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Waking Experience Affects Sleep Need in Drosophila

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Science  22 Sep 2006:
Vol. 313, Issue 5794, pp. 1775-1781
DOI: 10.1126/science.1130408

Abstract

Sleep is a vital, evolutionarily conserved phenomenon, whose function is unclear. Although mounting evidence supports a role for sleep in the consolidation of memories, until now, a molecular connection between sleep, plasticity, and memory formation has been difficult to demonstrate. We establish Drosophila as a model to investigate this relation and demonstrate that the intensity and/or complexity of prior social experience stably modifies sleep need and architecture. Furthermore, this experience-dependent plasticity in sleep need is subserved by the dopaminergic and adenosine 3′,5′-monophosphate signaling pathways and a particular subset of 17 long-term memory genes.

Sleep is critical for survival, as observed in the human, mouse, and fruit fly (13), and yet, its function remains unclear. Although studies suggest that sleep may play a role in the processing of information acquired while awake (4, 5), a direct molecular link between waking experience, plasticity, and sleep has not been demonstrated. We have taken advantage of Drosophila genetics and the behavioral and physiological similarities between fruit fly and mammalian sleep (2, 3) to investigate the molecular connection between experience, sleep, and memory.

Drosophila is uniquely suited for exploring the relation between sleep and plasticity. First, fruit flies sleep. This is evidenced by consolidated periods of quiescence associated with reduced responsiveness to external stimuli and homeostatic regulation—the increased need for sleep that follows sleep deprivation (6). Second, Drosophila has been successfully used to elucidate conserved mechanisms of plasticity. For example, exposure to enriched environments, including the social environment, affects the number of synapses and the size of regions involved in information processing in vertebrates and Drosophila (7, 8). In the fruit fly, these structural changes occur in response to experiential information received within a week of emergence from pupal cases (9). Although brain plasticity is not limited to this period, the first week of emergence does coincide with the development of complex behaviors in Drosophila, including sleep. Hence, daytime sleep, which accounts for about 40% of total sleep in adults, is highest immediately after eclosion and stabilizes to adult levels 4 days after emergence (3).

To assess the impact of waking experience during this period of brain and behavioral development, individuals from the wild-type C-S strain were exposed to either social enrichment or impoverishment immediately at eclosion and were tested individually for sleep 5 days later (Fig. 1A). Socially enriched individuals (E), exposed to a group of 30 or more males and females (1:1 sex ratio) before being tested, slept significantly more than their socially impoverished (I) siblings, who were housed individually (Fig. 1, B and C; P < 0.001). This difference in sleep [ΔSleep (E)] was restricted to daytime sleep. Socially enriched individuals consolidated their daytime sleep into longer bouts of ∼60 min compared with their isolated siblings, who slept in 15-min bouts (Fig. 1D, P < 0.0001). In contrast, nighttime sleep was unaffected by prior social experience [Fig. 1, B and C; P = 0.4328 for 1B by analysis of variance (ANOVA)], corresponding with observations that daytime sleep is more sensitive to sex, age, genotype, and environment, when compared with nighttime sleep (10). This effect of social experience on sleep persisted over a period of days (Fig. 1E). Moreover, it was a stable phenotype: When socially enriched, longer-sleeping individuals and socially impoverished, shorter-sleeping siblings were sleep-deprived for 24 hours, they defended their respective predeprivation baseline sleep quotas by returning to these levels after a normal homeostatic response (Fig. 1F and fig. S1).

Fig. 1.

Social experience changes Drosophila sleep patterns. (A) Experimental paradigm for juvenile exposure. (B) Sleep per hour, over a 24-hour period. (C) Total sleep, daytime sleep, nighttime sleep, and ΔSleep: The response in sleep to social enrichment, calculated as a difference in sleep between E individuals and their I siblings. (D) Daytime sleep bout number and duration. I, n = 24; E, n = 51. (E) Sleep during 12 days after social exposure. Fruit flies are transferred to fresh food on day 9 (d9). I, n = 39; E, n = 48. (F) Sleep after 24 hours of sleep deprivation. I, n = 16; E, n = 16. Baseline (no sleep deprivation), d1; 24-hour sleep deprivation, d2; recovery, d3; postrecovery, d4 and d5. (G) ΔSleep (E) in circadian, visual, olfactory, and auditory mutants. (H) Sleep in fruit flies reared in a 2-cc tube and a 40-cc fly vial. (I) Sleep in socially impoverished virgins (V) and mated (M) flies. (J) Activity per waking minute each hour over 24 hours. (K) Daytime sleep in C-S and blind mutants after exposure to increasingly larger social groups. N denotes size of social group. [(A) to (J)] *P < 0.005.

