. Ultrasound Produces Extensive Brain Ac=va=on via a Cochlear Pathway. Neuron 98, 1020-1030.e4\n(2018).\n15. Sato, T., Shapiro, M. G. & Tsao, D. Y. Ultrasonic Neuromodula=on Causes Widespread Cor=cal Ac=va=on via an\nIndirect Auditory Mechanism. Neuron 98, 1031-1041.e5 (2018).\n16. Braun, V., Blackmore, J., Cleveland, R. O. & Butler, C. R. Transcranial ultrasound s=mula=on in humans is associated\nwith an auditory confound that can be effec=vely masked. Brain S8mul. Basic Transl. Clin. Res. Neuromodula8on\n13, 1527–1534 (2020).\n17. Kidgell, D. J. et al. Different Current Intensi=es of Anodal Transcranial Direct Current S=mula=on Do Not\nDifferen=ally Modulate Motor Cortex Plas=city. Neural Plast. 2013, 603502 (2013).\n18. Agboada, D., Mosayebi Samani, M., Jamil, A., Kuo, M.-F. & Nitsche, M. A. Expanding the parameter space of anodal\ntranscranial direct current s=mula=on of the primary motor cortex. Sci. Rep. 9, 18185 (2019).\n19. Jamil, A. et al. Systema=c evalua=on of the impact of s=mula=on intensity on neuroplas=c a_er-effects induced\nby transcranial direct current s=mula=on. J. Physiol. 595, 1273–1288 (2017).\n20. van Boekholdt, L., Kerstens, S., Khatoun, A., Asamoah, B. & Mc Laughlin, M. tDCS peripheral nerve s=mula=on: a\nneglected mode of ac=on? Mol. Psychiatry 26, 456–461 (2021).\n21. Poreisz, C., Boros, K., Antal, A. & Paulus, W. Safety aspects of transcranial direct current s=mula=on concerning\nhealthy subjects and pa=ents. Brain Res. Bull. 72, 208–214 (2007).\n22. Murphy, K. R. et al. A prac=cal guide to transcranial ultrasonic s=mula=on from the IFCN-endorsed ITRUSST\nconsor=um. Clin. Neurophysiol. 171, 192–226 (2025).\n23. Thielscher, A., Antunes, A. & Saturnino, G. B. Field modeling for transcranial magne=c s=mula=on: A useful tool\nto understand the physiological effects of TMS? in 2015 37th Annual Interna8onal Conference of the IEEE\nEngineering in Medicine and Biology Society (EMBC) 222–225 (2015). doi:10.1109/EMBC.2015.7318340.\n24. Pilloni, G. et al. Tolerability and feasibility of at-home remotely supervised transcranial direct current s=mula=on\n(RS-tDCS): Single-center evidence from 6,779 sessions. Brain S8mul. Basic Transl. Clin. Res. Neuromodula8on 15,\n707–716 (2022).\n25. Simpson, E. A. et al. Remotely monitored transcranial direct current s=mula=on in pediatric cerebral palsy: open\nlabel trial protocol. BMC Pediatr. 22, 566 (2022).\n26. Lafon, B. et al. Low frequency transcranial electrical s=mula=on does not entrain sleep rhythms measured by\nhuman intracranial recordings. Nat. Commun. 8, 1199 (2017).\n27. Wang, J., Wei, Y., Wen, J. & Li, X. Skin Burn A_er Single Session of Transcranial Direct Current S=mula=on (tDCS). Brain S8mulat. 8, 165–166 (2015).\nsolidity of the data and explanations provided by the authors to address Rev. 2's comments\nRESPONSE TO REVIEWER COMMENTS\nWe thank Reviewer 1 for their additional comments. We have responded to all of review\ncomments raised and revise the manuscript further. Below, you will find our detailed responses to\neach reviewer comment in BLUE, with the original reviewer comments in BLACK. Revised or\nadded text in the manuscript / supplementary information is highlighted in RED.\nI have studied the comments of Rev. 2 and the response le8er of the authors. In my opinion, the\nauthors have sa;sfactorily answered all comments of Rev. 2. In par;cular:\n· tFUS: I agree with the authors that ac;ve sham is the accepted methodology to control for\nauditory confounds. Addi;onally, smoothed waveforms are also effec;ve to reduce auditory\neffects, as used in the addi;onal experiments in Fig. S10.\nI agree with the authors that earplugging is not effec;ve, because of bone conduc;on\n(earplugging might increase the auditory confounds, by crea;ng a closed ear canal cavity).\nI am less convinced by 30 kHz PRF ultrasound as a control for auditory confounds, because I\nbelieve that the sharp pulse onsets introduce lower frequency components to the waveform: the\nauthors men;on that the par;cipants could not hear these 30 kHz PRF waveforms, but there is\ns;ll significant A1 to deep brain Granger causality. However, this is an addi;onal experiment, that\nis interes;ng as addi;onal informa;on (Fig. S3): the main experiments use ac;ve sham. For these\nreasons, I believe that the methodology to control for auditory confounds is well mo;vated.