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.

Keywords: Caenorhabditis elegans; mechanosensation; mechanosensitive ion channels; neurostimulation; thermosensation; ultrasound.

Figure 1.

A system for delivering pulsed ultrasound to C. elegans nematodes. A, Schematic side view of the setup, showing a single wild-type adult hermaphrodite crawling on the surface of agar slab, tracked by a digital video camera, and maintained within the field of view by a copper sulfate boundary. A piezoelectric ultrasound transducer (10 MHz carrier frequency, line-focused) is coupled directly to the bottom of the agar slab by a column of degassed water. B, Simulation of the distribution of radiation force expected from the line-focused 10 MHz ultrasound transducer (see Materials and Methods). Animals were stimulated only during forward movement, as they entered the zone corresponding to the highest expected pressure. C, Schematic of a typical stimulus consisting of ultrasound pulses delivered at PRF of 1 kHz for a total duration of 200 ms at 50% duty cycle. In this study, we systematically varied the applied pressure, pulse duration, PRF, and duty cycle.

Figure 2.

Ultrasound elicits reversal behavior in a pressure- and stimulus time-dependent manner in wild-type C. elegans. A, B, Raster plots showing the response of 20 animals (10 trials/animals) to a 200 ms sham stimulus (0 MPa pressure, A) and a bona fide stimulus (1.0 MPa, B). Heading angle is encoded in color such that headings similar to the average angle in the 1 s window immediately preceding stimulus onset are blue and reversals are encoded in yellow. Rows correspond to single trials and blocks are 10 trials delivered to each animal; traces were smoothed with a zero-lag rectangular sliding 150 ms window. Top, Silhouettes depict representative responses to sham (A) and 1.0 MPa stimuli (B). C, Reversal frequency increases with applied pressure. Points indicate mean ± SEM (n = 20) for animals stimulated at each of the six pressure values for a total of 10 trials. Solid line indicates a Boltzmann fit to the data with an P_1/2 of 0.71 MPa, a slope factor of 0.15, and a maximum probability of 83%. Dotted line indicates the unstimulated reversal rate. Stimulus parameters: 1 kHz, 50% duty cycle, 200 ms pulse duration, variable pressure. D, Reversal probability increases with stimulus duration. Points indicate mean ± SEM (n = 20). Smooth line indicates an exponential fit to the data with a time constant of 90 ms. Stimulus parameters: 1 kHz, 50% duty cycle, variable pulse duration, 1.0 MPa pressure. C, D, Dotted line indicates baseline rate of responding (see Materials and Methods). Smooth line indicates an exponential fit to the data with a time constant of 90 ms.

Figure 3.

Loss of mechanosensation, but not thermosensation, disrupts ultrasound-evoked reversals. A, B, Pressure-response curves of wild-type N2 animals (blue) compared with a thermosensation-defective mutant (orange) and three mechanosensation-defective mutants (black): mec-3(e1338), mec-4(e1611), and mec-4(u253). Points indicate mean ± SEM (n = 20 animals tested in 10 trials/animals). Smooth lines indicate fit to the data according to a sigmoidal function. The data and fit for wild-type are the same as in Figure 2C. Fitting parameters for gcy-23(nj37)gcy-8(oy44)gcy-18(nj38) are (F_max, P_1/2, slope, base): 80%, 0.76 MPa, 0.10, 9%. Dotted lines indicate the average baseline response rate (see Materials and Methods) for each case. There was no significant effect of genotype on baseline reversal rates. Stimulus parameters: 1 kHz pulse frequency, 50% duty cycle, 200 ms duration, variable pressure. C, Response rate as a function of genotype. Error bars indicate mean (SEM; n = 20) reversal rate. Annotations below the graph indicate the nature of the known sensory deficit associated with each genotype. Animals were tested as young adult hermaphrodites and blind to genotype.

Figure 4.

Strains carrying deletions in the trp-4 NOMPC channel gene differ in their response to ultrasound stimulation. A, Pressure-response curves of wild-type (blue), VC1141 trp-4(ok1605) (magenta) mutants used in a previous study (Ibsen et al., 2015). Smooth curve fit to the trp-4(ok1605) data yielded F_max = 65%; P_1/2 = 0.83 MPa, slope = 0.13, base = 8%. B, Pressure response curves of three other trp-4 deletion mutants were indistinguishable from wild-type. VC818 trp-4(gk341), TQ296 trp-4(sy695), and GN716 trp-4(ok1605) mutants, which was derived from VC1141 by outcrossing four times with wild-type (N2) animals. C, Response rate in four trp-4 mutant strains. Error bars indicate mean ± SEM reversal rate evoked by ultrasound stimulation with the following parameters: 1 kHz, 50% duty cycle, 200 ms pulse duration, 1.0 MPa. Dotted line indicates the average baseline response rate (see Materials and Methods). The number of animals analyzed across 10 trials is indicated in parentheses. We used PCR to verify that all strains harbored the expected deletions in the trp-4 locus (Materials and Methods). Wild-type data are from Figure 2C.

Figure 5.

Ultrasound efficacy depends on pulse repetition frequency and duty cycle. A, The response (black circles, mean ± SEM) of wild-type animals to a train of ultrasound pulses plotted as a function of pulse repetition frequency. The duty cycle was held constant at 50% in all cases, ensuring that all stimuli deliver the same amount of energy. The smooth curve (green) shows a simulation of the sensitivity of TRN currents to sinusoidal mechanical indentations (Eastwood et al., 2015). B, The response (black circles, mean ± SEM) of wild-type animals as a function of duty cycle. Because the pulse repetition rate was 1 kHz in all cases, a duty cycle of 5, 10, 25, 50, 75, 100% corresponds to a pulse width of 50 μs, 100 μs, 250 μs, 500 μs, 750 μs, and 1 ms (continuous wave, no off epochs), respectively. The green curve shows the level of TRN activation expected from the frequency filtering shown in panel A (Materials and Methods for modeling details). Temperature increase (orange circles, mean ± SEM, n = 5) as a function of duty cycle. The smooth curve (orange) is a quadratic fit to the data and is included for visual clarity. A, B, The carrier frequency was 10 MHz, the stimulus amplitude was 1 MPa, and the stimulus duration 200 ms. The dotted line shows the unstimulated reversal frequency.