We thank Eric Roberts for advice on imaging. We also wish to thank Information Technology Services at Rhode Island College and in particular Michael Caine and Jake Douglas for videography. Monoclonal antibodies anti-dlg and anti-brp were developed by UC-Berkeley and Universitaetsklinikim Wuerzburg respectively, and were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242, USA. The research reported here was supported fully by the Rhode Island Institutional Development Award (IDeA) Network of Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes of Health under grant number [P20GM103430].

The procedures for preparing working solutions used in conjunction with this protocol are described in Table 1.
<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td colspan="3">Preparation</td>
			<td colspan="2">Storage</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td colspan="3">Make a working stock of 1x PBS from a 10x PBS stock solution by diluting in distilled water.&#160; The pH of the working PBS stock should be 7.2- 7.4</td>
			<td colspan="2">4 &#176;C for 1+ months until bacterial contamination is visible.</td>
		</tr>
		<tr>
			<td>PBT</td>
			<td colspan="3">1x PBS solution with 0.1% non-ionic surfactant.</td>
			<td colspan="2">4 &#176;C for 1+ months until bacterial contamination is visible.</td>
		</tr>
		<tr>
			<td rowspan="2">FA</td>
			<td colspan="3">3.7% formaldehyde solution made from a 37% formaldehyde stock and diluted in 1x PBS.</td>
			<td colspan="2" rowspan="2">Room temperature. Make fresh each day of dissection</td>
		</tr>
		<tr>
			<td colspan="3">CAUTION: Formaldehyde supplied as 37% stock solution is a potential carcinogen and should be diluted in a fume hood.</td>
		</tr>
		<tr>
			<td>5% NGS</td>
			<td colspan="3">5% normal goat serum diluted in PBT.&#160; The serum used should match the species of the secondary antibody to be used.</td>
			<td colspan="2">4 &#176;C for several weeks until bacterial contamination is visible</td>
		</tr>
	</tbody>
</table>
Table 1. Solutions for performing immunocytochemistry of the adult Drosophila leg. 
1. Initial fixation of tissues
<ol>
	<li>Select approximately 10 flies for each genotype and age. Anesthetize on a fly pad under carbon dioxide (Figure 1A, B). 
	NOTE: Begin with more flies than necessary to ensure a large enough sample size after dissection.</li>
	<li>Using a paintbrush, transfer flies to cold methanol in a glass well or dish for approximately 30 seconds to 1 minute (Figure 1C). The methanol solubilizes the cuticular hydrocarbons and flies can now be submerged rather than float in aqueous solutions.</li>
	<li>With forceps, carefully transfer flies to PBS. Rinse 3x in ice-cold PBS to remove excess methanol and keep flies in PBS on ice until dissection and fixation. At this point, dissect flies and fix within the shortest time possible (&#60;30 minutes).</li>
	<li>To isolate legs, transfer flies to a silicone elastomer dissection dish filled with cold PBS, remove the metathoracic legs at the coxa using two pairs of #5 Dumont forceps or cut the legs with Vanna scissors (Figure 1D). Transfer the legs to a well in a plastic 24 well plate filled with 1 mL of PBS and keep the plate on ice until all legs are removed and transferred to wells. 
	NOTE: Each well can hold at least 20 legs.</li>
	<li>Replace the PBS solution with 1 mL of FA solution and rotate on a nutator for 30 minutes (Figure 1E). The nutator setting should be set at a medium speed (17 rpm). Ensure the legs are completely submerged in FA solution during this time for adequate fixation.</li>
	<li>To remove FA solution, wash in 1 mL of PBS 3x quickly followed by an additional 3 washes for 5 minutes each in 1 mL of PBS. Hold tissues in 1 mL of PBS on ice prior to, and during the dissection steps described below.</li>
</ol>
2. Removing the Leg Cuticle and Antibody Staining
<ol>
	<li>Removing the leg cuticle
	<ol>
		<li>The dissecting forceps are critical for success. Introduce slight parallel bends in both prongs at the end of #5 super fine forceps to provide a bevel that allows the cuticle to be grabbed superficially rather than be poked, which can ruin the tissue (Figure 1F, G). 
		NOTE: Prongs bent in parallel should still make contact with each other throughout the length of the prong when closed (Figure 2).</li>
		<li>Transfer legs to a silicone elastomer dish in PBS for dissection. Orient a leg so that the anterior side is facing up (see Figure 3 for leg anatomy and orientation information). Using one pair of forceps, hold the tibia segment against the silicone elastomer dish. Using the other forceps held bevel side down, grab a piece of cuticle on the distal end of the femur and pull in the proximal direction toward the trochanter.</li>
		<li>Keep methodically removing cuticle until the naked muscle is visible throughout the proximal end of the femur (Figure 1F, G, H). 
		NOTE: Only make superficial contact with the legs using the beveled side of the forceps to avoid pulling muscle.</li>
