Optogenetics-Sleep-Deprivation/README.md

Optogenetics-Sleep-Deprivation

Analysis notebooks for a Drosophila melanogaster sleep-deprivation study using ChRmine-mediated optogenetic activation of candidate wake-promoting / sleep-modulating neurons. Behavioural recordings were collected with ethoscopes and analysed with the ethoscopy Python library.

Experimental design

Flies are tracked in ethoscopes for ~5 days. After a 48 h baseline period, a 24 h stimulation window of red (or green, as a control) LED light is delivered, followed by a recovery period. Sleep is quantified per fly and compared baseline vs. stimulus, split into 24 h, day (12 h) and night (12 h) windows.

Each notebook focuses on a single genotype or comparison:

#	Notebook	Purpose
1	1.CantonS_Baseline.ipynb	Wild-type Canton-S baseline sleep
2	2.CantonS_Red_Stimulus.ipynb	Canton-S response to red light (control)
3	3.CantonS_Green_Stimulus.ipynb	Canton-S response to green light (control)
4	4.CantonS_RedvsGreen.ipynb	ΔSleep red vs. green in Canton-S
5	5.UAS-ChRmine-attP2_Baseline.ipynb	UAS-ChRmine (attP2 insertion) baseline
6	6.UAS-ChRmine-attP5_Baseline.ipynb	UAS-ChRmine (attP5 insertion) baseline
7	7.UAS-ChRmine-attP5_Red_Stimulus.ipynb	UAS-only effector control under red light
8	8.11H05-GAL4_Red_Stimulus.ipynb	11H05-GAL4 driver-only control
9	9.60D05-GAL4_Red_Stimulus.ipynb	60D05-GAL4 driver-only control
10	10.11H05-GAL4_ChRmine_Red.ipynb	11H05 × ChRmine, no ATR
11	11.60D05-GAL4_ChRmine_Red.ipynb	60D05 × ChRmine, no ATR
12	12.11H05_ChRmine_Red_ATR.ipynb	11H05 × ChRmine, +ATR (functional optogenetics)
13	13.60D05_ChRmine_Red_ATR.ipynb	60D05 × ChRmine, +ATR (functional optogenetics)
14	14.11H05_ChRmine_noATRvsATR.ipynb	ΔSleep no-ATR vs. ATR for 11H05 cross
15	15.60D05_ChRmine_noATRvsATR.ipynb	ΔSleep no-ATR vs. ATR for 60D05 cross

ChRmine is a red-shifted channelrhodopsin that requires the cofactor all-trans retinal (ATR) to function; the no-ATR vs. ATR contrast (notebooks 14–15) therefore isolates the specific contribution of optogenetic activation from any non-specific effect of red light.

Genotypes

• Canton-S — wild-type background
• UAS-ChRmine in two insertion sites: attP2 and attP5
• GAL4 drivers: 11H05-GAL4, 60D05-GAL4 (Janelia FlyLight collection)
• Experimental flies: GAL4 > UAS-ChRmine crosses, raised ± ATR food

Analysis pipeline

Each notebook follows roughly the same structure:

1. Load metadata with etho.link_meta_index(...) and pull raw ethoscope data via etho.load_ethoscope(..., FUN=etho.sleep_annotation).
2. Build a behavpy object combining data + metadata, pickle it for reuse.
3. Baseline normalisation with df.baseline(column='baseline').
4. Visualisations — heatmap, plot_overtime, with the stimulation window shaded.
5. Quantification — total sleep over 24 h / 12 h day / 12 h night windows, ΔSleep = stimulus − baseline.
6. Statistics — paired tests (Wilcoxon / paired t-test) within group, Mann–Whitney U or independent t-test between groups; normality checked with Shapiro–Wilk.

Requirements

• Python ≥ 3.10
• ethoscopy
• pandas, numpy, scipy, matplotlib, seaborn
• Jupyter / JupyterLab

pip install ethoscopy pandas numpy scipy matplotlib seaborn jupyterlab

Data location

The notebooks read pickled behavpy objects from absolute paths under /home/rdingjin/… and raw ethoscope output from /mnt/ethoscope_results. Update these paths to point to your own metadata CSV and ethoscope data mount before re-running.

License

Research code — please contact the authors before redistribution.