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Forces applied during classical touch assays for Caenorhabditis elegans.
For decades, Caenorhabditis elegans roundworms have been used to study the sense of touch, and this work has been facilitated by a simple behavioral assay for touch sensation. To perform this classical assay, an experimenter uses an eyebrow hair to gently touch a moving worm and observes whether or not the worm reverses direction. We used two experimental approaches to determine the manner and moment of contact between the eyebrow hair tool and freely moving animals and the forces delivered by the classical assay. Using high-speed video (2500 frames/second), we found that typical stimulus delivery events include a brief moment when the hair is contact with the worm's body and not the agar substrate. To measure the applied forces, we measured forces generated by volunteers mimicking the classical touch assay by touching a calibrated microcantilever. The mean (61 μN) and median forces (26 μN) were more than ten times higher than the 2-μN force known to saturate the probability of evoking a reversal in adult C. elegans. We also considered the eyebrow hairs as an additional source of variation. The stiffness of the sampled eyebrow hairs varied between 0.07 and 0.41 N/m and was correlated with the free length of hair. Collectively, this work establishes that the classical touch assay applies enough force to saturate the probability of evoking reversals in adult C. elegans in spite of its variability among trials and experimenters and that increasing the free length of the hair can decrease the applied force
Forces applied by volunteers all exceeded the force required to produce the maximum probability of response from a worm.
<p>(A) Volunteers used an eyebrow hair to touch a force-sensing cantilever 30 times. A sensing circuit records the z-force applied to the cantilever as a function of time. The traces plotted here are the minimum, median, and maximum for touch events for the median volunteer (volunteer G). (B) Box plots of forces delivered by human volunteers. The central mark is the median, the edges of the boxes are 25th and 75th percentiles, and the whiskers indicate the minimum and maximum forces. The red line indicates the force at which the probability of a worm reversing is saturated (about 2 μN, F<sub>sat</sub>).</p
The mechanical properties of eyebrow hair tools.
<p>(A) Schematic of the method for measuring the stiffness of an eyebrow hair. (B) Representative trace of the deflection of the hair as a function of time. (C) Representative force-displacement curve. At least four curves were collected for each eyebrow hair. The slope of this curve is the stiffness. (D) Stiffness vs. length of the hair. Points are the measurements from five different hairs, and the smooth line was fit to the data according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178080#pone.0178080.e001" target="_blank">Eq 1</a>.</p
The <i>C. elegans</i> touch response encompasses the entire mechanotransduction pathway from mechanical stimulus to behavior.
<p>(A) In the classical touch assay, a stimulus is applied with an eyebrow hair, (B) force is transmitted through the worm, (C) ion channels regulate depolarization of a touch neuron, (D) interneurons pass the signal between the touch neuron and motor neurons, and (E) motor neurons activate body wall muscles to produce a behavioral response.</p
Representative examples of high-speed videos that indicate maximum force is applied to the worm.
<p>(A) Schematic representing the hair moving across the worm. (B) Cross-section showing when the hair first comes into contact with the worm. At this instant, it is also in contact with the agar and bacterial lawn. (C) As the hair moves across the worm, it lifts off the agar and is only in contact with the worm. (D-E) Images from representative videos of the hair sliding over the worm, temporarily lifting off the agar. (F) In this case, the worm is pushed across the agar laterally before the hair slides over the top of the worm. The dashed line reference shows lateral movement. Times are in reference to the time when the hair is only in contact with the worm as in panel C.</p
Forces applied during classical touch assays for <i>Caenorhabditis elegans</i>
<div><p>For decades, <i>Caenorhabditis elegans</i> roundworms have been used to study the sense of touch, and this work has been facilitated by a simple behavioral assay for touch sensation. To perform this classical assay, an experimenter uses an eyebrow hair to gently touch a moving worm and observes whether or not the worm reverses direction. We used two experimental approaches to determine the manner and moment of contact between the eyebrow hair tool and freely moving animals and the forces delivered by the classical assay. Using high-speed video (2500 frames/second), we found that typical stimulus delivery events include a brief moment when the hair is contact with the worm’s body and not the agar substrate. To measure the applied forces, we measured forces generated by volunteers mimicking the classical touch assay by touching a calibrated microcantilever. The mean (61 μN) and median forces (26 μN) were more than ten times higher than the 2-μN force known to saturate the probability of evoking a reversal in adult <i>C. elegans</i>. We also considered the eyebrow hairs as an additional source of variation. The stiffness of the sampled eyebrow hairs varied between 0.07 and 0.41 N/m and was correlated with the free length of hair. Collectively, this work establishes that the classical touch assay applies enough force to saturate the probability of evoking reversals in adult <i>C. elegans</i> in spite of its variability among trials and experimenters and that increasing the free length of the hair can decrease the applied force.</p></div