In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons
概要
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
Protocol
1. Preparation of Reagents
- 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
- 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.
- Pins: Pins used for dissection need to be extra short. The sharp bottom portion of minutien pins works best in dissection.
- Need one sharp microdissection spring scissors.
- Need two fine tipped forceps.
3. Collection of Larvae
- Wandering 3rd instar larvae that are expressing a GFP tagged vesicle protein are collected onto a petri dish.
- Larvae are washed in deionized water to get rid of all the food.
4. Live dissection of larvae
- A larva is transferred to the dissection gel cube on a glass slide. Larva is immersed in dissection buffer.
- Larva is pined at the anterior and posterior using two fine pins with the dorsal side up.
- Using microdissection scissors, a small cut is made at the midline near the posterior end. From this incision a cut is made through to the anterior. Using two pins, pin the cut cuticle on the lateral edges.
- Add a drop of dissecting buffer. Carefully remove the gut, intestines and fat bodies with forceps. Most of these tissues normally ooze out during the dorsal cutting.
- Replace the buffer with fresh dissecting buffer. The larva should never be dry and should constantly be in dissecting buffer.
- Incubations using in vivo markers MitoTracker or LysoTracker are done at this time, by diluting the in vivo marker in dissection buffer. After the incubation of the in vivo marker wash the dissected larva several times (5 quick washes) using fresh dissecting buffer.
- After replacing the last wash push the pins into the syligard and immediately coverslip. The larvae is now ready for observation.
5. Live imaging of dissected larvae
- Place the coverslip and gel onto the microscope stage and image using an inverted microscope.
- We use a 100x lens to image tagged vesicles within larval segmental nerves. GFP expression in the cell bodies of the ventral ganglion is first evaluated before segmental nerves are imaged. If robust GFP staining is observed in the cell bodies in the ventral ganglion, the nerves are then imaged and used for evaluation.
- Using a dual view system with filters for CFP/YFP or RFP/YFP and split view software we can simultaneously image two tagged protein in vivo.
6. Representative Results
Figure 1: A representative image showing many axonal vesicles on many axonal tracks within a larval segmental nerve. Note the blobs of GFP, representing axonal accumulations.
Figure 2: A representative image showing a single axonal track with many axonal vesicles within a larval segmental nerve.
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 genetics, GFP vesicle transport can be visualized within a single axon or within several axons by using a specific GAL4 driver to drive the expression of GFP. Additionally the dynamics of transport can also be observed in larvae carrying mutations for specific genes. Further two vesicles tagged with different GFP variants (CFP or RFP) can be simultaneously visualized.
開示
The authors have nothing to disclose.
Acknowledgements
SG is supported by funds from the State University of New York at Buffalo and from John R. Oishei Foundation.
MK was supported by a Student Research Award by CURCA at UB.
Materials
| Material Name | タイプ | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 1 Pair Micro Dissection Scissors | Fine Scientific | Spring scissors – 6mm blade. 0.125 mm tip diameter | ||
| 2 Pair Forceps | Fischer Scientific | Fisherbrand Dissecting Micro-Adson serrated tip forceps | ||
| Box of Dissection Pins | Fine Scientific | Minutien Pins 0.1mm diameter |
参考文献
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- Hurd, D. D., Saxton, W. M. Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in Drosophila. 遺伝学. 144, 1075-1085 (1996).
- Gunawardena, S., Her, L. S., Brusch, R. G., Laymon, R. A., Niesman, I. R., Gordesky-Gold, B., Sintasath, L., Bonini, N. M., Goldstein, L. S. Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila. Neuron. 40, 25-40 (2003).