\n· tDCS: The authors have improved the formula;on in the revised manuscript on the mo;va;on\nfor a subject specific tDCS dose. I agree with the correc;ons and with the mo;va;on for this\nmethodological choice. An important mo;va;on for this choice in this study is to avoid sensory\nconfounds as much as possible. Addi;onally, the authors included references that have or are\nplanning to use varied tDCS dosing and men;on the benefit of improved safety. I agree with the\nauthors that the choice for a subject specific tDCS dose is not a limita;on of this study, because\n(i) same dosing is used in within-subject comparisons, (ii) tDCS dose is not predic;ve of mean ROI\nac;vity in linear-effects model analysis (Fig. S7), (iii) an addi;onal supplementary experiment with\nfixed tDCS dose demonstrates similar results (Fig. S12).\nWe thank Reviewer 1 for their ;me, effort, and thoughZul responses.\nSmall minor textual comments, that I no;ced today:\n· Fig. S1 cap;on: “b) ipsilateral precision, c) ipsilateral recall” -> other way around\nWe thank the reviewer for catching this typo. We have fixed it in the document.\n· Fig S3 labels (C <-> D).\nWe thank the reviewer for catching this. We have fixed the labels in Fig S3.\n· P. 12 “[…] tDCS polarity and not tFUS PRF (Supplemental Figure S7).” -> Figure S8.\n· P. 13: “while etFUS not did” -> “did not”\nWe thank the reviewer for catching this typo. We have fixed it in the document.\n· P. 13: “Both of the spectral connec;vity changes occur ader 200 ms of sonica;on onset.” -> Ader\nsonica;on onset or post-s;mulus (as indicated by the cap;on, Fig. S5)?\nWe thank the reviewer for catching this typo. We have fixed it in the document.\n“Both of the spectral connectivity changes occur outside of the initial 200 ms window.\n· P. 22: It would be valuable if the authors could update the descrip;on of the par;cipants in the\nMethods sec;on with the addi;onal studies performed during the review. For the addi;onal\nexperiments (Fig. 5, S8, S10, S12), it would be interes;ng to have more informa;on on the\nultrasonic and/or tDCS dose (e.g., similar to Table S1).\nWe thank the reviewer for their comment. We have added par;cipant informa;on to the\nMethods for the addi;onal studies.\n“Subject recruitment and experiments were conducted in cohorts of 22 (subjects 101-122;\nages = 23.22 ± 1.10 years old), 11 (subjects 301-311; 5 F / 6 M; mean age = 24.2 ± 2.3 years\nold), 6 (subjects 401 – 406; 4 F / 2 M; mean ages = 23.6 ± 2.6 years old), and 8 (subjects\n501 – 508; 5 F / 3 M; mean ages = 23.6 ± 2.3 years old) par;cipants. Some subjects\npar;cipated in mul;ple cohorts of experimenta;on.”\nWe have also added dosing informa;on for the studies related to Fig 5 and S8 (Supplemental\nTable T2), Fig S10 (Supplemental Table T3), and S12 (Supplemental Table T4).\nSupplemental Table T2: Subject-specific Neuromodulation Dosage for Cohort 1 Follow Ups\nThe following subjects returned for the expanded tFUS parameter search (Figure 5) and tEAS\npolarity (Supplemental Figure S8) experiments. The sonication duration was normalized to 500\nms for each condition. I changed as a function of duty cycle (for the square pulses that were\nspta\nused in the experiments, I = I * DC).\nspta.T sppa.T\nSubject ID Isppa (W/cm2) MI Tested 60% DC Tested 0.6% Scalp Current\n.T .T\nDC (mA)\nSubj101 0.22 0.12 Yes No 1.2\nSubj105 0.16 0.10 Yes No 1.7\nSubj118 1.00 0.25 Yes No 1.8\nSubj104 1.02 0.25 No Yes 1.4\nSubj115 1.24 0.28 No Yes 1.1\nSubj106 0.18 0.11 No Yes 1.2\nSubj109 0.42 0.16 No Yes 1.0\nSubj107 0.21 0.11 No Yes 0.9\nSubj113 0.23 0.12 No Yes 0.8\nSupplemental Table T3: Subject-specific Neuromodulation Dosage for Smoothed Waveform\nFollow Up\nSubject ID Isppa (W/cm2) MI Scalp Current\n.T .T\n(mA)\nSubj401 0.71 0.27 0.7\nSubj402 0.14 0.12 0.7\nSubj403 0.26 0.16 0.7\nSubj404 0.14 0.12 0.7\nSubj405 0.63 0.25 0.5\nSubj406 0.33 0.18 1.3\nNote that Ispta is not well defined for a smoothed waveform.\nFollow Up\nSubject ID Isppa (W/cm2) Ispta MI Scalp Current\n.T .T .T\n(mW/cm2) (mA)\nSubj501 1.15 344.30 0.27 0.7\nSubj502 0.16 48.57 0.1 0.7\nSubj503 0.22 65.95 0.12 0.7\nSubj504 0.22 66.89 0.12 0.7\nSubj505 0.54 163.12 0.18 0.7\nSubj506 1.02 306.08 0.25 0.7\nSubj507 0.42 125.35 0.16 0.7\nSubj508 1.00 299.37 0.25 0.7\n· P. 23: “(0.45 mm resolu;on, 0.3 mm maximum step, and a ;me step of 1.0 / (speed of soundmax\n* \"1/dx2 + 1/dy2 + 1/dz2 )”. -> closing bracket\nWe thank the reviewer for the comment. We have added the closing bracket.\n4"}]

Decision letter