A Dynamic Deep Sleep Stage in Drosophila

Arousal thresholds

Transiently floored 11–40 Hz activity during sleep (Fig. 2C–E) suggested a deeper sleep stage, because the average LFP power is already lower during sleep than wake (Fig. 2A). To determine whether these dynamic sleep processes were behaviorally relevant, we investigated whether flies displayed different levels of behavioral responsiveness during sleep. To test and measure behavioral responsiveness efficiently in flies, we designed an apparatus to deliver brief (200 ms) mechanical stimuli of varying (randomized) intensities (0–1.2 g; see Materials and Methods) to groups of flies housed individually in small glass tubes (Fig. 3). Flies were filmed and their locomotor responses to the stimuli were tracked automatically (Fig. 3A). Startle responses increase with stimulus intensity in both males and females (Fig. 3B,C). By probing hourly for responses to robust vibration intensities (1.2 g), we were able to investigate how average behavioral responsiveness changed throughout several consecutive days and nights (Fig. 3D). As expected, responsiveness to the vibrations followed a robust day/night pattern, with decreased responsiveness at night and increased responsiveness during the day when flies were exposed to 12 h LD cycles (Fig. 3E–G). Flies kept in DD also displayed increased responsiveness during their subjective day (Fig. 3H), but flies kept in LL displayed an intermediate level of behavioral responsiveness that was not different between day and night (Fig. 3I). Our startle paradigm thus accurately probed fly arousal under the LD conditions that are traditionally used to study sleep in Drosophila.

Open in a separate window

Figure 3.

Stimulus-induced locomotion. A, Detection of a startle stimulus (brief vibrations, indicated by yellow flash; see Materials and Methods) increases fly locomotor activity (blue, baseline locomotion; red, startle locomotion). B, Startle responses (red) in wild-type males (± SEM) increase with g (n = 136). ***p < 0.001 by t test compared with mean baseline velocity (blue). C, Stimulus-induced locomotion, the difference between startle and baseline responses, is higher in wild-type males than females. ***p < 0.001 by t test of means. D, Sample experiment showing mean baseline velocity and startle responses to 1.2 g stimulus over 72 h for wild-type males (n = 17). E, Baseline and stimulus-induced velocity (± SEM) in response to hourly stimulation by 1.2 g, for 24 h (n = 136 male wild-type flies). A close correspondence was found between stimulus-induced velocity and the percentage of flies responding (black line, ± SEM; see Materials and Methods). F, Stimulus-induced locomotion (± SEM) over 24 h for wild-type males and females (n = 85 per group). G–I, Average daytime and nighttime stimulus-induced locomotion for wild-type males and females in 12 h LD cycles (n = 85) in DD after LD entrainment (n = 68), and in LL (n = 51). Night and day are subjective for DD and LL based on entrainment schedules. **p < 0.01; ***p < 0.001 by t test comparing means. Error bars show SEM for all graphs.

We therefore next examined arousal thresholds specifically in quiescent flies. By probing only nonmoving animals with increasing vibration intensities (Fig. 4A), we determined the minimal vibration intensity needed to evoke movement (arousal threshold; see Materials and Methods) in different individuals. By probing hourly over several days and nights, we then determined how arousal thresholds changed over 24 h (Fig. 4B). As expected, arousal thresholds were significantly lower during the day than at night in both males and females (p < 0.001, 1 df, H = 401.01, Kruskal–Wallis test for medians, n = 60, only male data are shown). Indeed, the arousal threshold profiles we found (Fig. 4B) closely resembled more conventional graphs plotting hourly sleep duration in Drosophila based on a 5 min inactivity threshold (Hendricks et al., 2000; Shaw et al., 2000; Huber et al., 2004; Andretic and Shaw, 2005; Fig. 8E), validating our approach as a reliable indicator of arousal state in flies (see also Materials and Methods, where we describe “BAs” per hour, which was found to be a reliable indicator of sleep intensity in flies, as shown in an earlier study: Huber et al., 2004).

Open in a separate window

Figure 4.

