Piezo1 and Piezo2 Mechanosensitive Ion Channels Regulate the Response of Rodents to Transcranial Focused Ultrasound Stimulation of the Motor Cortex

Academic Background

Transcranial Focused Ultrasound (TFUS) neuromodulation is a non-invasive, deep brain stimulation technique that shows great potential in the study of neural circuits and the treatment of brain diseases due to its high precision and safety. However, the exact mechanism of TFUS remains unclear. Studies have indicated that the mechanical effects of ultrasound, especially the Acoustic Radiation Force (ARF), may influence neuronal activity by acting on mechanosensitive ion channels. Therefore, elucidating the role of these ion channels in TFUS neuromodulation is crucial for developing new techniques for brain disease treatment.

Source

This research was co-authored by Tianqi Xu, Ying Zhang, Dapeng Li, Chunhao Lai, Shengpeng Wang, and Siyuan Zhang, from the Department of Biomedical Engineering, School of Life Science and Technology, and the Cardiovascular Research Center, Institute of Fundamental Medicine, both at Xi’an Jiaotong University. The paper has been accepted by the IEEE Transactions on Biomedical Engineering journal and is expected to be published in 2024.

Detailed Research Process

Research Objectives

This study aimed to investigate the role of Piezo1 and Piezo2 mechanosensitive ion channels in the mechanoreaction of the mouse brain cortex. The research team first knocked down Piezo1 and Piezo2 in the mouse brain cortex separately, compared the motor responses of different groups of mice under TFUS, and further observed ultrasound-induced neural activity through c-fos immunofluorescence.

Research Steps and Experimental Design

1. Virus Preparation and In Vivo Stereotactic Injection: Three types of AAV9 vectors were used: Piezo1 knockdown, Piezo2 knockdown, and control vector. The virus was injected into the motor cortex area of mice using stereotactic injection. Each mouse (a total of 78, all 8-week-old male C57BL/6J mice) had the virus injected at the site adjacent to the bregma (AP 0.50mm, ML -1.00mm, DV 1.00mm). Successive TFUS stimulation and assessment were carried out three weeks post-injection.

2. Expression Analysis: RT-PCR was employed to verify the knockdown effects of Piezo1 and Piezo2 mRNA. The experimental results showed that the expressions of Piezo1 and Piezo2 mRNA were significantly reduced in the knockdown groups.

3. Ultrasound Neuromodulation Protocol: A single-element concave transducer operating at 620 kHz was used, with a pulse duration of 2ms, frequency of 250Hz, and a total acoustic stimulation duration of 400ms. A self-designed dual-channel arbitrary waveform generator and a power amplifier excited the ultrasonic pulses. Ballistocardiogram data were recorded 20 times for each experimental group during ultrasound stimulation.

4. Immunofluorescence and Biosafety Assessment: c-fos immunofluorescence labeling was used to observe neural activity. Additionally, H&E staining, Nissl staining, and TUNEL staining were performed to assess the structural integrity and cellular safety post Piezo1 or Piezo2 knockdown.

Data Processing and Algorithms

The recorded potential data were processed using MATLAB software. Successful motor responses were defined as peak potential amplitude being more than three times the mean background noise. Data statistical analysis was conducted using GraphPad Prism software, applying t-tests and variance analysis methods.

Main Research Results

1. Motor Response Success Rate and Delay Time: The motor response success rate of C57BL/6J mice injected with the control virus was 85.69% ± 10.23%, while those in the Piezo1 and Piezo2 knockdown groups were 57.63% ± 14.62% and 73.71% ± 13.10%, respectively. The motor responses in the knockdown groups were significantly reduced compared to the control group. Additionally, the peak times in Piezo1 and Piezo2 knockdown groups were 0.62 ± 0.19 seconds and 0.60 ± 0.13 seconds, respectively, significantly higher than the control group’s 0.44 ± 0.12 seconds. This indicates that the absence of Piezo1 and Piezo2 affects the success rate and response time of cortical motor responses, independent of virus injection.

2. c-fos Immunofluorescence Results: The expression of c-fos post acoustic stimulation was significantly higher in each experimental group compared to the non-stimulated group. The control group’s c-fos expression was significantly higher than those in Piezo1 and Piezo2 knockdown groups, with p-values of 0.0105 and 0.0030, respectively. This shows that Piezo channels have a critical regulatory role in TFUS-induced neural activity.

3. Biosafety: H&E, TUNEL, and Nissl staining analyses revealed no structural damage or cellular apoptosis in mouse brain tissues and cells exposed to TFUS, regardless of Piezo knockdown status, thus confirming the safety of TFUS.

Conclusion and Significance

This study confirms the important role of Piezo1 and Piezo2 mechanosensitive ion channels in TFUS neuromodulation. The knockdown of these channels significantly reduces the success rate of motor responses and delays response times while significantly reducing TFUS-induced c-fos expression. These findings enhance our understanding of the mechanisms of non-invasive brain stimulation techniques and provide new targets and directions for brain disease treatments.

Research Highlights

1. First to clearly define the key roles of Piezo1 and Piezo2 in TFUS neuromodulation.
2. Systematically verified that the non-thermal effects of TFUS are realized through mechanical effects impacting mechanosensitive ion channels.
3. Proposed the functions of Piezo1 and Piezo2 in cortical motor responses. This provides new possibilities for future brain disease treatments.

This study not only reveals the working mechanism of TFUS but also provides scientific evidence for understanding the fundamental mechanisms of neuromodulation and brain function, while opening new research avenues for the development of brain disease treatment methods.