Ultrasound neuromodulation depends on pulse repetition frequency and can modulate inhibitory effects of TTX
Thomas J. Manuel, Jiro Kusunose, Xiaoyan Zhan, Xiaohui Lv, Ellison Kang, Aaron Yang, Zixiu Xiang, Charles F. Caskey
Scientific Reports 2020, 10 · 10.1038/s41598-020-72189-y
Abstract
Ultrasound is gaining traction as a neuromodulation method due to its ability to remotely and non-invasively modulate neuronal activity with millimeter precision. However, there is little consensus about optimal ultrasound parameters required to elicit neuromodulation and how specific parameters drive mechanisms that underlie ultrasound neuromodulation. We address these questions in this work by performing a study to determine effective ultrasound parameters in a transgenic mouse brain slice model that enables calcium imaging as a quantitative readout of neuronal activity for ultrasound neuromodulation. We report that (1) calcium signaling increases with the application of ultrasound; (2) the neuronal response rate to ultrasound is dependent on pulse repetition frequency (PRF); and (3) ultrasound can reversibly alter the inhibitory effects of tetrodotoxin (TTX) in pharmacological studies. This study offers mechanistic insight into the PRF dependence of ultrasound neuromodulation and the nature of ultrasound/ion channel interaction.
Abstract via europepmc.
Exposures
Exposure 1: Continuous-wave 250 kHz sonication of motor-cortex brain slices
Target: motor cortex, brain slice — “motor cortex (coronal brain slice)”
Device: other named manufacturer · NDT · 1-inch spherically focused transducer, 250 kHz ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 250 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | 100 | ✓✓✓ |
| Sonication duration (s) | 0.2, 1swept | ✓✓✓⚑ |
| Free-field pressure (kPa) | 80, 350swept | ✓✓✓ |
|---|---|---|
| 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 |
Brain slices were sonicated with continuous-wave parameters varying pressure (paper states 80 and 350 kPa, though the two values could not both be captured in one contiguous quote) and pulse length (matched cycle counts across 250/500 kHz). A single trial comprised a baseline measurement, sonication measurement, and a rest period of 30 s or greater.
Exposure 2: 500 kHz sonication of motor-cortex brain slices (continuous-wave and PRF/TTX pulsed trials)
Target: motor cortex, brain slice — “motor cortex (coronal brain slice)”
Device: other named manufacturer · NDT · 1-inch spherically focused transducer, 500 kHz ✓
| Waveform | continuous, pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 500 | ✓✓✓ |
| Pulse duration (ms) | 0.4, 2swept | ✓✓✓ |
| Pulse repetition frequency (Hz) | 300, 1,500swept | ✓✓✓ |
| Duty cycle (%) | 100, 60swept | ✓?⚑ |
| Sonication duration (s) | 0.1, 0.5, 100swept | ✓✓✓⚑ |
| Free-field pressure (kPa) | 80, 100, 350swept | ✓✓✓ |
|---|---|---|
| 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 |
Continuous-wave 500 kHz trials used matched pressures/cycle counts as the 250 kHz trials (100 ms and 500 ms pulse lengths). Separately, pulsed PRF-dependence and TTX trials used a fixed 100 kPa pressure with a slow 0.5 Hz trigger firing 50 bursts per trial (100 s total sonication time); duty cycle was 60% within each burst and 2% for the total sonication. The 1,500 Hz condition used 200 cycles per pulse (0.4 ms) with 100 pulses per burst; the 300 Hz condition used 1,000 cycles per pulse (2 ms) with 20 pulses per burst. TTX (0, 0.5 or 1.0 µM) was perfused before a subset of these pulsed trials.
Flags from extraction
exposures[0].timing.sonication_duration_s— Continuous-wave pressure sweep values (stated in text as 80 and 350 kPa) are split across a figure caption/page break in the source file and could not be captured in a single contiguous quote, so free_field.pressure_kpa for the 250 kHz exposure is left not_reported; only the 500 kHz pulsed-condition pressure (100 kPa) had a clean contiguous quote.exposures[1].timing.sonication_duration_s— This field mixes durations from two different sub-conditions sharing the same target/frequency per the one-exposure-per-target-x-frequency rule: continuous-wave burst lengths (0.1, 0.5 s) and the total pulsed/PRF sonication time (100 s, 50 bursts at 0.5 Hz).exposures[1].timing.duty_cycle_pct— The paper reports two different duty-cycle figures for the pulsed condition: 60% within each burst (which matches the stated pulse durations via PRF) and 2% for the total sonication including the 0.5 Hz inter-burst interval; only the burst-level 60% value is used alongside continuous-wave's 100%, with the nested 2% total duty cycle described in protocol_description.auditory_control— The paper argues that its ex vivo brain-slice model is inherently free of auditory-pathway confounds, but this is a property of the preparation rather than an explicit auditory_control method from the closed vocabulary.n_subjects— A total slice count (53) is given only for the continuous-wave experiment; slice counts for the PRF-dependence and TTX experiments are not aggregated to a total in the text (only per-condition cell/trial counts are given).