Ultrasound Elicits Behavioral Responses through Mechanical Effects on Neurons and Ion Channels in a Simple Nervous System

Abstract

Focused ultrasound has been shown to stimulate excitable cells, but the biophysical mechanisms behind this phenomenon remain poorly understood. To provide additional insight, we devised a behavioral-genetic assay applied to the well-characterized nervous system of nematodes. We found that pulsed ultrasound elicits robust reversal behavior in wild-type animals in a pressure-, duration-, and pulse protocol-dependent manner. Responses were preserved in mutants unable to sense thermal fluctuations and absent in mutants lacking neurons required for mechanosensation. Additionally, we found that the worm's response to ultrasound pulses rests on the expression of MEC-4, a DEG/ENaC/ASIC ion channel required for touch sensation. Consistent with prior studies of MEC-4-dependent currents, the worm's response was optimal for pulses repeated 300-1000 times per second. Based on these findings, we conclude that mechanical, rather than thermal, stimulation accounts for behavioral responses. Further, we propose that acoustic radiation force governs the response to ultrasound in a manner that depends on the touch receptor neurons and MEC-4-dependent ion channels. Our findings illuminate a complete pathway of ultrasound action, from the forces generated by propagating ultrasound to an activation of a specific ion channel. The findings further highlight the importance of optimizing ultrasound pulsing protocols when stimulating neurons via ion channels with mechanosensitive properties. How ultrasound influences neurons and other excitable cells has remained a mystery for decades. Although it is widely understood that ultrasound can heat tissues and induce mechanical strain, whether or not neuronal activation depends on heat, mechanical force, or both physical factors is not known. We harnessed nematodes and their extraordinary sensitivity to thermal and mechanical stimuli to address this question. Whereas thermosensory mutants respond to ultrasound similar to wild-type animals, mechanosensory mutants were insensitive to ultrasound stimulation. Additionally, stimulus parameters that accentuate mechanical effects were more effective than those producing more heat. These findings highlight a mechanical nature of the effect of ultrasound on neurons and suggest specific ways to optimize stimulation protocols in specific tissues.

Neuromodulation with transcranial focused ultrasound

“…It has been found that mutants that lack a pore-forming subunit of an ion channel that is critical for sensing gentle touch show a profound deficit in the responsiveness to FUS. 31 Thus, a mechanosensitive ion channel is required for the FUS response in this animal. Patch-clamp recordings in another recent study further revealed FUS effects on ion channels of the Piezo family.…”

“…It has been hypothesized that these effects are mediated by ion channels that can detect changes in membrane stretch following a propagating pressure wave. 30,31,49,57,58 Other mechanisms may also be at play; this topic is discussed in more detail below. Besides transient effects, FUS applied for a substantial time period (> 10 seconds) can induce long-term changes in neural activity of the stimulated circuits.…”

“…12,41 Thus, when using common neuromodulation protocols, heating is not considered to be a main driver of the effects. A recent study 31 using Caenorhabditis elegans specifically tested whether the FUS neuromodulatory effects can be of a thermal or mechanical nature. The animals rapidly reversed direction upon FUS impact.…”

“…Nonetheless, the displacement is distributed in sinusoidal fashion along the wavelength of the propagating wave. This creates a very small displacement gradient (e.g., 0.1 μm per 100 μm 31 ). It is questionable whether such a small gradient can cause significant enough deformation of a pore segment of an ion channel with regard to the channel dimensions.…”

“…This steady pressure may stretch a cell membrane to an extent that affects conformation states of ion channels or other active molecules tied to the membrane. 31 …”