Two pathways are required for ultrasound-evoked behavioral changes in Caenorhabditis elegans.

Ultrasound has been shown to affect the function of both neurons and non-neuronal cells, but, the underlying molecular machinery has been poorly understood. Here, we show that at least two mechanosensitive proteins act together to generate C. elegans behavioral responses to ultrasound stimuli. We first show that these animals generate reversals in response to a single 10 msec pulse from a 2.25 MHz ultrasound transducer. Next, we show that the pore-forming subunit of the mechanosensitive channel TRP-4, and a DEG/ENaC/ASIC ion channel MEC-4, are both required for this ultrasound-evoked reversal response. Further, the trp-4;mec-4 double mutant shows a stronger behavioral deficit compared to either single mutant. Finally, overexpressing TRP-4 in specific chemosensory neurons can rescue the ultrasound-triggered behavioral deficit in the mec-4 null mutant, suggesting that both TRP-4 and MEC-4 act together in affecting behavior. Together, we demonstrate that multiple mechanosensitive proteins likely cooperate to transform ultrasound stimuli into behavioral changes.

Introduction

Ultrasound has been shown to modify neuronal activity in number of animal models including humans [1, 2]. However, the direction of this action is somewhat controversial with some reporting activation [3-7], while others demonstrate inhibition [2, 8–10]. Moreover, the underlying mechanisms for ultrasound action on neuronal membranes have been suggested to include thermal [11-13], mechanical (direct or via cavitation [14-16]) or a combination of two [17]. Additionally, ultrasound neuromodulation has also been shown to include astrocyte signals in vitro [18] and auditory signals in vivo [19, 20]. To identify the underlying molecular mechanisms, we and others have been examining how ultrasound affects neurons in tractable invertebrate systems [16, 21–23] or mammalian cell [24, 25] and slice cultures [3, 26].

The nematode, C. elegans with just 302 neurons connected by identified chemical and electrical synapses generating robust behaviors with powerful genetic tools is ideally suited to probe the molecular effects of ultrasound on neuronal membranes [27-29]. We previously showed that the pore-forming subunit of the mechanosensitive ion channel TRP-4 is required to generate behavioral responses mediated by microbubbles activated by a single 10 ms pulse of ultrasound generated from a 2.25 MHz focused transducer [21]. This channel is specifically expressed in few dopaminergic (CEPs, and ADE) and interneurons (DVA and PVC) in C. elegans, where it has been shown to be involved in regulating the head movement and locomotion [30, 31]. Surprisingly, we found that ectopically expressing this TRP-4 in a neuron rendered that neuron sensitive to ultrasound stimuli, confirming ultrasound-triggered, microbubble-mediated control [21]. Moreover, a second mechanosensitive protein, a DEG/ENaC/ASIC ion channel MEC-4 was also shown to be required for behavioral responses to a 300 ms duration, 1 KHz pulse repetition frequency at 50% duty cycle generated from a 10 MHz focused ultrasound transducer [22]. These two studies confirm that ultrasound effects on C. elegans behavior is likely mediated by mechanosensitive proteins.

In this study, we used genetic tools in C. elegans to test whether TRP-4 and MEC-4 are both required to mediate the behavioral effects of ultrasound stimuli. We generated a trp-4 mec-4 double mutant and compared its ultrasound responses to both single mutants. Also, we found that ectopically expressing TRP-4 in specific chemosensory neurons can rescue the behavioral deficits in both trp-4 and mec-4 null mutants, confirming that these genes act together to drive ultrasound-evoked behavior. Our study demonstrates that multiple mechanosensory pathways act in concert to generate behavioral responses to ultrasound stimuli.

Results

To test the behavioral responses of C. elegans to various ultrasound stimuli, we aligned a transducer with a holder that positioned agar plates at the water level in a tank (). Animal responses were captured using a camera and analyzed (, See Methods for more details). Next, we evaluated both pressure and temperature changes at the agar surface for a single 10 ms pulse of ultrasound stimuli of different intensities. We assessed the area on the agar surface which was ensonified by the ultrasound stimuli and found that our system delivered mechanical, but not temperature changes (). We found that ultrasound stimuli delivering peak negative pressures greater than 0.75 MPa amplified by 1–10 μm-sized gas filled microbubbles generated robust responses in wild-type (WT) animals. We analyzed these responses and found that animals generated robust increases in their large reversals (events where the head bends twice or more), but not omega bends or small reversals (where the head bends only once) (Figs ). These data are consistent with previous studies showing that C. elegans generates dose-dependent responses to ultrasound stimuli [21, 22].

We then analyzed the behavioral responses of null mutants in both the TRP-4 and MEC-4 channels to ultrasound. We found that trp-4(ok1605) and mec-4(u253) mutants are both defective in their ultrasound-evoked large reversal behavior both with and without gas-filled microbubbles (). Also, we observe that mec-4(u253) null mutants have significant defects at higher (>0.92 MPa), but not lower pressures generated from a 2.25 MHz ultrasound transducer (). Similarly, we also found that an outcrossed allele of trp-4(ok1605 4X) was also defective in its response to ultrasound stimuli at higher pressures (). This result is consistent with our previous study, which identified a critical role for trp-4 in mediating ultrasound-evoked behavioral responses [21]. Additionally, we found that C. elegans can also respond to ultrasound stimuli even in the absence of gas-filled microbubbles, consistent with a previous study [22]. Moreover, we find that at least at lower ultrasound pressures (0.79 MPa), the probability of C. elegans’ responses are increased in the presence of microbubbles (). Importantly, we found that at high pressure, the trp-4(ok1605 4X); mec-4(u253) double mutant had a stronger defect in ultrasound-trigged large reversal responses compared to either single mutant (), both with and without microbubbles (), suggesting that these genes likely act together. Moreover, we do not observe any consistent change in small reversals, however changes in omega bends were similar to what we found with large reversals (). These data are consistent with previous studies showing that omega bends often occur together with large reversals [32, 33]. Collectively, these data indicate that MEC-4 and TRP-4 channel proteins might be acting together to mediate C. elegans behavioral responses to ultrasound stimuli.