		<li>Once all legs are dissected, replace PBS with FA to post-fix legs for 30 minutes with shaking on a nutator at medium speed (Figure 1I). Wash samples in 1 mL of PBS for 3 times quick and then 3 times for 5 minutes each in PBT (Figure 1J). 
		NOTE: If staining with monoclonal antibody NC82 (anti-bruchpilot) to label active zones, post-fix for 20 minutes as this antigen is sensitive to longer fixations.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62844/62844fig02v2.jpg" /> 
Figure 2. Modified forceps used for dissecting adult legs. (A) The ends of the forceps are bent and then flattened at the bottom (arrow) creating a bevel by filing on a sharpening stone. (B) In contrast, the prongs of unmodified forceps are not bent. Scale bar = 1 mm <a href="https://www.jove.com/files/ftp_upload/62844/62844fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Antibody staining
	<ol>
		<li>To block tissues for antibody staining, replace 1 mL of PBT with blocking solution consisting of 1 mL of 5% NGS diluted in PBT. Incubate dissected legs for 4 hours at room-temperature or overnight at 4 &#176;C while rocking on a nutator at medium speed (17 rpm). During all incubations, cover wells with sealing tape in addition to the plastic cover (Figure 1J). 
		NOTE: 24 well plates are used for immunocytochemistry rather than 1.5 mL or 2 mL microcentrifuge because previous attempts to use microcentrifuge tubes resulted in broken legs and damaged tissue.</li>
		<li>Remove blocking solution and add 300 mL of primary antibodies diluted in fresh blocking solution. The small volume of antibody used should be sufficient to cover the tissues. Re-seal wells with laboratory sealing tape and plastic lid and incubate overnight at 4 &#176;C with shaking on a nutator at medium speed (Figure 1J). The working antibody reagent information and concentrations used in for these studies are described in Table 2.</li>
		<li>Wash primary antibodies in 1 mL of PBT for 3 times briefly and then 3 times for 15 minutes each (Figure 1J). 
		NOTE: Diluted primary antibodies can be saved and reused if stored at 4 &#176;C for up to 2 weeks.</li>
		<li>Block tissues again in 1 mL of 5% NGS for at least 2 hours at room temperature or overnight at 4 C (Figure 1J).</li>
		<li>Remove the 5% NGS blocking solution and add 300 &#181;L of the appropriate diluted fluorescent conjugated secondary antibodies. Additionally, add 1:2000 dilution of fluorescence-conjugated phalloidin to label muscle (Figure 1J).</li>
		<li>For secondary antibody incubations, seal wells with laboratory sealing tape and lid. Also, wrap plates in aluminum foil to protect fluorophores from light. Incubate for 6-8 hours at room-temperature or overnight at 4 &#176;C.</li>
		<li>Wash secondary antibodies and phalloidin as described in step 2.2.3 above. Cover plates with aluminum foil in between washes to protect fluorophores from light.</li>
	</ol>
	</li>
</ol>
3. Mounting Legs
<ol>
	<li>Transfer legs to a slide using forceps and orient anterior side up. Cover the legs with mounting media (Figure 1K, L). 
	NOTE: The dissected legs can be aspirated with a P1000 pipette tip if the bottom bore is widened by cutting with a razor blade.</li>
	<li>Add clay spacers to a 22x22 mm2 coverslip (#1.5 thickness) by scraping the coverslip corners across a small ball of modeling clay. Each corner should have a small amount of clay 1-2 mm thick (Figure 1K).</li>
	<li>To cover the slide, add the coverslip with the clay spacers facing towards the slide and carefully push on the corners until the coverslip just touches the surface of the femur.</li>
	<li>To prevent evaporation, seal the edges of the coverslip with nail polish and let dry in a dark place (about 10 minutes) before storing at 4 &#176;C until imaging.</li>
</ol>
4. Imaging
<ol>
	<li>Image by confocal microscope (Figure 1M). Include a transmitted light channel to better assess the quality of the dissection and samples with visibly disrupted muscle fibers in the area of interest should be discarded.
	<ol>
		<li>Begin imaging z-stacks with 20x magnification with 2x zoom and a total image depth of ~ 40 mm corresponding to the thickness of the femur. For fluorescent signal detection, capture images at resolutions consistent with Nyquist sampling (we are using 1024 x 1024 pixels with a dwell setting of 8-10 &#956;s/pixel). The signal intensity should be in the linear range which is achieved by adjusting the high voltage gain settings. Once the gain settings are set for a series of samples within an experiment, they should not be altered so that signal intensities can be compared between samples.</li>
	</ol>
	</li>
	<li>For imaging synaptic boutons and other subcellular structure, capture confocal images at &#8805;60x magnification. The detector settings should be in the linear range, while the pixel density, dwell settings and z-depth should be similar for images captured at lower magnification (step 4.1).</li>
</ol>

