Imaging set-up delivers ultrasound waves to animals

To investigate the role of ultrasound in wild-type C. elegans neural activity and behaviour, we developed a set-up that aligns optical imaging with the ultrasound focal zone (Fig. 1a). Ultrasound with different peak negative pressures was generated from a transducer and focused onto an agar plate, where animals were corralled into a small area using a copper solution (Fig. 1b). The transducer focused the ultrasound wave to a 1-mm-diameter circular area at the agar surface (red circle in Fig. 1b). The entire set-up was placed in a large tank filled with water to facilitate uniform transduction of the ultrasound wave (Fig. 1a). Depending on solution or tissue gas concentrations, high ultrasound peak negative pressures (>2.5 MPa) can create inertial cavitation, with the resulting shockwaves compromising the integrity of cell membranes22,23. Consistently, we observed that animals exposed to multiple pulses of high ultrasound pressures were unable to maintain their normal body posture (Supplementary Fig. 1). Therefore, we chose to use low-pressure ultrasound, which does not cause these damaging effects, to stimulate animal behaviour.

Figure 1: Amplifying ultrasound signals using microbubbles modifies animal behaviour. (a) Schematic of the computer-controlled imaging and ultrasound exposure system (frontal view) and (b) the agar plate with animals (top view) corralled into a small area by a copper barrier (1.5 cm in diameter). (c) Image sequence showing that animals do not respond to low-pressure ultrasound (US) alone. (d) Schematic of a stabilized microbubble. (e) Images showing that animals exhibit reversals and omega bends upon ultrasound (US) stimulus (single 10-ms pulse, 2.25 MHz with peak negative pressure of 0.9 MPa) in the presence of microbubbles. Full size image

We found that a single 10-ms-duration ultrasound pulse of 2.25 MHz and peak negative pressures below 0.9 MPa had no effect on animal behaviour (Fig. 1c). The mechanical disturbances24 of the fluid and tissue in the ultrasound focal zone take the form of compression and expansion deformations as well as bulk tissue distortions caused by acoustic radiation forces, but at low pressures they were not large enough to influence C. elegans locomotion. Previous studies have shown that ultrasound waves can cause temperature changes in the focal zone25. We first estimated the temperature increase as a result of ultrasound exposure. In a previous study, a continuous 1.1-MHz-ultrasound pulse with a peak negative pressure of 2.6 MPa increased the temperature of the surrounding media at the rate of 35 °C s−1 (ref. 25). Using these data, we estimated the temperature increase around the worms on the agar surface to be 0.04 °C for a single ultrasound pulse at 0.9 MPa. Moreover, we also directly measured the magnitude of temperature change on the agar surface using a miniature thermocouple (Supplementary Fig. 2) and found that an ultrasound peak negative pressure of 0.7 MPa caused a temperature increase of less than 0.1 °C (see Methods). This is a temperature stimulus that animals including C. elegans are unlikely to detect26,27. Together, these results show that C. elegans is unlikely to respond to the temperature and mechanical changes induced by the low-pressure ultrasound wave.

Microbubbles amplify ultrasound mechanical deformations

To test whether the tiny mechanical deformations created by a single low-pressure ultrasound pulse could be amplified we included gas-filled microbubbles in our assay (Fig. 1d,e). We found that animals showed a dramatic response to ultrasound when surrounded by microbubbles (Fig. 1e). When the ultrasound wave was focused on the head of a worm surrounded by microbubbles, the animal immediately initiated a backward movement (termed ‘reversal’28) followed by a high-angled turn (identified as an ‘omega bend’28) (Figs 1e and 2a). These behaviours were scored as previously described (Methods, Fig. 2a)28 and are quantified as shown in Fig. 2b,c. Reversals were separated into two categories: ‘large’ reversals involving three or more head bends and ‘small’ reversals with two or less head bends (Fig. 2a)28. All behavioural data were analysed using a non-parametric Fisher’s exact test (two-sided), with error bars showing the standard error of the proportion. The animal’s behavioural responses were positively correlated with the pressure of the ultrasound wave in the presence of microbubbles (Fig. 2c). Importantly, using a thermocouple, we found that the presence of microbubbles did not cause any measurable increase in the ultrasound-induced temperature change at the agar surface (see Methods). Collectively, these results showed that C. elegans responds behaviourally to a single pulse of low-pressure ultrasound in the presence of microbubbles.

