Ultrasound Modulation of Visual Circuits in Mice Independent of Auditory Confound

Despite its growing potential as a noninvasive neuromodulation technology, the fundamental biophysical mechanism of focused ultrasound neuromodulation (FUN) remains elusive. Progress has been hampered by a persistent auditory confound, making it difficult to isolate the direct mechanical effects on neural circuits. To overcome this barrier, we employed two-photon calcium imaging (2PCI) in the primary visual cortex (V1) of deafened mice. Here, this study identifies a sparse (<1.5%) and spatially distributed population of neurons that is robustly and directly activated by ultrasound in a pressure-dependent manner. Crucially, this sparse, direct activation is sufficient to modulate the broader visual circuit, altering the response dynamics of neighboring neurons to visual stimuli. By unequivocally decoupling direct neuromodulation from auditory artifacts, the findings identify a key cellular substrate for FUN and establish a robust framework for future studies. This work paves the way for targeted investigations into the precise biophysical mechanisms governing the interaction between ultrasound and neurons.