The Drosophila fragile X mental retardation gene regulates sleep need

Abstract

Sleep need is affected by developmental stage and neuronal plasticity, but the underlying mechanisms remain unclear. The fragile X mental retardation gene Fmr1, whose loss-of-function mutation causes the most common form of inherited mental retardation in humans, is involved in synaptogenesis and synaptic plasticity, and its expression depends on both developmental stage and waking experience. Fmr1 is highly conserved across species and Drosophila mutants carrying dFmr1 loss-of-function or gain-of-function mutations are well characterized: amorphs have overgrown dendritic trees with larger synaptic boutons, developmental defects in pruning, and enhanced neurotransmission, while hypermorphs show opposite defects, including dendritic and axonal underbranching and loss of synapse differentiation. We find here that dFmr1 amorphs are long sleepers and hypermorphs are short sleepers, while both show increased locomotor activity and shortened lifespan. Both amorphs and hypermorphs also show abnormal sleep homeostasis, with impaired waking performance and no sleep rebound after sleep deprivation. An impairment in the circadian regulation of sleep cannot account for the altered sleep phenotype of dFmr1 mutants, nor can an abnormal activation of glutamatergic metabotropic receptors. Moreover, overexpression of dFmr1 throughout the mushroom bodies is sufficient to reduce sleep. Finally, dFmr1 protein levels are modulated by both developmental stage and behavioral state, with increased expression immediately after eclosure and after prolonged wakefulness. Thus, dFmr1 expression dose-dependently affects both sleep and synapses, suggesting that changes in sleep time in dFmr1 mutants may derive from changes in synaptic physiology.

Figure 1.

Inverse relationship between dFmr1 levels and daily sleep amount. A, B, Daily sleep amount from eclosure to day 42 and average values for the entire 42 d period. Allele combinations are arranged from left to right based on increasing levels of dFmr1 expression. C, Percentage change in sleep amount compared with wild-type flies over the first 42 d after eclosure. D, E, Waking activity (number of beam crossing/min) for the first 42 d after eclosure and average values for the entire 42 d period. Statistics were based in all cases on Kruskal–Wallis test for multiple comparisons, followed by post hoc analysis using Mann–Whitney test to compare mutant genotype to wild type. In A and D, significant differences (p < 0.05) relative to wild type are indicated by gray squares (amorphs) and black circles (hypermorphs). In B and E, black dots indicate significant differences relative to wild type. Number of flies for each genotype for day 1 and day 42 were as follows: males: Δ50M, n = 60/34; EP3517/Δ50M, n = 73/49; EP3517, n = 112/81; EP3517/+, n = 68/56; dFmr1+, n = 112/92; nSyb-GAL4->EP3517, n = 47/39; females: Δ50M, n = 45/21; EP3517/Δ50M, n = 51/25; EP3517, n = 85/56; EP3517/+, n = 62/56; dFmr1+, n = 117/102; nSyb-GAL4→EP3517, n = 38/32. F, DFMRP levels in amorphs, hypermorphs, and wild-type flies as measured by Western blot (β-tubulin was used as loading control). A black dot indicates a significant difference (p < 0.05) from wild type as determined by Student's t test.

Figure 2.

Effects of changes in dFmr1 expression on sleep parameters. Daily values from eclosure until day 42 (A, C, E) and means across the entire 42 d period (B, D, F). The number of brief awakenings is per hour of sleep. Number of flies and statistics are as in Figure 1.

Figure 3.

Effects of changes in dFmr1 expression on the diurnal sleep pattern. Hypnograms illustrating sleep amount (min/30 min) throughout the 24 h period. Sleep amounts were averaged over days 7–9 (top) and days 21–23 (bottom). Number of flies and statistics are the same as in Figure 1.

Figure 4.