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The authors declare no conflicts of interest.

The Drosophila adult leg is an ideal model to study neurodegeneration given relative simplicity with well-characterized MNs mapped from neuroblast lineages and stereotyped arborization patterns. Several reports have previously used leg MNs for the study of neurodegenerative disease21,22. These studies utilized GFP-expressing lines combined with mosaic analysis with a repressible cell marker (MARCM) to image through the cuticle and documented a series of morphological changes. Imaging adult NMJs by immunocytochemistry with resected cuticle enables further characterization with the ability to track complex molecular changes using a toolbox of antibodies available.
The immunocytochemistry portion of this protocol is relatively standard and can be implemented independent of genotype (see23 for an excellent description of general antibody staining methods for use with Drosophila). Furthermore, parameters such as fluorescence intensity, axon branch length, and bouton numbers and size can be determined using a variety of available ImageJ macros once images are captured and detailed methods for quantitative analysis have been published (for example, see24,25,26). Thus, the dissection technique is the main innovation described here. Prior to dissection, flies are submerged in an alcohol to strip cuticular hydrocarbons. Both ethanol and methanol are commonly used for this purpose; however, we have only used methanol. Critical to dissection success are several factors: First, using modified forceps with a bevel allows for very superficial contact with the cuticle. Second, using a dissecting microscope capable of 60-100x total magnification so that the surface of the cuticle is clearly visible. For microscopes with lower maximum magnification, 2x objectives are available for most common brands and should be sufficient when combined with existing lenses. Third, the initial fixation step makes the cuticle brittle and easier to pull away without damaging muscle underneath. Over-fixation at this step makes the entire leg too stiff for effective dissection. Therefore, the initial fixation should be limited to 30 minutes. The formaldehyde fixative will not penetrate enough to effectively crosslink the underlying tissue during this short period and thus a second fixation step is necessary. Prior to the second fixation, tissues should be kept on ice to prevent degradation and changes in morphology. Fourth, we have found dissecting samples while cold is also important, likely for similar reasons in that cuticle is brittle and a small piece can be more easily removed.
With practice, we find ~50% of dissections will be usable within no discernable tissue damage. Although this percentage may seem low relative to some other tissues, the dissection procedure is rapid, and many legs can be processed in 30- 60 minutes. Therefore, even if success rates are low initially, it is feasible to obtain 4-5 good samples for each experimental group. However, a limitation may be the number of flies available at any given time if genotypes and/or age result in substantial lethality.
Another limitation is that we have not been able to dissect other areas of the leg beyond the proximal region of the femur due to size. Thus, we can study identified MN arbors innervating the TILM reliably and it is possible to dissect cuticle above the tibia depressor muscle with small changes to the way the leg is oriented when dissecting. However, accessing other regions of the leg have proved more difficult without disrupting axonal architecture during dissection.
Here, we present dissection methods to detect changes at the adult NMJ for defined MNs innervating the tilm using immunocytochemistry. The leg is useful as a simple system, innervated by only ~50 MNs and containing 14 muscles with well-described anatomy. The dissected leg preparation can be used across genotypes and a suite of antibodies are available for NMJ visualization without the need for building genotypically complex stocks of reporter gene constructs in mutant backgrounds. This approach will enable a more detailed characterization of changes at the NMJ for MN diseases and other age-related conditions.