Figure 2: C. elegans behavioural responses to ultrasound in the presence of microbubbles. (ai-vii) Panels show an animal reversing and generating a high-angled omega bend on ultrasound stimulus. Reversals with greater than two head bends were scored as large, while those with fewer than two head bends were counted as small. Reversals and omega bends are shown with a red line overlaid on the animal tracks on the agar surface. Animal responses to ultrasound stimuli (single 10-ms pulse, 2.25 MHz) with varying peak negative pressures (b) without and (c) with microbubbles were quantified. n=30 for each of the conditions. Proportion of animals responding and standard error of the proportion are shown. **P<0.01 and *P<0.05 by Fisher’s exact test (two-sided). Full size image

To probe how microbubbles transduce the ultrasound wave and modify animal behaviour, we analysed their behaviour upon ultrasound stimulation. Previous studies have shown that the majority of the ultrasound energy propagates through water and soft tissue as a longitudinal wave with alternating compression and rarefaction phases. These two phases create pressures that are alternately higher and lower than the ambient pressure level, respectively17. We designed our microbubbles to respond to the mechanical deformations induced by an ultrasound pulse. We filled our microbubbles with a stabilizing mixture of perfluorohexane and air that allows the compression and rarefaction phases of the ultrasound wave to shrink and expand the microbubbles from one half to four times their original diameters29 in a process known as stable cavitation. This occurs at the driving frequency of the underlying ultrasound pulse30,31 (Fig. 3a). These size oscillations create mechanical deformations of the surrounding fluid31 as well as fluid microstreaming32 that can push against surfaces32,33. The microbubbles can also undergo lateral translations34 and complete collapse, creating large mechanical deformations that have been shown to propagate through water and tissue31. We found that ultrasound exposure affected the distribution of microbubbles in the focal zone on agar plates confirming these mechanical deformations (Fig. 3b,c). Next, we hypothesized that these large mechanical deformation events created by the ultrasound–microbubble interaction likely propagate from the location of the microbubbles into the body of the worm, thereby affecting behaviour. To test our hypothesis, we analysed the behaviour of microbubbles labelled with a fluorescent dye, DiO (Fig. 3d,e). We found that these microbubbles naturally distribute themselves around the animal, but are not attached to the animal’s body (Fig. 3f,g,j,k). On ultrasound stimulation, some microbubbles collapse, some merge together, while others move (Fig. 3h,l). These mechanical deformations occur all around the worm, including the worm’s head, and are specifically captured in the difference image (shown in red, Fig. 3i,m). Taken together, these results suggest that the ultrasound–microbubble interaction creates mechanical disturbances around the worm that result in increased reversal behaviour.

Figure 3: Ultrasound−microbubble interaction with C. elegans. (a) The microbubble (i) expands and (ii) contracts in size with the rarefaction (low-pressure) and compression (high-pressure) phases of the ultrasound pressure wave. This oscillation behaviour occurs at the frequency of the driving ultrasound, resulting in a variety of behaviours including (iii) microbubble collapse, (iv) fluid microstreaming and (v) merging of microbubbles. These microbubble behaviours create mechanical distortions that can propagate through the agar and the body of the animal. (b) Microbubbles are uniformly distributed on an agar surface and appear white. (c) Ultrasound stimulus (10 pulses lasting 10 ms each with a duty cycle of 1 Hz, 2.25 MHz with a peak negative pressure of 0.9 MPa) activates and destroys the microbubbles in the ultrasound focal zone of 1 mm diameter (white arrow). Microbubbles outside this focal zone (denoted by the red circle) appear undisturbed. Microbubbles were labelled with DiO, and (d) brightfield and (e) fluorescent images are shown. A whole-animal view showing (f) brightfield, (g) fluorescence before and (h) after ultrasound stimulus and finally, (i) the difference in red showing the locations of microbubble-induced mechanical deformations. A magnified view of the animal’s head in (j) brightfield, (k) before and (l) after ultrasound stimuli (US) and (m) the difference in red showing the locations of microbubble-induced mechanical deformations. The white arrow points to a large microbubble that is destroyed upon ultrasound stimulation. Full size image

