Sleep deprivation drives brain-wide changes in cholinergic pre-synapse 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 pre-synaptic 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 an flp-based genetic reporter to label pre-synaptic 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 pre-synapse abundance, across 37 neuropil regions of the central fly brain. Our results indicate that sleep loss, either by overnight (12h) 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 hours 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 the excitatory-inhibitory balance in the central brain. 

Methods
3-7 day old adult female flies were housed individually in 65mm borosilicate glass tubes (5mm diameter) containing fly food coated with paraffin wax on one end and a foam plug in the other. Locomotor activity was measured using Drosophila Activity Monitors from Trikinetics (Waltham MA, USA) and sleep was analyzed using Visual Basic macros in Microsoft Excel (1). Baseline sleep was monitored in all groups, and sleep deprivation was performed for 12 hours during the dark phase using mechanical stimulation via the SNAP method (1).

All specimens were imaged on a Zeiss 880 laser scanning confocal microscope using a 20x objective with an optical slice thickness of 0.98 μm. Matching image acquisition settings were used for each brain within individual experiments. Whole brain confocal stacks were registered to an adult brain template (JFRC2010) from Janelia Research Campus (2) by performing affine and warp transformations within the CMTK Registration plugin for Fiji (3-5). Registered images were excluded from further analysis when we observed visible distortion or misalignment with the template stack. Each experiment was replicated using two to three independent batches of flies. Any neuropil region was excluded from analysis for an individual brain when at least 30% of pixels within that region were not successfully registered.

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