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 Caenorhabditis elegans 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 in vivo , 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. SIGNIFICANCE STATEMENT 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 Caenorhabditis elegans 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.

Abstract via europepmc.

Flags from extraction

• exposures[0].timing.pulse_repetition_frequency_hz — PRF is described as a swept range (30 Hz-10 kHz) rather than an enumerated list of discrete tested values.
• exposures[0].timing.sonication_duration_s — Paper names specific durations tested (100, 200, 400 ms) within a duration sweep (Fig. 2D) but does not enumerate the complete set of values tested; recorded as the named examples.
• exposures[0].free_field.pressure_kpa — 0 MPa is the sham condition and is excluded from the active pressure list; remaining values (0.2-1.0 MPa) are the tested active pressures.
• n_subjects — n=20 is reported for the primary wild-type pressure-response cohort and is repeated (separately) for each additional mutant strain tested (thermosensory mutant, mec-3, mec-4(e1611), mec-4(u253), trp-4 alleles); no single total count across all strains/animals in the study is given.
• exposures[0].in_situ.method — Worm placed directly on an agar slab coupled to the transducer via water column (no skull); paper does not separately report an in-situ value distinct from the free-field hydrophone calibration, so left not_reported.
• n_sessions_per_subject — Each animal was tested in a single behavioral session comprising 10 stimulus trials; paper does not explicitly state a session count as a number.