Inducing Penetrating Traumatic Brain Injury in Adult Drosophila Flies

1. Penetrating traumatic brain injury (PTBI;  Figure 1)

1. Sort newly eclosed F1 flies. Select young males within 6 h post-eclosion. Place these males in clean vials containing food, with 40 or fewer flies per vial.
    NOTE: This is most easily accomplished in mid-morning by anesthetizing flies on the CO₂ pad and identifying adult males that still have meconium (visible as a dark greenish spot through the abdominal wall) in their guts.
2. Sanitize Minutien pins (see Table of Materials) for at least 5 min by placing ~100 pins in a 1.5 mL microcentrifuge tube filled with 70% ethanol.
3. Sanitize the CO₂ pad and a paintbrush by spraying 70% ethanol and wipe dry with a clean, lint-free tissue. Once the tools are clean and dry, transfer 40 or fewer sorted F1 males back onto the clean pad.
4. Separate the F1 males into 2 groups on the fly pad. One group will serve as a control, uninjured flies. The second experimental group will be subjected to PTBI.
5. Using forceps, pull 4-5 new Minutien pins out of the microcentrifuge tube and place them near the edge of the CO₂ pad. Under the dissecting scope, choose a straight Minutien pin with a sharp point.
    NOTE: Using a single Minutien pin for an experiment will reduce variability. Reuse sharp pins. Place damaged or blunt pins in a separate 1.5 mL microcentrifuge tube containing 70% ethanol for safe disposal.
6. If working with flies from the standard cross, switch on the stereo microscope light-emitting diode (LED) lamp equipped with the appropriate excitation and emission filters for green fluorescence protein (GFP). This permits excitation at 440-460 nM and allows visualization of 500-560 nM.
    NOTE: With this lamp and filter set, the cell bodies of the mushroom body will fluoresce green through the head cuticle (Figure 1C). For perma-twin flies or flies of other genotypes, a standard white light illuminator for the stereo microscope can be used to visualize landmarks on the head cuticle for targeting the injury to the mushroom body (Figure 1C).
7. Use the forceps to pick up and hold the selected Minutien pin in one hand (for right-handed people, this is usually the right hand) and the paintbrush in the other (usually left) hand. Choose a fly from the experimental group and position the fly such that you have a dorsal view of the head capsule with the fly's head to the right. Place the brush on the anterior of the dorsal thorax and push down gently to stabilize the fly.
8. Aim the Minutien pin's tip at the mushroom body's cell bodies on the right side of the head and penetrate the head capsule. If using landmarks, target the dorsal head cuticle between the ocelli and the dorsal rim of the eye (Figure 1).
9. After completing the injury, use the paintbrush to push the head off the Minutien pin gently.
10. If using the brain for RNA-Seq or qRT-PCR, make a second injury on the left side of the head.
11. Repeat steps 2.8-2.10 to injure all of the flies in the experimental group.
12. Once all flies have been injured, place the control and injured flies into labeled, separate vials containing food. Lay the vials horizontally (i.e., on their sides) while the flies recover from the anesthesia and during subsequent aging to prevent flies from getting caught in the food.
13. Place standard cross flies at 25 °C, and perma-twin flies at 30 °C to age.
14. For flies aged longer than 24 h, place them on clean food every 1-2 days.

Figure 1: Penetrating Traumatic Brain Injury (PTBI) to the adult Drosophila central brain. (A) Schematic of the exterior of an adult fly head. This is a frontal view. Thus, the right side of the animal is to the viewer's left. (B) Schematic of the interior of an adult Drosophila head with the injury trajectory indicated in grey. This is a posterior view. Thus, in this image and subsequent figures, the right side of the brain is to the right. Central brain PTBI impacts multiple brain structures, including the mushroom body (green) and tissues outside the brain, including the fat body (blue) and hemocytes (red). CB = central brain region. OL= optic lobe region. (C) Dorsal view of a live adult head in which mushroom bodies (arrowheads) are labeled with a green fluorescent protein (GFP). This is the 'standard genotype' (see text for details). The PTBI protocol reproducibly results in injury to the mushroom bodies.