The microbubble synthesis process creates a size distribution of microbubbles from 1.5 to 6.5 μm in diameter (see Methods). To better understand the effect of microbubbles from different size ranges we separated the mixed microbubbles into distinct large and small size categories based on settling time (see Methods, Fig. 4a). Both small and large microbubbles surround the animals completely (Fig. 4b). Interestingly, we found that the smaller microbubbles stimulated a significantly larger proportion of worms to generate large reversals when exposed to ultrasound, compared with larger microbubbles (Fig. 4c). To be consistent in the size distribution between different experiments we used well-mixed samples. This enabled us to ensure that worms were reproducibly exposed to the same microbubble size distribution for all experiments. We found that the proportion of wild-type animals that responded to the same ultrasound peak negative pressure (0.47 MPa) in the presence of microbubbles changed over several months. Recent work has shown that C. elegans detects humidity changes and modifies its locomotory behaviours35. We also tracked environmental conditions during the days that experiments were conducted and found that the wild-type animals’ reversal responses were related to the relative humidity level. At low humidities more animals responded, while at higher humidities fewer animals executed reversal responses (Fig. 4d). We suggest that the observed variability in wild-type behaviour is independent of the ultrasound stimulus and depends on environmental humidity. We controlled for this by testing wild-type animals along with the mutants or transgenics on the same day. We also repeated our analysis over multiple days for each genotype to assure reproducibility.

Figure 4: Microbubbles transduce ultrasound stimuli. (a) A bar and whisker plot showing the distribution of microbubbles fractionated based on their size. A one-way ANOVA test shows significance in the distribution (***P<0.001 using the LSD post hoc test). A mixed-size population was used for all experiments to maintain consistency. (b) Images showing animals incubated with small (1.5–2.5 μm; top) and large (4–6.5 μm; bottom) populations of microbubbles. (c) Behavioural responses of wild-type animals incubated with small and large microbubbles upon ultrasound stimulation with a single 10-ms pulse at 2.25 MHz with peak negative pressure of 0.9 MPa. Averages and standard error of the proportion are shown. ***P<0.001 using Fisher’s exact test. (d) A graph showing the effect of external humidity levels on animal reversal behaviour. We observed that at different times of the year the animals had different reversal behaviour in response to the same 0.47 MPa ultrasound exposure. Under low-humidity levels the animals would undergo more large reversals than under high-humidity conditions. We accounted for this variable behaviour by running a wild-type control for each of the genetically modified strains that was tested. These controls were run on the same day and under the same conditions as the tested strain. Error bars show standard error of the proportion. Full size image