Different levels of sleep intensity in quiescent flies. A, Increasing g levels were used to determine the minimal level required to induce locomotion in an inactive fly. B, Arousal thresholds (boxplots showing median g, black circles) ± 25–75th percentiles (gray boxes) and min/max values (whiskers) for wild-type males (n = 60) across 24 h. NR indicates nonresponding. C, D, Normalized response probability (fraction response) in wild-type males (C, n = 85) and females (D, n = 85) is plotted against minutes of inactivity before the stimulus (see Materials and Methods). A 1.2 g vibration stimulus was used to probe responsiveness every hour over several consecutive days. Gray circles indicate mean daytime responsiveness; black circles, mean nighttime responsiveness (± SEM). Gray and white shading indicate 5 min inactivity bins. E, F, Data from C and D were binned into 5 min epochs. Mean responsiveness levels (± SEM) were compared for different immobility durations within day (gray) or night (black) sleep bouts. *Bins that differed significantly (p < 0.05) from the first (1–5 min) bin by Tukey post hoc comparison.

Open in a separate window

Figure 8.

Effect of socialization on fly sleep intensity. A, B, Socialization increases deep sleep response probability (RP; ± SEM) in males during the day (blue, n = 51) compared with males kept in isolation (red, n = 51). **p < 0.05 by Wilcoxon rank-sum test. The vertical lines indicate the point of deepest sleep, from which deep sleep RP was calculated (see Materials and Methods). C, D, Socialization does not significantly change sleep intensity (deep sleep RP ± SEM) in females, day or night (blue, n = 51), compared with females kept in isolation (red, n = 51). E, Activity was measured using the DAMS (see Materials and Methods). Rest is defined as 5 min or longer without any detected activity in the DAMS, cumulative for every hour (± SEM, n = 32 flies per sex or condition). Socially reared adult flies (blue traces) are compared with 5- to 6-d-old flies that were isolated in plastic vials soon after eclosion (red traces) under the same conditions as in A–D. Flies are wild-type CS. F, Daytime and total sleep duration (± SEM) is decreased in both male and female flies that have been raised in isolation (red; socially reared flies are blue) and tracked by the DAMS system. Data are processed from flies in E. ***p < 0.001; **p < 0.01 by Wilcoxon rank-sum test.

Sleep intensity

Sleep duration and sleep intensity are not necessarily correlated, as suggested by our brain-recording experiments. To investigate sleep intensity in our assay, we applied our arousal-testing paradigm to examine responsiveness levels during extended bouts of inactivity (as determined by video tracking) during the day or night. We measured behavioral responsiveness to a robust vibration stimulus (1.2 g) every hour for 3 d, and then determined whether the duration of prior immobility (only inactive flies were analyzed) predicted responsiveness probability. We compiled all ranges of inactivity from 1 to 59 min for day versus night (see Materials and Methods). We confirmed that nighttime sleep was on average deeper than daytime sleep, but also found that responsiveness probability varied depending on the time elapsed (Fig. 4C,D). We confirmed that wild-type flies were significantly less responsive after 5 min of immobility (Shaw et al., 2000; Huber et al., 2004) and this was true in both males and females during the day and the night (Fig. 4E,F), but also that flies had already become significantly less responsive than moving flies after only 1 min of inactivity (p < 0.01 for males and females, day or night). Responsiveness decreased gradually in wild-type flies (i.e., sleep intensity increased) until a deeper sleep stage was achieved after ∼10 min of immobility, for daytime or nighttime sleep in males and females (Fig. 4C–F). The timing of this deeper sleep stage is consistent with an early observation of a distinct postural change occurring at 14 min in a similar preparation (Hendricks et al., 2000). Interestingly, after this first period of deepest sleep intensity, responsiveness levels then appear to change again: sleep became lighter at ∼20 min and a second deep sleep stage was evident at ∼30 min in males and females, day or night, ending in comparatively lighter sleep as the quiescent hour proceeded (Fig. 4C–F). Indeed, analysis of extended sleep bouts during the day revealed that male flies that had been quiescent for 45–60 min were as highly responsive as flies that had been quiescent for <5 min (Fig. 4E). Sleep intensity in flies thus depends on how long they have been asleep, but this relationship is nonlinear, with some evidence of what appears to be cycling behavior in arousability. Responsiveness differed significantly across the twelve 5 min bins (one-way ANOVA, males, day: F_(11,46) = 15.2, p < 0.001; males, night: F_(11,46) = 23.16, p < 0.001; females, day: F_(11,46) = 23.59, p < 0.001; females, night: F_(11,46) = 16.49, p < 0.001).

