Two-Photon Laser-Induced Neural Injury: A Method to Observe Axon Degeneration and Regeneration in Drosophila Larvae

Abstract

Source: Li, D., et al. A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System. J. Vis. Exp. (2018).

This video describes two-photon laser ablation of axons in a Drosophila melanogaster larva and an example protocol to induce injury and image the injury site.

Protocol

This protocol is an excerpt from Li et al., A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System, J. Vis. Exp. (2018). 1. Two-photon Injury and Confocal Imaging Microscope setup NOTE: A confocal laser scanning microscope with a two-photon laser was used for this experiment, but other systems with an equivalent setup will also suffice. The two-photon las…

Representative Results

Figure 1: Da neuron axon regeneration in the periphery displays class specificity. (A and B) Schematic drawing showing the position of the larvae. (C) Schematic drawing of class III da neurons. (D) Axons of the class III da neurons ddaF, labeled with 19-12-Gal4, UAS-CD4-tdGFP, repo-Gal80/+, fail to regrow. (E) Schematic drawing of class IV da neurons…

Materials

Diethyl ether, ACS reagent, anhydrous	Acros Organics	AC615080010
Halocarbon 27 Oil	Genesee Scientific	59-133
Propionic Acid	J.T.Baker	U33007
Cover Glasses: Rectangles	Fisher Scientific	12-544-D	50 mm X 22 mm
Zeiss LSM 880 laser scanning microscope	Zeiss
Zen software	Zeiss
Chameleon Ultra II	Coherent