D1 receptor activation in the mushroom bodies rescues sleep-loss-induced learning impairments in Drosophila

Abstract

Background: Extended wakefulness disrupts acquisition of short-term memories in mammals. However, the underlying molecular mechanisms triggered by extended waking and restored by sleep are unknown. Moreover, the neuronal circuits that depend on sleep for optimal learning remain unidentified.

Results: Learning was evaluated with aversive phototaxic suppression. In this task, flies learn to avoid light that is paired with an aversive stimulus (quinine-humidity). We demonstrate extensive homology in sleep-deprivation-induced learning impairment between flies and humans. Both 6 hr and 12 hr of sleep deprivation are sufficient to impair learning in Canton-S (Cs) flies. Moreover, learning is impaired at the end of the normal waking day in direct correlation with time spent awake. Mechanistic studies indicate that this task requires intact mushroom bodies (MBs) and requires the dopamine D1-like receptor (dDA1). Importantly, sleep-deprivation-induced learning impairments could be rescued by targeted gene expression of the dDA1 receptor to the MBs.

Conclusions: These data provide direct evidence that extended wakefulness disrupts learning in Drosophila. These results demonstrate that it is possible to prevent the effects of sleep deprivation by targeting a single neuronal structure and identify cellular and molecular targets adversely affected by extended waking in a genetically tractable model organism.

Figure 1. Sleep disruption impairs learning

A, Sleep Deprivation (SD)successfully eliminated 100% of baseline sleep (light-grey) during to 3, 6 and 12h of SD using the Sleep Nullifying Apparatus (SNAP). Sleep recovered in 24h after 3, 6 and 12h of sleep deprivation (SD) is proportional to sleep lost (dark-grey). Flies were sleep deprived until tested. SD was started at ZT 18 and ZT 21 for 3 h and 6 h SD. B, Learning was not impaired following 3 h SD but was significantly lower after 6 or 12 h of SD. C, 6 h of stimulation in the SNAP between ZT0-ZT5:59 (‘stimulated’) does not impair performance compared to untreated circadian-matched controls (black). D–E, Performance is impaired following 6 h of SD using the Sleep Interrupting Device (SLIDE). F, Total sleep and average sleep bout duration for Cs flies with consolidated sleep (black) and their spontaneously sleep-fragmented siblings (white). G, Learning is impaired in spontaneously fragmented flies (F) compared to siblings with consolidated sleep bouts (C). Learning was evaluated between ZT0-ZT3; (n=15/group). H. Experimental fragmentation was induced for three consecutive days in otherwise sleep consolidated flies by activating the SNAP for 5 minutes every 30 minutes. Controls were sleep deprived for 4 hours (ZT11-ZT15) and thus received the same number of stimuli (1440) and similar total sleep loss while obtaining consolidated sleep bouts. I Experimental sleep fragmentation (F) impairs performance compared to flies that are disturbed (D) but with consolidated sleep. (*: p<0.05).

Figure 2. The effect of sleep drive and recovery sleep on learning

A, Sleep min/h for each hour of the 24-h day in female Cs flies maintained on a 12:12 LD schedule for 3 days; the dark bar represents lights-out, the white bar indicates sleep deprivation. When recovery from 22h SD begins in the evening at ZT10 sleep homeostasis is delayed until the next morning. B, Although sleep rebound is a condition with high sleep-drive, flies show normal learning compared to untreated circadian-matched controls. C, Learning is restored in flies that had been sleep deprived for 12 h and allowed to sleep spontaneously for 2 h between ZT0 and ZT2 (white) compared to sleep deprived siblings (black). D, The duration of rebound sleep during the ‘nap’ was 98±2 min vs. 27±6 min in untreated flies and the average sleep bout duration was significantly longer than spontaneous sleep observed in untreated flies at this circadian time. E, When the dark vial contained dry filter paper with 10% sucrose 12 h SD does not disrupt learning (white). However, sucrose in the dark vial could not prevent learning impairments following 36 h SD (grey). Learning was assessed between ZT0-3 for both 12 h and 36 h SD groups (*: p<0.05).

