Many thanks to CSIC and PEDECIBA for the financial support given to this work; to Natalia Rosano for her manuscript corrections; to Marcelo Casacuberta that makes the video and to Nicol&#225;s Bolatto for lending his voice for it.

All animal procedures were performed according to the guidelines of the National Law N&#176; 18611 for Care of Animals Used for Experimental Purposes. The protocol was approved by the Institutional Ethical Committee (CEUA IIBCE, Protocol Number 004/09/2015).
1. Muscle dissection (Day 1)
NOTE: Before starting, make 40 mL of 0.5% paraformaldehyde (PFA), pH 7.4 in Dulbecco&#180;s phosphate saline (DPBS). Optionally, make 20 mL of 4% PFA. Prepare 5 mL aliquots and freeze at -20 &#176;C. On the day of dissection, defrost a 4% aliquot and add 35 mL of DPBS to obtain 40 mL of 0.5% PFA.
<ol>
	<li>Isolation of EDL (fast-twitch muscle)
	<ol>
		<li>Euthanize a rat. Place the animal with the abdomen facing upwards. Make an initial incision using a surgical blade between the toes towards the hind limb. 
		NOTE: In this case, an intraperitoneal injection of 90:10 mg/Kg ketamine:xylazine was given to euthanize the rat, but other options of anesthesia are also allowed.</li>
		<li>Peel off the skin, pulling it upwards until the rat knee is exposed.</li>
		<li>To find the EDL, follow the foot tendons up to the annular ligament. This ligament circles two tendons. Cut the ligament between the two tendons with uniband scissors (or similar). Identify the EDL tendon by lifting both and choose the one that makes the toes move upwards.</li>
		<li>Cut the tendon with uniband scissors. Then, while holding the tendon with fine biological tweezers, begin to slowly separate the EDL muscle from the tibialis anterior, the peroneus brevis, and peroneus longus40 (see Figure 1A). 
		NOTE: Ensure that the muscle is separated without any damage. Do this by cutting the rest of the lateral muscles to open a path between them (the EDL does not have a superficial location) while holding and lifting the EDL muscle.</li>
		<li>To completely isolate the muscle, cut the tendon attached to the rat knee with uniband scissors.</li>
		<li>Immerse the dissected muscle in 5 mL of 0.5% PFA and leave for 24 h at 4 &#176;C. Optionally, staple the muscle in a piece of cardboard and turn it with the muscle facing down. Then immerse it completely in 8 mL of the fixative solution. This step helps in maintaining the muscle elongated during the fixation process.</li>
	</ol>
	</li>
	<li>Isolation of soleus (slow-twitch muscle)
	<ol>
		<li>Flip the animal (the abdomen is now facing downwards). Through the skin, cut the calcaneal tendon using a surgical blade.</li>
		<li>With the help of the biological tweezers and uniband scissors, separate the gastrocnemius muscle from the bones, creating a &#34;muscular lid&#34;. The soleus will be on the internal side of the muscular lid. It can be identified because it is red in color and has a flat morphology.</li>
		<li>With a pair of biological tweezers, reach and lift the soleus tendon that lies above the gastrocnemius (see Figure 1B).</li>
		<li>Cut the tendon with uniband scissors. Lift the whole muscle while cutting some of the weak attachment points (i.e., neurovascular elements). Finally, to completely free the soleus muscle, cut the soleus fascicle that forms the calcaneal tendon with uniband scissors.</li>
		<li>Repeat step 1.1.6 to fix dissected soleus muscle.</li>
	</ol>
	</li>
</ol>
2. Teased fiber preparation (Day 2)
<ol>
	<li>After 24 h of fixation, rinse the muscles with 6 mL of DPBS solution 3x for 10 min each before isolating the muscle fibers.</li>
	<li>To isolate the fibers, put a muscle on a microscope slide. Using a stereomicroscope, gently hold one of the tendons with one pair of biological tweezers. Then, with the other biological tweezers, begin to pinch the tendon slowly to separate the muscle fibers. After that slowly pull the pinched tissue&#160;upwards towards the opposite muscle tendon. It will be necessary to repeat this action several times until obtaining multiple small, isolated bundles.</li>
	<li>Place them carefully on a pre-treated slide (silanized). It is necessary to keep all the fibers in order so that they do not overlap. At this point, air-dry the fibers for 24 h.</li>
</ol>
3. Immunofluorescence
<ol>
	<li>Primary antibody incubation (Day 3) 
	NOTE: All steps were performed at room temperature, except if otherwise stated. The most efficient way to remove the different solutions without detaching fibers from the slide is to use an insulin syringe.