To confirm whether these two mechanosensitive proteins are acting together, we tested combinations of transgenic animals ectopically expressing TRP-4 in various null mutant backgrounds. We expressed TRP-4 under ASH and AWC-chemosensory neuron selective promoters and analyzed the ultrasound responses of the resulting transgenics [21]. Neither ASH nor AWC expression of TRP-4 in wildtype animals altered its ultrasound-behavior (). However, at lower pressures (0.79 MPa) TRP-4 expression in AWC chemosensory neurons was able to partially rescue large reversals in both trp-4(ok1605) and mec-4(u253) mutants ( In contrast, we found that ASH expression of TRP-4 was able to partially rescue behavioral deficits in both the trp-4(ok1605) and mec-4(u253) null mutants only at higher pressures (> 0.92 MPa) (). These data suggest that TRP-4 protein likely functions in different neurons to affect ultrasound-evoked large reversals: AWC for lower pressures and ASH at higher pressures (). While AWC neurons are known to have an expanded fan-shaped cilia, ASH neurons have a rod shaped cilia [34]. We suggest that this difference in the shape of the cilia might result in AWC and ASH neurons having a different sensitivity to ultrasound-evoked stimuli. In addition, we found that AWC and ASH-selective expression of trp-4 was able to rescue the behavioral deficits observed in the trp-4(ok1605 4x) outcrossed strain () confirming that TRP-4 likely functions in these two chemosensory neurons to drive ultrasound-evoked large reversal behaviors. Also, while small reversals were not consistent, omega bends often matched large reversals (). Collectively, these data suggest that TRP-4 and MEC-4 likely act together to generate large reversals in response to ultrasound stimuli.

Discussion

We showed that a double mutant that deleted both MEC-4 and TRP-4 channels had stronger behavioral deficits compared to either single mutant alone. Additionally, we showed that AWC and ASH-specific expression of TRP-4 could partially rescue the deficit in both mec-4(u253) and trp-4(ok1604 4x) single mutants confirming that these two pathways act together to drive ultrasound-evoked changes in large reversals. We suggest that expression of TRP-4 channel in AWC and ASH neurons renders them sensitive to ultrasound stimuli. Activating these neurons has been previously shown to generate reversal behavior [35, 36]. We speculate that the ultrasound-driven activation of ASH and AWC neurons mediated by TRP-4 channels acts independent of the MEC-4 pathway to generate reversal behavior.

The nematode C elegans has provided insights into our understanding of how ultrasound affects animal behavior. We previously showed that ultrasound evoked behavioral changes required the pore-forming subunit of the TRP-4 mechanosensitive channel [21]. This protein is selectively expressed in a few dopaminergic and interneurons and is likely involved in generating head movement and coordinating locomotory behaviors. We suggested that delivering ultrasound to the head of the animal likely activates this channel resulting in reversal behavior [21]. Moreover, a second mechanosensitive channel, MEC-4 (DEG/ENaC/ASIC) has been shown to be required for ultrasound-evoked behavioral responses in C. elegans [22]. MEC-4 is a key component of the touch sensitive mechanosensitive ion channel and is expressed in the ALM, PLM, AVM, PVM, FLP and other touch-activated neurons [37, 38]. This study indicated that ultrasound delivered to the head of the animal would also generate a reversal response [22]. While we find that mec-4(u253) and trp-4(ok1605 4X) mutants are indeed defective under our stimulus conditions and likely act together to generate ultrasound-evoked behavioral changes, we are unable to observe ultrasound-evoked behavioral changes to 10 MHz ultrasound stimuli (). Although we were unable to achieve the magnitude of behavior reported in [22], we observed that, consistent with our other findings, introducing microbubbles to the assay did significantly increase worm responses to ultrasound stimuli ( Perhaps, differences in ultrasound delivery and stimulus parameters might explain the discrepancy between our study and previous studies, which have reported C. elegans responses to 10 MHz and ~28 MHz [22, 39, 40]. Additionally, while previous studies have shown that mec-4(u253) are defective to a broad range of ultrasound pressures delivered from 10 MHz transducer [22], we observe that these mec-4(u253) mutants are only defective at pressures greater than 0.92 MPa delivered from a 2.25 MHz transducer. These data might imply that distinct molecular machinery might sense ultrasound stimuli at different frequencies. Furthermore, we find that trp-4(ok1605 4x) has similar behavioral deficits compared to mec-4(u253) animals confirming that these two genes might act together to affect ultrasound-evoked behaviors.

Ultrasound has been used to non-invasively manipulate both neuronal and non-neuronal cells in a number of animals including humans. We show that animals missing two mechanosensitive proteins are defective in their responses to ultrasound stimuli, confirming a role for mechanosensation in mediating the biological effects of this modality. This is also consistent with multiple studies identifying other mechanosensitive proteins that can confer ultrasound sensitivity to mammalian cells in vitro and in vivo [24–26, 41–43]. This is particularly relevant to the method of using ultrasound to selectively and non-invasively manipulate cells within an animal (“Sonogenetics”). Furthermore, our study implies that ultrasound might affect at least two mechanosensitive ion channels to affect animal behavior. Identifying downstream signaling pathways of these and other ultrasound-sensitive mechanosensitive channels would provide a framework to decode ultrasound neuromodulation and enhance sonogenetic control.

