Introduction and aims
Sleep pressure, the process variable in sleep homeostasis, currently lacks a physical interpretation. Although prolonged waking is associated with numerous changes in the brain—of neuronal firing patterns, the strengths of synaptic connections, metabolite concentrations, and metabolic and gene expression programs —, it remains generally indeterminable whether these changes are causes or consequences of a growing need for sleep. Perhaps the only realistic opportunity for separating causation from correlation exists in specialist neurons with active roles in the induction and maintenance of sleep; in these cells, sleep’s proximate (and maybe also its ultimate) causes must interlock directly with the processes that regulate spiking.
Results and methods
To obtain a comprehensive, unbiased view of molecular changes in the brain that may underpin these processes, we have characterized the transcriptomes of single cells isolated from rested and sleep-deprived Drosophila melanogaster flies. Transcripts upregulated after sleep deprivation, in sleep-control neurons projecting to the dorsal fan-shaped body (dFBNs) but not ubiquitously in the brain, encode almost exclusively proteins with roles in mitochondrial respiration and ATP synthesis (Figure 1). These gene expression changes are accompanied by mitochondrial fragmentation, enhanced mitophagy, and an increase in the number of contacts between mitochondria and the endoplasmic reticulum, creating conduits for the replenishment of peroxidized lipids (Figure 3). The morphological changes are reversible after recovery sleep and blunted by the installation of an electron overflow in the respiratory chain (Figure 3). Inducing or preventing mitochondrial fission or fusion in dFBNs alters sleep and the electrical properties of sleep-control cells in opposite directions: hyperfused mitochondria increase, whereas fragmented mitochondria decrease, neuronal excitability and sleep (Figure 4). ATP levels in dFBNs rise after enforced waking because of diminished ATP consumption during the arousal-mediated inhibition of these neurons, which predisposes them to heightened oxidative stress (Figure 2). Consistent with this view, uncoupling electron flux from ATP synthesis relieves the pressure to sleep, while exacerbating mismatches between electron supply and ATP demand (by powering ATP synthesis with a light-driven proton pump) promotes sleep (Figure 2).
Conclusions
Sleep pressure control has thus mitochondrial origins, and conditions that allow or prevent electrons flowing through the respiratory chain of its feedback control neurons determine its accumulation. Parallels with hunger-control neurons in the mammalian hypothalamus suggest similar mechanisms have been adopted for the control of both such metabolism-directed behaviours. Sleep, like ageing, may thus be an inescapable consequence of aerobic metabolism.
Ethical standards, statistical and methodological details
All the work presented here has been conducted with Drosophila melanogaster flies, for which ethical approval is not needed.
The investigators were blind to sleep history and/or genotype in imaging experiments but not otherwise. Sample sizes in behavioral experiments (typically n=32 flies per genotype) were chosen to detect 2-h differences in daily sleep with a power of 0.9. All behavioral experiments were run at least three times, on different days and with different batches of flies.
For further statistical and methodological details, see our preprint: Sarnataro R, et al., 2024 Mitochondrial origins of the pressure to sleep. bioRxiv, 2024.02.23.581770; doi: https://doi.org/10.1101/2024.02.23.581770