Investigating sleep intensity stages by awakening animals with a stimulus, as we have done here, presents a fundamental conundrum: individual animals can only be awakened once during a sleep bout, after which they are obviously no longer asleep. So how can we investigate sleep dynamics in flies behaviorally? Our population assay suggests that, on average, flies are more deeply asleep after 10 and 30 min of immobility compared with other times, especially females (Fig. 4C,D). Although electrophysiology supported the view that sleep intensity levels may be dynamic throughout a sleep bout (Fig. 1, Fig. 2), it is nevertheless difficult to demonstrate behaviorally that this is happening in individual animals. To get around this problem, we investigated whether different groups of flies may be contributing disproportionally to different parts of the sleep intensity curves that we uncovered (Fig. 4C,D). In that experiment, we probed hourly for responsiveness and, for all animals that were immobile at the time of probing, we determined how long they had been immobile, thereby assigning them to an inactivity bin between 1 and 59 min. We found that responsiveness levels were normally distributed among all inactive flies tested (Lilliefors test for normality, p < 0.05; Fig. 5A,B). Plotting the contributions of each fly to different inactivity bins also revealed these to be evenly distributed across individuals (Fig. 5C,D). For example, any fly that had provided responsiveness data for 20–40 min inactivity bins also provided responsiveness data for 0–20 min inactivity bins (i.e., these were not different groups of flies). However, it is clear that all flies contributed less to 20–40 min inactivity bins compared with 0–20 min and 40–60 min inactivity bins (Fig. 5E,F). One explanation for this distribution of the data could be that sleep intensity levels were indeed cycling within an extended sleep bout: there may have been fewer 20–40 min events because flies that have slept through the end of their first sleep cycle will most likely initiate the next sleep cycle and therefore not wake up during the deeper stages (i.e., centered around 30 min) of their second sleep cycle. Most flies, however, would naturally awaken after 20 min, which would be consistent with our averaged data showing that sleeping flies are comparatively more responsive at that time (Fig. 4C,D) and with the average sleep bout length being ∼20–30 min in our wild-type strain (see Materials and Methods). We therefore focused on the first 20 min of sleep, which includes the first deep sleep stage, for the remainder of this study.

Open in a separate window

Figure 5.

Individual fly sleep data distribution. A, B, Distribution of average responsiveness levels (response probability) for wild-type male (n = 85) and female (n = 85) flies discussed in Figure 4C, D sorted from lowest to highest responders (only flies that were inactive before the startle test are represented). Responsiveness is normally distributed among flies (Lilliefors test, p < 0.05), indicating that the different sleep intensity levels after sleep onset (Fig. 4C,D) are unlikely to reflect subpopulations of flies behaving differently. C, D, Distribution of inactivity data for each fly (same flies as in A,B). The blue dots show, for each fly, whether it contributed to each inactivity bin (0–59 min). (E, F) By summing data from C and D, we determined the proportion (fraction) of flies contributing to each bin. Fewer flies contribute to middle-length inactivity bins (20–40 min), although data within this epoch are evenly distributed among flies.

To better identify the first deep sleep stage, we fit a third-order polynomial to smoothed responsiveness data (see Materials and Methods), with the first local minimum in the curve indicating the time of deepest sleep (Fig. 6A,B, vertical dashed lines). The first deep sleep stage was found to occur between 12 and 15 min of inactivity in wild-type flies regardless of time of day or sex, which suggests strongly regulated processes controlling sleep intensity in Drosophila. When we tested for responsiveness every 30 min instead of every hour in a replicate set of experiments, the first deep sleep stage seemed earlier and deeper, especially in males (Fig. 6C,D), suggesting that a 30 min testing regime may be increasing sleep drive (i.e., producing some sleep deprivation). Similarly, our brain-recording paradigm yielded a comparatively earlier onset of lower 10–40 Hz activity (Fig. 2E), suggesting that the tethered recording setup may have caused some sleep deprivation. We therefore focused on an hourly sleep probe in subsequent experiments.

Open in a separate window

Figure 6.

The first deep-sleep stage. A, B, To quantify deep sleep intensity and timing, third-order polynomials were fitted to smoothed mean responsiveness data from Fig. 4C, D: A, males; B, females, n = 85 in both groups) for the first 20 min of inactivity (see Materials and Methods). The local minimum in the curve indicates the first deep sleep stage (vertical dashed lines at ∼12–15 min). Curves show polynomials fitted to means ± SEM. A, Day, r² = 0.97; night, r² = 0.98. B, Day, r² = 0.96; night, r² = 0.98. C, D, A 1.2 g vibration stimulus was used to probe responsiveness every 30 min over three consecutive days in separate group of 85 wild-type males and females. Gray circles indicate mean daytime responsiveness; black circles, mean nighttime responsiveness (± SEM).

