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>
2. Dissection of Larvae:
<ol>
 <li>Select wandering third instar larvae that are crawling around the sides of the vial and have not begun to pupate.</li>
 <li>Place a larva on the dissection surface under a stereomicroscope. Wick away excess moister using a Kimwipe.</li>
 <li>Arrange each larva so that the dorsal side is facing up. You will be able to see two trachea, white lines along the dorsal side of the larvae. Try to center these in the middle as much as possible because this marks the dorsal midline.</li>
 <li>Place pin #1 just above the mouth hooks (Figure 1).</li>
 <li>Place pin #2 in the tail just above the spiracles between the tracheae.</li>
 <li>Add a small amount of PBS to the larvae to prevent drying of the cuticle.</li>
 <li>Repeat the above steps with all the larvae, making sure that the genotypes are separated spatially by placing a pin between each genotype.</li>
 <li>Make a small transverse cut right above pin #2 across both tracheas (Figure 1).</li>
 <li>Insert your scissors in the first cut and cut along the dorsal midline between the tracheas all the way up past pin #1.</li>
 <li>You will need to make other small cuts at the very top and bottom of each side of the cuticle, so that the left and right sides will open up allowing you to pin them down without pulling on the cuticle.</li>
 <li>Place pin #'s 3, 4, 5, and 6 at each corner.</li>
 <li>Fix the sample with 4% PFA for 20-25 minutes at room temperature</li>
 <li>Decant the PFA and rinse with PBS 3-4 times, decanting each time.</li>
</ol>
3. Removal of Tissue:
<ol>
 <li>Start by removing tissues at the posterior end because typically you want to protect the area where the brain is located.</li>
 <li>While using a steromicroscope and with the larvae under a drop of PBS, first grab the posterior end of one of the trachea and pull it out which may remove some of the fat body with it. Repeat for the second trachea.</li>
 <li>Grab some of the posterior fat body or intestine and pull them out of the prep. Once most of the posterior tissue has been removed, focus on removing the small tissues around the brain. If you are only interested in the muscles, or morphology of the NMJ you can remove the brain as well.</li>
 <li>Removal of the brain allows for better visualization of Muscle 31, which is a helpful marker for abdominal segment orientation.</li>
</ol>
4. Immunofluorescence for Active Zones:
<ol>
 <li>Add blocking agent (5% Normal Goat Serum in PBS) to the dissected preps in the dissection dish. Block for 1 hour at room temperature. If you would like to only make one solution for blocking and antibody dilutions, include 0.1% TritonX-100 to your blocking agent for tissue permeabilization.</li>
 <li>Apply the Primary antibody diluted in blocking reagent with 0.1% TritonX-100. The active zone antibody is attainable through the Developmental Studies Hybridoma Bank (DSHB, http://dshb.biology.uiowa.edu/). Use a reference antibody (e.g. horseradish peroxidase-Cy3 conjugate) to label the NMJ membrane and axons.</li>
 <li>Incubate the primary antibodies at 4&deg;C overnight. So that the dryness of the refrigerator will not evaporate the antibody solution, create a humidity chamber by putting some water in the bottom of a used pipette tip box. Cover the translucent lid with aluminum foil to reduce fluorescent bleaching. Place the dissection slide in the humidity chamber and put the whole box in the 4&deg;C incubator overnight.</li>
 <li>The next morning, wash with PBST (PBS with 0.2% Triton-X-100) 5-10 times for 5-10 minutes each.</li>
 <li>Incubate with the secondary antibody (e.g. Invitrogen Alexa Goat anti-Mouse-488) diluted in blocking reagent with 0.1% TritonX-100 at room temperature for 2-4 hours in the covered humidity chamber. Include a nuclear stain (e.g. DAPI) in the secondary, if you would like to identify the location of the nucleus in the muscles or brain.</li>
 <li>Wash with PBST (PBS with 0.02% Triton-X-100) 5-10 times for 5-10 minutes each.</li>
</ol>
5. Mounting the Larval Samples on Slides:
<ol>
 <li>Decant and wick off the last PBST wash.</li>
 <li>Pipette approximately 1mL 80% glycerol to cover the pinned down larval pelts.</li>
 <li>Remove 5 of the 6 pins in all the larvae.</li>
 <li>When you are ready to put the first genotype on a slide remove the last pin for all the larvae of that genotype.</li>
 <li>Prepare a "wash" slide by putting two drops of your mounting medium on a clean slide. This step is important to wash the water solution off the larvae for mounting and clearer imaging.</li>
 <li>Grab one larva by the tail with your blunt forceps, drag the larva around in the glycerol and then along the dry edges of your dissection dish, to remove a majority of the glycerol. Place the larvae in the first mounting medium drop.</li>
 <li>Repeat step 6 for all of the larvae of the same genotype.</li>
 <li>Once all the larvae are in the first mounting medium drop, grab the first larva by the tail and pull it through the drop and then along the dry edges of the dish to remove a majority of the medium. Then place the larvae in the second mounting medium drop.</li>
 <li>Repeat step 8 for all the larvae with the same genotype.</li>
 <li>Prepare your final slide by labeling it and placing a small "T" shape of mounting medium on the slide.</li>
 <li>As in step 5.8, grab the larva by the tail; pull the pelt through the second mounting medium drop and then along the dry sides of the "Wash" slide. After the majority of the medium has come off, place the larval pelt on the final slide in the mounting medium.</li>
 <li>Repeat for all larval pelts.</li>
 <li>Make sure that all the larval pelts are arranged as desired making sure that the cuticle side is down and the exposed muscle is facing up.</li>
 <li>Place a cover slip on the slide to cover the dissected larvae, with the starting edge in the upper line of the "T."</li>