Experience-dependent modifications in sleep have long been observed in humans, rats, mice, and cats (1113). But what is the nature of the experiential information that modifies sleep need in genetically identical Drosophila? Differences in sleep need in socially enriched and socially impoverished individuals were not a function of the space to which they were exposed: Flies reared in 2-cc tubes slept the same as those reared in 40-cc vials (Fig. 1H; P = 0.5407). Neither did it arise out of differences in reproductive state or sexual activity between the two groups: Socially impoverished mated and virgin individuals slept the same (Fig. 1I; P = 0.9450), as did socially enriched individuals from mixed-sex or single-sex groups (fig. S2). Further, differences in sleep were not a reflection of differences in overall activity (measured as infrared beam breaks) between the two groups (Fig. 1J; P =0.6386). Although social context can reset biological rhythms (14), mutations in clock (Clkjerk), timeless (tim01), and cycle (cyc01) disrupt circadian rhythms but had no effect on experience-dependent responses in sleep need (Fig. 1G).

Because social interaction requires sensory input, we next evaluated fly strains that were selectively impaired in vision, olfaction, and hearing. Blind norpA homozygotes failed to display a response in sleep to waking experience: Sleep need in norpA mutants did not increase after exposure to social enrichment (Fig. 1G; P = 0.8385). In contrast, norpA/+ heterozygotes with restored visual acuity slept more when previously socially enriched (Fig. 1G; P < 0.0001). Attenuating visual signals by rearing wild-type (C-S) flies in darkness also abolished the effect of waking experience on sleep (Fig. 1G; P = 0.7198). Compromising the sense of smell while retaining visual acuity also blocked experience-dependent changes in sleep need: Socially enriched smellblind1 mutants slept the same as their impoverished siblings (Fig. 1G; P = 0.8478). As confirmation, we specifically silenced neurons carrying olfactory input to the brain [Or83b-Gal4/UAS-TNT (15)] and observed that sleep in these flies was also not affected by prior waking experience (Fig. 1F; P = 0.7569). Auditory cues, however, did not affect the relation between experience and sleep (Fig. 1G; P < 0.0001). Finally, sleep need in individual Drosophila increased with the size of the social group to which they were previously exposed (Fig. 1K). Socially isolated flies slept the least, whereas those exposed to social groups of 4, 10, 20, 60, and 100 (1:1 sex ratio) showed proportionately increased daytime sleep need (Fig. 1K). When rendered blind, however, flies did not display this relation between sleep need and the intensity of prior social interactions (see norpA mutants in Fig. 1K).

If sensory stimulation received during a critical period of juvenile development directs the maturation of the adult sleep homeostat, then subsequent environmental exposure should not affect adult sleep time and consolidation. Alternatively, if experience-dependent modifications in sleep are a reflection of ongoing plastic processes, this phenomenon would persist in the adult. We observed that sleep in flies was modified by their most recent social experience regardless of juvenile experience. Shorter sleeping socially impoverished adults became longer sleepers when exposed to social enrichment (I⇒E) before being assayed (Fig. 2, A to C). Conversely, longer sleeping socially enriched flies became shorter sleepers after exposure to a period of social isolation (E⇒I; Fig. 2, D and E). Moreover, repeated switching of exposure between the two social environments consistently modified sleep, reflecting an individual's most recent experience (fig. S3).

Fig. 2.

Plasticity in sleep need and dopamine. (A) Experimental paradigm for adult plasticity. (B to E) Daytime sleep amount, sleep response (ΔSleep), and sleep bout number and duration in I⇒E (n = 20) and E⇒I (n = 55) fruit flies compared with their respective age-matched controls (I⇒I, n = 25; E⇒E, n = 23). (F) Dopamine content in whole brains. (G) ΔSleep in C-S flies. (H) ΔSleep in strains with aberrant dopaminergic transmission. In the case of E⇒I TH/TNT, flies show an aberrant increase in sleep. *P < 0.005.

An estimation of neurotransmitter levels in whole brains revealed that short-sleeping, socially impoverished individuals contained one-third as much dopamine as their longer-sleeping, socially stimulated isogenic siblings (Fig. 2F). Silencing or ablating the dopaminergic circuit in the brain [TH-Gal4/UAS-TNT and TH-Gal4/UAS-Rpr (16)] specifically abolished response to social impoverishment in individuals that were reared in social enrichment (Fig. 2H). We obtained similar results when endogenous dopamine levels were aberrantly increased, by disrupting the monoamine catabolic enzyme, arylalkylamine N-acetyltransferase, in Datlo mutants (17) (Fig. 2H). Hence, abnormal up- or down-regulation of the dopaminergic system prevented behavioral plasticity in longer sleeping, socially enriched individuals when switched to social impoverishment.

