Inducing Penetrating Traumatic Brain Injury in Adult Drosophila Flies

Take newly emerged, genetically modified adult Drosophila flies. 

Mushroom bodies in the fly's brain contain neurons that express green fluorescent proteins.

Mushroom bodies are structures containing complex neuronal networks essential for learning and memory.

Anesthetize the flies on a carbon dioxide pad.

Under a stereo microscope equipped with the required filters, visualize the mushroom bodies expressing green fluorescence.

Take a sanitized Minutien pin and push it through the head capsule into the mushroom body.

This causes damage to mushroom body neurons and disrupts neuronal networks, inflicting penetrating traumatic brain injury.

Next, gently remove the pin from the fly.

Place the injured flies in a vial with food and allow them to recover for further studies.

After anesthetizing the flies on a carbon dioxide pad, sort newly eclosed F1 young male flies within six hours of eclosion, and place them in clean vials containing food with 40 or fewer flies per vial. Next, sanitize minutien pins by placing approximately 100 pins in a 1.5-milliliter microcentrifuge tube filled with 70% ethanol for five minutes. Then, sanitize the carbon dioxide pad in paintbrush by spraying 70% ethanol and wiping dry with a clean, lint-free tissue.

Once the tools are clean and dry, transfer 40 or fewer sorted F1 males onto the clean pad and separate them into two groups. One group will serve as the control uninjured flies, and the second experimental group will be subjected to penetrating traumatic brain injury. Next, using forceps, pull four to five new minutien pins out of the microcentrifuge tube, and place them near the edge of the carbon dioxide pad. Then, under the dissecting scope, choose a straight minutien pin with a sharp point. Reuse sharp pins and place damaged or blunt pins in a separate tube containing 70% ethanol for safe disposal.

Next, for flies with the standard cross genotype, switch on the stereomicroscope LED lamp equipped with the appropriate excitation and emission filters for the green fluorescent protein, which permits excitation at 440 to 460 nanometers and allows visualization at 500 to 560 nanometers. Then, choose a fly from the experimental group, and position the fly such that the experimenter has a dorsal view of the head capsule with the fly's head to the right. Using forceps, pick up and hold the selected minutien pin in one hand, and the paintbrush in the other hand. Then, place the brush on the interior of the dorsal thorax, and push down gently to stabilize the fly.

Aim the minutien pin's tip at the mushroom bodies, 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. After completing the injury, use the paintbrush to push the head gently off the minutien pin. If using the brain for RNA sequencing or qRT-PCR, make a second injury on the left side of the head. Once all experimental flies have been injured, place the control and injured flies into separate labeled vials containing food.