Genetic and environmental factors impact age-related impairment of negative geotaxis in Drosophila by altering age-dependent climbing speed

Abstract

Age-related locomotor impairment in humans is important clinically because it is associated with several co-morbidities and increased risk of death. One of the hallmarks of age-related locomotor impairment in humans is a decrease in walking speed with age. Genetically tractable model organisms such as Drosophila are essential for delineating mechanisms underlying age-related locomotor impairment and age-related decreases in locomotor speed. Negative geotaxis, the ability of flies to move vertically when startled, is a common measure of locomotor behavior that declines with age in Drosophila. Toward further developing Drosophila as a model for age-related locomotor impairment, we investigated whether negative geotaxis reflects climbing or a combination of climbing and other behaviors such as flying and jumping. Additionally, we investigated whether locomotor speed in negative geotaxis assays declines with age in flies as found for walking speed in humans. We find that the vast majority of flies climb during negative geotaxis assays and that removal of hind legs, but not wings, impairs the behavior. We also find that climbing speed decreases with age in four wild type genetic backgrounds, in flies housed at different temperatures, and in control and long-lived flies harboring a mutation in OR83b. The decreases in climbing speed correlate with the age-related impairments in the distance climbed. These studies establish negative geotaxis in Drosophila as a climbing behavior that declines with age due to a decrease in climbing speed. Age-related decreases in locomotor speed are common attributes of locomotor senescence in flies and humans.

Figure 1. Contribution of climbing and jumping/flying behavior to negative geotaxis performance

Canton-S (CS), panels A and B; Samarkand (Sam), panels C and D. The percentage of flies that climbed or jumped/flew in RING assays was determined by careful manual inspection of video-taped Canton-S (A) and Samarkand (C) flies at 1- and 4-weeks of age. The percentage of flies that climbed was significantly greater than the percentage that jumped/flew (two-way ANOVA, p<0.0001, n=5). Age had no effect on the percentage of climbers or jumpers (n.s.). The distance climbed was decreased with age in Canton-S (B) and Samarkand (D) flies, but the behavior in flies that climbed (climbers) was indistinguishable from that of all flies combined (climbers, jumpers/fliers) (two-way ANOVA; effect of age, p<0.0001; effect of climbing, n.s.; n=5).

Figure 2. Negative geotaxis is impaired by removal of legs, but not wings

A. Negative geotaxis was lower in 4-week-old than in 1-week-old Canton-S flies, but was not affected by removal of the wings (two-way ANOVA; effect of age, p<0.0001; effect of wing removal, n.s.; n=10–20). B. The vertical distance climbed in one-week-old Canton-S flies with no hind legs was significantly less than in age-matched intact flies (unpaired two-tailed t test, p<0.0001, n=5).

Figure 3. Negative geotaxis time-courses across genetic background and age

Canton-S (CS, A and B); Oregon-R (OR, C and D); Lausanne-S (LS, E and F); Samarkand (Sam, G and H). Negative geotaxis was assessed during 4-second RING assays in females (left panels) and males (right panels) at the ages (weeks) indicated by arrows. Lines are best-fit linear regression. Data are mean ± S.E.M. (n=12). The results from statistical tests are summarized in Table 1, Table S1, Table S2 and Table S3.

Figure 4. Linear models for negative geotaxis time-courses

Linear models of mock negative geotaxis time-courses in young flies and aged flies with decreased climbing speed only (Age Δ Speed), increased climbing latency only (Age Δ Latency), or both (Age Δ Both). Climbing speed and latency are 1 cm/s and 0.5 seconds, respectively, in the Young model. Climbing speed is changed to 0.5 cm/s while the climbing latency is maintained at 0.5 s in the Age Δ Speed model. Climbing latency is changed to 2.5 seconds while the climbing speed is maintained at 1 cm/s in the Age Δ Latency model. Climbing speed is changed to 0.5 cm/s and climbing latency is changed to 2.5 s in the Age Δ Both model.

Figure 5. Effect of age on the distance climbed, climbing speed and climbing latency—genetic background

Data in Figure 4 were analyzed to determine the distance climbed at 4-seconds (A and B), climbing speed (C and D), and response latency (E and F) in females (left panels) and males (right panels). Individual two-way ANOVAs revealed that the distance climbed at 4 s and climbing speed decreased with age while climbing latency increased with age in all experiments (p<0.0001). Genetic background indications are the same as in Figure 4. Symbols indicating genetic background are the same in all panels.

Figure 6. Effect of age on the distance climbed, climbing speed and climbing latency—elevated housing temperature

The distance climbed at 4-seconds (A and B), climbing speed (C and D), and climbing latency (E and F) in Canton-S (left panels) and Samarkand (right panels) males. The distance climbed at 4-seconds and climbing speed decreased with age; the climbing latency increased with age (two-way ANOVAs, p<0.0001, n=8–10). Negative geotaxis at 4-seconds (A and B) and climbing speed (C and D) were lower in flies housed at 29°C than at 25°C (p<0.0001). Climbing latency (E and F) was increased at the elevated temperature (p≤0.0283).

Figure 7. Effect of age on the distance climbed, climbing speed and climbing latency—long-lived OR83b mutants

The distance climbed at 4-seconds (A) and climbing speed (B) decreased with age while climbing latency (C) increased with age (two-way ANOVAs, p<0.0001, n=20). The distance climbed at 4-seconds (A) and climbing speed (B) were significantly higher overall in OR83b mutants compared to w¹¹¹⁸ controls (p<0.0001, n=20). Climbing latency was significantly lower in OR83b mutants (C, p<0.0001).

Figure 8. Decomposition of age-dependent changes in climbing speed and climbing latency

Data are the proportional contribution of the changes in climbing speed and latency on the change in distance climbed determined by decomposition analyses of the data from Figure 5–Figure 7. The age-dependent change in climbing speed is the major contributor to age-dependent reductions in the distance climbed in control females (A), control males (B), flies aged at 25°C (CS-25 and Sam-25) or 29°C (CS-29 or Sam-29), and in Or83b mutants and controls. CS, Canton-S; OR, Oregon-R; LS, Lausanne-S; Sam, Samarkand. Data are mean and S.E.M. for all vials of flies for each group.