Name: whole mount preparation of the adult drosophila ventral nerve cord for giant fiber dye injection
Uploaded: 2011-06-04T14:00:00
Description: Watch this Scientific Journal Video about Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection at JoVE.com

The project described was supported by Grant Number R01HD050725 from the National Institute of Child Health and Human Development to T.A.G. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Child Health and Human Development or the National Institutes of Health. We thank members of the Godenschwege lab as well as Barbara Schreader for their input to the manuscript and video.

1. Dissection of the ventral nerve cord
Dissection and dye filling of the GF can be performed at room temperature. For all dissection steps cold (4&deg;C) saline should be used to slow down cell metabolism and keep the neurons alive longer.
<ol>
 <li>Anesthetize adult flies (4 days or older) with CO2 or chill them on ice until they are immobilized. Anesthetizing with ice will require more 
 time (up to 30 min) but will keep the flies immobilized for longer.</li>
 <li>Place one fly...

<ol>
	<li>Power, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thoracico-abdominal+nervous+system+of+an+adult+insect+Drosophila+melanogaster.">The thoracico-abdominal nervous system of an adult insect Drosophila melanogaster.</a> J. Comp. Neurol. 88, 347-409 (1948).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, T. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: development and function of the Drosophila giant fibre system.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Boerner, J., Duch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Average+shape+standard+atlas+for+the+adult+Drosophila+ventral+nerve+cord.">Average shape standard atlas for the adult Drosophila ventral nerve cord.</a> J. Comp. Neurol. 518, 2437-2455 (2010).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+synaptic+transmission+in+adult+Drosophila+Kenyon+cells+in+sity.">Cholinergic synaptic transmission in adult Drosophila Kenyon cells in sity.</a> J. Neurosci. 26, 265-272 (2006).</li><li>Boerner, J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+for+the+Drosophila+ventral+nerve+cord+standard+in+neuronal+circuit+reconstruction+and+in-depth+analysis+of+mutant+morphology.">Application for the Drosophila ventral nerve cord standard in neuronal circuit reconstruction and in-depth analysis of mutant morphology.</a> J. Neurogent. 24, 158-1567 (2010).</li><li>Godenschwege, T. A., Kristiansen, L. V., Uthaman, S. B., Hortsch, M., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conderved+role+for+Drosophila+Neuroglian+and+human+L1-CAM+in+central-synapse+formation.">A conderved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.</a> Curr. Biol. 16, 12-23 (2006).</li><li>Uthaman, S. B., Godenschwege, T. A., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+distinct+from+highwire+for+the+Drosophila+ubiquitin+conjugase+bendless+in+synaptic+growth+and+maturation.">A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation.</a> J. Neurosci. 28, 8615-8623 (2008).</li><li>Storkebaum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mutations+in+the+tyrosyl-tRNA+synthetase+gene+recapitulate+in+Drosophila+features+of+human+Charcot-Marie-Tooth+neuropathy.">Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot-Marie-Tooth neuropathy.</a> Proc. Natl. Acad. Sci. USA. 106, 11782-11787 (2009).</li><li>Allen, M. J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+Drosophila+giant+fiber+system+(GFS).">Electrophysiological recordings from the Drosophila giant fiber system (GFS).</a> Cold Spring Harb. Protoc. 2010, pdb.prot5453-pdb.prot5453 (2010).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. , (2011).</li></ol>

The nervous system can be dissected without extracting it from the fly body. This has two advantages, first, the dissection causes little damage to the nervous system, and second, most nerves stay attached to the muscles and sensory organs. Performing the dissection, as described, prepares the sample for straight forward dye-labeling of the GFs. Conveniently, the motoneurons postsynaptic to the GFs stay attached to the muscles. Axons, therefore, are undamaged, keeping the neurons alive longer. In addition, damage to the ...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>AmScope</td>
			<td>SM-2TZ</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vannas Scissors Superfine</td>
			<td>Academic Instruments</td>
			<td>VS1023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont Forceps Dumontstar 55</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Austerlitz Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>&Oslash; 0.1mm</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Electrodes</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vertical Pipette Puller 700c</td>
			<td>David Kopf Instruments</td>
			<td>Model 700C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lucifer Yellow CH dilithium salt</td>
			<td>Sigma</td>
			<td>L0259</td>
			<td>1% in H2O</td>
		</tr>
		<tr>
			<td>Fixed Stage Upright Microscope & Camera</td>
			<td>Nikon</td>
			<td>FN-1 & DS-U2 Camera</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plan Fluor 10X Air Objective</td>
			<td>Nikon</td>
			<td>CFI Plan Fluor 10X NA 0.3 WD 16mm</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluor 40X water dipping Objective</td>
			<td>Nikon</td>
			<td>CFI Fluor 40X W NA 0.80 WD 2.0mm</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3D hydraulic Micromanipulator</td>
			<td>Narishige International</td>
			<td>Model MW0-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Getting Instruments, Inc.</td>
			<td>Model 5A</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

whole mount preparation of the adult drosophila ventral nerve cord for giant fiber dye injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Watch this Scientific Journal Video about Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection at JoVE.com

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

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