Widespread changes in synaptic markers as a function of sleep and wakefulness in Drosophila

Abstract

Sleep is universal, strictly regulated, and necessary for cognition. Why this is so remains a mystery, although recent work suggests that sleep, memory, and plasticity are linked. However, little is known about how wakefulness and sleep affect synapses. Using Western blots and confocal microscopy in Drosophila, we found that protein levels of key components of central synapses were high after waking and low after sleep. These changes were related to behavioral state rather than time of day and occurred in all major areas of the Drosophila brain. The decrease of synaptic markers during sleep was progressive, and sleep was necessary for their decline. Thus, sleep may be involved in maintaining synaptic homeostasis altered by waking activities.

Fig. 1. Levels of synaptic proteins are high after sleep deprivation and low after sleep

(A) Presynaptic (pre), postsynaptic (post), Bruchpilot (BRP), Cysteine string protein (CSP), Discs-large (DLG), Synapsin (Syn), Syntaxin (Syx). (B) Daily pattern of sleep in Canton-S males sleep deprived (SD) for the last 6h of the night (green), 12h at night (red), or 24h (yellow) and control siblings left undisturbed (no SD, black line). Arrow shows when flies were collected. White and black bars: light and dark periods. Each group includes 12–16 flies and represents one of the 2–3 experiments used for panels C–H. (C–H) Representative immunoblots (E) and gel quantification (mean ± standard deviation; n of flies below each bar). SD values (color-coded as in B) expressed as % change relative to sleep (= 0%). (I–K) Canton-S females. *, p < 0.05; **, p < 0.01 (one-way ANOVA followed by Tukey’s HSD Post Hoc test).

Fig. 2. The expression of synaptic proteins increases due to sleep loss

(A–C, left) SD, sleep deprivation; S, sleep; W, spontaneous waking. Vertical arrows show when flies were collected. (A, right) Correlation between BRP levels and sleep deprivation efficiency during the last 24h in Canton-S males (Pearson correlation). Each dot represents 4 flies with similar SD efficiency. (B–C, right) Levels of synaptic proteins after SD or W, expressed as % change relative to sleep (= 0%). (mean ± standard deviation; n of flies below each bar). *, p < 0.05; **, p < 0.01 (Student's t-test).

Fig. 3. Synaptic markers decrease during sleep

(A–B, left) Rec, recovery sleep after SD. Vertical arrows show when flies were collected. (A, right) BRP levels expressed as % change relative to sleep at the end of the night (= 100%). (B, right) Levels of synaptic proteins after recovery sleep, expressed as % change relative to SD (=100%; mean ± standard deviation; n of flies is below each bar). *, p < 0.05 (Student's t-test).

Fig. 4. Widespread BRP increase after sleep loss

Representative examples of BRP immunofluorescence (IF) in controls and flies sleep deprived for 16h ending at light onset (sum of selected optical stacks, false colored using a quantitative scale). Immunoreactivity levels were measured in antennal lobes (AL), beta lobes of the mushroom bodies (MB), ellipsoid body of the central complex (CC) and central cerebrum (excluding the optic lobes, CB).