	<ol>
		<li>To start the immunostaining process, encircle the dried isolated muscle fibers with a pap pen to create a waterproof barrier.</li>
		<li>To hydrate the muscle fibers, add 200 &#181;L of distilled water for 5 min, and then change the water to 200 &#181;L of DPBS for the next 5 min incubation. At this point, start the immunofluorescence labeling.</li>
		<li>Incubate fibers with 300 &#181;L of a blocking buffer (BB) containing 50 mM glycine, 1% BSA, and 1% Triton X-100 for 30 min.</li>
		<li>Replace BB with the primary antibody at a concentration suggested by manufacturers. Use the BB to dilute the antibody. This experiment used 400 &#181;L of SMI 32 or SMI 31 that recognized non-phosphorylated (Nf) or phosphorylated (Phos-Nf) neurofilament H&#160;at 1/800 dilution to detect the NMJ presynaptic component. 
		NOTE: When deciding to recognize the whole presynaptic element instead of evaluating neurofilament phosphorylation, choose antibodies against synapsin, synaptophysin or SV2a that were previously successfully employed.</li>
		<li>Incubate the fibers with the primary antibody dilution at 4 &#176;C overnight (optionally, incubation can be performed at 37 &#176;C for 1 h). In both cases, it will be important to perform the incubation using a humid chamber.</li>
	</ol>
	</li>
	<li>Immunofluorescence: Secondary antibody incubation (Day 4)
	<ol>
		<li>Remove the primary antibody and rinse the fibers 3x for 10 min each with 500 &#181;L of DPBS and the last time with BB.</li>
		<li>Remove this last wash and add the fluorescently labeled secondary antibody diluted in BB. For this experiment, 500 &#181;L of goat anti-mouse Alexa Fluor 488 at 1/1000 dilution in BB was used. To view the postsynaptic element of the NMJs, 1:300 &#945;-Bungarotoxin (Btx)&#160;biotin-XX conjugate was added to the secondary antibody dilution. 
		&#8203;NOTE: The addition of Btx-biotin XX&#160;allows better signal-to-noise images once detected with streptavidin that amplifies the signal. In addition, streptavidin conjugation to different fluorophores increased color combinations in co-labeling assays. Alternatively, fluorescently labeled Btx allows obtaining images of similar quality.</li>
		<li>Incubate the secondary antibody and the Btx for 2 h at room temperature or 1 h at 37 &#176;C protected from light.</li>
		<li>Remove the secondary antibody and the Btx. Wash 3x for 10 min each with 500 &#181;L of DPBS and the last rinse with BB.</li>
		<li>Incubate with 500 &#181;L of Streptavidin Alexa Fluor 555 at 1/1000 dilution with BB for 1 h at room temperature. At this point, if desired, add a probe to counterstain the nuclei, such as diaminophenylindole at a final concentration of 1 &#181;g/mL.</li>
		<li>Remove the incubation solution and wash fibers 3x for 10 min each with 500 &#181;L of DPBS. Then, mount the fibers.</li>
	</ol>
	</li>
	<li>Mounting
	<ol>
		<li>Place 4 dots of transparent nail polish on the microscope slide as if they were the vertices of a square. Let them dry 1-2 min. These will be the points where the coverslips will rest, helping to avoid tissue crushing.</li>
		<li>Remove the last DPBS wash and add enough mounting media (~ 200 &#181;L) to cover the fibers; avoid drying. 
