The overall goal of this procedure is to induce mechanical injury of motor neurons in Drosophila larvae. This method can help answer key questions in the field of neurodegenerative research such as the temporal sequence of cellular events leading to neurodegeneration. The main advantages of this technique are that it is inexpensive and uses equipment that most Drosophila labs already have.
The implications of this technique extend towards the understanding of the molecular mechanisms that drive neurodegeneration. To begin the protocol, select 10 second or third instar larvae from a vial or bottle of Drosophila containing wandering third instar larvae that have been cultured at 25 degrees Celsius. Use forceps to carefully pick individual larvae up by forceps or a paintbrush without compressing them.
Directly place the 10 larvae into a glass dish containing cold 1x PBS to remove any food debris and to decelerate larval motility. Identify third instar larvae by their anterior branch spiracles and second instar larvae by their smaller size and club-like anterior spiracles. 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 two-thirds of the way down the length of the larva.
Once positioned, pinch approximately one-third of the ventral cuticle containing the segmental nerves with size 5 foreceps. 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 insure 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. Prior to injury, wild-type neuromuscular junctions, or NMJs, show the presynaptic active zone marker Brp in apposition to the postsynaptic marker Dlg throughout the entire NMJ.
Mechanical injury of segmental nerves can induce moderate to severe neurodegeneration in which butons stained with Dlg now lack the presynaptic active zone protein Brp. After its development, this technique paved the way for researchers studying neuronal injury to explore the temporal sequence of cellular events leading to the neurodegeneration at the neuromuscular junction in Drosophila.
