Mechanically Injuring Drosophila Larvae Segmental Nerves to Induce Neurodegeneration

Abstract

Source: Danella, E. B., et al. A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration. J. Vis. Exp. (2017).

The video demonstrates inducing neurodegeneration in Drosophila larva segmental nerves via mechanical injury. The ventral cuticle of anesthetized larvae is pinched using forceps to cause injury to the segmental nerves. The injured larvae are maintained with food, allowing the injury to cause degeneration of the motor neurons in the nerves, causing disruption of their neuromuscular junctions (NMJs).

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 au....

Materials

List of materials used in this article

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