A conserved role for sleep in supporting Spatial Learning in Drosophila

Abstract

Sleep loss and aging impair hippocampus-dependent Spatial Learning in mammalian systems. Here we use the fly Drosophila melanogaster to investigate the relationship between sleep and Spatial Learning in healthy and impaired flies. The Spatial Learning assay is modeled after the Morris Water Maze. The assay uses a "thermal maze" consisting of a 5 × 5 grid of Peltier plates maintained at 36-37°C and a visual panorama. The first trial begins when a single tile that is associated with a specific visual cue is cooled to 25°C. For subsequent trials, the cold tile is heated, the visual panorama is rotated and the flies must find the new cold tile by remembering its association with the visual cue. Significant learning was observed with two different wild-type strains-Cs and 2U, validating our design. Sleep deprivation prior to training impaired Spatial Learning. Learning was also impaired in the classic learning mutant rutabaga (rut); enhancing sleep restored learning to rut mutants. Further, we found that flies exhibited a dramatic age-dependent cognitive decline in Spatial Learning starting at 20-24 days of age. These impairments could be reversed by enhancing sleep. Finally, we find that Spatial Learning requires dopaminergic signaling and that enhancing dopaminergic signaling in aged flies restored learning. Our results are consistent with the impairments seen in rodents and humans. These results thus demonstrate a critical conserved role for sleep in supporting Spatial Learning, and suggest potential avenues for therapeutic intervention during aging.

Keywords: Drosophila; Spatial Learning; aging; plasticity; sleep.

Figure 1.

Spatial learning apparatus. The floor of the apparatus is made up of a 5 × 5 grid of Peltier plates, which are maintained at a temperature of 36–37°C (which is aversive to flies). The first trial begins when a single fly in placed into the apparatus and one of the tiles is cooled to 25°C via an Arduino Uno controller (not shown). Distal visual cues mark the cool spot. In subsequent trials, the location of the cool spot and the distal visual cues move in tandem such that the fly learns to associate the visual cue with the location of the cool spot. The location of the flies is monitored using a camera (see methods for details).

Figure 2.

Validation of our spatial learning apparatus. (A) Sleep in minutes per hour for the 2U wild-type strain maintained on a 12:12 light–dark schedule (LD) (n = 29 flies). (B) Total sleep time in minutes in 2U flies. (C) Average daytime sleep bout duration (a measure of sleep consolidation during the day) in 2U flies. (D) Sleep in minutes per hour for the Cs wild-type strain maintained on a 12:12 LD schedule (n = 32 flies). (E) Total sleep time in minutes in Cs flies. (F) Average daytime sleep bout duration in Cs flies. (G and I) Spatial Learning in two independent cohorts of 2U flies trained in the coupled condition. Spatial Learning is expressed as the “time to target” normalized to the time in the first trial. Flies reduced their “time to target” over 10 trials by ~80% (n = 9–11 flies/replicate, repeated measures ANOVA for trials, F_[9,162] = 15.36, p < 10⁻¹⁰). (K) Quantification of learning scores in G and I, expressed as percentage change in the time to target in trials 9 and 10, relative to trial 1. The two replicates of 2U flies exhibited similar Learning Indices (n.s. p = 0.09, two-tailed t-test). (H and J) Learning in two independent replicates of Cs flies trained in the coupled condition. Flies reduced their “time to target” over 10 trials by ~70% (n = 8–10 flies/replicate, repeated measures ANOVA for trials F_[9,160] = 12.28, p < 10⁻¹²). (L) Two independent cohorts of Cs flies exhibited similar Learning Indices (n.s. p = 0.84, two-tailed t-test). (M and N) In contrast to flies trained in the coupled condition, Cs flies in the uncoupled condition showed little to no improvement in their time to target (n = 7–12 flies/condition, two-way repeated-measures ANOVA for condition × trial F_[9,198] = 4.93, p ≤ 0.01). (O) Learning index of flies in the “coupled” condition is much higher than in the “uncoupled” condition (*p < 0.01, t-test).

Figure 3.

Sleep deprivation impairs Spatial Learning in Cs flies. (A) Sleep-deprived Cs flies lost 98% of their sleep and recovered ~60% of their lost sleep during the subsequent 48 h in recovery (n = 30 flies, repeated measures ANOVA for time, F_[70,1470] = 12.97, p < 10⁻¹⁵). (B) Sleep-deprived Cs flies (green) display impaired Spatial Learning compared with controls (blue) (n = 7–10 flies/condition,* p < 0.01, t-test). (C) Sleep deprivation did not impair heat avoidance (n = 10–11 flies/condition, n.s. p = 0.97, t-test).

Figure 4.

rut-dependent impairments in spatial learning are reversed by enhancing sleep. (A) Sleep profiles of rut²⁰⁸⁰ mutants was not different compared with Cs controls (n = 18–22 flies/genotype, n.s. p = 0.49, t-test). (B) Spatial Learning is impaired in rut²⁰⁸⁰ mutants compared with Cs controls (n = 9–10 flies/genotype,*p < 0.001, t-test). (C) rut²⁰⁸⁰ males are not impaired in heat avoidance (n = 10 flies/genotype, n.s. p = 0.27). (D) Gaboxadol increases total sleep in rut²⁰⁸⁰ flies compared with vehicle-fed siblings (n = 18–20 flies/condition, *p < 10⁻¹⁰, t-test). (E) Gaboxadol-fed rut²⁰⁸⁰ flies display increased average daytime sleep bout duration compared with vehicle-fed siblings (*p < 10^–4, t-test). (F) Gaboxadol restored spatial learning to rut²⁰⁸⁰ flies compared with controls (n = 9–10 flies/condition,*p < 0.01, t-test). (G) Gaboxadol did not impair the optomotor response of rut²⁰⁸⁰ flies (n = 19 flies/condition, n.s. p = 0.43, t-test).

