MULTISENSORY FUNCTIONAL ULTRASOUND IMAGING THROUGH CHEMICALLY ENHANCED SKULL ACOUSTIC TRANSPARENCY IN RATS

Functional ultrasound (fUS) imaging offers whole-brain coverage and high spatiotemporal resolution, making it a powerful tool for mapping distributed neural networks across sensory modalities. However, its application in rats has been limited by skull-induced acoustic attenuation, typically necessitating invasive cranial windows. Here, we demonstrate minimally invasive, transcranial fUS imaging of multisensory brain activity in rats enabled by brief, topical chelation of skull calcium with ethylenediaminetetraacetic acid (EDTA) combined with skull thinning. Seven animals underwent brief skull exposure and careful thinning with a dental drill. EDTA solution was applied using soaked sponges over the right hemisphere for up to 5 min per application, repeated for maximum of 20 min. Functional imaging was performed under light isoflurane anesthesia (≤1% in 90/10% Air/O₂) using an IcoPrime 4D MultiArray probe. Auditory, visual, and whisker stimulation were delivered sequentially in 5-s blocks separated by 15-s rest, repeated 10 times (~5 min total). EDTA-treated skulls enabled deep transcranial fUS recordings across all sensory modalities. Each modality evoked robust, stimulus-locked hemodynamic responses in the expected cortical regions: Au1 during auditory stimulation, V1 during visual stimulation, and M1 and S1BF during whisker stimulation. Additionally, strong activation was observed in modality-specific subcortical nuclei, including the MGB and IC for auditory stimulations, the dorsal LGN and SC for visual stimulation, and the VPM for whisker stimulation. ROI analyses within these defined regions confirmed the temporal locking of hemodynamic responses to stimulus presentation. Overall, this reversible protocol establishes a practical route to longitudinal, multisensory fUS imaging in large rodents.