This article describes a method for inducing and studying neurodegeneration in third instar Drosophila larvae by injuring segmental nerves. The process involves anesthetizing the larvae, injuring the nerves, and monitoring the effects on motor neuron function and neuromuscular junctions.
Take third instar Drosophila larvae and anesthetize them using carbon dioxide.
Under a microscope, position the larvae with the ventral surface facing up to visualize the segmental nerves.
These nerves contain motor neurons that innervate the body wall muscles and control larval movement.
The axon terminals of these neurons connect with muscle fibers to form neuromuscular junctions or NMJs.
The neurons release neurotransmitters that bind to receptors on the muscle fibers, causing ion influx and triggering signal transmission.
Using forceps, pinch the ventral cuticle to injure the segmental nerves, thereby inhibiting movement.
Place the injured larvae on an agar plate with larval food to ensure survival.
Place the plate in a dish, cover it with a moist paper towel to maintain moisture, and store it in a container to allow larval recovery.
With time, the injury leads to motor neuron death, known as neurodegeneration, which disrupts the NMJs.
Place each larva onto a CO2 anesthetizing apparatus. Under a dissecting microscope, carefully roll each larva onto its dorsal side to visualize the segmental nerves throughout the cuticle.
It is essential to carefully roll each larva onto their dorsal side in order to visualize the segmental nerves through the cuticle before injury.
Starting at the mouth hooks, position size 5 forceps approximately one-half to 2/3rds of the way down the length of the larvae. Once positioned, pinch approximately one-third of the ventral cuticle containing the segmental nerves with size 5 forceps. To ensure that larval segmental nerves are injured, apply sufficient force to crush the segmental nerves, but leave the cuticle intact. To determine the correct amount of force, crush 10 larvae and ensure the death rate after five hours is under 50%.
After injury, carefully transfer the larvae to a fruit juice agar plate containing approximately 0.5 grams of yeast paste. Place each larva so its anterior end is on the yeast paste for continued feeding. Place agar plates containing yeast paste, and 10 injured larvae onto the bottom of an empty 100-by-15 millimeter Petri dish. Cover the Petri dish with a moist paper towel, ensuring that the paper towel does not touch the larvae or the agar plates. Then, place the Petri dish into an airtight container at room temperature. Finally, retrieve larvae for dissection after specified periods post-injury.
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