Ultrasound Mediated Cellular Deflection Results in Cellular Depolarization
Aditya Vasan, Jeremy Orosco, Uri Magaram, Marc Duque, Connor Weiss, Yusuf Tufail, Sreekanth H Chalasani, James Friend
Advanced Science 2022, 9 · 10.1002/advs.202101950
Abstract
Ultrasound has been used to manipulate cells in both humans and animal models. While intramembrane cavitation and lipid clustering have been suggested as likely mechanisms, they lack experimental evidence. Here, high-speed digital holographic microscopy (kiloHertz order) is used to visualize the cellular membrane dynamics. It is shown that neuronal and fibroblast membranes deflect about 150 nm upon ultrasound stimulation. Next, a biomechanical model that predicts changes in membrane voltage after ultrasound exposure is developed. Finally, the model predictions are validated using whole-cell patch clamp electrophysiology on primary neurons. Collectively, it is shown that ultrasound stimulation directly defects the neuronal membrane leading to a change in membrane voltage and subsequent depolarization. The model is consistent with existing data and provides a mechanism for both ultrasound-evoked neurostimulation and sonogenetic control.
Abstract via europepmc.
Exposures
Exposure 1: HEK293 cell membrane deflection imaging
Target: other — “HEK293 cells (coverslip-adherent, in perfusion chamber)”
Device: custom-built
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 6,720 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 0.05 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | ⚑ |
|---|---|---|
| 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 |
For imaging, coverslips were mounted in a custom perfusion chamber with a built-in ultrasound transducer. Each recording consisted of a 25 ms baseline, a 50 ms ultrasound stimulus, and a 25 ms post-stimulus dwell; no pressure amplitude was reported for this imaging experiment.
Exposure 2: Primary rat neuron membrane deflection imaging and current-clamp electrophysiology
Target: cultured neurons — “rat primary cortical/hippocampal neurons in culture”
Device: custom-built
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 6,720 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 0.05 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| 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 | |
| Pressure, domain unspecified (kPa) | 500 | ✓✓✓⚑ |
Each DHM recording consisted of a 25 ms baseline, a 50 ms ultrasound stimulus, and a 25 ms post-stimulus dwell. The same ultrasound delivery rig was used for whole-cell current-clamp electrophysiology; recordings were carried out at peak pressures of 0.5 MPa because access resistance could not be maintained at higher pressures.
Flags from extraction
exposures[0].free_field.pressure_kpa— No pressure amplitude was reported for the DHM imaging experiments (only 6.72 MHz frequency and 50 ms pulse duration were stated); the 0.5-1 MPa values elsewhere in the paper are from the theoretical model or the separate patch-clamp experiment.exposures[1].unspecified_domain.pressure_kpa— The paper does not state whether the 0.5 MPa peak pressure used for patch-clamp recordings is a free-field or in-situ value; recorded as unspecified domain.n_subjects— Paper reports N=30 for each of the two cell types (HEK293, neurons) in the DHM imaging experiments; no separate n is given for the patch-clamp electrophysiology experiments, and totals are not summed per instructions.