In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Summary

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Abstract

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method ¹ to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

Protocol

1. Preparation of Reagents

1. Prepare the dissection buffer preparation by adding the following compounds: 128 mM NaCl, 1mM EGTA, 4 mM MgCl₂, 2mM KCl, 5mM HEPES, and 36 mM Sucrose in 1000 mL, PH to 7.2 and filter sterilize.

2. Preparation for the Dissection

1. Siligard cube: The gel is cut to approximately one inch wide, 1 inch long and about 1cm high. The gel cube is placed on a glass slide during dissection. Cubes may be used more than once.
2. Pins: Pins used for dissection need to be extra short. The sharp bottom portion of minutien pins works best in dissection.
3. Need one sh....

Discussion

In vivo imaging of synaptic vesicle transport within Drosophila larval segmental nerves is a powerful tool for studying the mechanisms in axonal transport. We have previously used this protocol to evaluate the transport dynamics within larval nerves expressing expanded polyQ repeats to elucidate how expansion of repeats affect the dynamics of vesicle transport ³. Using this protocol the velocity of vesicle movement can be determined by converting the video streams to a kymograph. Using geneti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 Pair Micro Dissection Scissors	Fine Science Tools		Spring scissors – 6mm blade. 0.125 mm tip diameter
2 Pair Forceps	Fisher Scientific		Fisherbrand Dissecting Micro-Adson serrated tip forceps
Box of Dissection Pins	Fine Science Tools		Minutien Pins 0.1mm diameter