		NOTE: To prepare 10 mL of the mounting media mix, use Tris-HCl 1,5 M pH 8,8: glycerol in a 4:1 (v:v).</li>
		<li>Use a 200 &#181;L pipette tip to carefully spread the mounting media over the fibers to ensure a complete immersion of all of them. Before placing the cover glass, remove air bubbles.</li>
		<li>Place the coverslip onto the samples trying to prevent the generation of air bubbles. This movement can be done with the aid of forceps.</li>
	</ol>
	</li>
</ol>
4. Microscopy and morphometric analysis
<ol>
	<li>Image the teased fiber preparations using laser confocal microscopy.</li>
	<li>Take a series of optical sections (30 &#181;m Z stacks) across the entire synapse using a one-micrometer interval. To set the stack, use the Btx signal. Image at least 15-20 NMJs for each muscle condition with a 2048 px x 2048 px resolution. This method was chosen to obtain images with sufficient optical quality and resolution for the morphometric analysis.</li>
	<li>To perform the measurements, use the projection images of stacks with any analysis software. 
	NOTE: Although the explained morphometric analysis was done manually, there are two recently developed Image J-based tools for studying many different aspects of pre and postsynaptic NMJ morphology33,34.</li>
	<li>To obtain the Total NMJ area values, select the external border (external outline of the stained area, see Figure 2) of the endplate using the Photoshop&#160;Magnetic lasso tool and determine the number of selected pixels in the Histogram window. Then, the pixel areas can be converted to square micrometers using the scale specified by the confocal software.</li>
	<li>With the same lasso tool, select the internal border (outlines of the stained area within the endplate, see Figure 2) and determine the non-stained area in the whole structure (or empty area) as detailed above (step 4.4.). The Postsynaptic Area values will be obtained after subtracting the total area from the empty area of each NMJ.</li>
	<li>Obtain the Presynaptic Area, that is defined as the area with anti-neurofilament immunofluorescence, in a similar way that the Total NMJ area. 
	NOTE: All these parameters were used to determine the Post-Synaptic Density index (Postsynaptic Area/Total Area) and the NMJ Coverage (Presynaptic Area/Postsynaptic Area). A higher Postsynaptic Density index implies a denser -or more compact- endplate, whereas a smaller NMJ Coverage reflects a much poorer interaction between nerve terminal and endplate (both compatible with a denervation process). In the case shown here, since phosphorylated and non-phosphorylated forms of neurofilaments were detected at the nerve terminal level, the phosphorylated/non-phosphorylated presynaptic signal ratio and the phosphorylated/non-phosphorylated coverage ratio were calculated.</li>
</ol>

The authors have nothing to disclose.

In this article, we present a detailed protocol for the dissection of two rat skeletal muscles (one slow-twitch and the other fast-twitch), fiber muscle isolation and immunofluorescence detection of pre and postsynaptic markers to quantitatively assess&#160;NMJ changes as well as assembly/disassembly processes. This kind of protocol can be useful in rodent models41,42 to evaluate NMJ during physiological or pathological processes as exemplified here in a model of motor neuron degeneration such as found in the ALS hSOD1G93A rats.
To obtain success and beneficial results, it is necessary to keep in mind some tips. For example, the protocol described allows immediate use of dissected muscles. However, when necessary to preserve the muscle longer the use of DPBS with 0.05% sodium azide plus 4 &#176;C storage allows prolonging excellent muscle preservation up to 4 weeks. When using this option, extensive washes with DPBS must be done (5x, 10 min each) before starting the staining process. Another helpful option to obtain good quality images includes lowering the PFA concentration at 0.5% because it reduced the muscle autofluorescence. This must be accompanied by longer fixation up to 24 h at 4 &#176;C.
Regarding fiber isolation, smaller groups of fibers produce better immunodetection qualities. The fibers dried on silanized slides must be used within 2 to 3 days. After this time, the immunodetections are not of good quality.