 <li>Put a small weight (e.g. 9 volt battery) on the cover slip, making sure that the mounting medium spreads completely under the cover slip.</li>
 <li>To reduce possible fluorescent quenching, place the slide in a drawer or dark space until the mounting medium has cured.</li>
 <li>Clean up the mounting medium that overflowed the edged of the cover slip. Clean off excess dry mounting medium on the cover slip or slide with 70% ethanol.</li>
 <li>To create a final seal, seal with fingernail polish.</li>
</ol>
6. Representative Results:
An example slide of dissected and immunostained larvae is shown in Figure 2. When the larvae dissections, stains, and mounting are done properly, the user can identify the larvae muscle of interest (Figure 3) in segments A2-A6. Hoanget al. provides a detailed description of muscle position as well as characterization of specific synaptic boutons (Hoang and Chiba, 2001). Using a high power objective the user can focus on a single synapse and take z-stack image. A representative maximum projection image of a portion of synapse 6/7 is shown (Figure 4). Then using imaging software, the active zones can be quantitatively evaluated for characteristics that are particular to your mutant phenotype. Examples of features that may be altered in a particular mutant phenotype include the total number, flourescence intensity, and spacing between active zones.
Potential Pitfalls:
Immunofluorescence evaluation of the larval neuromuscular junction is a valuable system that can provide valuable insight into synaptic biology, although there are potential pitfalls that can limit the utility of this system. For example, frequently it is desirable to image the staining pattern of two or more primary antibodies in the same sample. The choice of appropriate primary antibodies, appropriate fluorophores, and appropriate negative controls is essential to success.
If imaging two antigens in your experiment, be certain that the primary antibodies are from different species so that each protein of interest can be uniquely identified with a fluorescently labeled secondary antibody. A recommended way to reduce background is to use a blocking agent containing the normal serum of the species from which the secondary antibodies were derived (e.g. use normal goat serum as a blocking agent when using goat anti-rabbit or anti-mouse secondary antibodies).
After planning the primary and secondary antibodies that you will use in your experiment, you must also consider including controls in your staining sets to ensure that the fluorescent signal that you are seeing is real and not just background. If possible, include negative control samples that do not contain the protein of interest. This is easily done in studies of exogenous transgene overexpression by including a wild type animal, but in some cases can be accomplished by using a homozygous mutant of your protein of interest. Another essential negative control, useful for measuring the background levels of your fluorescent signal, is to use the secondary antibody without including the primary. When imaging your resulting preps, it is important to understand that all of the fluorescent signal that you are seeing might not be real, and might be background from your secondary antibodies or from endogenous tissue background. You may need to normalize your images to consider background fluorescence when assaying for changes between fluorescence intensity.
Choice of fluorophore must also be made with care. Consideration of the excitation and emission spectra for each fluorophore and the ability to uniquely detect each signal is essential to avoid channel bleed through. If there is any uncertainty, a simple way to determine whether the extent of bleed through is to immunostain with a single fluorophore-labeled antibody at a time and check the image with each channel that you will be using in your final experiment. This should reveal if bleed through is occurring with your microscope filter set.
 <img src="/files/ftp_upload/2676/2676fig1.jpg" alt="Figure 1" /> 
Figure 1. Flow chart of cuticle cutting and placement of pins 1-6. <a href="https://www.jove.com/files/ftp_upload/2676/2676fig1_large.jpg">Click here to view a larger image</a>
 <img src="/files/ftp_upload/2676/2676fig2.jpg" alt="Figure 2" /> 
Figure 2. Placement of larvae on the final slide. Make sure that the larvae preps are exposed muscle side up and cuticle side down. Leave at least a larval body width in-between each sample.
 <img src="/files/ftp_upload/2676/2676fig3.jpg" alt="Figure 3" /> 
Figure 3. When imaging, locate the muscles and segments of interest. Common neuromuscular junctions characterized innervate muscles 6/7, muscle 13, muscle 12, or muscle 4. On the opposite side of the ventral midline, there is a mirror image muscle structure of the shown hemi-segment. Use Muscle 31 as a marker to identify which segment you are imaging. Image the same synapses and segments for the rest of your samples, collecting many synaptic images per genotype that can be used for quantification.
 <img src="/files/ftp_upload/2676/2676fig4.jpg" alt="Figure 4" /> 
Figure 4. A representative maximum projection image of a portion of the NMJ innervating muscle 6/7 is shown. NC82 (Bruchpilot) staining is shown in green. HRP pre-synaptic stain is shown in red. Note the active zone punctae that can be further quantified by imaging software.

<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>

No conflicts of interest declared.

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 ...

dissection-imaging-active-zones-drosophila-neuromuscular

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Neuroscience

15.3K Views.  St. Jude Children’s Research Hospital. Нервно-мышечном соединении (NMJ) из Дрозофилы Является важной модельной системой для изучения нормальной синаптической функции, а также возмущения в синаптической функции найдены в некоторых неврологических заболеваний. Мы представляем протокол вск?...

Video: Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...