Two-Photon Laser-Induced Neural Injury: A Method to Observe Axon Degeneration and Regeneration in Drosophila Larvae

- Begin by mounting a clean, anesthetized larva under a glass cover slip with its head up. Next, expose the area of interest by gently pushing the cover slip to roll the larva and adjust its position. Use confocal imaging to locate the larva neural cells. Identify a specific axon on one of the target neural cells as the site for injury.

Then expose the target axon to a two-photon laser at a higher power than used for imaging to induce damage. A two-photon laser is used due to its ability to penetrate and precisely localize damage within live tissue with minimal damage to surrounding tissue.

Stop the laser exposure immediately when there is damage to the target neuron, resulting in axotomy, or severing of the axon. Lastly, image the injured neuron to view its degeneration and subsequent regeneration at the desired time points.

In the example protocol, we will mount a larva for microscopy, induce damage to larval sensory neurons, and track neural regeneration.

- Begin with an anesthetizing the larvae. In a fume hood, place a 60-millimeter glass dish into a 15-centimeter plastic petri dish. Then fold a piece of tissue paper and place it in the glass dish. Place the grape agar plate on the tissue after the diethyl ether is added.

Next, onto a glass slide place one drop of halocarbon 27 oil at the center, and place a spot of vacuum grease on each of the four corners. Then use forceps to transfer one larva onto the agar plate and cover the glass dish to anesthetize the larva. As soon as the lava stops moving, carefully transfer it to the halocarbon oil with its head upright. Then place a cover slip over the slide and press it down gently until it touches the larva.

Next, use gentle force to slide the cover slip to roll the cells to be ablated to where the two-photon laser will most easily hit them. The location will vary depending on what neurons are being targeted. Now secure the assembly on the two-photon microscope stage and focus on the cells of interest using a 40x oil immersion objective.

In the software, switch to the scanning mode and load the saved protocol. Make sure the pinhole is opened all the way. Then, in live mode, get a good image of the region of interest.

Next, stop the live scan so that the crop button will become available. Using the crop function, adjust the scan window to focus the target area on just the prospective site of injury. Then open a new imaging window. Now reduce the scan speed and increase the laser intensity. Then toggle the continuous button to start and stop the scan. Watch carefully. As soon as there is a drastic increase in fluorescence, end the scan.

Next, switch back to the original imaging window and select the live mode, and find the region that was just targeted by adjusting the focus.

- A good indication of successful injury is the appearance of a small crater, ring-like structure, or localized debris right on the injury site. If the laser power was too high, a large damaged area would be visible, which can be lethal.

- Now carefully remove the cover slip, and transfer the injured larva onto a new plate with yeast paste. Put the plate into a 60 millimeter dish along with a propionic acid-soaked tissue. Then return the plate to culturing temperature.

For subsequent imaging of the larva, make use of the saved confocal setup and collect z stack images with a 25x objective. Be sure to include the normalization point so the regeneration can be quantified.