Methods

Ultrasound imaging assay

A schematic of the system for imaging ultrasound-triggered behavior appears in Fig 1A. An immersible 2.25 MHz central frequency point focused transducer (V305-SU-F1.00IN-PTF, Olympus NDT, Waltham, MA) was positioned in a water bath below a 60 mm 2% agar-filled petri dish, and connected via waterproof connector cable (BCU-58-6W, Olympus). A single 10ms pulse was generated using a TTL pulse to trigger a multi-channel function generator (MFG-2230M, GWINSTEK, New Taipei City, Taiwan); the amplitude of the signal was adjusted through a 300-W amplifier (VTC2057574, Vox Technologies, Richardson, TX) to achieve desired pressures. C. elegans behavior was captured through a high speed sCMOS camera (Prime BSI, Photometrics, Tuscon, AZ) and a 4x objective (MRH00041, Nikon, Chicago, IL). Worm movement was compensated via a custom joystick-movable cantilever stage (LVP, San Diego, CA) controlled by Prior Box (ProScanIII, Prior, Cambridge, UK). All components were integrated via custom MetaMorph software (Molecular Devices, San Jose, CA). A goose-neck lamp (LED-8WD, AmScope, Irvine, CA) at ~45° provided oblique white light illumination.

Fig 1

Recording C. elegans behavior in response to ultrasound at 2.25 MHz.

(A) Schematic of 2.25 MHz ultrasound imaging system with transducer, water bath, and 4x objective over agar plate. (B) Top view of agar plate with animals corralled by copper sulfate barrier (1.5 cm in diameter) on agar plate with polydisperse microbubbles. (C) Fiberoptic hydrophone measurements at perpendicular distance from focal point of transducer show peak negative pressures ~1 MPa, with (D) negligible temperature changes at 10ms ultrasound pulses at t = 0. Individual points connected via spline fit. (E) Relative head positions (yellow dots) of each animal at time of stimulation, some example points highlighted for visibility.

The ultrasound transducer was focused in the Z-plane to the focal plane of the camera, allowing for X-Y motion of the stage and petri dish using the joystick-movable stage all in the focal plane of both the ultrasound transducer and camera. The petri dish was coupled to the ultrasound transducer via degassed water in the water bath.

For 10 MHz experiments, the 2.25 MHz transducer was replaced with a 10 MHz line-focused transducer (A327S-SU-CF1.00IN-PTF, Olympus) coupled via plastic 20 mL syringe and degassed water as previously described [22]. The plastic portion of the petri dish was removed to couple the transducer directly to the agar slab, and a 2x objective (MRD00025, Nikon) replaced the 4x objective to allow for full imaging of the larger focal area of the transducer.

Behavioral assays

For experiments with microbubbles, polydisperse microbubbles (PMB, Advanced Microbubbles, Newark, CA) were diluted to a concentration of ~4x107 and added to an empty 2% agar plate 20 minutes before imaging to allow for absorption/evaporation of the solvent media, leaving microbubbles on the surface of the agar. A dry filter paper with a 1 cm hole previously soaked with 200mM copper sulfate solution was placed around the microbubble lawn, and a young adult C. elegans was moved from a home plate to the imaging plate using an eyelash. The agar plate was moved around using the motorized stage to place the worm into the focal zone of the transducer where it was stimulated with a single ultrasound pulse of appropriate amplitude. Videos were recorded for 10 seconds at 10 frames/second, with ultrasound stimulation (described above, via TTL pulse) occurring at 1.5 seconds.

Reversals with more than two head bends were characterized as large reversals, those with fewer than two head bends were characterized as small reversals, and omega bends were those which led to a high-angled turn that lead to a substantial change in direction of movement [21]. Wildtype animals were tested daily to monitor and maintain a baseline level of reversal behavior, and comparisons between strains were made for animals recorded within same days of testing. Where possible, wildtype, mutants and rescue animals were tested on the same day. For 10 MHz ultrasound stimulation, all deviations from the baseline are plotted in S3C Fig. Behavioral data were collected over at least three days to confirm reproducibility, the data were then pooled for final statistical analysis, shown in relevant figures.

C. elegans

Wildtype C. elegans–CGC N2; VC1141 trp-4(ok1605); GN716 trp-4(ok1605) outcrossed four times [22]; TU253 mec-4(u253) [22]; IV903 trp-4(ok1605); mec-4(u253), made by crossing GN716 and TU253.

ASH rescues: IV133 ueEx71 [Psra-6::trp-4; Pelt-2::GFP] made by injecting N2 with 50ng/μL Psra-6::trp-4, 10ng/μL elt-2::gfp for ASH overexpression of trp-4. IV160 trp-4(ok1605) I; ueEx88 [Psra-6::trp4; Pelt-2::GFP] made by injecting VC1141 with 50ng/μL Psra-6::trp-4, 10ng/μL elt-2::gfp for ASH rescue of trp-4. IV840 mec-4(u253) X; ueEx71 [Psra-6::trp-4; Pelt-2::GFP] made by crossing IV133 and TU253.

AWC rescues: IV157 ueEx85 [Podr-3::trp-4; Pelt-2::GFP] made by injecting N2 with 50ng/μL Podr-3::trp4, 10ng/μL elt-2::gfp for AWC overexpression of trp-4. IV162 trp-4(ok1605) I; ueEx89 [Podr-3::trp-4; Pelt-2::GFP] made by injecting VC1141 with 50ng/μL Podr-3::trp-4, 10ng/μL elt-2::gfp for AWC rescue of trp-4. IV839 mec-4(u253) X; ueEx85 [Podr-3::trp-4; Pelt-2::GFP] made by crossing IV157 and TU253.

