Dataset DOI: 10.5061/dryad.fttdz097t

Description of the data and file structure

Dataset overview

This supplementary dataset contains raw image files that support key findings of our study. To determine whether ethanol or starvation alters memory consolidation in Drosophila, we utilized the CaLexA system in which integrated cellular calcium activity is visualized with a genetically encoded GFP reporter. Here, the UAS-GAL4 system was used to express UAS-CaLexA in subsets of mushroom body (MB) neurons using specific GAL4 driver lines. Following behavioral manipulations, Drosophila brains were dissected and imaged using a Zeiss 980 confocal microscope. The resulting confocal stacks depict calcium changes marked by GFP, while the structures were visualized via a coexpressed RFP. In addition, the dataset contains image files that depict changes in NPF expression following ethanol exposure. Here, dissected brains were stained with an NPF antibody and then imaged using a confocal microscope. Together, the data suggest that ethanol exposure induces a switch in memory pathways, from sleep-dependent to sleep-independent, by increasing neuropeptide F (NPF) signaling.  

Data acquisition

A standard protocol was used to fix and stain dissected fly brains. Primary antibodies used were: mouse anti-GFP (1:200; Roche, Cat. #11814460001), rabbit anti-dsRed (1:200; Takara Bio Cat. #632475), and rabbit anti-NPF (1:400; Ray Biotech Cat. # RB-19-0001-20). Secondary antibodies were: Alexa Fluor 488 donkey anti-mouse (1:200; ThermoFisher Cat. # A-21202), Cy5 donkey anti-rabbit (1:200; Jackson ImmunoResearch Cat. # 711-175-152), and Alexa Fluor 488 goat anti-rabbit (1:200; ThermoFisher Cat. # A-11008). The brain samples were imaged using a Zeiss 980 confocal microscope with a 40x water-immersion objective. 

Data analysis

The dissected fly brains from the experimental and control groups were stained simultaneously using identical reagent preparations. Acquisition parameters were consistent across brains for a given experiment. Image analysis was performed using Fiji 2.0. For NPF quantification, the maximum-intensity projection was used to draw ROIs around NPF cell bodies. Background correction was performed by subtracting the mean fluorescence intensity of a background region adjacent to the target ROI for each sample to account for baseline fluorescence variability. The background-subtracted mean intensity values were summed across slices in which the cell bodies were visible to obtain a single intensity value for each brain. To assess the CaLexA-based calcium signal, a standardized threshold was first established, and the respective values were then applied across all brains. An ROI was drawn manually around the mushroom body in each brain hemisphere using the maximum intensity projection for the red reference marker. Integrated density from the slices containing the mushroom body was summed for each channel, and relative fluorescence was calculated as the ratio of green to red signal. Relative intensity was evaluated by normalizing individual values by the mean intensity of the corresponding control groups.

Folders and files

All raw image files are stored in the standard OME-TIFF format. The primary folders denote distinct experiments with corresponding data acquisition dates, while subfolders are categorized by specific experimental manipulations. The details are listed below:

Impact of ethanol on α’β’ medial MB neuron activity in trained sleep-deprived and control flies

Raw images depicting differences in the activity of α’β’ medial mushroom body neurons in ethanol-fed flies that were either sleep-deprived or left undisturbed (control) after training.

• 20250507_The_impact_of_ethanol_feeding_and_sleep_deprivation_on_the_activity_of_alpha’beta’_medial_MB_neurons_in_trained_flies.zip

Impact of gaboxadol on α’β’ medial MB neuron activity in trained and starved flies

Gaboxadol, a GABAA agonist, induces sleep in flies. These images depict the impact of gaboxadol-induced sleep on the activity of α’β’ medial mushroom body neurons in trained and starved flies.

• 20250607_The_impact_of_gaboxadol_on_the_activity_of_alpha’beta’_medial_MB_neurons_in_trained_and_starved_flies.zip
• 20250613_The_impact_of_gaboxadol_on_the_activity_of_alpha’beta’_medial_MB_neurons_in_trained_and_starved_flies.zip

Impact of brief ethanol on NPF expression

Neuropeptide F, a mammalian homolog of NPY, mediates ethanol-related behaviors in flies. These images depict changes in NPF expression when flies are exposed to ethanol for 30 min.

• 20250828_The_impact_of_brief_ethanol_on_NPF_expression.zip
• 20260312_The_impact_of_brief_ethanol_on_NPF_expression.zip

Impact of sleep deprivation on α’β’ medial MB neuron activity in untrained flies

These images depict the impact of sleep deprivation on the activity of α’β’ medial mushroom body neurons in untrained flies.

• 20260330_The_impact_of_sleep_deprivation_on_the_activity_of_alpha’beta’_medial_MB_neurons_in_untrained_flies.zip

Impact of sleep on α’β’ medial MB neuron activity in untrained and starved flies

These images depict the impact of gaboxadol-induced sleep on the activity of α’β’ medial mushroom body neurons in untrained and starved flies.

• 20260404_The_impact_of_gaboxadol_on_the_activity_of_alpha’beta’_medial_MB_neurons_in_untrained_and_starved_flies.zip
• 20260420_The_impact_of_gaboxadol_on_the_activity_of_alpha’beta’_medial_MB_neurons_in_untrained_and_starved_flies.zip

Code/software

Fiji 2.0 can be used to visualize and analyze raw image files.