Motor neuron (MN) diseases encompass a group of heterogenous conditions that include progressive degeneration leading to muscle wasting and paralysis as a primary clinical phenotype1. Although rare with a global prevalence of 4.5 per 100,000, this prevalence is expected to increase with an aging population2. Amyotrophic lateral sclerosis (ALS) is the most common MN disease (MND) and is typically fatal within a short time of diagnosis with no existing disease-modifying treatments available3. MNDs share in common a protracted presymptomatic phase with early molecular biomarker changes and functional imaging changes seen in patients4. Early presymptomatic cellular pathology is also observed in non-human disease models5,6,7,8. The study of early changes at the neuromuscular junction is important for understanding MN disease pathogenesis and may aid in developing early diagnostics and potential therapeutics.
A wealth of genetic and molecular tools exists in Drosophila to dissect the structure and function of the neuromuscular junction (NMJ, see9 for a review of the well-characterized larval NMJ). These tools combined with a short lifespan make Drosophila an excellent model to study neurodegenerative changes at the NMJ. Specifically, MNs innervating adult muscles are present throughout the ~90-day adult lifespan and are subject to normal aging processes10,11,12,13. The adult MNs therefore provide an opportunity to study slow degenerative changes in contrast to larval NMJs which exist for only a short ~1 week time-period prior to metamorphosis14,15.
Here, we describe a dissection procedure that allows us to conduct immunocytochemical analysis of MNs in the adult leg. Each adult leg is innervated by ~50 MNs, which synapse onto the associated leg muscular to drive locomotion. The leg anatomy, mechanical physiology, and neurobiology has been well described16,17,18. Axon arbors of leg MNs have previously been characterized by imaging through cuticle in back-filled or genetically labeled cell populations using the bipartite Gal4/UAS system and imaging methods have been published previously19. The dissection methods presented here preserves axon branching morphology and allows us to exploit a diverse range of antibodies to label different molecular components of the NMJ. Our previous work has focused on projections of a defined MN in the metathoracic (3rd) leg, which innervates the tibia levator muscle (tilm) and shows consistent arborization patterns and bouton numbers. Initially we studied age-dependent changes in Drosophila superoxide dismutase 1 (dsod1) mutants and found alterations consistent with dismantling of the NMJ20. These dissection methods offer the opportunity to better characterize slow degenerative changes at the NMJ for other ALS models, basic studies of aging and other MN associated diseases.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62844/62844fig01.jpg" /> 
Figure 1. Workflow summary for dissecting legs. See protocol for detailed steps. (A,B) Flies are selected and anesthetized. (C) Flies are transferred to methanol and washed with PBS. (D) The metathoracic legs are removed at the base of the coxa while visualized with a dissecting microscope (~30x magnification); scale bar = 500 &#956;m. (E) The legs are then fixed in 3.7% formaldehyde/PBS (FA) solution for 30 minutes within wells of 24-well plates and then FA is removed by washes with PBS. (F, G, H) Legs are transferred to silicone elastomer dissecting trays and a piece of cuticle is removed from the proximal femur using beveled forceps while visualized under a dissecting microscope at 80x; scale bar = 50 &#956;m. (I) Legs are post-dissection fixed in FA and washed in PBS and then PBT (PBS+ 0.1% non-ionic surfactant). (J) Legs are subjected to immunocytochemical staining. (K,L) Legs are transferred to a glass slide, cleared in mounting media, and covered with a coverslip containing clay spacers; scale bars = 2 mm and 500 &#956;m. (M)&#160;Legs are imaged by confocal microscopy. <a href="https://www.jove.com/files/ftp_upload/62844/62844fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>BP3991</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>Corning</td>
			<td>3473</td>
			<td>Hydrophobic, ultra-low attachment surface</td>
		</tr>
		<tr>
			<td>2x objective accessory</td>
			<td>Olympus</td>
			<td>110AL2X</td>
			<td>Screw-on attachment</td>
		</tr>
		<tr>
			<td>Anti-ATP5A primary antibody</td>
			<td>Abcam</td>
			<td>ab14748</td>
			<td>Mouse monoclonal</td>
		</tr>
		<tr>
			<td>Anti-bruchpilot primary antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82</td>
			<td>Mouse monoclonal</td>
		</tr>
		<tr>
			<td>Anti-discs large primary antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td>Mouse monoclonal</td>
		</tr>
		<tr>
			<td>Anti-hrp primary antibody</td>
			<td>Jackson Immuno Research</td>
			<td>123-605-021</td>
			<td>Alexa Fluor 647 conjugated polyclonal</td>
		</tr>
		<tr>
			<td>Anti-polyubiquitin (FK2) primary antibody</td>
			<td>Millipore Sigma</td>
			<td>04-263</td>
			<td>Mouse monoclonal</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>Objectives (NA):&#160; 10x (0.4), 20x (0.85), 40x (1.20), 60x (1.42), 100x (1.40)</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Corning</td>
			<td>285022</td>
			<td>160-190 mm thickness</td>
		</tr>
		<tr>
			<td>Dissecting forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>Dumont #5SF</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>BP531-500</td>
			<td>37% stock stabilized with methanol</td>
		</tr>
		<tr>
			<td>Goat anti-mouse secondary antibody</td>
			<td>Jackson Immuno Research</td>
			<td>115-545-146</td>
			<td>Alexa Fluor 488 conjugated</td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Novus Biologicals</td>
			<td>NB036768</td>
			<td>0.2 mm filtered</td>
		</tr>
		<tr>
			<td>Laboratory sealing tape</td>
			<td>Fisher Scientific</td>
			<td>03-448-254</td>
			<td>Parafilm M</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A413</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-123</td>
			<td>76mm x 25mm</td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Molecular Probes</td>
			<td>S36972</td>
			<td>Slowfade Diamond mounting media</td>
		</tr>
		<tr>
			<td>Nonionic surfactant</td>
			<td>Acros Organics</td>
			<td>215680010</td>
			<td>Triton-X 100</td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>Fisher Scientific</td>
			<td>S06622</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Invitrogen</td>
			<td>A34055</td>
			<td>Alexa Fluor 555- conjugated</td>
		</tr>
		<tr>
			<td>Sharpening stone</td>
			<td>Fine Science Tools</td>
			<td>29008-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>Electron Microscopy Sciences</td>
			<td>2423610</td>
			<td>Sylgard 184</td>
		</tr>
	</tbody>
</table>