Behavioral ultrasound pressure and temperature measurements

Pressure and temperature measurements were collected through 2% agar plates using a Precision Acoustics Fiber-Optic Hydrophone connected to a 1052B Oscilloscope (Tektronix, Beaverton, OR). The hydrophone probe was moved sub-mm distances using the same stage used for animal recordings while the petri dish was held in place using a three-prong clamp. The noise floor for this instrument is 10kPa [44], and the uncertainty of the instrument is 10% in the frequency range used in this study [45], allows us to adequately measure pressures used in this study (>500kPa). The full width at half maximum (FWHM) reported was determined by interpolating the perpendicular distances at which half of the maximum pressure was measured using the fiberoptic hydrophone, and averaging over the amplifier gains used to achieve various pressures.

Statistical analysis

Data were analyzed by combining several days’ recordings within several days’ experimental sessions, with sample sizes chosen to reflect those described previously [21]. All behavioral data were plotted as proportion of response plus/minus standard error of the proportion. For significance tests, two-proportion z-tests were used with Bonferroni corrections for multiple comparisons. All sample sizes were >30, and animals were chosen at random from their broader population. The observer was not blind to the genotype of the group being tested. Animals were excluded from the study if they showed visible signs of injury upon transfer to the assay.

Frequency of small reversals, defined as fewer than two head bends, and omega bends in different strains at different peak negative pressures.

(A, C, E) Small reversals and (B, D, F) omega bends from Fig 2 recordings. n = 45 for each condition (n = 90–135 in c-d single mutants). Proportion of animals responding with standard error of the proportion are shown. *** p < .001, ** p < .01, * p < .05 by two-proportion z-test with Bonferroni correction (c = 5) for multiple comparisons.

Fig 2

Mutants in mechanosensitive proteins are defective in their responses to ultrasound. Frequency of large reversals (more than two head bends) with and without microbubbles.

Large reversal frequency (A) without microbubbles and (B) with microbubbles at various peak negative pressures are quantified. n = 90–135 for each condition, n = 45 for double mutant strains. At certain pressures, the double mutant trp-4;mec4 responds significantly less than each of the individual mutants, which respond less than WT animals. At 0.99 MPa, the outcrossed double mutant trp-4 4x;mec-4 shows a significant defect in large reversals both without (C, n = 43) and with (D, n = 55) microbubbles. Proportion of animals responding with standard error of the proportion are shown. ***p < .001, **p < .01, *p < .05 by two-proportion z-test with Bonferroni correction for multiple comparisons, with c = 2 for 2A, c = 5 for 2B.

(TIF)

Click here for additional data file.

(A, C, E) Small reversals and (B, D, F) omega bends from Fig 3 recordings. n = 45 for each condition, Proportion of animals responding with standard error of the proportion are shown. *** p < .001, ** p < .01, * p < .05 by two-proportion z-test with Bonferroni correction (c = 3) for multiple comparisons.

Fig 3

MEC-4 and TRP-4 act in parallel to mediate ultrasound-evoked behavioral changes.

Frequency of large reversals (more than two head bends) in strains ectopically expressing TRP-4. Large reversal frequency with (A) AWC::trp-4 and (B) ASH::trp-4 in different backgrounds at different peak negative pressures are quantified. At certain conditions, trp-4 expression significantly increases large reversal frequency in both trp-4 knockout and mec-4 knockout animals. (C) At 0.99 MPa, TRP-4 expression in both ASH and AWC rescues large reversal behavior in the outcrossed trp-4(ok1605)4x mutant. n = 45 for each condition. Proportion of animals responding with standard error of the proportion are shown. *** p < .001, ** p < .01, * p < .05 by two-proportion z-test.

(TIF)

Click here for additional data file.

Schematic of 10 MHz behavioral imaging setup, modified from Kubanek et al. 2018 [22].

(A) Experimental setup has agar slab with C. elegans corralled by a copper sulfate barrier resting on top of a 20 mL syringe. Degassed water couples the piezoelectric line-focused transducer (10 MHz) to the agar slab. (B) Hydrophone measurements at different perpendicular positions relative to the transducer line focus, with peak negative pressures reaching 1 MPa at highest amplifier settings. Yellow bar represents line focus of highest pressure, points connected via spline fit, FWHM ~ 0.52mm. (C) C. elegans exhibits minimal behavioral responses to 10 MHz ultrasound stimuli, although these are significantly enhanced in the presence of microbubbles. (D) Example image of C. elegans on agar slab approaching ultrasound focal line. Yellow dots (some highlighted for visibility) represent head positions of each of n = 224 worms, indicating a significant proportion of ultrasound stimulations occurred when the head was positioned within the high-pressure band (yellow).

(TIF)

Click here for additional data file.

WT C. elegans performing a large reversal (and omega bend) in response to ultrasound.

10 second video recorded at 10 fps, with ultrasound pulse at 1.5 s (15 frames). Ultrasound label temporally extended for visibility. After ultrasound pulse, worm reverses with >2 head bends and completes an omega bend reorientation.

(MP4)

Click here for additional data file.

Minimal data set for Magaram et al. 2022.

An excel file with minimal data for Figs 1–3 and S1–S3.

(XLSX)

Click here for additional data file.