Our observation that dopaminergic transmission affects experience-dependent plasticity in sleep need is particularly compelling, given its role as a modulator of memory (18). We thus screened mutations in 49 genes implicated in various stages of learning and memory (1921) to assess their impact on experience-dependent changes in sleep need. Of these, only mutations in short- and long-term memory genes affected experience-dependent plasticity in sleep need (Fig. 3). Mutations in dunce (dnc1) and rutabaga (rut2080) have opposite effects on intracellular levels of adenosine 3′,5′-monophosphate (cAMP), but are both correlated with short-term memory loss. In dnc1 mutants, waking experience had no impact on subsequent sleep need (Fig. 3A). This effect was partially rescued in dnc1/+ heterozygotes, but complete rescue was only achieved when a fully functional dunce transgene (22) was introduced into the null mutant background (Fig. 3A). rut2080, however, selectively abolished the ability of socially enriched adults to demonstrate decreases in sleep after exposure to social impoverishment (Fig. 3A), which was reminiscent of aberrant dopaminergic modulation. Similarly, of the long-term memory genes screened, 17 (∼40%) specifically disrupted the change in sleep need in socially enriched adults after exposure to social impoverishment (Fig. 3B). For example, overexpression of the Drosophila CREB gene repressor, dCREB-b, resulted in socially enriched flies that continued to be longer sleepers even after exposure to social impoverishment (Fig. 3B). As a control, overexpression of the dCREB-a activator yielded wild-type phenotypic read out (Fig. 3B). It is noteworthy that not all long-term memory mutants had a disrupted relation between experience and sleep. Instead, the particular subset of genes identified, only half of which are expressed in the mushroom bodies (21), may specifically contribute to pathways that underlie sleep-dependent consolidation of memories.

Fig. 3.

Long- and short-term memory mutants are resistant to experience-dependent changes in sleep. (A) ΔSleep in short-term memory mutants, dnc1, rut2080, and controls. (B) ΔSleep (E⇒I) in w118/CJ-1 wild-type background strain, dCREB-a (memory activator) and dCREB-b (memory repressor) heat-inducible strains, and 17 of 43 long-term memory mutants that demonstrated disrupted experience-dependent changes in sleep. Underlined genes are not expressed in the mushroom bodies. *P < 0.001.

Finally, to assess the correlation between sleep and memory, male flies trained for a courtship conditioning task that generated long-term memories were measured for sleep after training. Males whose courtship attempts are thwarted by nonreceptive, recently mated females or by males expressing aphrodisiac pheromones form long-term associative memories as evidenced by subsequently reduced courtship of a receptive virgin female (23, 24). Trained males that formed long-term memories slept significantly more than their untrained siblings and wake controls (ones that were sleep-deprived while the experimental flies were being trained; Fig. 4, A to D). Exposure to a virgin female did not alter sleep need. As before, this increase in sleep was associated with longer daytime sleep bouts in trained individuals compared with controls (Fig. 4C). Further, sleep deprivation for 4 hours immediately after training abolished training-induced changes in sleep-bout duration (24 ± 4 min in trained versus 18 ± 3 min in naïve controls, P = 0.3617), as well as courtship memory (Fig. 4, A and E). Although these results are intriguing, invertebrate memory is particularly sensitive to extinction by mechanical perturbations. However, gentle handling that ensured wakefulness, but not mechanical stimulation, immediately following training, also abolished subsequent courtship memory (fig. S4). Furthermore, sleep deprivation per se did not affect the formation of long-term memory: Trained flies that were allowed to sleep unperturbed for 24 hours and then subjected to 4 hours of sleep deprivation retained courtship memory (Fig. 4, A and E).

Fig. 4.

Formation of associative memories is correlated with posttraining increases in sleep. (A) Schematic of experimental design. (B) Sleep following training for courtship conditioning in trained (T) and untrained (U) males. (C) Daytime sleep amount and bout duration. (D) ΔSleep in trained and untrained flies [ΔSleep (U⇒T)] compared with ΔSleep in untrained wake controls (WC) and unperturbed controls (C) [ΔSleep (WC⇒C)]. (E) Courtship index (ratio of the percentage of time spent courting to total time of exposure) in T and U flies, after training and following sleep deprivation (SD).

In summary, we demonstrate a rapid and dynamic relation between prior social experience and sleep need in a genetically tractable model organism, Drosophila melanogaster. In particular, we report that experience-dependent changes in sleep need require dopaminergic modulation, cAMP signaling, and a particular subset of long-term memory genes—supporting the hypothesis that sleep and neuronal activity may be inexorably intertwined. These observations are compelling given two recent studies (25, 26) demonstrating a central role of the mushroom bodies in sleep regulation and emphasize the importance of establishing Drosophila as a model system to investigate the molecular pathways underlying sleep and plasticity.

Supporting Online Material

www.sciencemag.org/cgi/content/full/313/5794/1775/DC1

Materials and Methods

Figs. S1 to S4

References and Notes

References and Notes

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