Alrm-Gal4 stocks were obtained from the Bloomington Drosophila Stock Center (Indiana University). The mb247-DsRed; mb247-splitGFP11, UAS-splitGFP1-10/TM3sb flies were obtained from T. Riemensperger and A. Fiala (University of Göttingen).
Flies were cultured at 25°C, 60% humidity, maintained on a 12:12 Light:Dark cycle, on Nutri-fly Bloomington Formulation fly food (Genesee Scientific, San Diego, CA). Newly eclosed virgin female flies were collected from culture vials daily under CO2 anesthesia and housed in groups of approximately 30 prior to experimentation.
Female flies 4–7 days after eclosion were used for all sleep studies. Flies were mouth aspirated into 5 mm × 65 mm (outside diameter × length) polycarbonate recording tubes (Trikinetics, Waltham, MA) with food (Bloomington Nutri-fly formula) on one end and yarn plugs on the other. Sleep parameters were continuously evaluated using the Trikinetics Drosophila activity monitoring system (DAMS; Trikinetics, Waltham, MA) as described previously. One acclimatization day was followed by 2 days of baseline sleep recording, one 24 h period of mechanical sleep deprivation, and 72 h of recovery sleep. Sleep deprivation was performed using a Sleep Nullifying Apparatus (SNAP), which produces waking without nonspecifically activating stress responses, as described previously.
Drosophila brains were dissected in phosphate-buffered saline (0.9% NaCl, 10 mm NaH2PO4, pH 7.2) containing 0.3% Triton X-100 (PBS-T) and fixed in 4% paraformaldehyde, washed, and mounted on cover slips. Optical sections were collected with a Leica DMi8 laser scanning confocal microscope. For each experiment, calibration on the microscope was held constant by establishing a signal threshold value for the control group. GRASP intensity levels were measured using Corrected Total Cell Fluorescence (CTCF). The corrected total fluorescence = Integrated Fluorescence density – (Area of ROI multiplied by Mean Fluorescence of background) and was calculated in max projected image stacks with the region of interest (ROI) around the mushroom bodies.
Stress and starvation
Virgin female flies were collected as described above. 4–7 days after eclosion, animals were mouth aspirated into 5 mm × 65 mm (outside diameter × length) polycarbonate recording tubes (Trikinetics, Waltham, MA) containing 0.1 mM Paraquat in minimal media (2% agar, 5% sucrose in ddH2O) or starvation food (2% agar in ddH2O). Animals were housed for 24 h on these media and afterwards were rapidly dissected at ZT0 for imaging of GRASP signal.
To standardize the environmental conditions during critical periods of brain development, virgin female flies were collected upon eclosion and maintained in same-sex vials containing approximately 30 flies for 2 days. This protocol kept environmental conditions constant between subsequently isolated and enriched flies for the first 2 days of adult life. 3 day old flies were then divided into a socially isolated group, in which flies were individually housed in 5 mm × 65 mm plastic tubes, and a socially enriched group, consisting of 50 female flies housed in a single vial. After 5 days of social enrichment/isolation, flies were placed into clean 5 mm × 65 mm plastic tubes and sleep was recorded for 3 days using the Trikinetics DAMS.
Statistics were calculated using Graphpad Prism software. Student’s t-test, one-way ANOVA, two-way ANOVA, and Tukey post-hoc analysis were used for analyses. Sleep data were analyzed by averaging across multiple experiments. Flies that did not survive the entire experimental paradigm were removed from data analysis.