9 Dec 2021

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Reviewer #1: Uri and colleagues report in this manuscript a follow-up study on the molecular underpinning of C. elegans response to ultrasounds. The group published that the mechanosensory TRP channel TRP-4 is at least in part responsible for the worm response to ultrasounds. Later though, another group reported that another mechanosensory channel of the DEG/ENaC superfamily called MEC-4 was underlying the animals’ response to ultrasounds. Thus, the question that the authors try to address here is whether these two channels function within the same or two parallel molecular pathways. I believe that the authors have data supporting the conclusion that these two channels function in parallel pathways. However, I think the manuscript could be substantially improved.

1. 0.92 MPa seems to be already at the plateau of the response. It would be useful to include a point between 0.79 and 0.92 MPa. This could help in teasing out differences between the mutants.

2. The trp-4 x4 strain should be used for the experiments and the rescues since it is the strain with the least likelihood of carrying other mutations in the background. It is not clear what strain was used for the rescues for example.

3. Rescue experiments both in mec-4 and trp-4 mutants should be performed using mec-4 DNA.

4. Each figure legend should contain the exact number of animals used in each experiment and how many times the experiment was repeated. Also, the figures should show the individual data points and the averages +/- SE or SD.

5. It is intriguing that the expression of TRP-4 in AWC rescues reversals at 0.79 MPa but expression of the same channel in ASH rescues at 0.92 MPa. The authors should comment on this finding and propose a hypothesis.

6. The authors should carefully review the text and the labels of the figures for the C. elegans genetic nomenclature. There are several errors.

7. Please labels the panels within the figures so that it is clear which experiments were done with and which without microbubbles.

8. Line 131, the authors need to explain why they were “unable to replicate this study”. Was it part of the study? The entire study? This is somewhat concerning.

Reviewer #2: In this study, the authors exposed C. elegans to 10 ms pulses of 2.25 MHz, mid-intensity ultrasound with the aid of gas-filled microbubbles. In some conditions, responses without the bubbles were also tested.

The authors replicated previous findings [22, 38] that mutations of the mec-4 pore-forming subunit diminish the responsiveness to ultrasound. The authors also replicated previous findings [22, 38] that mutations of the trp-4 pore-forming subunit have small but likely significant effect on the responses. For one pressure level, but not others, there was a significant increase in responsiveness when trp-4 was overexpressed in AWC neurons.

This study provides a useful replication of previous studies and thus should be eventually published.

There are, nonetheless, several points that should be addressed:

1) The trp-4 (ok1605 4X) effect is small, like in [22] and [38]. This should be acknowledged. There is a suggestion in this paper that the effect may reach significance. The significance values should be provided in the text. Importantly, there are many comparisons made among the numerous bars in the plots. Such multiple comparisons should be corrected for (e.g., Bonferroni), or an omnibus statistical analysis (n-way ANOVA) performed.

2) Fig. 3A versus B reveal enormous variability (20% versus 70%) in the response of WT animals at 0.79 MPa. This raises questions regarding the reliability of assessing the responses, and so the reliability of the plots. It seems the videos were quantified by eye. This should be specified and the reasons for the variability addressed.

3) The rescue AWC::trp-4 was performed on the trp-4 (ok1605), which has many other mutations present, unless these are outcrossed (Figure 2 and [22, 38] confirm this). There can be a complex interactions between the trp-4 and the other affected genes. Therefore, the AWC::trp-4 overexpression may not be conclusive. This should be acknowledged.

4) It seems only Fig. 2A and Suppl. Fig. 2 show data without microbubbles. The authors should specify which datasets used the bubbles.

Additional, less pressing issues:

A. The mec-4 effect appears weaker compared to [22] and [38]. Possible reasons could be provided.

B. "While we are unable to replicate this study" [22]

There are several important technological differences in the present study:

1) There is a relatively broad focus (Figure S2B), likely activating the entire animal. In this case, there may both reversal and acceleration tendencies, cancelling each other out.

2) The slab/dish sit directly over the syringe top, which may not have allowed the assurance that there is continuous water coupling. Water evaporation degrades the coupling over time.

3) [22] used longer duration (300 ms) and pulsed stimuli (1 kHz), which are both critical.

These differences should be acknowledged.

Moreover, the effects of [22] were replicated in two other studies ([38], [39]). These two studies should be cited.

C. mec-4 and trp-4 likely "act in parallel".

The finding of an increased effect does not necessarily imply parallel (or serial) engagement. I suggest to rephrase as "summed" or "compounded" effect, or "both act".

Minor:

i. "indirectly include astrocyte signals"

- based on my read of the study, the astrocytic stimulation is direct

ii. "We previously showed that the pore-forming subunit of the mechanosensitive ion channel TRP-4 is required to generate behavioral responses to a single 10 ms pulse of ultrasound generated from a 2.25 MHz focused transducer [21]"

-> "We previously showed that the pore-forming subunit of the mechanosensitive ion channel TRP-4 is required to generate behavioral responses mediated by microbbubles activated by a 10 ms pulse of ultrasound generated from a 2.25 MHz focused transducer [21]"

iii. "confirming ultrasound-triggered control (Sonogenetics)"

-> "confirming ultrasound-triggered, microbubble-mediated control"

iv. "We generated a trp-4 mec-4 double mutant and compared its ultrasound responses to both single mutants."

-> missing "We found ..." sentence following this sentence.

v. "lower ultrasound intensities (0.79 MPa)"

-> "pressures"

Refs:

[38] "Ultrasound neuro-modulation chip: activation of sensory neurons in Caenorhabditis elegans by surface acoustic waves"

[39] "Ultrasound activation of mechanosensory ion channels in Caenorhabditis elegans"

**********

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9 Feb 2022

Responses (in black) to the reviewer’s concerns (in blue)

Reviewer #1: Uri and colleagues report in this manuscript a follow-up study on the molecular underpinning of C. elegans response to ultrasounds. The group published that the mechanosensory TRP channel TRP-4 is at least in part responsible for the worm response to ultrasounds. Later though, another group reported that another mechanosensory channel of the DEG/ENaC superfamily called MEC-4 was underlying the animals’ response to ultrasounds. Thus, the question that the authors try to address here is whether these two channels function within the same or two parallel molecular pathways. I believe that the authors have data supporting the conclusion that these two channels function in parallel pathways. However, I think the manuscript could be substantially improved.

