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 stimulati..., 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 we..., , # Sleep deprivation drives brain-wide changes in cholinergic pre-synapse abundance in *Drosophila melanogaster*

[https://doi.org/10.5061/dryad.q573n5tqv](https://doi.org/10.5061/dryad.q573n5tqv)

Image segmentation and analysis code for fly brain images registered to JFRC2010 anatomical template (1). Python scripts were prepared in Jupyter Notebooks 6.4.3. Example confocal stacks imaged on Zeiss LSM 880, then registered to JFRC2010 template using affine and warp transformations in the CMTK Registration Template for FIJI (2-4).

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