Visual experience drives sleep need in Drosophila

Abstract

Sleep optimizes waking behavior, however, waking experience may also influence sleep. We used the fruit fly Drosophila melanogaster to investigate the relationship between visual experience and sleep in wild-type and mutant flies. We found that the classical visual mutant, optomotor-blind (omb), which has undeveloped horizontal system/vertical system (HS/VS) motion-processing cells and are defective in motion and visual salience perception, showed dramatically reduced and less consolidated sleep compared to wild-type flies. In contrast, optogenetic activation of the HS/VS motion-processing neurons in wild-type flies led to an increase in sleep following the activation, suggesting an increase in sleep pressure. Surprisingly, exposing wild-type flies to repetitive motion stimuli for extended periods did not increase sleep pressure. However, we observed that exposing flies to more complex image sequences from a movie led to more consolidated sleep, particularly when images were randomly shuffled through time. Our results suggest that specific forms of visual experience that involve motion circuits and complex, nonrepetitive imagery, drive sleep need in Drosophila.

Keywords: Drosophila; optogenetics; sleep; visual behaviour.

Figure 1.

Optomotor-blind mutants have reduced and fragmented sleep. (A) Sleep across 24 hours in omb^H31 and wild-type (CS) flies. (B) Total sleep duration during the day and the night. (C) Average walking speed during wakefulness. (D) Scatterplots depicting day sleep consolidation (left panel) and night sleep consolidation (right panel), where consolidated sleep is indicated by high bout duration and low bout number, whereas fragmented sleep is indicated by low bout duration and high bout number. Averaged data points for each condition are indicated by larger circles with black outline. (E) Total sleep averaged across 24 hours in wild type, omb^H31, omb^H31/d(1)rb13B, and omb^H31/d(1)rb5 flies. n = 60 flies per group for all panels. ***p < 0.001, by t tests in (B and C) and one-way analysis of variance with Tukey’s multiple comparisons in (E). Error bars show the SEM.

Figure 2.

Visual responses to motion and objects in wild-type and optomotor-blind flies are modulated differently by temporal frequency. (A) HS and VS neurons reside in the lobula plate of the fly optic lobe, and detect horizontal and vertical motion, respectively (left panel). Example images of a normal VS cell in a wild-type fly, and an undeveloped VS cell in an omb^H31 mutant (right panel). Scale bar = 25 µm. (B and C) Example responses of wild-type and omb^H31 flies to temporal frequencies of motion (B) and object flicker (C). (D and E) Quantification of optomotor responses (D) and object fixation behavior (E) in wild-type and omb^H31 mutants. (F) Optomotor responses to 3 Hz (left panel) or 16 Hz (right panel) moving gratings in wild type, omb^H31, omb^H31/d(1)rb13B, and omb^H31/d(1)rb5 flies. (G) Object fixation for stationary objects (left panel) or 3 Hz flickering objects (right panel) in wild type, omb^H31, omb^H31/d(1)rb13B, and omb^H31/d(1)rb5 flies. *p < 0.05, t tests between wild-type and omb^H31 mutant at each temporal frequency in (D and E) and ***p < 0.001, ****p < 0.0001, one-way analysis of variance with Tukey’s multiple comparisons in (F and G). n = 10 flies per condition in (D and E) and 10 flies per group in (F and G). Error bars show the SEM.

Figure 3.

