Low-intensity ultrasound directly modulates neural activity of the cerebellar cortex
Ruo-Shui Xu, Xue-Mei Wu, Zhi-Qi Xiong
Brain Stimulation 2023, 16, 918-926 · 10.1016/j.brs.2023.05.012
Abstract
Background Low-intensity ultrasound is a noninvasive neuromodulation technique with the potential to focally manipulate deep brain activity at millimeter-scale resolution. However, there have been controversies over the direct influence of ultrasound on neurons, due to an indirect auditory activation. Besides, the capacity of ultrasound to stimulate the cerebellum remains underestimated. Objective To validate the direct neuromodulation effects of ultrasound on the cerebellar cortex from both cellular and behavioral levels. Methods Two-photon calcium imaging were used to measure the neuronal responses of cerebellar granule cells (GrCs) and Purkinje cells (PCs) to ultrasound application in awake mice. And a mouse model of paroxysmal kinesigenic dyskinesia (PKD), in which direct activation of the cerebellar cortex leads to dyskinetic movements, was used to assess the ultrasound-induced behavioral responses. Results Low-intensity ultrasound stimulus (0.1 W/cm 2 ) evoked rapidly increased and sustained neural activity in GrCs and PCs at targeted region, while no significant changes in calcium signals were observed responding to off-target stimulus. The efficacy of ultrasonic neuromodulation relies on acoustic dose modified by ultrasonic duration and intensity. In addition, transcranial ultrasound reliably triggered dyskinesia attacks in proline-rich transmembrane protein 2 (Prrt2) mutant mice, suggesting that the intact cerebellar cortex were activated by ultrasound. Conclusion Low-intensity ultrasound directly activates the cerebellar cortex in a dose-dependent manner, and thus serves as a promising tool for cerebellar manipulation.
Abstract via europepmc.
Exposures
Exposure 1: Cerebellar cortex GrC/PC imaging and dyskinesia induction (88 kHz, CW, 5-10 s)
Target: cerebellar cortex — “cerebellar cortex, lobule VI-VIII (granule cell boutons and Purkinje cell dendrites); cerebellar hemisphere (dyskinesia induction)”
Device: other named manufacturer · Hainertec · HNN–4SS-0890 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 88 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | 100 | ✓?⚑ |
| Sonication duration (s) | 5, 10swept | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | 0.1 | ✓✓✓ |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not reported | |
| In-situ pressure (kPa) | not reported | |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
Continuous-wave (CW) ultrasonic stimuli of 5 or 10 s at 0.1 W/cm^2 were delivered to the cerebellar cortex (imaging window over lobule VI-VIII) during two-photon calcium imaging of GrC boutons; for Purkinje cell dendrite imaging, 5-s pulses were delivered every 30 s for a total of 10 trials. For dyskinesia induction, transcranial CW ultrasound (10 s, 0.1 W/cm^2) was applied to the cerebellar hemisphere; an off-target stimulus (10 s, 0.1 W/cm^2, same pattern) was applied to dental cement over the cerebrum. Each stimulation pattern was repeated 5 times, interleaved with at least 8 h for recovery.
Exposure 2: Cerebellar cortex acoustic dose-response (127 kHz tone bursts, 80/500 ms)
Target: cerebellar cortex — “cerebellar cortex (Purkinje cell dendrites); cerebellar hemisphere (500-ms on-target dyskinesia test)”
Device: custom-built
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 127 | ✓✓✓⚑ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | 100 | ✓?⚑ |
| Sonication duration (s) | 0.08, 0.5swept | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | 0.14, 1.32, 4.4swept | ✓✓✓ |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not reported | |
| In-situ pressure (kPa) | not reported | |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
Single uninterrupted tone bursts of 80 ms or 500 ms at 0.14, 1.32 or 4.4 W/cm^2 were delivered once per 20-s trial, for a total of 14-20 trials, to assess dose-dependence of Purkinje cell dendrite responses. A 500-ms, 4.4 W/cm^2 on-target burst was also tested for its ability to trigger dyskinesia in Prrt2-mutant mice.
Flags from extraction
n_subjects— Many separate sub-experiments (imaging, dose-response, dyskinesia) each report different, non-overlapping animal/session/bouton counts; no single study-wide total of animals exposed to ultrasound is stated.n_sessions_per_subject— Number of sessions per subject varies by sub-experiment (single imaging session vs. 5 repeated dyskinesia trials with recovery); no single value applies to the whole study.exposures[1].fundamental_frequency_khz— Paper states the second transducer was driven 'around 127 kHz', an approximate rather than exact value.exposures[0].unspecified_domain.isppa_w_cm2— Domain (free-field vs. in-brain) of the reported intensity is not stated in the main text; a figure legend indicates intensities were measured with a hydrophone at 3 mm from the horn surface (suggesting a free-field-like calibration), but this is not stated as such in running text.exposures[1].unspecified_domain.isppa_w_cm2— Domain (free-field vs. in-brain) of the reported intensities is not stated in the main text.exposures[0].timing.duty_cycle_pct— Duty cycle of 100% is inferred from the paper's designation of the stimulus as continuous wave (CW); not independently stated as a percentage.exposures[1].timing.duty_cycle_pct— Duty cycle of 100% is inferred from the single uninterrupted tone-burst-per-trial description; not independently stated as a percentage.