TRP-4 ion channels sensitize neurons to ultrasound

We hypothesized that mechanotransduction channels transduce the mechanical deformations of the ultrasound–microbubble interactions to stimulate individual neurons. Previous studies have identified TRP-4 as a stretch-sensitive, pore-forming cation mechanotransduction channel in C. elegans36,37. This channel is specifically expressed in a few C. elegans neurons, the four CEPs (CEPDL, CEPDR, CEPVL and CEPVR) and the two ADE (ADEL and ADER) dopaminergic neurons and the DVA and DVC interneurons36,37. Moreover, TRP-4 is both necessary and sufficient to generate mechanoreceptor currents in CEP neurons36. We tested whether TRP-4 was required to transduce the ultrasound–microbubble mechanical stimulus and modify animal behaviour. To probe ultrasound-induced behaviours we examined the effects of three different ultrasound pressure levels and found the responses to be dose dependent. We consistently found larger wild-type responses to ultrasound (see wild-type data in Figs 4d and 5a). In contrast, we found that animals lacking functional TRP-4 have reduced large reversal responses to specific peak negative pressures (0.41 and 0.47 MPa) of ultrasound stimulation, suggesting that this channel increases the probability of large reversals (Fig. 5a). However, with an ultrasound pulse at 0.6 MPa peak negative pressure, trp-4 mutants have similar behavioural responses compared with wild type (Fig. 5a), suggesting that there is an alternate pathway that detects ultrasound at these higher pressures. Moreover, these trp-4 mutants also show a significant increase in small reversal behaviours on ultrasound stimulation at 0.41 and 0.46 MPa (Supplementary Fig. 3a), but no change in their omega bend behaviours (Supplementary Fig. 3b). Collectively, these results suggest that TRP-4 might be activated in response to ultrasound with peak negative pressure levels less than 0.5 MPa and modifies neurons involved in generating both small and large reversals.

Figure 5: TRP-4 expression activates ASH and AWC neurons in the presence of microbubbles. (a) Large reversal responses to low-pressure ultrasound require the pore-forming TRP-4 channel. n=30 for each condition. Transgenic animals expressing TRP-4 in (b) ASH neurons and (c) AWC neurons execute more large reversals on low-pressure ultrasound stimulation (single pulse, 2.25 MHz, 10 ms). n>30 for each genotype and condition. The proportion and standard error of the proportion are shown in all data panels. **P<0.01, while *P<0.05 by Fisher’s exact test (two-sided). (d) Schematic identifying chemosensory neurons ASH and AWC in C. elegans. False-coloured images showing changes in GCaMP fluorescence in AWC neurons on ultrasound stimulation. Warmer colours indicate increased calcium and neural activity. (e) Average AWC calcium responses upon ultrasound stimulation at t=5 s (AWC::GCaMP2.2b n=20, AWC::GCaMP2.2b;AWC::trp4 n=23). (f) Average responses binned by distinct times for AWC::GCaMP2.2b and AWC::GCaMP2.2b;AWC::trp4 animals. Averages and s.e.m. are shown. *P<0.05 by t-test (two-sided, equal variances not assumed, AWC::GCaMP2.2b n=20, AWC::GCaMP2.2b;AWC::trp4 n=23). Full size image

To test whether ultrasound sensitivity could be conferred to other neurons, we analysed the behaviour of transgenic animals misexpressing TRP-4 in specific chemosensory neurons. We tested for dose-dependent responses using three different ultrasound pressure levels. We initially misexpressed the TRP-4 channel in ASH, a well-studied polymodal nociceptive neuron38, whose activation leads to large reversals39. Consistently, we found that ASH expression of TRP-4 generated a significant increase in large reversals at ultrasound peak negative pressures of 0.47 and 0.6 MPa (Fig. 5b). Moreover, we found that these ASH::trp-4 transgenics show a decrease in small reversals (Supplementary Fig. 3c), but not omega bends (Supplementary Fig. 3d), indicating that activating ASH via ultrasound and TRP-4 specifically modifies the neural circuits driving small- and large-reversal behaviours. Next, we tested the effects of TRP-4 misexpression on the function of the AWC sensory neurons and AWC-driven behaviour. Previous results have implied that AWC activation is correlated with an increase in the animal’s ability to generate large reversals21. We found that animals misexpressing TRP-4 in AWC neurons also initiated significantly more large reversals at the ultrasound peak negative pressure of 0.47 MPa (Fig. 5c), but we did not observe a change in small reversals or omega bends (Supplementary Fig. 3e,f). These results indicate that misexpressing the mechanotransduction channel, TRP-4, in ASH or AWC renders the neurons sensitive to low-pressure ultrasound, increasing the frequency of large reversals.