Inverse relationship between dFmr1 levels and daily sleep amount in constant darkness. Flies were kept in a 12 h LD cycle until day 6 after eclosure, then kept in DD until day 20. A, Number of beam crossings in 5 min intervals for the last 3 d in DD (days 18–20). B, Daily sleep amount in LD and DD. C, Sleep pattern during the last 3 d in DD. In B and C, significant differences (p < 0.05) relative to wild type are indicated by gray squares (amorphs) and black circles (hypermorphs). D, Daily sleep amount averaged over the last 3 d in DD for all genotypes tested (arranged from left to right as in Fig. 1). A black circle indicates significant difference from wild type. Number of flies: Δ50M, n = 29; EP3517/Δ50M, n = 34; EP3517, n = 59; EP3517/+, n = 39; dFmr1+, n = 35; nSyb-GAL4→EP3517, n = 29.

Figure 5.

Complementation analysis. A, Daily sleep amount and waking activity for the first 21 d after eclosure in Δ50M/Δ113M recombinants (gray line), EP3517 homozygotes (gray dots), and wild-type flies (black line). Average values over the 21 d period are shown on the right bar graphs. Numbers of flies: Δ50M/Δ113M, n = 20; Δ50M/EP3517 or Δ113M/EP3517 combined, n = 34; EP3517, n = 33; dFmr1+, n = 112. Kruskal–Wallis test followed by Mann–Whitney test; square boxes indicate days when Δ50M/Δ113M is significantly different from wild type. Black dots in the bar graphs indicate significant difference (p < 0.05 Mann–Whitney) from wild type. B, Daily sleep amount and waking activity for the first 21 d after eclosure after combining Δ50M with Df(3R)Exel6265. After recording sleep data, flies were genotyped by PCR: EP3517/Df(3R)Exel6265, n = 28; Df(3R)Exel6265/Δ50M, n = 28; EP3517/+, n = 28; +/Δ50M, n = 32. Kruskal–Wallis test followed by Mann–Whitney test. Gray boxes indicate significant difference between Df(3R)Exel6265/Δ50M and EP3517/+ (no other differences between genotypes were found). Black dots in the bar graphs indicate significant difference (p < 0.05 Mann-Whitney) from EP3517/+.

Figure 6.

Effects of changes in dFmr1 expression on the response to sleep deprivation. A, Hypnograms displaying sleep patterns over a 24 h period the day before sleep deprivation (baseline) and the day after 24 h of sleep deprivation (recovery). Only flies (all males) that lost >90% of their sleep during sleep deprivation were included in this analysis. Amorphs (n = 18) are Δ50M homozygotes, wild-type (n = 12) are dFmr1⁺ homozygotes, and hypermorphs (n = 30) have nSyb-GAL4 driving EP3517. Black circles indicate significant differences between baseline and recovery (p < 0.05 Mann–Whitney). B, Amount of sleep recovered during the entire 24 h of recovery expressed as percentage of sleep lost during sleep deprivation (same flies as in A). Horizontal bars indicate differences between mutants and wild type (*p < 0.05, Mann–Whitney). Only wild-type flies showed a significant increase in sleep duration during recovery relative to baseline (vertical bar, p < 0.05 signed rank test). C, Arousal thresholds measured as the percentage of sleeping flies that react to a complex stimulus of low intensity (Huber et al., 2004; Cirelli et al., 2005) (*p < 0.05, Mann–Whitney). D, Waking performance measured as the percentage increase in the number of beam crossings during the minute following the delivery of the stimulus relative to the minute before the stimulation. All flies had been active (i.e., awake) during the minute before the delivery of the stimulus (*p < 0.05, Mann–Whitney). In C and D, the number of flies (all males) is amorphs = 27, wild-type = 62, hypermorphs = 74. Flies were tested during the first 6 h of the light period in baseline (BL) and recovery (REC).

Figure 7.

