Two-Photon Laser-Induced Neural Injury: A Method to Observe Axon Degeneration and Regeneration in Drosophila Larvae
Transcription
– 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 à roll the larva and adjust its position. Use confocal imaging à 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 à a two-photon laser at a higher power than used for imaging à induce damage. A two-photon laser is used due à its ability à penetrate and precisely localize damage within live tissue with minimal damage à surrounding tissue.
Stop the laser exposure immediately when there is damage à the target neuron, resulting de axotomy, or severing of the axon. Lastly, image the injured neuron à view its degeneration and subsequent regeneration at the desired time points.
In the example protocol, we will mount a larva for microscopy, induce damage à 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 de 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 à transfer one larva onto the agar plate and cover the glass dish à anesthetize the larva. As soon as the lava stops moving, carefully transfer it à 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 à slide the cover slip à roll the cells à be ablated à 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 à the scanning mode and load the saved protocol. Make sure the pinhole is opened all the way. Then, de 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 à 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 à start and stop the scan. Watch carefully. As soon as there is a drastic increase de fluorescence, end the scan.
Next, switch back à 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 à 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 à include the normalization point so the regeneration can be quantified.