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.

Figure 4 shows a representative example of a metathoracic leg stained with anti-hrp, anti-dlg, and phalloidin. For dissections that remove cuticle from the proximal portion of the femur, stereotyped arbors will be apparent near the tendon which is detected easily by autofluorescence. Note that antibody penetration into the leg occurs for a short distance beyond the region in which cuticle has been removed (Figure 4A). These regions can be imaged effectively when strong fluorescence signal is present. Imaging at low magnification (20x with a 2x zoom) allows an easy determination of 1) how much cuticle is removed, and 2) whether damage occurred during dissection. Increased magnification (60x) shows stereotyped projections onto the tilm (Figure 4B). Our work has focused on one MN, likely derived from the I- MN lineage which innervate the tilm in the proximal femur (box, Figure 4B and Figure 4C). Increasing magnification further (100x with 2x zoom) allows for effective visualization of synaptic boutons (Figure 4D).
To study morphological changes at the adult NMJ over time, we have previously used dsod1 mutants as a model of ALS. Bouton swelling occurs in aged dsod1H71Y&#160;mutants relative to dsod1null and dsod1+ (Figure 5A, B). At the larval NMJ, monoclonal antibody NC82 is often used to label active zones and this these structures can be easily visualized at the adult NMJ (Figure 5C). Weakly-positive HRP axon branches are abundant in dsod1H71Y mutants and these branches often show weak and diffuse BRP localization (arrows).
Important for neurodegeneration, commercially available antibodies can detect ubiquitinated proteins often found in aggregates, and we have detected ubiquitinated aggregates within terminal axons of MNs in aged mutant dsod1 flies as well as within muscle (Figure 6A). Also, antibodies labeling mitochondria can also detect morphological changes with age (Figure 6B).
For some preparations, muscle damage during the dissection makes the sample unusable. At first, such damage can be a common occurrence, but improvement occurs with practice. Examples of good dissections are show Figure 7A, C while poor dissections are shown in Figure 7B, D. Poor dissections cause disorganization of muscle fibers as detected by phase contrast microscopy (Figure 7B) or missing muscle fibers visualized by fluorescent-conjugated phalloidin (Figure 7D, arrow). The combination of using phalloidin to label muscle, and transmitted light images can help to detect such muscle damage which may not be apparent when viewing under a dissecting microscope.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62844/62844fig03.jpg" /> 
Figure 3. Anatomy of the Drosophila leg. (A) Diagram of the anterior side of a metathoracic leg, characterized by the presence of bristles in contrast to the prominent naked cuticle present on the posterior side. The segmented leg is comprised of the coxa (Cx), trochanter (Tr), femur (Fe), tibia (Ti), 5 tarsal segments (Ta1-5), and claw (Cl) in order from proximal (Pr) to distal (Di). Dorsal (D) and ventral (V) sides of the femur are also indicated. Scale bar =100 &#956;m. (B) Diagram of the femur containing the tibia levator (tilm), tibia depressor (tidm), long tendon muscle 2 (ltm2) and tibia reductor (tirm) muscles and axon projections in the proximal femur innervating the tilm. These stereotyped axonal projections are presumed to be derived from I-lineage neuroblasts16, and the second arborization is the easiest to access by dissection (boxed). Scale bar = 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/62844/62844fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62844/62844fig04.jpg" /> 
Figure 4. Dissected metathoracic femur revealed stereotyped MN architecture innervating the tilm. Immunocytochemistry was used to mark neurons (HRP), discs-large (DLG) and muscle (phalloidin). Z-stacks were captured by confocal microscopy imaging through the entire femur and shown as a maximum series projection. (A) A transmitted light channel was also included to illustrate no detectable muscle damage occurred during dissection. Image was captured at 20x magnification, scale bar =50 &#956;m. (B) Identified arbors associated with the tilm (boxed area, center), scale bar =20 &#956;m; and (C) at 60x magnification, scale bar = 20 &#956;m. (D) DLG surrounding boutons was apparent in wild type animals when imaged at 100x magnification with 2x zoom; scale bar = 2 &#956;m <a href="https://www.jove.com/files/ftp_upload/62844/62844fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62844/62844fig05.jpg" /> 
