Two pathways are required for ultrasound-evoked behavioral changes in Caenorhabditis elegans
Uri Magaram, Connor Weiss, Aditya Vasan, Kirthi C. Reddy, James Friend, Sreekanth H. Chalasani
PLOS ONE 2022 · 10.1371/journal.pone.0267698
Abstract
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.
Abstract via europepmc.
Exposures
Exposure 1: 2.25 MHz single ultrasound pulse
Target: c elegans neurons — “the head of the animal”
Device: Olympus / Panametrics · Olympus NDT · V305-SU-F1.00IN-PTF
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 2,250 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 0.01 | ✓✓✓ |
| Free-field pressure (kPa) | 790, 990swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | ⚑ |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable |
A single 10 ms pulse was generated using a TTL pulse to trigger a multi-channel function generator; amplifier gain was adjusted to achieve desired peak-negative pressures. Worms were stimulated with a single ultrasound pulse while corralled on an agar plate, with and without polydisperse gas-filled microbubbles.
Exposure 2: 10 MHz single ultrasound pulse
Target: c elegans neurons — “the head of the animal”
Device: Olympus / Panametrics · Olympus · A327S-SU-CF1.00IN-PTF
| Waveform | not reported | |
|---|---|---|
| Fundamental frequency (kHz) | 10,000 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | |
| Duty cycle (%) | not reported | |
| Sonication duration (s) | not reported |
| Free-field pressure (kPa) | 1,000 | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | ⚑ |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable |
For 10 MHz experiments the 2.25 MHz transducer was replaced with a 10 MHz line-focused transducer coupled via a plastic syringe and degassed water; C. elegans exhibited minimal behavioural responses to 10 MHz stimulation, significantly enhanced in the presence of microbubbles.
Flags from extraction
exposures[0].in_situ.pressure_kpa— Domain uncertain: pressures were measured with a hydrophone through the 2% agar plate covering the worm (no skull/bone path); classified as in_situ by analogy to a measured-through-tissue value since the paper does not use the free-field/in-situ vocabulary.exposures[0].in_situ.pressure_kpa— List gives two of several pressures explicitly tested (0.79 and 0.99 MPa); the paper also reports a general threshold ('>0.75 MPa') and mutant-specific thresholds ('>0.92 MPa') that are inequalities rather than discrete stated test values and were not included in the list.exposures[1].in_situ.pressure_kpa— Value is stated only in the S3 Fig legend text ('peak negative pressures reaching 1 MPa at highest amplifier settings'), not in the main body text for the 10 MHz experiment.exposures[1].timing— The Methods section does not restate pulse duration/PRF/waveform for the 10 MHz experiments after describing the transducer swap; it is unclear whether the single-10-ms-pulse protocol used for the 2.25 MHz experiments also applied here, so these fields are left not_reported rather than assumed.n_subjects— Sample sizes vary by condition and genotype across figures (e.g., n=90-135, n=45, n=224); the paper never states a single total number of animals exposed to ultrasound.