High resolution ultrasonic neural modulation observed via in vivo two-photon calcium imaging
Zongyue Cheng, Chenmao Wang, Bowen Wei, Wenbiao Gan, Qifa Zhou, Meng Cui
Brain Stimulation 2022, 15, 190-196 · 10.1016/j.brs.2021.12.005
Abstract
Neural modulation plays a major role in delineating the circuit mechanisms and serves as the cornerstone of neural interface technologies. Among the various modulation mechanisms, ultrasound enables noninvasive label-free deep access to mammalian brain tissue. To date, most if not all ultrasonic neural modulation implementations are based on ∼1 MHz carrier frequency. The long acoustic wavelength results in a spatially coarse modulation zone, often spanning over multiple function regions. The modulation of one function region is inevitably linked with the modulation of its neighboring regions. Moreover, the lack of in vivo cellular resolution cell-type-specific recording capabilities in most studies prevents the revealing of the genuine cellular response to ultrasound. To significantly increase the spatial resolution, we explored the application of high-frequency ultrasound. To investigate the neuronal response at cellular resolutions, we developed a dual-modality system combining in vivo two-photon calcium imaging and focused ultrasound modulation. The studies show that the ∼30 MHz ultrasound can suppress the neuronal activity in awake mice at 100-μm scale spatial resolutions, paving the way for high-resolution ultrasonic neural modulation. The dual-modality in vivo system validated through this study will serve as a general platform for studying the dynamics of various cell types in response to ultrasound.
Abstract via europepmc.
Exposures
Exposure 1: 30 MHz (also 20 and 40 MHz) ultrasound to visual cortex
Target: primary visual cortex — “visual cortex (V1)”
Device: Olympus / Panametrics · Olympus NDT · PI35-2-R0.50 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 20,000, 30,000, 40,000swept | ✓✓✓⚑ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | 100 | ✓?⚑ |
| Sonication duration (s) | 60 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | 2.17, 4.46swept | ✓?⚑ |
| 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 |
The typical recording session was 300 s long: the first 60 s the ultrasound was off (baseline), the next 60 s the ultrasound was on (modulation), and the last 180 s the ultrasound was off (recovery). Amplitude-modulated (pulsed) waveforms were also tested at a 10% duty cycle with modulation frequency varied from 100 Hz to 100 kHz, and with sinusoidal amplitude modulation from 0.5 to 2 MHz, but none of these modulated waveforms suppressed calcium transients as effectively as the CW signal.
Consistency checks: f0 out of range.
Exposure 2: ultrasound to motor cortex (parameters not restated)
Target: motor cortex — “motor cortex”
Device: Olympus / Panametrics · Olympus NDT · PI35-2-R0.50 ✓
| Waveform | not reported | |
|---|---|---|
| Fundamental frequency (kHz) | not reported | ⚑ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | |
| Duty cycle (%) | not reported | |
| Sonication duration (s) | not reported |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| 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 |
We also tested the motor cortex which showed the same effects (Supplementary Fig. 5).
Flags from extraction
n_subjects— 4 mice specifically reported for the primary calcium-suppression phenomenon ('the statistics of 4 mice and 180 neurons'); additional, unspecified numbers of mice ('at least three mice ... in each experiment') were used in other sub-experiments (frequency comparisons, motor cortex, transgenic controls) not included in this count.exposures[0].fundamental_frequency_khz— 20 and 40 MHz were also tested at the same target as the primary 30 MHz condition, with only a qualitative comparison of suppression strength reported (no separate intensity/timing values); combined into a single exposure per the frequency-sweep rule.exposures[0].free_field.ispta_w_cm2— Intensity was measured with a hydrophone 'at the sound focus', typically a free-field/water measurement technique, but mice had a cranial window (skull removed, plastic coverslip with ~90% acoustic transmission) rather than an intact skull, so domain assignment (free-field vs. delivered-at-brain) is uncertain.exposures[0].timing.duty_cycle_pct— A pulsed/amplitude-modulated condition at 10% duty cycle and modulation-frequency range 100 Hz-100 kHz is also described in text, but was a secondary comparison to the primary CW protocol; not entered as separate list values to avoid conflating with the CW dose-response data.exposures[1].fundamental_frequency_khz— Motor cortex is mentioned only briefly in Methods with reference to a supplementary figure; no frequency, intensity, or timing values were restated for this target, so it is recorded as not_reported rather than assumed identical to the visual cortex protocol.sham_type— No sham condition is described; the closest control is moving the ultrasound transducer 5 mm away from the imaging/recording site, coded as 'other'.