Figure 5. Example of age-dependent changes in bouton morphology which can be detected using the leg dissection technique. Bouton swelling is seen in aged dsod1H71Y mutants. (A) Representative images of HRP stained boutons from young (newly eclosed, day 0 adults) and old (day 10) flies, scale bar = 1 &#956;m. (B) Bouton sizes were quantified from respective genotypes using the measure function within ImageJ. ***p&#60;0.0001 (2-way ANOVA with post-hoc Tukey test). (C)&#160;Active zones within synaptic boutons labeled with monoclonal antibody NC82 (anti-bruchpilot); scale bar = 1 &#956;m. Figure modified from20 <a href="https://www.jove.com/files/ftp_upload/62844/62844fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62844/62844fig06.jpg" /> 
Figure 6. Common markers associated with neurodegenerative disease used in Drosophila leg preparations. (A)&#160;Subcellular polyubiquitinated aggregates are detected in terminal axons and muscle of aged dsod1H71Y flies. Immunocytochemistry using anti-polyubiquitin (FK2) detects puncta (arrows) in aged dsod1H71Y/H71Y, scale bar = 20 &#956;m (left) and 5 &#956;m (right). (B) Swollen mitochondria can be detected using anti-ATP5A, scale bar = 1 &#956;m. Figure modified from20 <a href="https://www.jove.com/files/ftp_upload/62844/62844fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62844/62844fig07v2.jpg" /> 
Figure 7. Examples of good and poor dissections. (A) Dissection which did not disrupt the underlying muscle architecture. (B) An example of a poor dissection which showed frayed muscle fibers (arrow) and could not be used for analysis. Both samples were imaged by phase-contrast microscopy. Scale bar = 50 &#956;m. (C)&#160;Example of a good dissection imaged by confocal microscopy with HRP (green) and phalloidin (blue). (D)&#160;A poor dissection missing dorsal muscle fibers of the TILM missing (arrow). Scale bar = 50 &#956;m <a href="https://www.jove.com/files/ftp_upload/62844/62844fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody/Stain</td>
			<td>Dilution*</td>
		</tr>
		<tr>
			<td>Anti-ATP5A</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Anti-bruchpilot (nc82)</td>
			<td>1:20</td>
		</tr>
		<tr>
			<td>Anti-cysteine string protein (ab49)</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Anti-discs large (4F3)</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Anti-hrp</td>
			<td>1:550</td>
		</tr>
		<tr>
			<td>Anti-polyubiquitin (FK2)</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Anti-repo (8D12)</td>
			<td>1:5</td>
		</tr>
		<tr>
			<td>Goat anti-mouse secondary antibody</td>
			<td>1:800</td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td></td>
			<td>*Dilute in 5% NGS</td>
		</tr>
	</tbody>
</table>
Table 2. Antibodies and dilutions. The commercial suppliers of the antibodies used in these studies are listed in the Materials List.

Watch this Scientific Journal Video about Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron at JoVE.com

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.

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 ...

dissection-immunohistochemistry-drosophila-adult-leg-to-detect

Nanyang Technological University

Research

JoVE Journal

Neuroscience

4.3K Views.  Rhode Island College. 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. 

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

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.

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.

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

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

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 ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

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 ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

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 ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.