Experimental conditions and procedures.

(A) Main conditions: on-target TUS of the left-hemispheric hand motor area (Exp. I-III), active control TUS of the right-hemispheric face motor area (Exp. I-II), sound-only sham (Exp. I-III), and inactive control TUS of the white matter ventromedial to the hand motor area (Exp. IV). (B) Ultrasonic stimulation protocol. (C) Experimental timing. (D) TUS-TMS clamp (DOI: 10.5281/zenodo.6517599).

Ultrasonic stimulation parameters. f = fundamental frequency, PD = pulse duration, PRF = pulse repetition frequency, DC = duty cycle, SD = stimulus duration, Isppa = spatial-peak pulse-average intensity in free- water, P = pressure, MI = derated mechanical index. For estimated intracranial indices for Experiments I & II see Supplementary Figure 2.

Non-specific motor inhibitory effects of TUS.

A significant suppression of MEP amplitude relative to baseline (gray) was observed for on-target TUS (green), but also for stimulation of a control region (cyan), and presentation of a sound alone (sound-sham; blue) indicating a spatially non-specific and sound-driven effect on motor cortical excitability. Bar plots depict condition means, error bars represent standard errors, clouds indicate the distribution over participants, and points indicate individual participants. Square-root corrected MEP amplitudes are depicted for Experiments I, II, and IV, and Relative MEP amplitude is depicted for Experiment III (see Methods). *p < 0.05, **p < 0.01, ***p < 0.001.

No dose-response effects of TUS.

(A) Acoustic (top) and thermal (bottom) simulations for a single subject in Experiment II. The acoustic simulation depicts estimated pulse-average intensity (Ipa) above a 0.15 W/cm2 lower bound, with the dotted line indicating the full-width half-maximum of the pressure. The thermal simulation depicts maximum estimated temperature rise. (B) There is no significant effect of free-water stimulation intensity on MEP amplitude. Values are expressed as a percentage of baseline MEP amplitude (square root corrected). Remaining conventions are as in Fig. 2. (C) On-target TUS MEP amplitude as a percentage of active control MEP amplitude against simulated intracranial intensities at the two applied free- water intensities: 6.35 W/cm2 (top) and 19.06 W/cm2 (bottom). The shaded area represents the 95% CI, points represent individual participants. (D) Temporal autocorrelation, operationalized as the slope of the linear regression between trial t and baseline trial t-1, differed significantly as a function of stimulation site and intensity for masked trials. Individual points represent the differential autocorrelation compared to the active control site. Autocorrelation was not modulated by sound-only sham, but was significantly higher for on-target TUS at 6.35 W/cm2, and significantly lower for on-target TUS at 19.06 W/cm2 compared to active control TUS. *p < 0.05, **p < 0.01, ***p < 0.001.

Sound-driven effects on corticospinal excitability.

(A) Longer (auditory) stimulus durations resulted in lower MEP amplitudes, regardless of TUS administration, indicating a sound-duration-dependency of motor inhibitory outcomes (Exp. I). (C) A significant effect of auditory stimulus duration was also observed in Experiment III. (B) Less MEP attenuation was measured during continuous masking, particularly for lower stimulation intensities (i.e., auditory confound volumes), pointing towards a role of TUS audibility in MEP attenuation. (D-E) There were no significant effects of time-locked masking, indicating that audible differences between stimulation sites did not obscure or explain the absence of direct neuromodulation. (F) The pitch of auditory stimuli affected MEPs, where lower amplitudes were observed following a 1 kHz tone. There was no effect of TUS. Conventions are as in Figs. 2-3B.

Auditory cueing of TMS.

There was a significant reduction in MEP amplitude when participants were first presented with a 500 ms stimulus (initial trials) in Experiment I (left) and Experiment II (right), following by a stabilization of MEP amplitude during the rest of the experiment (following trials), indicating a learning process by which TUS acts as a cue signaling the onset of TMS. The solid line depicts the loess regression fit, and the shaded area represents the 95% confidence interval.

Contribution diagram.

This figure depicts the involvement of each author using the CRediT taxonomy (Brand et al., 2015) and categorizes their contributions according to three levels represented by color: ‘none’ (gray), ‘substantial contribution’ (light green), ‘leading contribution’ (dark green).