This protocol is significant because it demonstrates a central brain injury method that triggers adult neurogenesis in Drosophila, where there normally is none. We anticipate that using this technique to understand the molecular mechanisms underlying adult neurogenesis will be relevant to human neural regeneration. Learning this technique requires patience and perseverance.
We recommend practicing on five to 10 brains daily for several weeks before collecting brains for analysis. 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.
If planning to label dividing cells with EdU prepare 200 microliters of 50 mill or more EdU in 10%sucrose and place the prepared solution on a 23 millimeter round grade three filter paper in an empty vial, then place the male flies in the vial for pre-feeding six hours before the injury. Next, sanitize Minutien Pins by placing approximately 100 pins in a 1.5 milliliter micro centrifuge tube filled with 70%ethanol for five minutes. Then sanitize the carbon dioxide pad and 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 micro centrifuge 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 stereo microscope LED lamp equipped with the appropriate excitation and emission filters for the green fluorescence protein which permits excitation at 440 to 460 nanometers and allows visualization at 500 to 560 nanometers. Then choose a fly the experimental group and position the fly such that the experimenter has a dorsal view of the head capsule with the flies 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 anterior 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 us 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.
Lay the vials horizontally to prevent flies from getting caught in the food. Place the standard cross flies at 25 degree Celsius and permatwin flies at 30 degree Celsius to age. If aging longer than 24 hours transfer the flies on clean food every one to two days.
A significant increase in proliferation was observed 24 hours post-injury using anti pH3 a marker for cells actively undergoing mitosis. Approximately three pH3 positive cells are observed in each of control central brains. And 11 pH3 positive cells in each of the injured central brains.
By seven days, an average of two EdU positive cells are present in each of the control cell brains. And 11 EdU positive cells in each of the injured central brains, which is similar to the results obtained 24 hours post-injury with pH3 antibody. At 14 days controls averaged one EdU positive cell per central brain, and injured brains averaged 29 EdU positive cells per central brain.
Demonstrating that cell proliferation continues at least into the second week following a penetrating traumatic brain injury. The strongest proliferative response in the central brain is observed when brains are injured within the first 24 hours after eclosion. By seven days Post-eclosion a penetrating injury still causes a significant increase in proliferation.
However, by 14 days, the ability of the cells to divide decreases significantly to one dividing cell per brain. Which is similar to that of control brains. Using the permatwin labeling system more permatwin clones were detected in injured samples at two days, in two weeks, compared to controls.
With the number of clones increasing over time following injury. Two weeks post-injury there were new mushroom body neurons in 50%of the injured brains. These new neurons projected their dendrites approximately to the mushroom body calyx, and axons to the mushroom body lobes.
Other areas of the brain that appeared to regenerate include the ellipsoid body Antennal lobes and lateral horn. Be sure to use a sharp Minutien pin when doing the brain injuries as use of a dull or bent pin will increase the mortality. Also be sure not to push too hard on the flies with the paint brush as this will also increase mortality.
Following this procedure, immunohistochemistry can be used to quantify self proliferation and also to identify new neurons and glia.