To test whether ultrasound could directly stimulate AWC neurons, we expressed the calcium indicator GCaMP3 (ref. 40) in these neurons and recorded their activity levels upon ultrasound exposure (Fig. 5d). Consistent with our behavioural data, we found that ultrasound stimulation in the presence of microbubbles activated AWC neurons, as indicated by an increase in the fluorescence signal from the calcium sensor. The AWC calcium responses were greatly increased in transgenic animals expressing TRP-4 specifically in AWC neurons (Fig. 5d–f, Supplementary Fig. 4a–c). We also observed that AWC calcium responses were variable, with many transients lasting more than 30 s (the duration of our recording) and a few returning back to baseline (Supplementary Fig. 4c). Variability in AWC calcium responses has also been previously observed using odour stimuli21. To test whether AWC responses were related to artefacts in our imaging set-up, we recorded AWC calcium in the absence of ultrasound stimulus and found no consistent responses, suggesting that AWC responded to ultrasound stimulus (Supplementary Fig. 4d,e–g). Both wild-type AWC neurons and those misexpressing TRP-4 showed a response lasting about 2–3 s immediately on exposure to a single ultrasound pulse in the presence of microbubbles. However, we also observed that AWC neurons misexpressing TRP-4 show a significant increase in their activity starting at 7 s after ultrasound exposure (t=12 s in Fig. 5f) and lasting for at least 5 s, which is not observed in wild-type neurons. This sustained increase in AWC calcium levels likely represents the activity of TRP-4, which could potentiate calcium entry into the neuron via other calcium channels. Interestingly, large reversals take approximately 10–20 s to complete, a time window where we also observe sustained AWC calcium activity in the AWC::trp-4 transgenics. We suggest that the sustained AWC calcium activity observed in these AWC::trp-4 transgenics is likely correlated with the increased frequency of large reversals generated by these animals after ultrasound stimulation. Taken together, these results show that TRP-4 channels are sensitive to low-pressure ultrasound, and ectopic expression of these channels in sensory neurons causes correlated changes in neuronal activity and behaviour.

New roles for PVD sensory and AIY interneurons

To test whether our method of ultrasound activation of neurons can reveal unidentified behavioural roles, we misexpressed TRP-4 in the poorly understood PVD neurons (Fig. 6a). PVD neurons have extensive, regularly spaced and non-overlapping dendritic arbours that extend throughout most of the animal, excluding the head and the neck41. PVD shares promoters with the FLP, IL2 and PVC neurons42 and so the TRP-4 proteins were expressed in multiple neurons in the PVD::trp-4 transgenic animals. We find that expressing TRP-4 in PVD, FLP, IL2 and PVC neurons leads to a significant decrease in the animals’ reversal responses on ultrasound stimulation (two independent transgenics, Fig. 6b). We did not observe changes in FLP or other neurons in response to the ultrasound stimulus (Supplementary Fig. 5a,b). We hypothesize that misexpressing TRP-4 channels activates PVD neurons upon ultrasound stimulation, which in turn suppresses reversals. To test our hypothesis we monitored PVD neuron activity in response to ultrasound stimulation. We observed a sharp increase in PVD calcium over many seconds (Fig. 6c,d and Supplementary Fig. 6a). The initial decline is likely an artefact of the ultrasound stimulus as it was observed in both AWC and PVD recordings (Figs 5e and 6d). Interestingly, we find a strong correlation between PVD activity and animal movement. In particular, we find that PVD neurons reach their maximum response when the animal has finished reversing (Fig. 6d). Moreover, we find that PVD neurons are more likely to be activated when the animal is moving backward rather than when moving forward in response to the ultrasound stimulus (Supplementary Fig. 6b–d). These results suggest that ultrasound stimulation activates TRP-4-expressing PVD neurons and causes premature suppression of backward movement, leading to fewer reversals.

