Transcranial ultrasonic stimulation (TUS) is rapidly emerging as a promising non-invasive neuromodulation technique. TUS is already well-established in animal models, and now stimulation protocols that optimize neuromodulatory efficacy for human application are required. One promising protocol, pulsed at 1000 Hz, has consistently resulted in motor cortical inhibition. At the same time, a parallel research line has highlighted the potentially confounding influence of peripheral auditory stimulation arising from pulsing TUS at audible frequencies. Across four experiments, one preregistered, at three independent institutions, we employed tightly matched control conditions to disentangle direct neuromodulatory effects of TUS from those driven by the salient auditory confound in a combined transcranial ultrasonic and magnetic stimulation paradigm. We replicated motor cortical inhibition following TUS, but showed through both controls and manipulation of stimulation intensity, duration, and auditory masking conditions that this inhibition was driven by peripheral auditory stimulation rather than direct neuromodulation. This study highlights the substantial impact of the auditory confound, invites a reevaluation of prior findings, and calls for appropriate control conditions in future TUS work. Only when direct effects are disentangled from those driven by peripheral confounds can TUS fully realize its potential for neuroscientific research and clinical applications.

Sleep is an indispensable part of our life and plays a critical role in our physical and mental well-being. During sleep, despite the paucity of behavior, our brain stays active and exhibits a wide range of coupled brain oscillations. This activity in sleep-characteristic brain oscillations has been linked to various functions of sleep. However, whether these sleep oscillations mediate these functions or reflect mere epiphenomena is not yet fully understood. To disentangle the causality of these relationships, experiments utilizing non-invasive stimulation techniques have been essential. In particular, auditory stimulation aligned with neural activity in a closed-loop controlled fashion has drawn increasing attention during the last years due to its specificity and practical advantages. In this review, we summarize closed-loop auditory stimulation experiments that generated evidence to support a causal role of slow oscillations in information reprocessing functions of sleep. Furthermore, we provide technical details and guidelines regarding best practices in closed-loop auditory stimulation, from how to perform the stimulation to analysis strategies. Besides discussing important caveats and open questions, we also touch on various areas in which closed-loop auditory stimulation is applicable, from fundamental investigations on memory processing and endocrine function, to its potential for clinical applications. Eventually, we propose potential topics for future research.