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.
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.
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.