Name: dissection and imaging of active zones in the drosophila neuromuscular junction
Uploaded: 2011-04-27T16:00:00
Description: Watch this Scientific Journal Video about Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction at JoVE.com

We thank Dr. Nael Alami and Dr. Nam Chul Kim for their helpful comments about this manuscript.

1. Preparation for Immunofluorescence:
<ol>
 <li>To create a dissecting surface, pour Sylgard 184 Silicone Elastomer Base into a small tissue culture plate. Make sure not to fill the plate completely so that the dissection area is lower than the rim.</li>
 <li>Trim dissection pins to a length of approximately 3.5mm for increased ease of manipulation, and make sure to have at least 6 pins per larva. </li>
 <li>You will need blunt forceps that will allow to you grasp the pins for the dissection.</li>
</ol>
<p class="jov...

<ol>
	<li>Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+maturation+and+structural+plasticity+at+Drosophila+neuromuscular+junctions.">Synapse maturation and structural plasticity at Drosophila neuromuscular junctions.</a> Curr Opin Neurobiol. 6, 858-867 (1996).</li><li>Fouquet, W., Owald, D., Wichmann, C., Mertel, S., Depner, H., Dyba, M., Hallermann, S., Kittel, R. J., Eimer, S., Sigrist, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+active+zone+assembly+by+Drosophila+Bruchpilot.">Maturation of active zone assembly by Drosophila Bruchpilot.</a> J Cell Biol. 186, 129-145 (2009).</li><li>Hoang, B., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Analysis+of+Drosophila+Larval+Neuromuscular+Synapses.">Single-Cell Analysis of Drosophila Larval Neuromuscular Synapses.</a> Developmental Biology. 229, 55-70 (2001).</li><li>Keshishian, H., Kim, Y. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchestrating+development+and+function:+retrograde+BMP+signaling+in+the+Drosophila+nervous+system.">Orchestrating development and function: retrograde BMP signaling in the Drosophila nervous system.</a> Trends in Neurosciences. 27, 143-147 (2004).</li><li>Kittel, R. J., Wichmann, C., Rasse, T. M., Fouquet, W., Schmidt, M., Schmid, A., Wagh, D. A., Pawlu, C., Kellner, R. R., Willig, K. I., Hell, S. W., Buchner, E., Heckmann, M., Sigrist, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruchpilot+Promotes+Active+Zone+Assembly,+Ca2++Channel+Clustering,+and+Vesicle+Release.">Bruchpilot Promotes Active Zone Assembly, Ca2+ Channel Clustering, and Vesicle Release.</a> Science. 312, 1051-1054 (2006).</li><li>Koh, Y. H., Gramates, L. S., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+neuromuscular+junction:+Molecular+components+and+mechanisms+underlying+synaptic+plasticity.">Drosophila larval neuromuscular junction: Molecular components and mechanisms underlying synaptic plasticity.</a> Microscopy Research and Technique. 49, 14-25 (2000).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann N Y Acad Sci. 1184, e1-e20 (2010).</li><li>Ratnaparkhi, A., Lawless, G. M., Schweizer, F. E., Golshani, P., Jackson, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+ALS:+human+ALS-associated+mutation+in+VAP33A+suggests+a+dominant+negative+mechanism.">A Drosophila model of ALS: human ALS-associated mutation in VAP33A suggests a dominant negative mechanism.</a> PLoS One. 3, e2334-e2334 (2008).</li><li>Wagh, D. A., Rasse, T. M., Asan, E., Hofbauer, A., Schwenkert, I., D&#252;rrbeck, H., Buchner, S., Dabauvalle, M. -. C., Schmidt, M., Qin, G., Wichmann, C., Kittel, R., Sigrist, S. J., Buchner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruchpilot,+a+Protein+with+Homology+to+ELKS/CAST,+Is+Required+for+Structural+Integrity+and+Function+of+Synaptic+Active+Zones+in+Drosophila.">Bruchpilot, a Protein with Homology to ELKS/CAST, Is Required for Structural Integrity and Function of Synaptic Active Zones in Drosophila.</a> Neuron. 49, 833-844 (2006).</li><li>Zweier, C., Jong, E. K. d. e., Zweier, M., Orrico, A., Ousager, L. B., Collins, A. L., Bijlsma, E. K., Oortveld, M. A., Ekici, A. B., Reis, A., Schenck, A., Rauch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNTNAP2+and+NRXN1+are+mutated+in+autosomal-recessive+Pitt-Hopkins-like+mental+retardation+and+determine+the+level+of+a+common+synaptic+protein+in+Drosophila.">CNTNAP2 and NRXN1 are mutated in autosomal-recessive Pitt-Hopkins-like mental retardation and determine the level of a common synaptic protein in Drosophila.</a> Am J Hum Genet. 85, 655-666 (2009).</li></ol>

For neurons, the synaptic terminal area is of critical importance, and is the bridge for proper communication between the post- and pre-synaptic cells. A powerful way to investigate the health of the neuron in disease models is to analyze proteins of the synaptic terminal by immunofluorescence. The immunofluorescence method presented here enables the researcher to examine many larvae simultaneously while limiting the environmental differences between groups. The central nervous system ofDrosophila third-instar ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Base</td>
			<td>Dow Corning</td>
			<td>68037-59-2</td>
			<td>After mixing allow for bubbles to rise slowly out by putting on slow rotator or allowing to sit for 30 minutes or more.</td>
		</tr>
		<tr>
			<td>Stainless Steel Minutien PIns</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>Trim to approx. 3-4mm in length with regular scissors</td>
		</tr>
		<tr>
			<td>Laminectomy Forceps (Blunt- Used for grasping pins)</td>
			<td>Fine Science tools</td>
			<td>11223-20</td>
			<td>Use as blunt forceps for grasping pins</td>
		</tr>
		<tr>
			<td>Dissection Forceps</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFine Vannas Scissors, 8cm long</td>
			<td>World Precision Instruments</td>
			<td>501778</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Brp antibody</td>
			<td>DSHB</td>
			<td>NC82</td>
			<td>Use 1:50 dilution</td>
		</tr>
		<tr>
			<td>Cy3 Affinipure Goat Anti-Horseradish Peroxidase</td>
			<td>Jackson Immunoresearch</td>
			<td>123-165-021</td>
			<td>Use at 1:200 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td>Use approx. 1:200 dilution</td>
		</tr>
	</tbody>
</table>

dissection and imaging of active zones in the drosophila neuromuscular junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Watch this Scientific Journal Video about Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction at JoVE.com

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

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