Behavioral effects on sleep intensity

We next examined the effect of sleep drive on sleep intensity dynamics. In humans, SWS duration and intensity increase proportionally to prior wake time and decrease as sleep is restored throughout the night (Borbély and Achermann, 1999), evidence that sleep is a homeostatic process. Some of our data already suggest a homeostatic process: quiescent male flies were less responsive in the first hours of the night compared with equally quiescent flies in the last hours of the night (Fig. 4B). As in humans, extended wakefulness in Drosophila males and females results in increased sleep drive (pressure to sleep) (Hendricks et al., 2000; Shaw et al., 2000). To determine more directly whether increased wakefulness alters sleep intensity profiles, we sleep deprived flies for 24 h (see Materials and Methods) and measured sleep intensity for the following day or night. We found that 24 h of sleep deprivation precipitated the onset of deep sleep in males and females (Fig. 7A,B, vertical dashed lines). To quantify sleep intensity at this time, we averaged the (non-smoothed) data for a 5 min window around the point of deepest sleep (Fig. 7C), termed the deep sleep response probability. Deep sleep response probability was significantly lower in sleep-deprived flies, but only for nighttime sleep (Fig. 7D). Importantly, sleep intensity returned to comparatively lighter levels after a full day of recovery for both day and night in males (Fig. 7E, one-way ANOVA F_(1,135) = 5.42, p = 0.02; and F_(1,135) = 11.78, p < 0.001, respectively). Female recovery from sleep deprivation was less clear; only daytime responsiveness appeared to recover on the second day (Fig. 7F, one-way ANOVA F_(1,135) = 11.14, p = 0.001).

Open in a separate window

Figure 7.

Effect of sleep deprivation on the first deep sleep stage. A, B, After 24 h of sleep deprivation (SD), sleep initiation occurs faster and sleep is deeper when recovery sleep occurs during the day or the night (A, males; B, females, n = 68 in both groups). A, Day, r² = 0.92; night, r² = 0.84. B, Day, r² = 0.96; night, r² = 0.89. C, Schema showing how deepest sleep timing is identified, with corresponding response probability (RP) at that time. Deep sleep RP was determined by averaging (non-smoothed) responsiveness data for a 5 min epoch centered around the deepest sleep time. D, Deep sleep RP averages (± SEM) for data from A and B comparing daytime and nighttime sleep intensity and SD and non-SD controls. *p < 0.05 by Wilcoxon rank-sum test. E, Average responsiveness during day and night for three consecutive days after 24 h of sleep deprivation in wild-type males (n = 68). A significant increase in responsiveness can be observed between days 1 and 2 after sleep deprivation, suggesting that decreased responsiveness on day 1 was due to deeper rebound sleep and not to damage caused by the sleep deprivation procedure. *p < 0.05, F_(1,135) = 5.42; ***p < 0.001. F, Average responsiveness during day and night for three consecutive days after 24 h of sleep deprivation in wild-type females (n = 68). **p < 0.01.

Other behavioral manipulations predicted to increase sleep intensity are socialization and learning. The synaptic homeostasis hypothesis for sleep (Tononi and Cirelli, 2006) describes an interaction between wake experience and sleep drive: information encoded during wake leads to an overall increase in synaptic strength in the brain, which would then be proportionally normalized (i.e., downscaled) during the deeper stages of sleep. Because socialization has been proposed as one way to increase sleep drive in Drosophila (Ganguly-Fitzgerald et al., 2006), we tested the effect of this simple manipulation on sleep intensity in our arousal paradigm. Socialized male flies (males kept with other males for 4 d; see Materials and Methods) slept more deeply subsequently during the day compared with similarly handled male flies kept in isolation (Fig. 8A,B). In contrast, sleep intensity in female flies was not affected by intrasexual socialization (Fig. 8C,D), even though sleep duration was altered (Fig. 8E,F), as measured by more traditional (i.e., infrared beam crossing) sleep metrics. This result highlights an important point: longer sleep duration does not necessarily mean that the flies were sleeping more deeply. The dimorphism in sleep intensity between males and females may be due to different behavioral interactions among males or females, with more complex male social interactions such as aggression and courtship producing a greater sleep drive during the day. These social interactions may, for example, engage learning and memory pathways that affect sleep intensity in males.

