Name: drosophila larval nmj dissection
Uploaded: 2009-02-04T00:00:00
Description: Watch this Scientific Journal Video about Drosophila Larval NMJ Dissection at JoVE.com

Before you start
<ol>
<li>All cultures should be grown at 25&deg; C.</li>
<li>HL3.1 dissection buffer may be prepared in advance. Take a 50 ml aliquot of HL3.1 and keep it on ice.</li>
<li>Prepare 15 ml of fresh 3.5% Formaldehyde in HL3.1. Keep it on ice.</li>
</ol>
Larval Dissection
<ol>
<li>Put a drop of cold HL3.1 on the dissection plate. This will keep the animal from drying and stun the animal making it easier to work with.</li>
<li>Using the long forceps, select a wande...

<ol>
	<li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+larval+neuromuscular+junction+in+Drosophila+Melanogaster.">Properties of the larval neuromuscular junction in Drosophila Melanogaster.</a> J. Physiol. 262, 189-214 (1976).</li><li>Johansen, J., Halpern, M. E., Johansen, K. M., Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotypic+morphology+of+glutamatergic+synapses+on+identified+muscle+cells+of+Drosophila+larvae.">Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae.</a> J Neurosci. 9, 710-725 (1989).</li><li>Kesheshian, H., Broadie, K., Chiba, A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+neuromuscular+junction:+a+model+system+for+studying+synaptic+development+and+function.">The Drosophila neuromuscular junction: a model system for studying synaptic development and function.</a> Annu Rev Neurosci. 19, 545-575 (1996).</li><li>Vactor, D. V., Sink, H., Fambrough, D., Tsoo, R., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+that+control+neuromuscular+specificity+in+Drosophila.">Genes that control neuromuscular specificity in Drosophila.</a> Cell. , 73-1137 (1993).</li><li>Feng, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18 (2), 377-402 (2004).</li></ol>

The dissection technique demonstrated in this video can be used to prepare Drosophila larvae for a variety of experimental techniques. If fluorescent protein tags are present, the larvae can be mounted and imaged immediately. Otherwise, immunostaining can be performed in order to mark specific synaptic compartments. In addition, electrophysiology can easily be performed on the dissected larvae to evaluate the functioning of neurotransmission.
The NMJ of Drosophila has gained...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>Stereomicroscope &#x201C;Stemi&#x201D; 2000</td>
<td>&nbsp;</td>
<td>Carl Zeiss MicroImaging Inc.</td>
<td>495101-9804-000</td>
<td>&nbsp;</td>
<tr>
<td>Light Source KL 1500 LCD</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>000000-1063-181</td>
<td>&nbsp;</td>
<tr>
<td>3mm Vannas Spring Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools Inc.</td>
<td>15000-0</td>
<td>&nbsp;</td>
<tr>
<td>Dumont SS Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools Inc.</td>
<td>11200-33</td>
<td>Long forceps</td>
</tr>
<tr>
<td>Dumont #5 Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools Inc.</td>
<td>11252-20</td>
<td>Short forceps</td>
</tr>
<tr>
<td>SylGard 182 Silicone Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corp. </td>
<td>3097358-1004</td>
<td>Used to make dissection plates</td>
</tr>
<tr>
<td>Formaldehyde </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich Inc.</td>
<td>252549-25ML</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Minutien Pins -0.1 MM Diameter</td>
<td>&nbsp;</td>
<td>Fine Science Tools Inc.</td>
<td>26002-10</td>
<td>&nbsp;</td>		</tbody>
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			HL3 Dissection Buffer: (in mM) 70 NaCl, 5 KCl, 0.2 CaCl2, 20 MgCl2, 10 NaHCO3, 5 trehalose, 115 sucrose, and 5 HEPES, pH 7.3. 

Sylgard Dissecting Plate: Mix gently (to avoid bubbles) 10:1in a beaker. Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37C incubator.
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drosophila larval nmj dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

Watch this Scientific Journal Video about Drosophila Larval NMJ Dissection at JoVE.com

Drosophila Larval NMJ Dissection

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

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