Single cell electrophysiological alterations under dynamic loading at ultrasonic frequencies

The use of ultrasound as a non-invasive means to modulate neuronal electrophysiological signals in experimental in vivo and in vitro models has recently been gaining momentum. Paradoxically, the intrinsic mechanisms linking high-frequency minute mechanical vibrations to electrophysiological alterations at the cellular scale are yet to be identified in this context. To this end, this work combines patch clamp and nanoindentation to study the action potential alterations induced by direct mechanical vibrations at ultrasonic frequencies of dorsal root ganglion-derived neuronal single cells. The characteristics of the action potentials are studied under oscillatory shear loadings of 25 and 50 nm displacement amplitudes at frequencies ranging from 250 kHz to 1 MHz. Results show significantly narrower action potentials, with faster depolarisations and shorter rising and falling phases when induced after 1 MHz. The faster action potential dynamics appearing once the oscillation is removed points towards a cumulative or lagged effect of mechanical stimulation at ultrasonic frequencies, also observed in ultrasound neuromodulation studies. It is hypothesised here that this action potential modulation arises as a consequence of remarkable membrane properties changes induced above a threshold frequency, situated between 370 kHz and 960 kHz, and possibly related to membrane stiffening and membrane phase state alterations. These observations demonstrate the ability of mechanical cues at the cellular level to modify the neuronal signal and assert the importance of the direct mechanical vibrations induced by ultrasound stimulation protocols in assisting the observed neuromodulatory effects. Statement of Significance: In the last few decades, transcranial ultrasound stimulation (TUS) has established itself as one of the most promising non-invasive neuromodulating techniques. In particular, by avoiding both the lack of spatial specificity and surgical needs plaguing other established techniques, TUS offers new avenues for the treatment of neurological diseases. In order to enhance its specificity and efficacy, and, ultimately, optimise the sonication parameters for a given application, a better understanding of the underlying mechanisms linking mechanical vibrations to electrophysiological alterations is needed. By focusing on this coupling down to the cellular scale, this work demonstrates at the cellular scale that a transition between ∼ 400 kHz and ∼ 1 MHz exists above which mechanical vibrations are able to modulate the neuronal action potential by accelerating its dynamics