mechanically injuring drosophila larvae segmental nerves to induce neurodegeneration

Mechanically Injuring Drosophila Larvae Segmental Nerves to Induce Neurodegeneration

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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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

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1. Preparation of Reagents and Equipment 
<ol>
	<li>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).</li>
	<li>Prepare 1x Phosphate-Buffered Saline (PBS).</li>
	<li>Prepare 1x PBT using 1x PBS with 0.01% Triton X-100.</li>
	<li>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).</li>
	<li>Obtain size 5 forceps that allow one to mechanically injure the larvae.</li>
	<li>Use standard Drosophila CO2 based anesthesia apparatus.</li>
</ol>
2. Selecting Drosophila Larvae 
<ol>
	<li>Take a vial or bottle of Drosophila containing wandering third instar larvae that have been cultured at 25 &#176;C.</li>
	<li>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 &#176;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.</li>
</ol>
3. Preparing Drosophila Larvae for Mechanical Injury
<ol>
	<li>Carefully pick individual larvae up with forceps or a paintbrush, being cautious not to compress it in any way.</li>
	<li>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.</li>
</ol>
4. Mechanical Injury and Treatment of Drosophila Larvae
<ol>
	<li>Place each individual larva onto a CO2 anesthetizing apparatus.</li>
	<li>Under a dissecting microscope, carefully roll each larva onto their dorsal side in order to visualize the segmental nerves through the cuticle.</li>
	<li>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. &#8203;
	<ol>
		<li>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%.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
</ol>

Source: Danella, E. B., et al. <a href="https://app.jove.com/v/56128">A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration.</a> 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).

1. Preparation of Reagents and Equipment 

	Prepare 1x Dissection Buffer (70 mM NaCl, 5 mM KCl, 0.02 mM CaCl2, 20 mM MgCl2, 10 mM NaHCO3, 115 mM sucrose, ...

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.

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Mechanically Injuring Drosophila Larvae Segmental Nerves to Induce Neurodegeneration

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