Research ArticleNEUROPHYSIOLOGY

A deep sleep stage in Drosophila with a functional role in waste clearance

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Science Advances  20 Jan 2021:
Vol. 7, no. 4, eabc2999
DOI: 10.1126/sciadv.abc2999
  • Fig. 1 PEs occur during a deep sleep stage.

    (A) A PE consists of a full extension of the proboscis (red arrow), immediately followed by a full retraction. This process takes ~1.4 s. (B) Raster plot showing PE (each bar, 1 PE) and inactivity (gray blocks, 60-s inactivity threshold) for 1 hour (61 to 120 min after being tethered). Most inactivity bouts contain one or more PEs. (C) During PE bursts, inter-PE intervals are highly regular, with one PE occurring every 3 s (inset; average PE frequency is 0.34 Hz). (D) Arousal thresholds for flies that were inactive or inactive and making PE. After 5, but not 1, min of inactivity, flies making PE have greatly increased arousal thresholds. n = 8 to 13 per group; **P < 0.01, t test. n.s., not significant. (E) Representative 1-s traces for LFPs during wake, sleep, and PE. (F) Average power spectra of LFPs for flies that were awake, asleep, or producing PE show that LFP power is lower during PE compared to wake or sleep (inset). The data have been notch-filtered at 50 Hz (Australian line noise). n = 9; *P < 0.05, **P < 0.01, and ***P < 0.001 [analysis of variance (ANOVA) with Bonferroni test]. (G) Feeding flies Gaboxadol food (0.2 mg/ml) induces sleep (H) and decreases sleep latency (occurrence of first sleep bout after being tethered). (I) PEs per hour are greatly increased. (J) PE latency, the time to detection of the first PE after being tethered, is decreased. (K) Amount of PE per hour of sleep is increased after Gaboxadol administration. n = 15 control and n = 9 Gaboxadol; *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test. Error bars indicate SEM. All error bars indicate SEM.

  • Fig. 2 PEs are under circadian and homeostatic control.

    (A) Sleep in tethered flies in constant darkness follows a circadian pattern, where sleep is higher during the night than during the day (n = 15 flies per time point). (B) PEs in tethered flies increase during the day and are highest at the start of the subjective dark phase (CT12) and gradually dissipate throughout the night, although sleep remains high (n = 15 flies per time point). (C) After sleep deprivation (SD), sleep increases (*P < 0.05; ANOVA with Bonferroni test), (D) while sleep latency decreases (*P < 0.05, t test). (E) Likewise, the amount of PE is strongly increased for the first 2 hours of rebound sleep (*P < 0.05, n.s., ANOVA with Bonferroni test) (F), and the latency to detecting the first PE after being tethered decreases (*P < 0.05, t test). (C to F) n = 10 per group.

  • Fig. 3 PEs facilitate recovery from injury.

    (A) Full-body injury was induced using the HIT assay (30), where flies are loaded in a vial attached to a spring. By pulling back the spring (A1) and releasing it, the vial slams on a rubber block, causing full-body impact injuries (A2). (B) Flies were immediately tethered after injury and sleep, and PEs were measured over 3 hours postinjury, showing no effect on sleep (n.s., t test) (C) but a strong increase in PEs per hour (**P < 0.01, t test; n = 9 per group). (D) To test whether PE affects survival rate, the proboscis was immobilized with UV-cured glue. Sham-treated flies received glue on top of the head, covering the ocelli but leaving the proboscis free. Preventing PE increases the proportion of flies that die within 24 hours after injury. The proportion of flies immediately killed does not differ between groups. (E) Immobilizing the proboscis increases 24-hour mortality (n = 119, n = 88, and n = 130; ***P < 0.001, chi-square test). (F) Constitutively activating neurons controlling feeding (NP5137-Gal4) using NaChBac increases sleep and (G) decreases PE, compared to parental controls (n = 50 per group; **P < 0.01 and ***P < 0.001, ANOVA with Bonferroni t test), (H) and increases mortality after injury (n = 50 per group; *P < 0.05, chi-square test). All error bars indicate SEM.

  • Fig. 4 PEs facilitate clearance from hemolymph.

    (A) Model of how PE facilitates waste clearance. As the proboscis is extended, hemolymph moves forward, reversing when the proboscis is retracted. PE drives convective hemolymph along the MTs, where waste is absorbed, shunted into the gut, and excreted. (B) After transfer to nonluciferin food, luminescence generated by OK107>luc flies gradually decreases. This is slower in glue-immobilized flies, compared to sham-treated controls (n = 36 to 48 per group, two replicates). (C) As indicated by increased luminescence half-life (*P < 0.05, t test). (D) Glue-immobilizing the proboscis reduces the rate at which injected dye is cleared. However, the total amount of excreted dye in 24 hours is not different (n = 8 to 12 per group, nine replicates). (E) Within seconds after being injected, flies smurfed (inset). Immobilizing the proboscis caused flies to remain smurfed longer. Twenty-four hours after injection, no smurfed flies were detected. (F) Sleep deprivation decreases the rate at which injected dye is cleared (n = 8 to 12 per group, eight replicates) and (G) causes flies to remain smurfed longer. (H) Sleep deprivation before injection increases the rate at which injected dye is cleared (n = 8 to 10 per group, eight replicates). (I) Rebound sleep causes flies to lose their smurfed status sooner. All error bars indicate SEM. (D to I) *P < 0.05, **P < 0.01, and ***P < 0.001, n.s., ANOVA followed by t tests.

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