Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.
Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.
1. Preparation of Reagents
2. Preparation for the Dissection
3. Dissection of 3rd Instar Larvae
4. Fixation of the Dissected Larva
5. Antibody Staining of the Dissected Larva
6. Mounting and Visualization of the Dissected Larva
7. Representative Results
Figure 1: A representative image of larval segmental nerves from a wild type larva using antibodies against the synaptic vesicle protein cysteine string protein (CSP, Zinsmaier et al.1994 ). Note that the segmental nerves are smoothly stained. Bar=20μm
Figure 2: A representative image of larval segmental nerves from a motor protein mutant larva using antibodies against the synaptic vesicle protein cysteine string protein (CSP). Note that the segmental nerves show massive accumulations that stain brightly for CSP (arrows).
The Drosophila larval segmental nerves are a powerful system to study mechanisms in axonal transport. If the 3rd instar larval dissection is performed correctly, all components of the CNS, PNS, and associated tissue, such as the muscles and NMJs, can be examined within a single larva. We have adapted this protocol from the one published by Hurd and Saxton (1996). This protocol can also be adapted to test other neuronal antibodies, but optimization of antibodies should be done. The primary and secondary antibody incubation times can be changed or a blocking step can be added. We have successfully used this protocol to investigate the role of the amyloid precursor protein 1 and Huntingtin 2 in axonal transport.
The authors have nothing to disclose.
SG is supported by funds from the State University of New York at Buffalo and from John R. Oishei Foundation.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Dumont #5 Forceps | Fine Science Tools, Inc. | 11252-20 | ||
Minutien Pins, Stainless Steel | Fine Science Tools, Inc. | 26002-10 | ||
Mcpherson-Vannas Straight Blade 8cm Scissors | Fisher Scientific | 50822236 | ||
Vectashield, Mounting Medium | Vector Laboratories, Inc. | H-1000 | ||
Sylgard 184 Silicone elastomer | Dow Corning Corporation | 4026148 | ||
formaldehyde, 16% | Electron Microscopy Sciences | 15710 | ||
dCSP3 | Developmental Studies Hybridoma Bank | |||
Alexa Fluor 568 Goat Anti-Mouse IgG | Invitrogen | A-11004 |