The Mechanosensitive Ion Channel Piezo1 Significantly Mediates In Vitro Ultrasonic Stimulation of Neurons
Zhihai Qiu, Jinghui Guo, Shashwati Kala, Jiejun Zhu, Quanxiang Xian, Weibao Qiu, Guofeng Li, Ting Zhu, Long Meng, Rui Zhang, Hsiao Chang Chan, Hairong Zheng, Lei Sun
iScience 2019, 21, 448-457 · 10.1016/j.isci.2019.10.037
Abstract
Ultrasound brain stimulation is a promising modality for probing brain function and treating brain disease non-invasively and with high spatiotemporal resolution. However, the mechanism underlying its effects remains unclear. Here, we examine the role that the mouse piezo-type mechanosensitive ion channel component 1 (Piezo1) plays in mediating the in vitro effects of ultrasound in mouse primary cortical neurons and a neuronal cell line. We show that ultrasound alone could activate heterologous and endogenous Piezo1, initiating calcium influx and increased nuclear c-Fos expression in primary neurons but not when pre-treated with a Piezo1 inhibitor. We also found that ultrasound significantly increased the expression of the important proteins phospho-CaMKII, phospho-CREB, and c-Fos in a neuronal cell line, but Piezo1 knockdown significantly reduced this effect. Our findings demonstrate that the activity of mechanosensitive ion channels such as Piezo1 stimulated by ultrasound is an important contributor to its ability to stimulate cells in vitro.
Abstract via europepmc.
Exposures
Exposure 1: Ultrasound stimulation of Piezo1-expressing HEK293T cells, primary cortical neurons, and CLU199 hippocampal cells
Target: cultured neurons, other — “primary mouse cortical neurons; HEK293T cells (heterologous Piezo1/TRPC1/TRPV4 expression); CLU199 mouse hippocampal cell line”
Device: Olympus / Panametrics · Olympus · I7-0012-P-SU ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 500 | ✓✓✓ |
| Pulse duration (ms) | not reportedimplied by duty cycle ÷ PRF: 0.4 ms (not stated by the paper) | ⚑ |
| Pulse repetition frequency (Hz) | 1,000 | ✓✓✓ |
| Duty cycle (%) | 40 | ✓✓✓ |
| Sonication duration (s) | not reported | ⚑ |
| Free-field pressure (kPa) | 100, 500swept | ✓✓✓ |
|---|---|---|
| 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 |
Each stimulus was composed of 200 tone burst pulses at a center frequency of 500 kHz with a duty cycle of 40% at a pulse repetition frequency (PRF) of 1 kHz. The output intensity was limited to 0.1-0.5 MPa, with an interval of 10 s between pulses. For Western blot/c-Fos experiments, CLU199 cells or primary neurons were treated with ultrasound at 0.1, 0.3 or 0.5 MPa for 20 min inside a humidified incubator; calcium imaging used single stimuli, e.g. 0.3 MPa for primary neurons and 0.1-0.3 MPa for HEK293T cells.
Flags from extraction
exposures[0].timing.pulse_duration_ms— Paper states 200 tone burst pulses at PRF 1 kHz with 40% duty cycle but does not explicitly state the duration of a single pulse or of the whole sonication train in ms/s; pulse duration could be derived as duty cycle/PRF (0.4 ms) but this was not computed per protocol and is left not_reported.exposures[0].timing.sonication_duration_s— The total duration of one 200-pulse stimulus (implied ~200 ms from pulse count and PRF) is not explicitly stated in the text; left not_reported rather than computed.exposures[0].unspecified_domain.pressure_kpa— Acoustic pressure measured by hydrophone at the culture dish; paper does not describe this as a free-field/water-tank or in-situ tissue value (cells are directly exposed with no intervening skull/tissue), so placed in unspecified_domain.target— Three distinct cell/tissue types (primary cortical neurons, HEK293T with various transfected channels, and CLU199 cell line) were all stimulated with the same 500 kHz protocol; combined into a single exposure entry rather than splitting by cell type since parameters are identical.n_subjects— Paper reports numbers of independent experiments/cells per figure (e.g., n=3-15) but no single total count of cultures or replicates exposed to ultrasound across the study; recorded as not_reported.