Figure 6: PVD neurons inhibit reversals and AIY neurons stimulate omega bends in the presence of microbubbles. (a) Schematic showing the PVD and AIY neurons in C. elegans. (b) Two independent transgenic strains expressing TRP-4 in PVD neurons show reduced reversals when stimulated with a single 10-ms 2.25 MHz 0.47 MPa peak negative pressure ultrasound pulse. n>46 for each genotype. The proportion and standard error of the proportion are shown. **P<0.01, while *P<0.05 by Fisher’s exact test (two-sided). (c) False-coloured images showing changes in GCaMP3 fluorescence in PVD neurons upon ultrasound stimulation. Warmer colours indicate increased calcium and neural activity. (d) Average PVD calcium responses (n=16) along with average distance moved by the animal shown as a function of time. Peak PVD response occurs when the animal has stopped moving. (e) Two independent transgenics expressing TRP-4 in AIY neurons show increased incidences of omega bends when stimulated with a single 10 ms 2.25 MHz 0.41 MPa peak negative pressure ultrasound pulse. n>40 for each genotype. (f) AIY calcium responses to ultrasound stimuli. A representative trace showing the ratio of change in fluorescence to the baseline is shown. Ultrasound stimulus was given at t=5 s and neurons that responded within a 5.5-s window after the stimulus were counted as responders. Bar graphs show % responders with and without ultrasound stimuli for AIY::GCaMP and AIY::GCaMP;AIY::trp-4. Numbers on the bars indicate the number of animals analysed in each condition. *P<0.05 by Fisher's exact test. (g) Schematic showing the neural circuit that responds to ultrasound stimuli amplified by microbubbles. ASH and AWC neurons promote reversals, while PVD neurons inhibit reversals. AIY neurons promote omega bends. Full size image

We then tested whether our approach can manipulate the function of an interneuron, whose processes do not contact the external cuticle of the animal. We misexpressed TRP-4 in AIY interneurons, which are at least 25 μm from the cuticle19, and analysed the behaviour of these animals upon ultrasound stimulation. Optogenetic studies have previously shown that activating AIY interneurons reduces reversals and omega bends43. In contrast, we find that AIY::trp-4 transgenics are significantly more likely to initiate high-angled omega bends on ultrasound stimulation (two independent transgenics, Fig. 6e). It is possible that expressing TRP-4 in AIY neurons has altered that neuron’s function, leading to increased omega bends. Also, animals with genetically altered AIY function have been shown to have increased turns in a local search assay21,28. We found that these AIY::trp-4 transgenics did not show any defects in local search (Supplementary Fig. S7a–c), confirming that the AIY neurons were not altered in these animals. These data suggest that AIY can initiate different behaviours based on type of stimulation, ultrasound or light.

To confirm whether ultrasound stimulus is activating AIY interneurons, we used calcium imaging. AIY neural activity is typically measured from a bulb in the AIY neurite21. Consistent with previous observation21, we found that AIY is a noisy neuron that shows a number of transients during our recordings (Supplementary Fig. 8). We collected a number of AIY recordings from wild-type animals and defined the relevant transient. We counted all animals whose neurons responded within a 5.5s time window after the ultrasound pulse as responders. Using these criteria, AIY neurons in wild-type animals did not show a significant response to ultrasound stimulus (4/29) (Fig. 6f and Supplementary Fig. 8a,b). In contrast, we observed that a significant number of AIY neurons in AIY::trp-4 transgenics (11/28 animals) had a positive response (Fig. 6f and Supplementary Fig. 8e,f). The increased proportion of AIY responders in the AIY::trp-4 transgenics suggests that ultrasound stimulus activates AIY interneurons. These results show that mechanical deformations from the ultrasound–microbubble interaction can penetrate at least 25 μm into the worm and influence the function of AIY interneurons. Moreover, we find that misexpressing TRP-4 can influence both reversal and omega bend neural circuitry, suggesting that our sonogenetic approach is broadly applicable for manipulating circuit activity. Further, our results show that AIY interneurons are likely to have at least three activity states with one suppressing turns21, one promoting forward turns (as revealed by optogenetic stimulation)43 and one increasing omega bends (as revealed by ultrasound stimulation). These studies validate our approach of using sonogenetics to reveal novel roles for both PVD and AIY neurons in modifying turn behaviour.