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Characterization of Ultrasound‐Driven Localized Neural Activity In Vitro Using a Piezoelectric Micromachined Ultrasound Transducer: Investigating Neuromodulatory Mechanisms in the Auditory Cortex

Runo Kitahara, Takashi Tateno

IEEJ Transactions on Electrical and Electronic Engineering 2026 · 10.1002/tee.70263

ex vivo tissuehealthycellular imaging

Abstract

Ultrasound stimulation is a promising, low‐invasive technique that can activate deep brain regions with high spatial resolution; it holds potential for the treatment of various neurological disorders. However, the fundamental cellular mechanisms by which ultrasound induces neural activity remain incompletely understood. To address this issue, we developed a compact ultrasound stimulation device that was optimized for the local stimulation of individual neurons in vitro . We evaluated the ability of the device to induce neural activity through numerical simulations using a three‐dimensional model, and then validated these results through experimental measurements. We also analyzed calcium ion dynamics in the mouse auditory cortex in response to ultrasound stimulation; we identified four distinct patterns of calcium responses, including both excitatory and inhibitory activity. To explore the underlying mechanisms, we administered pharmacological agents that inhibit mechanosensitive ion channels. Collectively, our findings provide new insights into the biophysical mechanisms of ultrasound‐induced neural activation. © 2026 Institute of Electrical Engineers of Japan and Wiley Periodicals LLC.

Abstract via crossref.

Speciesmouse (C57BL/6J)
Subjects16 preparations
Sessions per subjectnot applicable
Randomisednot applicable
Blindingnot applicable
Sham / controlinactive transducer
Auditory controlnot reported
Readout timingboth
Anaesthesianot applicable
Readoutscellular imagingFluo-4AM calcium imaging of auditory-cortex brain-slice neurons/glia, with pharmacological block of mechanosensitive channels using ruthenium red
Direction of effectbidirectionalFour distinct calcium-response patterns were identified, including both excitatory (influx during stimulation followed by post-stimulus efflux) and inhibitory (efflux during stimulation, in some cases followed by delayed influx) activity across cells.
Adverse eventsnot applicableDirectly measured tissue temperature increased by only 0.0062 ± 0.010 °C during the 200-ms stimulation period, indicating negligible thermal effects; acoustic pressure was reported to be well below the cavitation threshold.

Exposures

Exposure 1: Continuous-wave ultrasound stimulation of auditory cortex brain slice

Target: auditory cortex — “auditory cortex (acute coronal brain slice)
Device: custom-built · piezoelectric micromachined ultrasound transducer (PMUT)

Pulse timing
Waveformcontinuous
Fundamental frequency (kHz)360✓✓
Pulse duration (ms)not applicable
Pulse repetition frequency (Hz)not applicable
Duty cycle (%)100?
Sonication duration (s)0.2✓✓
Pressure and intensity, by domain
Free-field pressure (kPa)92.8✓✓
Free-field Isppa (W/cm²)0.291✓✓
Free-field Ispta (W/cm²)not reported
In-situ estimatenot applicable
In-situ pressure (kPa)not applicable
In-situ Isppa (W/cm²)not applicable
In-situ Ispta (W/cm²)not applicable
Protocol, in the paper’s words

A single 200-ms continuous-wave ultrasound stimulation was delivered per trial, 3 s after the start of fluorescence recording (10 s total per trial); ten trials were conducted per slice with 30-s intertrial intervals. Responses were compared against sham (0 Pa) stimulation trials.

Flags from extraction

  • exposures[0].free_field.pressure_kpa92.8 kPa and 291 mW/cm2 were measured via hydrophone in water/calibration conditions at the stimulation voltage (60 Vpp); the paper does not separately report a pressure value measured with the brain slice in place, so domain is recorded as free-field rather than in-situ.
  • n_subjectsn_subjects is recorded as the 16 brain slices analysed (from 8 mice); subject_unit is 'preparation' since ultrasound was delivered to slices, not directly to the source animals.