A Technique to Inflict Closed-Head Traumatic Brain Injury in Drosophila Flies

Take control and test adult Drosophila flies under anesthesia.

Place the test fly in a holder and gently tap it to position the fly's head outside the tip. Ensure the fly's mouthparts and body remain inside the holder to avoid non-target injuries.

Attach the holder to a strike device with the fly's head facing downward.

The device is connected to a controlled gas supply and contains a gas-propelled impactor to inflict injury.

Deliver a gas burst to propel the impactor and strike the fly's head, inducing a closed-head traumatic brain injury without external damage.

The resulting mechanical stress damages neurons, impairing their function.  

Detach the holder to release the fly, then transfer it to a vial for recovery.

For the control fly, apply a gas burst without striking it with the impactor.

The control and test flies are now ready to assess the impact of the injury.     

Anesthetize a single two-day-old adult female fly using carbon dioxide.

From the carbon dioxide pad, use a fine brush to gently transfer the fly into the holder.

Then, gently tap the holder until the head protrudes from the tip.

If the proboscis is exposed, use a blunted 1 milliliter syringe needle to gently tuck it back inside the tip. It is critical to keep the fly body, especially the mouth part, inside the holder. Otherwise, the fly may die from damage to the internal organs or from not being able to ingest food.

Next, set the gas pressure to 100 kilopascals and adjust the flow as needed. Once the fly is loaded, tighten the holder to the syringe barrel using the connector, such that the fly head faces downward.

Then, using the toggle switch, send a burst of gas that moves the impactor to strike the fly once and only once.

Now, detach the fly holder and dump the fly back onto the carbon dioxide pad.

Brush the fly into an empty vial until it recovers.

Keep one fly per vial. The recovery takes only a few minutes.

Repeat the process to test four flies per experimental group. Processing two groups, a test and a sham, should only take 20 to 30 minutes. Treat shams just like test animals, except do not include an aerosol barrier in the impactor tube.