Sleep deprivation drives brain-wide changes in cholinergic presynapse abundance in Drosophila melanogaster.

Sleep is an evolutionarily conserved state that supports brain functions, including synaptic plasticity, in species across the animal kingdom. Here, we examine the neuroanatomical and cell-type distribution of presynaptic scaling in the fly brain after sleep loss. We previously found that sleep loss drives accumulation of the active zone scaffolding protein Bruchpilot (BRP) within cholinergic Kenyon cells of the Drosophila melanogaster mushroom body (MB), but not in other classes of MB neurons. To test whether similar cell type-specific trends in plasticity occur broadly across the brain, we used a flp-based genetic reporter to label presynaptic BRP in cholinergic, dopaminergic, GABAergic, or glutamatergic neurons. We then collected whole-brain confocal image stacks of BRP intensity to systematically quantify BRP, a marker of presynapse abundance, across 37 neuropil regions of the central fly brain. Our results indicate that sleep loss, either by overnight (12-h) mechanical stimulation or chronic sleep disruption in insomniac mutants, broadly elevates cholinergic synapse abundance across the brain, while synapse abundance in neurons that produce other neurotransmitters undergoes weaker, if any, changes. Extending sleep deprivation to 24 h drives brain-wide upscaling in glutamatergic, but not other, synapses. Finally, overnight male-male social pairings induce increased BRP in excitatory synapses despite male-female pairings eliciting more waking activity, suggesting experience-specific plasticity. Within neurotransmitter class and waking context, BRP changes are similar across the 37 neuropil domains, indicating that similar synaptic scaling rules may apply across the brain during acute sleep loss and that sleep need may broadly alter excitatory-inhibitory balance in the central brain.

Examining Sleep Modulation by Drosophila Ellipsoid Body Neurons.

Recent work in Drosophila has uncovered several neighboring classes of sleep-regulatory neurons within the central complex. However, the logic of connectivity and network motifs remains limited by the incomplete examination of relevant cell types. Using a recent genetic-anatomic classification of ellipsoid body ring neurons, we conducted a thermogenetic screen in female flies to assess sleep/wake behavior and identified two wake-promoting drivers that label ER3d neurons and two sleep-promoting drivers that express in ER3m cells. We then used intersectional genetics to refine driver expression patterns. Activation of ER3d cells shortened sleep bouts, suggesting a key role in sleep maintenance. While sleep-promoting drivers from our mini-screen label overlapping ER3m neurons, intersectional strategies cannot rule out sleep regulatory roles for additional neurons in their expression patterns. Suppressing GABA synthesis in ER3m neurons prevents postinjury sleep, and GABAergic ER3d cells are required for thermogenetically induced wakefulness. Finally, we use an activity-dependent fluorescent reporter for putative synaptic contacts to embed these neurons within the known sleep-regulatory network. ER3m and ER3d neurons may receive connections from wake-active Helicon/ExR1 cells, and ER3m neurons likely inhibit ER3d neurons. Together, these data suggest a neural mechanism by which previously uncharacterized circuit elements stabilize sleep-wake states.

Bidirectional Regulation of Sleep and Synapse Pruning after Neural Injury.

Following acute neural injury, severed axons undergo programmed Wallerian degeneration over several following days. While sleep has been linked with synaptic reorganization under other conditions, the role of sleep in responses to neural injuries remains poorly understood. To study the relationship between sleep and neural injury responses, we examined Drosophila melanogaster following the removal of antennae or other sensory tissues. Daytime sleep is elevated after antennal or wing injury, but sleep returns to baseline levels within 24 h after injury. Similar increases in sleep are not observed when olfactory receptor neurons are silenced or when other sensory organs are severed, suggesting that increased sleep after injury is not attributed to sensory deprivation, nociception, or generalized inflammatory responses. Neuroprotective disruptions of the E3 ubiquitin ligase highwire and c-Jun N-terminal kinase basket in olfactory receptor neurons weaken the sleep-promoting effects of antennal injury, suggesting that post-injury sleep may be influenced by the clearance of damaged neurons. Finally, we show that pre-synaptic active zones are preferentially removed from severed axons within hours after injury and that depriving recently injured flies of sleep slows the removal of both active zones and damaged axons. These data support a bidirectional interaction between sleep and synapse pruning after antennal injury: locally increasing the need to clear neural debris is associated with increased sleep, which is required for efficient active zone removal after injury.