Mechanically Injuring Drosophila Larvae Segmental Nerves to Induce Neurodegeneration

Protocol

1. Preparation of Reagents and Equipment

1. Prepare 1x Dissection Buffer (70 mM NaCl, 5 mM KCl, 0.02 mM CaCl₂, 20 mM MgCl₂, 10 mM NaHCO₃, 115 mM sucrose, 5 mM trehalose, 5 mM hydroxyethylpiperazine ethane sulfonic acid [HEPES]; pH 7.2).
2. Prepare 1x Phosphate-Buffered Saline (PBS).
3. Prepare 1x PBT using 1x PBS with 0.01% Triton X-100.
4. Prepare fruit juice agar plates.14 Briefly, mix 30 g agar in 700 mL of H₂O and autoclave. Dissolve 0.5 g of methyl paraben in 10 mL of ethanol and add solution to 300 mL of fruit juice concentrate (grape or apple). Mix concentrate into an autoclaved agar solution and pour into the lids of 10 mm x 35 mm Petri dishes.
   NOTE: Grape agar power premix can also be purchased from various companies (see Table of Materials).
5. Obtain size 5 forceps that allow one to mechanically injure the larvae.
6. Use standard Drosophila CO₂ based anesthesia apparatus.

2. Selecting Drosophila Larvae

1. Take a vial or bottle of Drosophila containing wandering third instar larvae that have been cultured at 25 °C.
2. Select ten individual 2nd or 3rd instar larvae based on appearance. Third-instar larvae can be identified by the appearance of anterior branched spiracles, while second instar larvae are smaller and have more club-like anterior spiracles.
   NOTE: Larval stages can also be identified by timing. At 25 °C, the second instar larvae appear approximately 48 h after hatching, and third instar larvae molt approximately 24 h later. If interested in examining neurodegeneration at the NMJ, second instar larvae need to be injured.

3. Preparing Drosophila Larvae for Mechanical Injury

1. Carefully pick individual larvae up with forceps or a paintbrush, being cautious not to compress it in any way.
2. Directly place ten larvae into a glass dish containing cold 1X PBS or dissection buffer to remove any food debris and to decelerate larval motility.

4. Mechanical Injury and Treatment of Drosophila Larvae

1. Place each individual larva onto a CO₂ anesthetizing apparatus.
2. Under a dissecting microscope, carefully roll each larva onto their dorsal side in order to visualize the segmental nerves through the cuticle.
3. Position size 5 forceps approximately 1/2 to 2/3 way down the length of the larva starting at the mouth hooks. Once positioned, pinch approximately 1/3 of the ventral cuticle containing the segmental nerves with size 5 forceps. ​
   1. 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 5 h is under 50%.
4. After injury, carefully transfer larvae to a fruit juice agar plate containing approximately 0.5 g of yeast paste. Place each larva so its anterior end is on the yeast paste, for continued feeding despite disrupted motility.
   NOTE: To ensure that larval segmental nerves have been injured, disrupted larval motility can be observed after transfer to the agar plate. Injured larvae are effectively paralyzed posterior to the site of injury, but can still move their mouth hooks and feed. A high death rate of animals after injury is common so it is best to injure 20-50% more animals than is necessary for an experiment.
5. Place agar plates containing yeast paste and 10 injured larvae into the bottom of an empty 100 x 15 mm petri dish. Cover this petri plate, now containing the injured larvae on an agar plate with a moist paper towel, ensuring that the paper towel does not touch the larvae or the agar plates. Place this Petri dish into a larger air-tight container, at room temperature (RT).
6. Retrieve larvae for dissection after specified periods (6, 12, or 24 h) post-injury.
   NOTE: To examine immediate effects of injury on the axonal cytoskeleton, larvae can be dissected directly after injury. To examine axonal transport defects, larvae can be dissected approximately 6 h after injury. About 12 h after injury a build-up of ubiquitinated proteins can be observed, and 24 h after injury, degeneration of motor neurons at the NMJ can be observed.

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Materials

Name	Company	Catalog Number	Comments
Micro-dissecting scissors	Fine Science Tools	15000-08
Dumont #3 Forceps	Fine Science Tools	11231-30	Some people prefer size 3, while others prefer size 5
Dumont #5 Forceps	Fine Science Tools	11251-30	Some people prefer size 3, while others prefer size 5
CO2 Air Tank	Tech Air	UN 1013	Various tank sizes can be purchased
CO2 Anesthetizing Apparatus	s Genesee Scientific	59-114
Stainless-steel pins, size 0.1	Fine Science Tools	26002-10
SylGard 184 Silicone Elastomer, Base and Curing Agent	Dow Corning	3097358-1004	To pour dissecting plates
Dissecting Stereo MIcroscope	AmScope	SM-1BZ
Light Source	AmScope	HL150-AY-220V
Flystuff Grape Juice Agar Premix	Genesee Scientific	47-102
Microscope slides	Genesee Scientific	29-101
Glass Coverslips	Fisher Scientific	12-545-87
Thermo Scientific Nalgene Utility Box	Fisher Scientific	03-484C	Used to create humid chamber for larval recovery