We thank the reviewer for their positive feedback.

1. 0.92 MPa seems to be already at the plateau of the response. It would be useful to include a point between 0.79 and 0.92 MPa. This could help in teasing out differences between the mutants.

We have included data from 0.88 MPa in this revised manuscript.

2. The trp-4 x4 strain should be used for the experiments and the rescues since it is the strain with the least likelihood of carrying other mutations in the background. It is not clear what strain was used for the rescues for example.

We have clarified the use of original versus 4x strain in the manuscript, and have included new data in the trp-4 outcrossed strain in the revised manuscript as suggested by the reviewer.

3. Rescue experiments both in mec-4 and trp-4 mutants should be performed using mec-4 DNA.

We agree with the reviewer that this would be an excellent experiment. However, the site of action for the MEC-4 protein has not been identified. We feel that mapping the site of MEC-4 action is beyond the scope of this study.

4. Each figure legend should contain the exact number of animals used in each experiment and how many times the experiment was repeated. Also, the figures should show the individual data points and the averages +/- SE or SD.

We have included the exact number of animals in each experiment in the revised manuscript. However, the animals either respond or don’t to a given ultrasound stimuli, making it difficult to present individual data points. We suggest that the proportion of responses is a better approach to display these results. Error bars represent standard error of the proportion, indicated in the figure legends.

5. It is intriguing that the expression of TRP-4 in AWC rescues reversals at 0.79 MPa but expression of the same channel in ASH rescues at 0.92 MPa. The authors should comment on this finding and propose a hypothesis.

We agree that this is an surprising finding. We have included a hypothesis in line 133-135. We suggest that AWC and ASH sensory neurons might have different sensitivities to mechanical stimuli.

6. The authors should carefully review the text and the labels of the figures for the C. elegans genetic nomenclature. There are several errors.

We have fixed the errors in C. elegans nomenclature in the revised text, figures, and legends.

7. Please labels the panels within the figures so that it is clear which experiments were done with and which without microbubbles.

We have included the labels within the panels of each figure to indicate the presence or absence of microbubbles.

8. Line 131, the authors need to explain why they were “unable to replicate this study”. Was it part of the study? The entire study? This is somewhat concerning.

We are unable to replicate C. elegans behavioral responses to 10 MHz ultrasound. We suspect that this might be a result of differences in assay setups and/or ultrasound delivery. We have included this explanation in our revised manuscript (line 217-220). We are able to replicate other parts of the Kubanek et al manuscript (C. elegans responds to ultrasound without microbubbles, mec-4 and trp-4 mutants are defective in their ultrasound-evoked behaviors).

Reviewer #2: In this study, the authors exposed C. elegans to 10 ms pulses of 2.25 MHz, mid-intensity ultrasound with the aid of gas-filled microbubbles. In some conditions, responses without the bubbles were also tested.

The authors replicated previous findings [22, 38] that mutations of the mec-4 pore-forming subunit diminish the responsiveness to ultrasound. The authors also replicated previous findings [22, 38] that mutations of the trp-4 pore-forming subunit have small but likely significant effect on the responses. For one pressure level, but not others, there was a significant increase in responsiveness when trp-4 was overexpressed in AWC neurons.

This study provides a useful replication of previous studies and thus should be eventually published.

We thank the reviewer for their positive feedback.

There are, nonetheless, several points that should be addressed:

1) The trp-4 (ok1605 4X) effect is small, like in [22] and [38]. This should be acknowledged. There is a suggestion in this paper that the effect may reach significance. The significance values should be provided in the text. Importantly, there are many comparisons made among the numerous bars in the plots. Such multiple comparisons should be corrected for (e.g., Bonferroni), or an omnibus statistical analysis (n-way ANOVA) performed.

Our study shows that the trp-4(ok1605 4x) has significant behavioral defects to specific ultrasound pressures. We have added a description of these data in the revised manuscript (line 98). Also, we add appropriate statistical measures corrected for multiple comparisons, described in all relevant figure legends.

2) Fig. 3A versus B reveal enormous variability (20% versus 70%) in the response of WT animals at 0.79 MPa. This raises questions regarding the reliability of assessing the responses, and so the reliability of the plots. It seems the videos were quantified by eye. This should be specified and the reasons for the variability addressed.

We thank the reviewer for catching this discrepancy. Indeed, this was a mistaken data point (small reversals were plotted instead of large reversals) and the panel has been updated with the correct data that shows consistent N2 behavior across the AWC and ASH rescue experiments.

3) The rescue AWC::trp-4 was performed on the trp-4 (ok1605), which has many other mutations present, unless these are outcrossed (Figure 2 and [22, 38] confirm this). There can be a complex interactions between the trp-4 and the other affected genes. Therefore, the AWC::trp-4 overexpression may not be conclusive. This should be acknowledged.

We include rescue experiments in the trp-4(ok1605 4x) outcrossed strain in the revised manuscript (new data). We find that the expressing trp-4 in AWC or ASH does indeed rescue the behavioral deficits (at 0.99 MPa ultrasound) of the outcrossed strain.

4) It seems only Fig. 2A and Suppl. Fig. 2 show data without microbubbles. The authors should specify which datasets used the bubbles.

