Drosophila Melanogaster Stocks and Conditions
Hiw mutant hiwΔN and control hiwWT (FRT19A;;) flies were obtained from Marc Freeman (University of Massachusetts). Newly enclosed flies were collected daily, separated by sex, into vials of 20-35 flies, and aged for experimental use. All experiments were conducted on flies aged 1-4 days unless otherwise stated. All flies were maintained at a constant 25°C temperature and humidity, in glass vials with standard agar/cornmeal/yeast feed. Flies were exposed to a 12h light-dark cycle. Feed was changed in all vials once every 14 days or sooner as required. All experiments were conducted exclusively on male flies in order to avoid confounding effects relating to the female reproductive cycle.
High impact trauma device, injury calibration, incapacitation rates, and intestinal barrier dysfunction
Flies were subjected to a standardised impact with the HIT device. After injury, vials were laid on their side and flies were given a minimum of 10 minutes to recover motility before being transferred to a glass vial containing standardised feed. All polystyrene vials were discarded after a single use. The severity of injury was calibrated in hiwWT flies by assessing the death rates 24 hours following HIT when the angle of initial deflection, and thus recoil force, was adjusted. Incapacitation rates were recorded by assessing the percentage of flies that failed to show signs of purposeful movement within 20 seconds of initial impact. To evaluate intestinal barrier dysfunction following HIT flies were transferred to feed containing dissolved Brilliant Blue FCF dye (#80717, Sigma). After 24 hours the percentage of flies that had blue food dye dispersed outside of the abdominal cavity and proboscis were counted.
Early death rate and long-term survival assay
To assess for variation in early death rates we exposed flies to a HIT at a standardized time of day. Any flies dying immediately or within the first 24 hours of a HIT were considered to have died of the undifferentiated primary effects of a HIT. Dead flies were removed and all remaining live flies were transferred to new vials and long-term survival was monitored. A daily count of number of fly deaths was conducted in all vials for the lifetime of all flies. Dead flies were discarded every day.
Rapid iterative negative geotaxis (RING) assay and flight assay
A custom made rapid iterative negative geotaxis (RING) device was manufactured and used to measure negative geotaxis/climbing ability as a behavioural measure of motor function.[36, 37] Flies were gently transferred to fresh empty polystyrene vials without anaesthesia with a maximum density of 25 flies per vial. Groups of up to 6 vials were inserted into the RING device, and after 5 minutes for the flies to adjust to the environmental change the device was tapped three times to settle flies to the bottom of the vials. Exactly 5 seconds after the last tap a picture was taken to assess the height climbed. The head of the fly was the reference point for the climbing height achieved. Maximum height achieved was graded into 5mm intervals, flies that climbed less than 5mm were scored zero, and any fly that exceeded 50mm was awarded the maximum score was 5cm. The average height achieved for the vial was calculated. This was repeated 3 times at 60 second intervals and an average score given for that vial. The reduction in climbing ability was calculated on a vial by vial basis by subtracting the baseline height climbed preinjury from the final height climbed at 45 days. For the flight assay, flies were anaesthetised on ice for exactly 5 minutes then the flat of a 30G 1” needle (#Z192368, Sigma) was attached to the anterior notum of a fly just posterior to the neck using clear nail varnish, leaving flight muscles unimpeded. Flies were given 15 minutes to fully recover. Needles were fixed in place under a video microscope. If required then a gentle mouth-blown puff of air was used to stimulate flight and the flying time was recorded for 30 seconds per fly for analysis. This was repeated 3 times per fly and the average of time spent in flight was calculated for each condition. All RING and flight assays were conducted at the same time of day in a quiet room with standardized light and environmental conditions.
Haematoxylin and eosin histology, and vacuole counting
Anaesthetised flies were submerged in cold 1x PBS, the proboscis and rostral trachea were removed, and the amputated heads gently rocked in fresh ice cold 4% paraformaldehyde solution for 45 minutes. The tissue was alcohol dehydrated, xylene washed, and embedded in paraffin for serial sectioning with a microtome (Leica RM2235) at a thickness 7μΜ. Sections were mounted on poly-L-lysine coated slides (P0245, Sigma). Wax was removed with a xylene bath then alcohol washes before haematoxylin and eosin staining, and application of coverslips. After blinding, three representative coronal sections were examined from a central brain region that included the medulla using brightfield microscopy. The average number of ³5μΜ vacuoles per slice in each brain was calculated.
Fly brains were dissected in cold 1x PBS and fixed in 4% paraformaldehyde-PBS for 30 minutes. Samples were washed in 1x PBS with 0.3% Triton X-100 (#T8787, Sigma) and blocked for 1 hour at room temperature in 1x PBS with 0.3% Trition X-100 and 1% BSA (#9647, Sigma). Brains were incubated in primary antibody diluted with blocking solution for 72 hours. After washing and incubating in a fluorescent secondary antibody solution for 4 hours, samples were washed and mounted between two coverslips in ProLong diamond antifade mountant (#P36965, ThermoFisher). Confocal images were acquired on a Leica imaging system, at z-stack intervals not greater than 0.6μΜ and blinded for analysis. Primary antibodies used were mouse Tyrosine-Hydroxylase (anti-TH) antibody 1:100 (TH-antibody, #22941, Immunostar Inc.) for the PPL1 cluster. Secondary antibodies were goat anti-mouse IgG (H+L) Alexa Fluor 488 (#A11034, ThermoFisher), and donkey anti-rabbit IgG (H+L) Alexa Fluor 594 (##21207).
Fifteen whole fly heads were collected and homogenized in Laemmli sample buffer and centrifuged 13,000 rpm for 5 minutes. Supernatant protein lysates were resolved by SDS-PAGE on Mini-protean 4-15% SDS resolving gel (#4561086, Bio-Rad) and transferred to Immobilon-P PVDF membrane (#IPVH00010, Merck). They were blocked in a 1% BSA solution (#9647, Sigma), then probed with the following primary antibodies: Bruchpilot 1:5000 (nc82, #2314866, DSHB), Discs-large 1:10,000 (4F3, #528203, DSHB), Neuroglian 1:5000 (BP104, #528402, DSHB), and β tubulin 1:5000 (E7, #2315513, DSHB). Bands were detected with goat anti-mouse, and goat anti-rabbit horseradish peroxidase-linked secondary antibodies (#1706515 and #1721011, Bio-Rad) and Supersignal West Dura extended duration chemiluminescence substrate (#34075, ThermoFisher). Density of bands was quantified using Image J software (v1.51n).
Five fly brains were dissected in cold 1xPBS solution and transferred to dissection media containing 7.5mls of DMEM (high glucose, HEPES, phenol-red free, #21063029, ThermoFisher), 2.5mls of 10x trypsin (#15400054, ThermoFisher), and 1% BSA (#9647, Sigma). Brains were washed in trypsin free dissection media, gently triturated using a 200μL pipette 30 times, then filtered through a 70μM strainer. The resulting solution was mixed in a 1:1 ratio with Annexin V & dead cell solution (Annexin V & 7-AAD, #MCH100105, Merck), incubated at room temperature for 20 minutes, then processed on the Muse Cell Analyser (Merck) using inbuilt analysis software. Dilutions in trypsin free dissection media we made as required. For positive controls brains were incubated in 200mM of Actinomycin D (#A1410, Sigma) at 37°C for 6 hours before processing. Gating was kept constant for all experiments.