Figure 3. Performance is dependent on previous waking experience

A, Sleep min/h for each hour of the 24-h day in female Cs flies maintained on a 12:12 LD schedule. The dark bar represents lights-out; inset number show average sleep bout during the day and night. B, Performance is impaired as waking accumulates across the biological day. Morning test (ZT0-3:59) Afternoon (ZT4-7:59) and Evening (ZT8-11:59). C, Learning was evaluated in three groups of flies at the same circadian time (ZT4-7:59). These flies differed in the amount of waking obtained since their last episode of consolidated sleep (defined as average sleep bout duration >30min). ‘0 minutes of waking’, (n=23) were sleep deprived from ZT12 until ZT2 and allowed to sleep unperturbed for 2 h (observed sleep time and sleep bout duration: 77 ± 6 min and 35 ± 8 min, respectively). ‘180 minutes of waking’, (n=17) were allowed 4 h of spontaneous sleep between ZT0-ZT3:59 (observed wake time, sleep time and sleep bout duration: 182 ± 17 min, 58 ± 17 min and 13.5 ± 1.65 min respectively). ‘360 min waking’, (n=17) were sleep deprived from ZT22 until ZT3:59 (observed sleep time =0 min). D, Learning was evaluated 3 h after lights-off in spontaneously sleeping flies compared to circadian-matched waking-controls that had been kept awake until ZT15 using the SNAP. Sleeping flies obtained 155 ± 5 minutes of nighttime sleep that was consolidated into bouts of 101 ± 17 min. Sleeping flies exhibited learning scores identical to those achieved after a full nights sleep but at an opposite circadian phase (schematic). In contrast, the flies that were not allowed to sleep in the evening were significantly impaired at the same circadian time. (*: p<0.05).

Figure 4. Learning requires the mushroom bodies

A, HU treatment results in ablation of the MBs: whole mount immuno-stainings of representative vehicle-control and HU treated brains with anti-fasciclin-2. B, Sleep time and sleep bout duration are reduced in MB ablated flies (HU short), but a minority of individual have normal sleep (HU long) compared to their vehicle-control (C). C, Performance is impaired in MB ablated flies regardless of sleep time. (*: p<0.05).

Figure 5. DA, extended waking and learning

A, Whole head DA levels, measured by HPLC, are increased following 12 h SD compared to untreated circadian matched controls. B, dDA1 transcripts are down regulated by 12 h SD whereas mRNA levels for the other D1 like receptor DAMB and for the D2 receptor (D2R) remain stable. All three receptors are transcriptionally down regulated in the flies with spontaneous sleep fragmentation described in Figure 1F (data are presented as % change from controls, one-sample t-test). C, Performance impairments after 12hSD are reversed when flies are fed methamphetamine (1mg/mL), L-DOPA (5mg/mL), Ritalin (2.5mg/mL) or the D1 agonist SKF-82958 (3mg/mL). Learning was evaluated between ZT0-ZT3:59; D, Blocking DA synthesis by feeding flies 3IY (10mg/mL) for 36 h results in performance impairments. Co-administering flies 3IY and L-DOPA (10mg/mL) rescues learning. E, Total sleep time (TST in minutes), average sleep bout duration (Bout in minutes) and locomoter activity during waking (C/W) are significantly increased in flies on 3IY. F, Feeding flies the D1 receptor antagonist SCH-23390 (1mg/mL) but not the D2 antagonist eticlopride (1mg/mL) for 2 h (ZT0-ZT2) before the test impairs performance. (*:p<0.05).

Figure 6. The D1-like receptor dDA1 is required for learning after sleep deprivation

A–C, dDA1 immunolocalization in controls (w¹¹¹⁸), dumb² and dumb³ mutant brains. In both dumb² and dumb³ dDA1 expression in the mushroom bodies is strongly reduced. dDA1 is ectopically expressed in glia and in the optic lobes in dumb³ mutant brains. D, The D1 like receptor hypomorph mutant dumb² shows performance decrements compared to wild type controls (C). Impairments are still observed in dumb²/Df(3R)red1 (dumb²/Df) and in dumb² flies fed with a D1 agonist SKF-82958. E, The misexpression mutant dumb³ also shows performance decrements as homozygotes or over Df(3R)red1 (dumb³/Df) compared to wild type controls. Feeding dumb³ a D1 agonist does not improve performance. F, Learning was evaluated in MB-Switch::dumb² flies fed 100µg/mL RU486 (RU+) or vehicle (RU-) for 2 days. Both RU+ and RU-groups show normal performance under baseline conditions. However, MB-Switch::dumb² RU+ flies show normal learning after SD in contrast to vehicle-control siblings (RU-). G, RU486 does not prevent learning impairments following sleep deprivation in MB-Switch/+ and dumb2/+ parental lines. Learning was evaluated in all flies between ZT0-ZT3:59. (*:p<0.05).