We have included labels within the panels of each figure to indicate the presence or absence of microbubbles.

Additional, less pressing issues:

A. The mec-4 effect appears weaker compared to [22] and [38]. Possible reasons could be provided.

Our experiments use ultrasound at 2.25 MHz, while [22] uses 10 MHz ultrasound. [38] makes no reference to mec-4, but uses 28 MHz ultrasound. Also, [39] tests a different allele of trp-4. It is possible that there are different mechanisms at play at different ultrasound frequencies. We have included this explanation in our revised manuscript (line 220-226).

B. "While we are unable to replicate this study" [22]

There are several important technological differences in the present study:

1) There is a relatively broad focus (Figure S2B), likely activating the entire animal. In this case, there may both reversal and acceleration tendencies, cancelling each other out.

2) The slab/dish sit directly over the syringe top, which may not have allowed the assurance that there is continuous water coupling. Water evaporation degrades the coupling over time.

3) [22] used longer duration (300 ms) and pulsed stimuli (1 kHz), which are both critical.

These differences should be acknowledged.

Moreover, the effects of [22] were replicated in two other studies ([38], [39]). These two studies should be cited.

We agree that there are differences in ultrasound delivery between our present study and these previous studies. We have included these explanations in our revised manuscript and cited these two studies (line 220-224).

C. mec-4 and trp-4 likely "act in parallel".

The finding of an increased effect does not necessarily imply parallel (or serial) engagement. I suggest to rephrase as "summed" or "compounded" effect, or "both act".

We have rephrased “act in parallel” to “summed” or “both act”.

Minor:

i. "indirectly include astrocyte signals"

- based on my read of the study, the astrocytic stimulation is direct

This study says that the effect on the neurons is indirect as it is a result of a change in astrocyte signals. Edited this line.

ii. "We previously showed that the pore-forming subunit of the mechanosensitive ion channel TRP-4 is required to generate behavioral responses to a single 10 ms pulse of ultrasound generated from a 2.25 MHz focused transducer [21]"

-> "We previously showed that the pore-forming subunit of the mechanosensitive ion channel TRP-4 is required to generate behavioral responses mediated by microbbubles activated by a 10 ms pulse of ultrasound generated from a 2.25 MHz focused transducer [21]"

Fixed.

iii. "confirming ultrasound-triggered control (Sonogenetics)"

-> "confirming ultrasound-triggered, microbubble-mediated control"

Fixed.

iv. "We generated a trp-4 mec-4 double mutant and compared its ultrasound responses to both single mutants."

-> missing "We found ..." sentence following this sentence.

Fixed.

v. "lower ultrasound intensities (0.79 MPa)"

-> "pressures"

Fixed.

Refs:

[38] "Ultrasound neuro-modulation chip: activation of sensory neurons in Caenorhabditis elegans by surface acoustic waves"

[39] "Ultrasound activation of mechanosensory ion channels in Caenorhabditis elegans"

Submitted filename: Reviewer_responses.docx

Click here for additional data file.

4 Mar 2022

19 Mar 2022

Responses (in black) to the reviewers comments (in blue)

Reviewer #1: This is a revised manuscript from Maragam and colleagues, in which they explored the idea that mechanosensitive channels mec-4 and trp-4 might function together in detecting ultrasounds. The authors have addressed the concerns we raised in the first round of reviews. There are a few minor points that still need to be addressed.

1. In the abstract, results and discussion, the authors removed the word “parallel” and replaced it with “together”. However, “parallel pathways” is still listed under keywords. It should be removed.

Fixed.

2. Figure 2B, 0.88 MPa group. Is there statistical difference between the strains? if so, statistics need to be added.

There is no statistical differences between the strains in the 0.88 MPa group. We have edited the figure appropriately.

3. Trp-4 over-expression in AWC neurons of mec-4 mutants rescues the phenotype. These two channels belong to two different families. Based on what it is known about their mechanosensitivity and physiological properties, can the authors add some discussion on how they envision this rescue being possible?

Included a comment. Line 130-134

4. There should be a space between number and unit. Check for the presence of the space throughout the manuscript. For example the space is missing at these two locations: Line 145: 28MHz, line 169: 2.25MHz.

Fixed.

Reviewer #2: The authors addressed most of the comments, and this will be an interesting study.

There are two final points to be addressed:

A) The Bonferroni correction should say how many comparisons c were considered, e.g., by what value was the p-value divided by to yield the ultimate p/c value, stars for which are shown in the figures.

We have added the Bonferroni correction along with the number of comparisons (c) in the appropriate figures. We have made changes to other figures to show the corrected p-values.

B) A set of points associated with the 10 MHz data (Suppl Fig. 3).

Given the difficulties in obtaining robust responses, it is important to stress the differences from [28] so that others can replicate these findings:

i) SFig. 3A shows a white plastic piece below the agar slab. In [28], no such piece was present---the slab was sitting directly over the "syringe top". In the present study, the presence of this piece may complicate coupling (air pockets forming within it) and its monitoring.

ii) Sfig. 3B shows that the setup produced a relatively [28] large focus. The full-width half-maximum (FWHM) values should be described. It appears that the average FWHM was on the order of the animal's length. This way, most of the animal was stimulated, precluding effective frontal stimulation that leads to reliable reversals.

iii) Sfig. 3C would be stronger if it included all responses; not just large reversals; ([28] used all responses).

iv) In [28], responses were quantified objectively using an algorithm run on recorded videos. Thus, experimenters were blinded to the results. In the present study, the calls were made by the naked eye.

v) Besides the subjective judgements in the present study, the eye may not be sensitive enough to spot effects. The algorithm in [28] assessed *any* deviation from baseline; not just reversals.