Activation of motion circuits increases nighttime sleep. (A) Flies expressing a red-light-activated channelrhodopsin (Chrimson) were placed in the recording set up and sleep was analyzed under baseline conditions on day 1 (normal white light from 8 am to 8 pm, followed by 12 hours darkness at night), an activated condition on day 2 (normal white light + 12 hours red light illumination from 8 am to 8 pm, followed by 12 hours darkness at night), and recovery conditions (same as baseline). (B and C) Total sleep duration was unchanged in UAS-Chrimson/+ (genetic control) or in GMR-Gal4/+>UAS-Chrimson/+ (flies expressing Chrimson in photoreceptors). (D–F) Upper panel: whole-mount brain immunostaining of three motion circuits: 3A-Gal4>UAS-GFP (HS and VS neurons), R27B03-Gal4>UAS-GFP (HS cells), and R79D04/+>UAS-GFP (T5 neurons). Brains were immunostained with anti-GFP (green) and anti-Bruchpilot (BRP, nc82, magenta). Scale bar = 100 µm. Lower panel: total daytime and nighttime sleep for flies expressing Chrimson in the aforementioned circuits, under baseline, activated and recovery conditions. n =31 flies in (B), 33 flies in (C), 33 flies in (D), 30 flies in (E), and 47 flies in (F). Error bars indicate the SEM. *p < 0.05, **p < 0.01, one-way analysis of variance with Tukey’s multiple comparisons.

Figure 4.

Activation of T5 motion-detection neurons consolidates nighttime sleep. (A) Flies expressing a red-light-activated channelrhodopsin (Chrimson) were placed in the recording set up and sleep was analyzed under baseline conditions on day 1 (normal white light from 8 am to 8 pm, followed by 12 hours darkness at night), an activated condition on day 2 (normal white light + 12 hours red light illumination from 8 am to 8 pm, followed by 12 hours darkness at night), and recovery conditions (same as baseline). Analyses in this figure are from the same dataset as in Figure 3. (B–F) Sleep bout duration during the day and night across all three conditions for the UAS-Chrimson/+ control (B) and red-light-activated optic lobe circuits (C–F). (G) Summary graph showing sleep consolidation for the aforementioned strains, viewed as a change in sleep bout number/duration following red-light activation (night 2), normalized to baseline sleep conditions (night 1). Activation of T5 neurons (cyan) led to consolidation of nighttime sleep as observed by an increase in sleep bout duration and decrease in sleep bout number. n = 31 flies in (B), 33 flies in (C), 33 flies in (D), 30 flies in (E), and 47 flies in (F). Error bars indicate the SEM. **p < 0.01, ***p < 0.001, one-way analysis of variance with Tukey’s multiple comparisons.

Figure 5.

Simple visual stimuli have little effect on sleep consolidation in CS flies. (A) Visual stimuli were presented on LED panels while sleep was recorded with a webcam to measure fly locomotion in tubes. Three different visual stimuli were presented to flies: control stimulus (constant blue light), 3 Hz (slow moving) gratings, and 16 Hz (fast moving) gratings. The order of the movies was shuffled across three independent experiments. Visual stimuli were presented during the day (8 am–8 pm) across three consecutive days, and sleep was analyzed across the day and night. (B) Total sleep duration for flies exposed to the different visual stimuli during the day (left panel) and following visual stimulation, at night (right panel). (C and D) Scatterplots showing sleep consolidation for wild-type flies during the day (C) and night (D). Averaged data points for each condition are indicated by larger circles with black outline. (E–H) Quantification of sleep bout number for the day (E) and night (F), as well as sleep bout duration for the day (G) and night (H). n > 70 flies, three experiments. *p < 0.05, one-way analysis of variance with Tukey’s multiple comparisons. Error bars show the SEM.

Figure 6.

Complex image sequences drive sleep consolidation. (A) Visual scenes from the movie “Terminator” were presented to flies in three different formats: normal movie, spatial shuffle (pixel positions were randomly shuffled), and temporal shuffle (movie frames were randomly shuffled through time). The order of the movies was alternated across three independent experiments. (B) Total sleep duration for day time sleep (during the movies, left panel) and nighttime sleep (following movies, right panel). (C and D) Scatterplots depicting day time sleep consolidation (C) and nighttime sleep consolidation (D), where consolidated sleep is indicated by high bout duration and low bout number, whereas fragmented sleep is indicated by low bout duration and high bout number. Averaged data points for each condition are indicated by larger circles with black outline. (E–H) Sleep bout number during the day (E) and night (F) and sleep bout duration during the day (G) and night (H) for the flies that observed the different movies. *p < 0.05, **p < 0.01, one-way analysis of variance with Tukey’s multiple comparisons. n = 150 flies, three experiments.