The mechanosensitive ion channel Piezo1 contributes to ultrasound neuromodulation
Jiejun Zhu, Quanxiang Xian, Xuandi Hou, Kin Fung Wong, Tingting Zhu, Zihao Chen, Dongming He, Shashwati Kala, Suresh Murugappan, Jianing Jing, Yong Wu, Xinyi Zhao, Danni Li, Jinghui Guo, Zhihai Qiu, Lei Sun
Proceedings of the National Academy of Sciences 2023, 120 · 10.1073/pnas.2300291120
Abstract
Transcranial low-intensity ultrasound is a promising neuromodulation modality, with the advantages of noninvasiveness, deep penetration, and high spatiotemporal accuracy. However, the underlying biological mechanism of ultrasonic neuromodulation remains unclear, hindering the development of efficacious treatments. Here, the well-known Piezo1 was studied through a conditional knockout mouse model as a major mediator for ultrasound neuromodulation ex vivo and in vivo. We showed that Piezo1 knockout (P1KO) in the right motor cortex of mice significantly reduced ultrasound-induced neuronal calcium responses, limb movement, and muscle electromyogram (EMG) responses. We also detected higher Piezo1 expression in the central amygdala (CEA), which was found to be more sensitive to ultrasound stimulation than the cortex was. Knocking out the Piezo1 in CEA neurons showed a significant reduction of response under ultrasound stimulation, while knocking out astrocytic Piezo1 showed no-obvious changes in neuronal responses. Additionally, we excluded an auditory confound by monitoring auditory cortical activation and using smooth waveform ultrasound with randomized parameters to stimulate P1KO ipsilateral and contralateral regions of the same brain and recording evoked movement in the corresponding limb. Thus, we demonstrate that Piezo1 is functionally expressed in different brain regions and that it is an important mediator of ultrasound neuromodulation in the brain, laying the ground for further mechanistic studies of ultrasound.
Abstract via europepmc.
Exposures
Exposure 1: Right motor cortex (Piezo1 conditional knockout vs control; ex vivo slices and in vivo)
Target: motor cortex — “right motor cortex”
Device: custom-built · 0.5-MHz lithium niobate ultrasound transducer (ex vivo: 0.5-MHz plane transducer) ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 500 | ✓✓✓ |
| Pulse duration (ms) | 0.5 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 1,000 | ✓✓✓ |
| Duty cycle (%) | 50pulse duration × PRF gives 50% | ✓✓✓ |
| Sonication duration (s) | 0.05, 0.25, 0.3, 0.5swept | ✓✓✓ |
| 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 | |
| Pressure, domain unspecified (kPa) | 30, 60, 120, 200, 250, 300, 350, 400, 450, 800swept | ✓✓✓⚑ |
Across sub-experiments (ex vivo calcium imaging, forelimb/hind limb video tracking, EMG, fiber photometry, and an ipsilateral-vs-contralateral smoothed-waveform test), the same 0.5 MHz / 1 kHz PRF / 50% duty-cycle protocol was used with pressures, sonication durations and inter-stimulus intervals (5-15 s, or randomised 10-50 s for the smoothed-waveform test) varied by assay; the fiber-photometry assay in Ctrl/P1KO cortex used a separately stated pressure range of 0.02-0.12 MPa (300 ms duration) not included in the numeric pressure list here.
Exposure 2: Central amygdala (CEA), neuronal vs astrocytic Piezo1 knockout
Target: central amygdala — “central amygdala (CEA)”
Device: custom-built · 0.5-MHz transducer with water-tube wave-guide ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 500 | ✓✓✓ |
| Pulse duration (ms) | 0.5 | ✓?⚑ |
| Pulse repetition frequency (Hz) | 1,000 | ✓✓✓ |
| Duty cycle (%) | 50pulse duration × PRF gives 50% | ✓✓✓ |
| Sonication duration (s) | 0.3 | ✓✓✓ |
| 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 | |
| Pressure, domain unspecified (kPa) | 30, 60, 120, 250swept | ✓✓✓ |
Four trials of ultrasound (0.03, 0.06, 0.12 or 0.25 MPa), each trial 3-6 stimuli with 5-s inter-stimulus interval, and 50 s rest between trials, delivered to the CEA of anesthetised mice while recording GCaMP6s fiber photometry in four viral groups (neuronal or astrocytic Piezo1 knockout and their controls).
Flags from extraction
n_subjects— Study uses many separate small cohorts across sub-experiments (e.g. N=5 Ctrl/4 P1KO for behaviour, N=5/5 for fiber photometry, 24 mice for CEA groups, 3/3 for c-Fos); no single total exposed to ultrasound is stated.n_sessions_per_subject— Not explicitly stated for any sub-experiment; appears to be a single recording/testing session per assay.exposures[0].unspecified_domain.pressure_kpa— List merges discrete pressures reported across several sub-experiments (ex vivo calcium imaging, forelimb/hind limb video, EMG, and the smoothed-waveform ipsi/contralateral test); the fiber-photometry cortex assay additionally reports a 0.02-0.12 MPa range not folded into this list.exposures[0].domain— Paper does not state whether applied pressures were measured in free field or at the brain surface/in situ; placed in unspecified_domain.exposures[1].timing.pulse_duration_ms— 500-µs tone burst is explicitly stated for the ex vivo/behavioural sub-experiments using the same fixed device protocol (1 kHz PRF, 50% duty cycle) but is not separately restated for the CEA fiber-photometry assay.auditory_control— Paper describes a 'smoothed waveform' with randomised pulse timing to exclude auditory confound, which is analogous to but not verbatim 'ramped pulses'.