Figure 5.

Age-dependent declines in spatial learning can be reversed by enhancing sleep. (A) Sleep, in minutes per hour, was reduced in 21–24 days old flies (green) compared with 5-day-old controls (blue) (n = 20–27 flies/group, repeated measures ANOVA age × time; F_[23,966] = 5.49, p < 0.001). (B) Total sleep was reduced in 21–24 days old flies compared with 5-day-old flies;*p < 0.01, t-test. (C) Aging reduced average daytime sleep bout duration; p < 0.05, t-test. (D) Waking activity was not impaired in 21–24 days old flies (n.s. p = 0.83). (E) Spatial learning was impaired in 21–24 days old flies compared with 5-day-old flies (n = 10–14 flies/group, *p < 10⁻⁴). (F) Age did not disrupt heat avoidance (n = 10 flies/condition, n.s. p = 0.49). (G) Age did not impair optomotor responses (n = 51–62 flies/condition, *p < 10^–5). (H) Gaboxadol (Gab) increased sleep in 21–24 days old flies (n = 20 flies/group, *p < 10⁻¹⁰). (I) Gaboxadol increased average daytime sleep bout duration in 21–25 days old flies (green) compared with age-matched controls (blue) (*p < 10⁻⁴, t-test). (J) Spatial learning was restored to Gaboxadol-fed 21–24 days old flies (green) compared with age-matched vehicle-fed controls (n = 9–10 flies/condition, *p < 10⁻⁴, t-test). (K) Spatial learning was significantly higher in 21–24 days old R23E10-GAL4/+>UAS-NaChBac/+ flies compared with age-matched R23E10-GAL4/+ and UAS-NaChBac/+parental controls (n = 8–10 flies/genotype, One-way ANOVA for genotype F_[2,49] = 4.59, p < 0.05, *p < 0.01, modified Bonferroni test).

Figure 6.

Reducing dopamine signaling impairs learning. (A) 3-Iodo-l-tyrosine (3IY)-fed flies display an increase in sleep compared with vehicle-fed controls (Sleep in minutes per hour, n = 20–21 flies/group, two-way repeated-measures ANOVA for drug × time F_[23,782] = 5.49, p < 10⁻⁶). (B) 3IY-fed flies display more total sleep than age-matched vehicle-fed siblings (*p < 10^–6, t-test). (C) 3IY increased average daytime sleep bout duration (*p < 0.001, t-test). (D) 3IY did not impair waking activity compared with vehicle-fed controls (*p < 0.01, t-test). (E) Vehicle-fed Cs controls displayed spatial learning. (F) In contrast to vehicle-fed flies, 3IY-fed Cs flies were impaired in spatial learning. (n = 8 flies/group, two-way ANOVA drug × trial, F_[9,126] = 2.33, p < 0.05). (G) Learning index of 3IY-fed flies was greatly reduced compared with vehicle-fed controls (*p < 0.01, t-test). (H) Schematic of temperature-shift experiment for sleep. Sleep is recorded over 24 h of 5-day-old TH-GAL4/+, tubpGAl80^ts, UAS Kir/+, and TH-GAL4/+ > tubpGAl80^ts, UAS Kir/+ flies maintained at 18°C and of their sibling flies that are reared for 3 days at 18°C, and then shifted to the elevated temperature of 30°C for 2 days prior to testing. (I) TH-GAL4/+>GAL80^ts, UAS Kir/+ flies displayed an increase in sleep at 30°C compared with siblings maintained at 18°C; sleep in TH-GAL4/+ or the tubP GAL80^ts, UAS Kir/+ parental controls was similar at both 18°C and 30°C (n = 20–30 flies/group, two-way ANOVA for genotype × temperature, F_[2,131] = 7.28, p < 0.01; *p < 0.001, modified Bonferroni test). (J) Schematic of temperature-shift experiment for learning. Spatial learning is evaluated in 5-day-old TH-GAL4/+, tubpGAl80^ts, UAS Kir/+, and TH-GAL4/+ > tubpGAl80^ts, UAS Kir/+ flies maintained at 18°C and of their sibling flies that are reared for 3 days at 18°C, and then shifted to the elevated temperature of 30°C for 2 days prior to testing. (K) Spatial learning is impaired in TH>GAL80^ts, UAS Kir flies at 30°C compared with siblings maintained at 18°C; temperature did not impact spatial learning in either TH-GAL4/+ or the tubP GAL80^ts, UAS Kir/+ parental controls (n = 8–12 flies/group, Two-way ANOVA for genotype × temperature, F_[2,116] = 4.96, p < 0.01, *p < 0.05, modified Bonferroni test).

Figure 7.

Enhancing dopamine signaling ameliorates age-related cognitive decline. (A) Nighttime sleep was reduced in levodopa (l-Dopa, green) fed 21–24 days old Cs flies compared with age-matched vehicle-fed (blue) controls (n = 10–12 flies/group, *p < 0.01, t-test). (B) Spatial learning was elevated in old l-Dopa fed Cs flies (green) compared with age-matched controls (blue) (n = 9–10 flies/group, *p < 0.01). (C) Heat avoidance was not changed in old flies fed l-Dopa or Gaboxadol (green) compared with age-matched controls (n = 10 flies/condition, n.s. p > 0.25, modified Bonferroni test). (D) Spatial learning was elevated in old R15B07>dumb² flies compared with age-matched R15B07-GAL4/+ and dumb²/+ parental controls (n = 8 flies/genotype, One-way ANOVA for genotype F_[2,45] = 5.82, p < 0.01; *p < 0.01, planned comparisons, modified Bonferrsoni test).