Challenges and lessons learned in attempts to develop in vitro models for ultrasound neuromodulation using local field potential measurements in hippocampal tissue
Martin Loynaz Prieto, Daniel V. Madison, Merritt Maduke
Biophysical Journal 2025 · 10.1016/j.bpj.2025.06.039
Abstract
Ultrasound neuromodulation is a rapidly developing tool for noninvasive control of brain activity. An in vitro model recapitulating the effects of ultrasound on neural tissue in vivo would be extremely valuable in guiding the development of this tool for optimal implementation. Yet, there are relatively few studies of ultrasound on neural activity in vitro. Here, we describe our attempts to measure neuromodulatory outcomes using local field potential measurements in two in vitro models of hippocampal activity. First, we measured the effects of ultrasound at 1 MHz and 100-600 kPa on a mouse hippocampal in vitro model of sharp wave ripples. Our primary protocol involved brief ultrasound pulses delivered at intervals shorter than the mean interval between sharp wave ripple events, with other stimulus protocols tested with small sample size. No set of parameters produced detectable effects on the amplitude or frequency of sharp wave ripples. We considered whether missing synaptic connections or the relatively small volume in brain slices might account for the lack of effect in our experimental setup. To test these hypotheses, and to examine ultrasound's effects in another system, we measured the effects of ultrasound on theta oscillations in the intact rat hippocampus in vitro. We found that ultrasound at 1 MHz and 500 kPa, applied continuously for 2 s, produced no detectable effects on the amplitude or frequency of in vitro theta oscillations. Finally, we considered a novel mechanism for ultrasound's effects on neural activity, in which acoustic pressure causes microscale phase transitions in the pores of ion channels, such as nicotinic receptor channels, that exhibit hydrophobic gating. To test this hypothesis, we repeated our experiments on the intact hippocampus in the presence of 5 μM nicotine; however, as with the other experimental systems, we found no detectable effects of ultrasound in our experimental setup.
Abstract via europepmc.
Exposures
Exposure 1: SWR hippocampal slices (mouse)
Target: CA1, brain slice — “hippocampal slice, CA1 layer”
Device: Olympus / Panametrics · Olympus · A303
| Waveform | continuous, pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | 0.5, 20swept | ✓✓✓ |
| Pulse repetition frequency (Hz) | 1.43, 5swept | ✓✓✓⚑ |
| Duty cycle (%) | not reported | |
| Sonication duration (s) | 300 | ✓✓✓⚑ |
| Free-field pressure (kPa) | 100, 600swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | 0.33, 12.3swept | ✓✓✓ |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable |
Brief single pulses (0.5-20 ms) repeated at 200-700 ms intervals for 5 min, flanked by 5 min control recordings; tone bursts (e.g. 50 ms burst, 200 Hz PRF, 0.5 or 1 ms pulses) tested in two slices and a 5 s stimulus in one slice.
Exposure 2: Intact rat hippocampus in vitro (theta)
Target: hippocampus — “intact hippocampus in vitro”
Device: Olympus / Panametrics · Olympus · A303
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 2 | ✓✓✓ |
| Free-field pressure (kPa) | 500 | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable |
2 s continuous-wave ultrasound repeated every 20 s for 5 min, also repeated with 5 uM nicotine in the bath.
Flags from extraction
exposures[0].free_field— Pressures were calibrated with a hydrophone in the recording chamber (solution, no tissue or skull); entered as free field. Intensities are the paper's stated equivalents.exposures[0].timing.pulse_repetition_frequency_hz— Pulses repeated at intervals of 200-700 ms (and 200 Hz PRF within tone bursts); PRF not recorded as a single number.exposures[0].timing.sonication_duration_s— Trains of single pulses repeated for 5 min; no single sonication duration.n_subjects— Seven slices (mouse) and three intact hippocampi (rat); number of animals not stated. Numbers stated as words.exposures[0].timing.waveform— Single brief continuous-wave pulses mainly; two slices received tone bursts.