Effects of focused ultrasound in a "clean" mouse model of ultrasonic neuromodulation.

Recent studies on ultrasonic neuromodulation (UNM) in rodents have shown that focused ultrasound (FUS) can activate peripheral auditory pathways, leading to off-target and brain-wide excitation, which obscures the direct activation of the target area by FUS. To address this issue, we developed a new mouse model, the double transgenic Pou4f3+/DTR × Thy1-GCaMP6s, which allows for inducible deafening using diphtheria toxin and minimizes off-target effects of UNM while allowing effects on neural activity to be visualized with fluorescent calcium imaging. Using this model, we found that the auditory confounds caused by FUS can be significantly reduced or eliminated within a certain pressure range. At higher pressures, FUS can result in focal fluorescence dips at the target, elicit non-auditory sensory confounds, and damage tissue, leading to spreading depolarization. Under the acoustic conditions we tested, we did not observe direct calcium responses in the mouse cortex. Our findings provide a cleaner animal model for UNM and sonogenetics research, establish a parameter range within which off-target effects are confidently avoided, and reveal the non-auditory side effects of higher-pressure stimulation.

Introduction

Purpose Transcranial ultrasound stimulation

Study Objective To develop and validate a double-transgenic mouse model enabling inducible deafening and calcium imaging to eliminate auditory confounds and clarify the direct effects of focused ultrasound neuromodulation.

Animal model / Human subject Mouse, double transgenic Pou4f3+/DTR × Thy1-GCaMP6s, age None, sex None

Disease model Deafness (inducible deafening/hearing loss)

Targeted brain region(s) Mouse Cortex

Cargo name and characteristics Diphtheria toxin (protein toxin used to inducibly ablate auditory hair cells and produce deafening) and GCaMP6s (genetically encoded calcium indicator protein expressed via Thy1 transgene for fluorescent calcium imaging)

Outcomes and Safety

Summary of Outcomes In a deafened Pou4f3+/DTR × Thy1-GCaMP6s mouse model, low-to-moderate focused ultrasound pressures (a defined lower-pressure range) eliminated auditory confounds and avoided off-target brain-wide activation, whereas higher pressures produced focal fluorescence dips, non-auditory sensory confounds, tissue damage and spreading depolarization; no direct cortical calcium responses were observed under the acoustic conditions tested.

Safety-related matter The authors report that at higher focused ultrasound (FUS) pressures there can be tissue damage leading to spreading depolarization and elicitation of non-auditory sensory confounds; FUS also activates peripheral auditory pathways causing off-target, brain-wide excitation. They state a specific pressure range in which these adverse off-target effects are minimized or eliminated.

Brain Region

Ultrasound Parameters

Focal Characteristics Focal depth: None; Focal length: None; Aperture size: None

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