Optogenetic manipulation of sleep intensity

To investigate a possible link between learning and sleep intensity in flies (Seugnet et al., 2008), we manipulated acutely a molecular pathway that has been associated with synaptic plasticity and memory formation. Learning and memory processes are closely tied to cyclic adenosine monophosphate (cAMP) signaling pathways in neurons, where increased cAMP activity results in increased synaptic efficacy (Huang et al., 1994) and in the transcription of genes involved in upregulating synaptic strength (Dash et al., 1990). Fly memory mutants defective in cAMP regulation, such as dunce¹ (which has chronically increased cAMP levels), sleep less (Hendricks et al., 2001) and do not show experience-dependent changes in sleep duration (Ganguly-Fitzgerald et al., 2006). We therefore sought to specifically increase cAMP activity only during the day, when most learning behavior presumably occurs, to determine whether acute molecular control of plasticity mechanisms in awake flies altered sleep intensity. To best achieve this, we used an optogenetic approach: a transgenic adenylyl cyclase that is activated by blue light, PACα (Schröder-Lang et al., 2007), under control of the UAS/Gal4 expression system (Brand and Perrimon, 1993). Under blue light, PACα converts ATP into cAMP in Gal4-specified neurons (Fig. 9A), initiating a signaling cascade that modulates synaptic function (Huang et al., 1994; Schröder-Lang et al., 2007). We found that acute, pan-neuronal activation of PACα (in elav-Gal4/UAS-PACα flies) significantly increased arousal thresholds (decreased responsiveness) to levels similar to that in dunce¹ mutants (Fig. 9B, p < 0.01, Kruskal–Wallis test comparing medians, 3 df, H = 12.63). When adenylyl cyclase activation was continued for the flies' entire day (but turned off at night), elav-Gal4/UAS-PACα flies displayed chronically decreased responsiveness to mechanical stimuli during both day and night compared with controls also exposed to blue light (Fig. 9C). However, sleep intensity was only significantly increased at night, after blue light was turned off, compared with both genetic controls (Fig. 9D–F). This suggests that cAMP-related signaling that accrues during the day has long-lasting effects on sleep intensity at night.

Open in a separate window

Figure 9.

Adenylyl cyclase activity increases nighttime sleep intensity. A, cAMP synthesis and degradation pathway. A transgenic adenylyl cyclase (PACα) is activated by blue light, resulting in increased levels of cAMP and consequent plasticity signaling mechanisms (PKA and CREB). Increased cAMP levels are also achieved by the phosphodiesterase gene mutation, dunce¹. B, Acute blue light activation of PACα increases arousal thresholds to levels similar to dunce¹ mutants. Arousal thresholds (the minimal vibration, g, required to arouse a quiescent fly; see Materials and Methods) for wild-type (+, n = 42), UAS-PACα/+ (n = 36), elav-Gal4/+ (n = 34), UAS-PACα/elav-Gal4 (n = 37), and dunce¹ (n = 71). White boxplots indicate white light exposure; blue boxplots, 10 min blue light exposure. **p < 0.01 by Kruskal-Wallis comparison of medians (red) adjusted for multiple comparisons. C, Baseline and stimulus-induced velocity (± SEM) for UAS-PACα/+ control male flies and UAS-PACα/elav-Gal4 male flies in response to hourly stimulation by 1.2 g for 72 h averaged over a 24 h period. Blue light (458 ± 10 nm) was turned on throughout the day and turned off at night for both. D, E, Acute activation of PACα with blue light during the day increases sleep intensity at night in males. The sleep intensity profiles for UAS-PACα/+ (gray, n = 85), elav-Gal4/+ (black, n = 85), and the blue-light-activated UAS-PACα/elav-Gal4 (blue, n = 85) is shown (± SEM). Third-order polynomials were fitted to normalized, smoothed mean responsiveness data for the first 20 min of inactivity for daytime or nighttime sleep. Day: UAS-PACα/+, r² = 0.85; elav-Gal4/+, r² = 0.84; UAS-PACα/elav-Gal4, r² = 0.97. Night: UAS-PACα/+, r² = 0.88; elav-Gal4/+, r² = 0.77; UAS-PACα/elav-Gal4, r² = 0.94. F, Deep sleep response probability (RP) data and statistics. *p < 0.05 by Wilcoxon rank-sum test.