Sleep deprivation drives brain-wide changes in cholinergic pre-synapse abundance in Drosophila melanogaster
Data files
Mar 11, 2024 version files 2.41 GB
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general_files_for_setup.zip
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README.md
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sample_images.zip
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sample_outputted_data.zip
Abstract
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.
README: Sleep deprivation drives brain-wide changes in cholinergic pre-synapse abundance in Drosophila melanogaster
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).
- A. Jenett, et al., A GAL4-driver line resource for Drosophila neurobiology. Cell Reports 2, 209 991–1001 (2012).
- A. Ostrovsky, S. Cachero, G. S. X. E. Jefferis, Clonal Analysis of Olfaction in Drosophila: Image Registration. Cold Spring Harb Protoc 2013, pdb.prot071738-pdb.prot071738 (2013).
- S. Cachero, A. D. Ostrovsky, J. Y. Yu, B. J. Dickson, G. S. X. E. Jefferis, Sexual Dimorphism in the Fly Brain. Curr Biol 20, 1589–1601 (2010).
- J. Schindelin, et al., Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676–682 (2012).
Description of the data and file structure
Sample Images.zip contains experimental data from one cohort of registered images from Chat-2A-Gal4>STaR flies that were either allowed ad libitum sleep (con) or 12h of overnight sleep deprivation (SD).
Sample outputted data.zip contains files produced by using our Python scripts to analyze the image data contained within Sample Images.zip. Output files include:
1) Individual pixel intensity histograms:
- histobins_con_051923.csv - relative pixel fluorescence intensity for bins used to generate a histogram for control brains ( values normalized to mean pixel intensity from the control group)
- histobins_sd_051923.csv - relative pixel fluorescence intensity for bins used to generate a histogram for SD brains (values normalized to mean pixel intensity from the control group)
- histovalues_con_051923.csv - distribution of relative pixel intensities within bin sizes from "histobins_con_051923.csv"
- histovalues_sd_051923.csv - distribution of relative pixel intensities within bin sizes from "histobins_sd_051923.csv"
- histogram_sd_vs_con_normalizedScalar_051923.jpg - image file plotting histograms of normalized pixel intensities using values in files listed above
2) Neuropil region mean values and variances:
- sample_region_means_051923.csv - mean normalized pixel intensity values for each brain (rows) across local neuropil regions (columns). The second column denotes the experimental group (SD or con) for each brain. All values are normalized to the mean pixel intensity from the control group.
- sample_region_vars_051923.csv - variance values for each brain (rows) across local neuropil regions (columns). The second column denotes the experimental group (SD or con) for each brain.
- sample_con_region_means_051923.csv - mean normalized pixel intensity values for each brain (rows) across local neuropil regions (columns) for samples from the rested control group (con). All values are normalized to the mean pixel intensity from the control group.
- sample_con_region_vars_051923.csv - variance values for each brain (rows) across local neuropil regions (columns) for samples from the rested control group (con).
- sample_SD_region_means_051923.csv - mean normalized pixel intensity values for each brain (rows) across local neuropil regions (columns) for samples from the SD group. All values are normalized to the mean pixel intensity from the control group.
- sample_SD_region_vars_051923.csv - variance values for each brain (rows) across local neuropil regions (columns) for samples from the SD group.
- sample_batchnormalized_combinedbilaterals_region_means_con_051923.csv - mean normalized pixel intensity values for each brain (rows) across local neuropil regions (columns) for samples from the rested control group (con). In this file, neuropil regions with bilateral structures (L and R) are pooled into a single value. All values are normalized to the mean pixel intensity from the control group.
- sample_batchnormalized_combinedbilaterals_region_means_SD_051923.csv - mean normalized pixel intensity values for each brain (rows) across local neuropil regions (columns) for samples from the SD group. In this file, neuropil regions with bilateral structures (L and R) are pooled into a single value. All values are normalized to the mean pixel intensity from the control group.
- sample_batchnormalized_combinedbilaterals_region_vars_con_051923.csv - variance values for each brain (rows) across local neuropil regions (columns) for samples from the rested control group (con). In this file, neuropil regions with bilateral structures (L and R) are pooled into a single value. All values are normalized to the mean pixel intensity from the control group.
- sample_batchnormalized_combinedbilaterals_region_vars_SD_051923.csv - variance values for each brain (rows) across local neuropil regions (columns) for samples from rested control group (con). In this file, neuropil regions with bilateral structures (L and R) are pooled into a single value. All values are normalized to the mean pixel intensity from the SD group.
3) Matrix files
- justcon_filled_051923.npy - Matrix file compiling individual pixel intensity values for each sample in the rested control group (con).
- justsd_filled_051923.npy - Matrix file compiling individual pixel intensity values for each sample in the rested control group (SD).
- justcon_filled_normalizedScalar_051923.npy - Matrix compiling individual pixel intensity values for each sample in the rested control group (con). Values are normalized to the overall mean pixel intensity value from the con group samples.
- justsd_filled_normalizedScalar_051923.npy - Matrix compiling individual pixel intensity values for each sample in the SD group (SD). Values are normalized to the overall mean pixel intensity value from the con group samples.
general files for setup.zip contains files required to segment whole brain pixel values into localized data for each neuropil region.
user guide.txt includes step-by-step instructions for analyzing example image data using included Python code files.
Code:
Included Python code files (see user guide.txt for step-by-step usage instructions):
- 1_Feature_Construction_SDvsControl_050423.ipynb - accesses registered confocal images, reads pixel intensity values from each brain
- 2_mean_variance_comparisons_SDvsControl_050423.ipynb - normalizes pixel intensity values to mean intensity from control samples, segments whole brain data to regional neuropils, outputs region-by-region results
- 3_pixel_intensity_distributions_050423.ipynb - plots pixel intensity distributions into histograms
- functions_feature_construction.ipynb - functions required to transform pixel intensity values from registered images into data vectors, sort values by neuropil region
- Mean_and_Variance_Comparisons.ipynb - functions to generate mean and variance output files by neuropil region
- Region_Mean_Variable_Loadings_Central.ipynb - functions related to PCA analysis of whole brain data
- All Python code was generated, edited, and run in Jupyter Notebook.
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.
References:
- P. J. Shaw, G. Tononi, R. J. Greenspan, D. F. Robinson, Stress response genes protect against lethal effects of sleep deprivation in Drosophila. Nature 417, 287–291 (2002).
- A. Jenett, et al., A GAL4-driver line resource for Drosophila neurobiology. Cell Reports 2, 991–1001 (2012).
- A. Ostrovsky, S. Cachero, G. S. X. E. Jefferis, Clonal Analysis of Olfaction in Drosophila: Image Registration. Cold Spring Harb Protoc 2013, pdb.prot071738-pdb.prot071738 (2013).
- S. Cachero, A. D. Ostrovsky, J. Y. Yu, B. J. Dickson, G. S. X. E. Jefferis, Sexual Dimorphism in the Fly Brain. Curr Biol 20, 1589–1601 (2010).
- J. Schindelin, et al., Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676–682 (2012).