Comparing Caenorhabditis elegans gentle and harsh touch response behavior using a multiplexed hydraulic microfluidic device

Abstract

The roundworm Caenorhabditis elegans is an important model system for understanding the genetics and physiology of touch. Classical assays for C. elegans touch, which involve manually touching the animal with a probe and observing its response, are limited by their low throughput and qualitative nature. We developed a microfluidic device in which several dozen animals are subject to spatially localized mechanical stimuli with variable amplitude. The device contains 64 sinusoidal channels through which worms crawl, and hydraulic valves that deliver touch stimuli to the worms. We used this assay to characterize the behavioral responses to gentle touch stimuli and the less well studied harsh (nociceptive) touch stimuli. First, we measured the relative response thresholds of gentle and harsh touch. Next, we quantified differences in the receptive fields between wild type worms and a mutant with non-functioning posterior touch receptor neurons. We showed that under gentle touch the receptive field of the anterior touch receptor neurons extends into the posterior half of the body. Finally, we found that the behavioral response to gentle touch does not depend on the locomotion of the animal immediately prior to the stimulus, but does depend on the location of the previous touch. Responses to harsh touch, on the other hand, did not depend on either previous velocity or stimulus location. Differences in gentle and harsh touch response characteristics may reflect the different innervation of the respective mechanosensory cells. Our assay will facilitate studies of mechanosensation, sensory adaptation, and nociception.

Fig. 1

Microfluidic device for assaying touch response behavior. (a) A touch channel (magenta) inflates upon pressurization, partially closing the worm channel (blue). (b) Schematic of the device containing 64 sinusoidal worm channels and 16 control channels. (c) Dark field image of C. elegans crawling in the worm channels with photomask design overlaid.

Fig. 2

Stimulus measurement and calibration. (a) Optical transmission of dye-filled touch valves is used to monitor worm channel height in the valve. (b) Worm channel ceiling deflection when control solenoid is driven by a 25 Hz square wave with an amplitude of 40 psi and a 50% duty cycle. Red lines denote pressure on. (c) Maximum deflection as a function of pressure. Points (mean ± SD) show the average of 3–5 trials. Colors show 4 different devices.

Fig. 3

Proportion responding of WT and mec-4 as a function of stimulus amplitude. Vertical error bars are 95% confidence intervals based on a binomial fit, and hor izontal error bars are standard deviations of the calibration measurements for the device. Each point represents data from n=21–57 animals.

Fig. 4

Gentle touch response fields. For both plots, responses are grouped into five bins by body coordinate (0 = head, 1 = tail) of the center of stimulus. Average ± 95% CI of the mean is plotted for each bin. Asterisks denote significant difference from zero for the responses in the bin (Z test, p < 0.05 after Bonferroni correction). Pale dots show responses of individual animals. (a) Velocity of WT animals after gentle touch stimuli. (b) Velocity of egl-5(u202) animals after gentle touch stimuli.

Fig. 5

Relationship between pre-stimulus velocity and post stimulus velocity for gentle touch in WT worms (a) and harsh touch in mec-4 worms (b). Responses are classified by touch location (green circles = anterior, blue squares = middle, black diamonds = posterior). Large shapes represent averages of animals moving backward or forward prior to the stimulus. Small tinted shapes represent individual animals in the same categories. In no case does velocity prior to the stimulus have a significant effect on velocity after the stimulus (p > 0.05, two-tailed t-test with Bonferroni correction)

Fig. 6

Relationship between preceding stimulus location and post-stimulus velocity for gentle touch in WT worms (a) and harsh touch in mec-4 worms (b). Responses are grouped into three bins by body coordinate of the current touch (0 = head, 1 = tail) and colored by the location of the preceding touch (green = previous touch to the anterior half, red = previous touch to the posterior half). 95% CI of the mean is shown for each bin. Pale dots show responses of individual animals. The asterisk denotes a significant difference between the two groups in the location bin (two-tailed t-test, p = 0.0315 after Bonferroni correction).