Changes in DFMRP levels due to developmental stage and behavioral state. A, Relative dFmr1 levels in sleeping males 1, 3, 7, 14, and 21 d after eclosure. Males were harvested 8 h after lights off and selected for maximal sleep (asleep >90% of the previous 8 h). To exclude potentially sick flies, mean waking activity (crossings/min of waking) the day before harvesting was required to be >0.7. B, The three experimental groups. C, Average sleep amounts during the last 8 h before harvesting. S flies were required to spend >90% of the previous 8 h asleep. SD-D flies were required to stay awake >90% of the last 8 h, with no sleep in the last 4 h before harvesting. Since flies mainly sleep at night SD-N is more difficult to enforce than SD-D. Criteria for SD-N flies were therefore less stringent: > 80% awake over the last 8 h, with <13% of sleep in the last 2 h before harvesting. Mean waking activity the day before harvesting was >0.7 in all flies. C, Relative DFMRP expression levels as measured in Western blots. Black squares indicate, within the same genotype, significant differences relative to S flies (p < 0.05, Student's t test). Black circles indicate, within the same behavioral state, significant differences relative to wild-type (p < 0.05, Student's t test).

Figure 8.

Effects of changes in dFmr1 expression on lifespan. A, Longevity curves for amorph (Δ50M), wild-type (dFmr1⁺), and hypermorphic (nSyb-GAL4 driving EP3517) flies. B, Average lifespan for all male and female dFmr1 mutants tested. Genotypes are arranged from left to right in order of increasing dFmr1⁺ expression as in Fig. 1 (same number of flies). Black dots indicate significant differences from wild type (p < 0.05, Mann–Whitney test).

Figure 9.

Sleep effects of dFmr1 overexpression in the adult. A, Daily sleep amount and waking activity from eclosure to day 42 and average values for the entire 42 d period. All flies shared the same genotype (w¹¹¹⁸; ELAV-GeneSwitch/EP3517), but their food contained either vehicle (control) or RU486. Gray squares (in A) indicate significant difference from controls (p < 0.05, Mann–Whitney test). Number of flies for day 1 and day 42 were as follows: RU486 = 26/24, Control = 36/29. B, Western blot analysis using an antibody that binds DFMRP (β-tubulin was used as loading control).

Figure 10.

MPEP, an mGluR antagonist, does not rescue the long sleeping phenotype due to loss of dFMR1. Age-matched flies, harvested within 24 h of eclosure, were tested on cornmeal molasses media for 2 d. On the third day (at lights on), flies were transferred to tubes with sucrose media containing vehicle or 86 μm MPEP, tested for 6 d, and then transferred to fresh sucrose media and monitored for seven additional days. A, Daily sleep amount (min/24 h) before, during, and after MPEP treatment. B, Waking activity (number of beam crossings/min) before, during, and after MPEP treatment. Solid circles indicated significant difference between MPEP-treated Δ50M amorphs and MPEP-treated EP3517 hypomorphs; triangles represent significant difference between untreated Δ50M amorphs and untreated EP3517 hypomorphs (p < 0.05, Mann–Whitney test). Number of flies were as follows: MPEP-treated EP3517 = 48, MPEP-treated Δ50M = 39, untreated EP3517 = 41, untreated Δ50M = 32.

Figure 11.

Regional specificity of the effects of dFMR1 on sleep duration. Males flies were generated by crossing w: GAL4-driver/+ to w: EP3517/+. Progeny were selected based on eye color and genotype confirmed using PCR after testing. A, Sleep time (min/24 h) for each of the 5 d tested (days 15–19 posteclosure). B, Waking activity represents the number of beam crossings per minute. C, D, Sleep time (min/12 h). E, F, Number and duration of sleep episodes throughout the 24 h period. G, Number of brief awakenings per hour of sleep. H, Hypnograms illustrating sleep amount (min/30 min) throughout the 24 h period, averaged over days 15–19. Number of flies tested were as follows (GAL4-driver driving EP3517, EP3517): 30Y = 16, 19; 201Y = 25, 20; 238Y = 17, 20; c309 = 7, 10. Significant differences (p < 0.05, Mann–Whitney test) are indicated by black circles (A–G) and gray squares (H).