Name: In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons (Video) | JoVE Journal
Uploaded: 2010-10-15T16:00:00.000+00:00
Duration: 5 min 58 s
Description: Learn live dissection of Drosophila larvae for imaging GFP-tagged synaptic vesicle transport in axons using fluorescence microscopy. This method enables real-time visualization of axonal dynamics in neuroscience research.

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5 minutes

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.

1. Preparation of Reagents
<ol>
	<li>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.</li>
</ol>
2. Preparation for the Dissection
<ol>
	<li>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.</li>
	<li>Pins: Pins used for dissection need to be extra short. The sharp bottom portion of minutien pins works best in dissection.</li>
	<li>Need one sharp microdissection spring scissors.</li>
	<li>Need two fine tipped forceps.</li>
</ol>
3. Collection of Larvae
<ol>
	<li>Wandering 3rd instar larvae that are expressing a GFP tagged vesicle protein are collected onto a petri dish.</li>
	<li>Larvae are washed in deionized water to get rid of all the food.</li>
</ol>
4. Live dissection of larvae
<ol>
	<li>A larva is transferred to the dissection gel cube on a glass slide. Larva is immersed in dissection buffer.</li>
	<li>Larva is pined at the anterior and posterior using two fine pins with the dorsal side up.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Replace the buffer with fresh dissecting buffer. The larva should never be dry and should constantly be in dissecting buffer.</li>
	<li>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.</li>
	<li>After replacing the last wash push the pins into the syligard and immediately coverslip. The larvae is now ready for observation.</li>
</ol>
5. Live imaging of dissected larvae
<ol>
	<li>Place the coverslip and gel onto the microscope stage and image using an inverted microscope.</li>
	<li>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.</li>
	<li>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.</li>
</ol> 
6. Representative Results
<img src="/files/ftp_upload/2151/2151fig1.jpg" alt="Fluorescence microscopy image displaying protein distribution along a cellular structure, 10 µm scale." data-altupdatedat="1788235205000" data-alt="Figure 1"> 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.
<img src="/files/ftp_upload/2151/2151fig2.jpg" alt="Microscope image of neuron axon with fluorescent markers in a 10 µm scale, fluorescence microscopy." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2: A representative image showing a single axonal track with many axonal vesicles within a larval segmental nerve.

<ol><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li>
<li>Hurd, D. D., Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinesin+mutations+cause+motor+neuron+disease+phenotypes+by+disrupting+fast+axonal+transport+in+Drosophila%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in Drosophila.</a> Genetics. 144, 1075-1085 (1996).</li>
<li>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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+axonal+transport+by+loss+of+huntingtin+or+expression+of+pathogenic+polyQ+proteins+in+Drosophila%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila.</a> Neuron. 40, 25-40 (2003).</li>
</ol>

No conflicts of interest declared.

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...

<table><tbody><tr><td>1 Pair Micro Dissection Scissors</td><td>Fine Science Tools</td><td></td><td>Spring scissors &amp;#8211; 6mm blade. 0.125 mm tip diameter</td></tr><tr><td>2 Pair Forceps</td><td>Fisher Scientific</td><td></td><td>Fisherbrand Dissecting Micro-Adson serrated tip forceps</td></tr><tr><td>Box of Dissection Pins</td><td>Fine Science Tools</td><td></td><td>Minutien Pins 0.1mm diameter</td></tr></tbody></table>

in vivo visualization of synaptic vesicles within drosophila larval segmental axons

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

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.

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

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

To study whether defects in long distance axonal transport are present in neurological diseases such as Alzheimer's disease or Huntington's disease. Axonal transport of transport vesicles is imaged in live esophagal lava. Here wondering.Third in star larvae that express A GFP tagged physical protein are dissected on a gel cube, then transferred to the microscope for in vivo imaging. Fluorescence microscopy is then used to track sical transport within the axon. The main advantage of using this technique over existing methods is to visualize live undisrupted movement of synaptic vesicles and larval axons.Prepare for the dissection by ensuring that the following supplies and reagents are at hand. Dissection buffer. A Sill guard cube cut to one inch wide, one inch long, and one centimeter high placed on a glass slide.Extra short dissection pins. The sharp bottom portion of manum pins works best in dissection. The correct length can be attained by inserting longer pins into the gel and cutting off any portion of the pin that protrudes.You will also need one pair of sharp microdissection spring scissors, and two sets of fine tipped forceps. Use A small metal spatula to transfer wandering. Third in star larvae expressing A GFP tagged SLE protein to a Petri dish.Next, apply deionized water to the larvae in the Petri dish to get rid of all the food. Then using forceps, transfer Them to a dissecting gel cube to perform The live dissection. Use forceps to transfer a larvae to the dissection gel cube.On a glass slide, add dissection buffer to the slide to immerse it. Next, pin the lava at the anterior and posterior ends with the dorsal side up. Using microdissection scissors, make a small cut at the midline near the posterior end.From this incision, make another cut through to the anterior. The gut intestines and fat bodies will ooze out using two pins. Fix the cut cuticle the lateral edges to the sil guard cube.Use a pipette to add a drop of dissecting buffer. Then using forceps carefully remove the gut intestines and fat bodies. Apply a kim wipe to one side of the lava to remove the buffer while simultaneously adding fresh dissecting buffer with a pipette.The lava should never be dry. To label mitochondrial or lysosomal structures in the lava, replace the dissecting buffer with dissecting buffer containing mito tracker or lyo tracker. Place the dissecting cube in a square humidified chamber and cover it in aluminum foil.Incubate for 10 minutes at room temperature. After the incubation of the in vivo marker, repeatedly soak up dissection buffer surrounding the larvae and add more dissection buffer with a pipette. Repeat this five times to wash off any remaining marker.After replacing the last wash, push the pins completely into the S guard and immediately place a cover slip to avoid larva movement. The larva Is now ready for observation To perform live imaging of the dissected lava. Place the sample onto the stage of an inverted microscope to image tagged vesicles.Within larval segmental nerves. Perform imaging using a 100 times objective lens. If robust, GFP staining is observed in the cell bodies in the ventral ganglion image, the segmental nerves using a dual view system with filters for C-F-P-Y-F-P or R-F-P-Y-F-P and split view software two in vivo tagged proteins can be viewed simultaneously.In this time lapse series are many axonal vesicles on many axonal tracks within a larval segmental nerve. Note that GFP representing axonal accumulations also note the bidirectional movement of vesicles. Here, the movement of axonal vesicles on a single track within a larval segmental nerve are seen.Note, the bidirectional movement movement of these sles are on average one micron per Second. After watching this video, you should have a good understanding of how to complete a clean dissection for in vivo viewing of GFP tagged vesicles and gsof larval axons.

in-vivo-visualization-synaptic-vesicles-within-drosophila-larval

Research

JoVE Journal

Neuroscience

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Learn live dissection of Drosophila larvae for imaging GFP-tagged synaptic vesicle transport in axons using fluorescence microscopy. This method enables real-time visualization of axonal dynamics in neuroscience research.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

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