These points should be stressed in the Methods and the Results.

We do not have a plastic piece between the agar surface and the 10 MHz transducer (clarified in the methods). The image in Supplementary Figure 3A is showing the top of the plastic syringe. We have now marked this in the figure for clarity. We only scored animals where we stimulated the head. In Supplementary figure 3B, the area shaded is about 0.4 mm at the maximum, which is half the length of the animal. Further, in Supplementary Figure S3D, we are showing all the positions of the head of the animal in our assay. These data indicate a broad distribution, but we were unable to observe any significant behavioral effects. Additionally, the Kubanek et al 2018 study used a hydrophone to confirm that the agar did not attenuate the ultrasound pressure, the shape of the radiation force was plotted used a simulation (which is likely to be imprecise), while we are reporting measurements from a Precision Acoustics fiber optic probe across the agar surface.

We now plot all responses and not just large reversals in Supplementary Figure S3C. While we did score these behaviors by eye, we did not observe any significant deviation from the baseline. Furthermore, in Figure 2B from Kubanek et al 2018, the reversals observed appear to last multiple seconds, which we would have definitely identified in our new analysis.

However, we do include a comment in the methods section. Line 215.

Submitted filename: Magaram_reviewer responses.docx

Click here for additional data file.

4 Apr 2022

I believe that you can readily address reviewer #2's comments on the quantification of Supplementary Fig. 3B.

12 Apr 2022

Dear Dr. Kim

Please find attached a reviewer response file along with the manuscript file with and without tracked changes. We would like you to consider the comments from reviewer 2 in totality with this being our third revision. They seem to be resistant to our manuscript and are bringing up newer concerns in each subsequent review (for example, they are now asking for an estimate of the accuracy of our hydrophone, edit our figure title, etc.).

We would request you to make a decision on whether this reviewer is being reasonable. Also, we would like an opportunity to discuss our concerns with you.

Sincerely

Sreekanth Chalasani and Uri Magaram

Salk Institute

La Jolla, CA

Responses in black to the reviewers comments in blue

1. The PA fiber-optic hydrophone is inaccurate for measurements of the relatively small pressured used in this study. Estimates of its accuracy for these pressures should be provided. A dB value suffices.

We have updated the manuscript based on the reviewers recommendation and confirming that the manufacturer has guaranteed operation in frequency range used in this study. Further, additional peer-reviewed work by several groups have used this system in the desired frequency range with similar levels of acoustic pressure [1,2].

There is no direct conversion to dB as the uncertainty depends on the original measurement amplitude. However, for a 1 MPa signal, the uncertainty would be 100 kPa. We have included the relevant citations in the methods section which justify the use of the PA fiber-optic hydrophone.

2. The 10 MHz setup differs from that of Kubanek et al. 2018. In the present study, the agar sits directly on a syringe. This way, there is no way to validate good and continued coupling.

At the very least, the caption of SF3 should be changed from

"Schematic of 10 MHz behavioral imaging setup as described in Kubanek et al 2018."

to

Schematic of 10 MHz behavioral imaging setup."

Edited the caption

3. My previous suggestion to quantify the FWHM in SF3B does not seem to be taken. Yet, the focality of the field is critical for effective C. elegans responses and thus should be quantified.

As recommended by the reviewer, the FWHM in SF3B has been added to the figure legend and relevant methods section. The FWHM we observed is on the order of half the body length of the animal, which we believe gives us enough precision to be able to stimulate the head without the tail as we know is necessary from our own experience and previous literature. Additionally, for this reason we went back and marked the head positions of all the worms in the assay at the moment of stimulation, displayed in SF3D to ensure that the ultrasound stimulus was delivered to the head of the animal as best as possible.

4. In SF3C, it is worth to indicate effect significance (e.g., using stars).

Added as recommended. We find that behavioral responses in the presence of microbubbles is significantly more than those in their absence and also referenced this in the manuscript lines 148-151.

References

1. Lakshmanan A, Jin Z, Nety SP, Sawyer DP, Lee-Gosselin A, Malounda D, Swift MB, Maresca D, Shapiro MG. Acoustic biosensors for ultrasound imaging of enzyme activity. Nat Chem Biol. 2020 Sep;16(9):988-996. doi: 10.1038/s41589-020-0591-0. Epub 2020 Jul 13. Erratum in: Nat Chem Biol. 2020 Jul 23;: PMID: 32661379; PMCID: PMC7713704.

2. Vasan, A., Allein, F., Duque, M., Magaram, U., Boechler, N., Chalasani, S.H. and Friend, J. (2022), Microscale Concert Hall Acoustics to Produce Uniform Ultrasound Stimulation for Targeted Sonogenetics in hsTRPA1-Transfected Cells. Adv. NanoBiomed Res. 2100135. https://doi.org/10.1002/anbr.202100135

Submitted filename: reviewer_responses.docx

Click here for additional data file.

14 Apr 2022

Two pathways are required for ultrasound-evoked behavioral changes in Caenorhabditis elegans

PONE-D-21-33415R3

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Reviewer #2: All comments have been addressed

**********

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: No

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Reviewer #2: I appreciate the authors' response, and this interesting article can now be published.

A final suggestion for an improvement:

"C. elegans exhibits minimal behavioral responses to 10 MHz ultrasound stimuli"

In SF3, I meant to indicate the significance of both bars (instead of a paired test). It would be good to know if the "minimal behavioral response" was significant.

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Reviewer #2: No

27 Apr 2022

PONE-D-21-33415R3

Two pathways are required for ultrasound-evoked behavioral changes in Caenorhabditis elegans

Dear Dr. Chalasani:

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