Mechanosensitive Ion Channels Piezo1 and Piezo2 Mediate Motor Responses In Vivo During Transcranial Focused Ultrasound Stimulation of the Rodent Cerebral Motor Cortex

Tianqi Xu, Ying Zhang, Dapeng Li, Chunhao Lai, Shengpeng Wang, Siyuan Zhang

IEEE Transactions on Biomedical Engineering 2024, 71, 2900-2910 · 10.1109/tbme.2024.3401136

rodenthealthyemg mepcellular imaginghistology molecular

Abstract

Objective Transcranial focused ultrasound (tFUS) neuromodulation offers a noninvasive, safe, deep brain stimulation with high precision, presenting potential in understanding neural circuits and treating brain disorders. This in vivo study investigated the mechanism of tFUS in activating the opening of the mechanosensitive ion channels Piezo1 and Piezo2 in the mouse motor cortex to induce motor responses. Methods Piezo1 and Piezo2 were knocked down separately in the mouse motor cortex, followed by EMG and motor cortex immunofluorescence comparisons before and after knockdown under tFUS stimulation. Results The results demonstrated that the stimulation-induced motor response success rates in Piezo knockdown mice were lower compared to the control group (Piezo1 knockdown: 57.63% ± 14.62%, Piezo2 knockdown: 73.71% ± 13.10%, Control mice: 85.69% ± 10.23%). Both Piezo1 and Piezo2 knockdowns showed prolonged motor response times (Piezo1 knockdown: 0.62 ± 0.19 s, Piezo2 knockdown: 0.60 ± 0.13 s, Control mice: 0.44 ± 0.12 s) compared to controls. Additionally, Piezo knockdown animals subjected to tFUS showed reduced immunofluorescent c-Fos expression in the target area when measured in terms of cells per unit area compared to the control group. Conclusion This in vivo study confirms the pivotal role of Piezo channels in tFUS-induced neuromodulation, highlighting their influence on motor response efficacy and timing. Significance This study provides insights into the mechanistic underpinnings of noninvasive brain stimulation techniques and opens avenues for developing targeted therapies for neural disorders.

Abstract via europepmc.

Speciesmouse (C57BL/6J)

Subjects9, 10, 11swept animals

Sessions per subjectnot reported

Randomisednot reported

Blindingnot reported

Sham / controlinactive transducer

Auditory controlnot reported

Readout timingboth

Anaesthesianot reported

Readoutsemg mep, cellular imaging, histology molecular — EMG success rate and peak time of motor response; c-Fos immunofluorescence for neuronal activity; RT-PCR for Piezo1/Piezo2 mRNA expression; H&E, Nissl and TUNEL staining for safety

Direction of effectexcitatorytFUS stimulation of the motor cortex evoked motor responses (EMG) and increased c-Fos expression; Piezo1 and Piezo2 knockdown reduced (but did not abolish) the success rate of evoked motor responses and prolonged response latency, indicating Piezo channels mediate but are not solely required for the excitatory tFUS effect.

Adverse eventsnone observedH&E, Nissl, and TUNEL staining showed no significant tissue damage, decrease in Nissl bodies, or apoptosis (no TUNEL-positive cells) following tFUS stimulation in Piezo knockdown or control mice.

Exposures

Exposure 1: tFUS to cerebral motor cortex (M1)

Target: primary motor cortex — “cerebral motor cortex (M1)”
Device: other named manufacturer · Chongqing Haifu Medical Technology · single-element concave transducer (100 mm aperture, 80 mm focal length) ✓

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	620	✓✓✓
Pulse duration (ms)	2	✓✓✓
Pulse repetition frequency (Hz)	250	✓✓✓
Duty cycle (%)	not reportedpulse duration × PRF gives 50%	⚑
Sonication duration (s)	0.4	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	not reported
Free-field Isppa (W/cm²)	not reported
Free-field Ispta (W/cm²)	not reported
In-situ estimate	deratingsingle value
In-situ pressure (kPa)	570	✓✓✓⚑
In-situ Isppa (W/cm²)	not reported
In-situ Ispta (W/cm²)	not reported

Protocol, in the paper’s words

Ultrasound neurostimulation parameters were prepared using a previously described protocol: Ultrasound pulse duration was 2 ms, pulse repetition frequency (PRF) was 250 Hz, and total sonication duration (SD) was 400 ms. Twenty ultrasound stimulations were performed for each condition. For c-Fos immunofluorescence, ultrasound was targeted to stimulate the cerebral motor cortex for 30 min at an interval of 9 s.

Flags from extraction

n_subjects — 78 mice total were purchased but only subsets were used per experiment; the clearest enumerated group sizes exposed to tFUS for the main EMG comparison (control/Piezo1_KD/Piezo2_KD) are reported as a list; other analyses (c-Fos, safety staining) used smaller overlapping subsets of n=5
exposures[0].in_situ.pressure_kpa — Pressure was measured with a hydrophone in degassed water but reported as the 'intracranial peak rarefactional pressure', so the exact derivation (measurement vs derating/simulation) is unclear
exposures[0].timing.duty_cycle_pct — Not explicitly stated in the text; could be computed as pulse_duration x PRF but this was not done since it was not stated by the paper
anaesthesia — Isoflurane anaesthesia is described for the stereotaxic virus injection, but the anaesthesia state during the tFUS/EMG recording session itself is not explicitly stated