The Effect of Stress on Motor Function in <i>Drosophila</i>

<div><p>Exposure to unpredictable and uncontrollable conditions causes animals to perceive stress and change their behavior. It is unclear how the perception of stress modifies the motor components of behavior and which molecular pathways affect the behavioral change. In order to understand how stress affects motor function, we developed an experimental platform that quantifies walking motions in <i>Drosophila</i>. We found that stress induction using electrical shock results in backwards motions of the forelegs at the end of walking strides. These leg retrogressions persisted during repeated stimulation, although they habituated substantially. The motions also continued for several strides after the end of the shock, indicating that stress induces a behavioral aftereffect. Such aftereffect could also be induced by restricting the motion of the flies via wing suspension. Further, the long-term effects could be amplified by combining either immobilization or electric shock with additional stressors. Thus, retrogression is a lingering form of response to a broad range of stressful conditions, which cause the fly to search for a foothold when it faces extreme and unexpected challenges. Mutants in the cAMP signaling pathway enhanced the stress response, indicating that this pathway regulates the behavioral response to stress. Our findings identify the effect of stress on a specific motor component of behavior and define the role of cAMP signaling in this stress response.</p></div

Effects of combining amputation and suspension on fly walking.

<p><b>A</b>. Retrogression at different times after release from suspension. Amputated and suspended flies display a robust increase in backtracking that gradually decreases. Recovery of retrogression was fit to a monoexponential decay with a time constant of 11±2 min. n = 15. * p<0.05, Wilcoxon signed-rank test. <b>B</b>. Onset time course of walking adaptations. Flies were suspended for increasing durations and the retrogression was determined upon release at each time point. Data were fit to a monoexponential association with a time constant of 10 min. n≥10. p = 0.0025, Kruskal-Wallis test. * p<0.05 ** p<0.01, Mann-Whitney test with Bonferroni correction for multiple comparisons. <b>C</b>. Habituation of the response to combined amputation and suspension. The initial response upon release from suspension (blue) decreases to the baseline level of flies that have recovered from stress (red). p<0.05, Friedman test. * p<0.05, Wilcoxon signed rank test with bonferroni correction of p values. <b>D</b>. Sustained retrogressions could be repeatedly toggled. Retrogression at 0 min (red bars) and 30 min (blue bars) after two consecutive two hour suspension treatments (Susp1 and Susp2). n = 19–21. p = 0.0009, Friedman test. ** p<0.01 * p<0.05, Wilcoxon signed-rank test. Error bars are mean ±SEM.</p

Effects of leg tip amputation on fly walking.

<p><b>A</b>. Leg trajectories immediately after amputation of front left leg (0 min) and after 2 hours of recovery (120 min). Black dots are touchdowns. Blue lines represent forward motions in the amputated leg, while red lines represent retrogression. <b>B</b>. Retrogression at different times after amputation. Note that the retrogression following amputation (initial vs. final value) is not statistically significant. The inset is a schematic of the amputated front leg showing the amputated section in black shading. n = 17. <b>C</b>. Slippery surfaces amplify the effects of electric shock. Δ retrogression is the difference between retrogression during the first four steps of recovery from electric shock and retrogression prior to the electric shock, and controls for changes in baseline retrogression between the three conditions tested. n = 12–17. p = 0.0033, Kruskal-wallis test. * p<0.05, ** p<0.01, Mann-Whitney test with bonferroni correction of p values. Error bars are mean ±SEM.</p

Walking analysis parameters.

<p>The table lists the parameters that are calculated from the frame-by-frame acquisition of coordinates. Reference appears whenever a parameter has been used before.</p><p>Walking analysis parameters.</p

Effects of suspension on fly walking.

<p><b>A</b>. Trajectory of leg motions (blue lines) in unperturbed flies (Con). Black dots are touchdowns. Immediately after suspension (“0 min”), the legs retrogress (red line). After 2 hours (120 min) retrogression is reduced. <b>B</b>. Backtracking at different times after release from suspension. Note that the initial retrogression gradually subsides. Dotted line represents retrogression in Con. n = 12. * p<0.05, One way repeated measures ANOVA with post-hoc Dunnett multiple comparison test. <b>C</b>. The effects of suspension and amputation are additive. The effect of suspension (Susp), amputation(Amp) and their combination (Susp+Amp) was defined as the difference between the initial (0 min) and the final (120 min) values. n = 12–17. p<0.0001, Kruskal-Wallis test. * p<0.05 *** p<0.001, Mann-Whitney test with bonferroni correction of p values. Error bars are mean ±SEM.</p

The effects of cAMP pathway mutants on retrogressions.

<p><b>A</b>. cAMP pathway mutants demonstrate a stronger aftereffect following the end of electric shock. Retrogression was measured over the first four steps following an electric shock of 30 seconds. n = 16–20. p = 0.0097, Kruskal-Wallis test. * p<0.05, Mann-Whitney test with bonferroni correction for multiple comparisons. <b>B</b>. cAMP pathway mutants demonstrate a stronger aftereffect following the end of combined amputation and 2 hour suspension. n = 20–26. p = 0.0037, Kruskal-Wallis test. * p<0.05 ** p<0.01 Mann-Whitney test with bonferroni correction of p values. <b>C</b>. cAMP pathway mutants accumulate stress more rapidly than wild type flies. In order to compare the kinetics of stress induction between mutants, retrogression was determined immediately following different suspension times, and normalized to the retrogression levels after a 2 hour hanging period for each group. n≥8. Error bars are mean ±SEM.</p