One alternative to dried fibers on silanized slides is to generate a set of partially isolated fibers that are held together by the tendon tissue in one of the fibers end (a &#34;tuft&#34; of muscle fibers) and carry out incubations in microcentrifuge tubes by the &#34;free-floating&#34; method. Using this option is necessary to increase the antibody incubation times by several hours (24-48 h) as well as the number of washes (at least 6 of 10 min each).
In relation to fiber isolation without damage, it is necessary to perform the &#34;tease&#34; by removing the ends where the tendon is gently pull in opposite directions to achieve fiber separation.
Another critical step to improve immunodetection is the addition of 1% Triton X-100 and 50 mM glycine in the BB. This is important to obtain preparations that have homogeneous staining and a little background. It is also worth mentioning that having a little background can be beneficial when unstained fibers need to be imaged. The fiber diameter is good to be measured far from the NMJ field since it is described that it may be increased at this level. Therefore, determine the fiber diameter at a distance equal to the value of an NMJ profile.
Using the procedure presented it is possible identifying and quantitating NMJ components under physiological conditions. It also allows studying early key pathological events as NMJ dismantlement24 and loss of phosphorylated neurofilament in terminal endings39 as described in ALS pathogenesis. For this reason, the protocol described offers an alternative and simple approach to help to get a better understanding of the NMJ remodeling, suggesting that morphological features visualized can be useful tools to diagnose degenerating state of NMJs or to assess different therapeutic approaches. It is also applicable to other disease models such as those of Charcot-Marie-Tooth41 and spinal muscular atrophy42 that present NMJ disruption.
In addition, approaches described in this work can be employed to identify the Schwann cells that integrate this tripartite synapse and eventually to assess some of its roles during NMJ remodeling43. Studies can be done in neonates until adults, under physiological conditions or as a response to different treatments impacting on the neuromuscular system.
Finally, in spite of the excellent handling needed to dissect and label samples together with time consumed to make manual measurements, methodology presented here enables an optimal labeling of the different NMJ components because of the facilitated penetration of antibodies and probes. Although better penetration can be obtained in sectioned muscle preparations, teasing preserves the 3D morphological integrity of all of the synaptic components that can be lost in muscle sections. It also avoids all of the preparation needed to make sections such as the inclusion of the muscle piece and further antigen retrieval to obtain feasible recognition. Furthermore, when compared to whole mount preparations that preserve the complete innervation patterns, teasing enables the study of isolated fibers. This can be a significant advantage when necessary to assess the fiber heterogeneity reported in pathological conditions.

The mammal neuromuscular junction (NMJ) is a large cholinergic tripartite synapse made up of the motor neuron nerve ending, the postsynaptic membrane on the skeletal muscle fiber, and the terminal Schwann cells1,2,3. This synapse exhibits high morphological and functional plasticity4,5,6,7,8, even during adulthood when NMJs can undergo dynamic structural modifications. For example, some researchers have shown that motor nerve endings continually change their shape at the micrometer scale9. It has also been reported that the morphology of the NMJ responds to functional requirements, altered use, aging, exercise, or variations in locomotor activity4,10,11,12,13,14,15. Thus, training and lack of use represent&#160;essential stimulus to modify some characteristics of the NMJ, such as its size, length, dispersion of synaptic vesicles and receptors, as well as nerve terminal branching14,16,17,18,19,20.
Furthermore, it has been shown that any structural change or degeneration of this vital junction could result in motor neuron cell death and muscle atrophy21. It is also thought that altered communication among nerves and muscles could be responsible for the physiological age-related NMJ changes and possibly for its destruction in pathological states. Neuromuscular junction dismantling plays a crucial role in the onset of Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that constitutes one of the best examples of impaired muscle-nerve interplay3. Despite the numerous studies conducted on motor neuron dysfunction, it is still debated whether the deterioration observed in ALS occurs due to the direct damage into the motor neuron and then extends to the cortico-spinal projections22; or if it should be considered as a distal axonopathy where degeneration begins in the nerve endings and progresses toward the motor neuron somas23,24. Given the complexity of ALS pathology, it is logical to consider that a mix of independent processes occurs. As NMJ is the central player of the physiopathological interplay between muscle and nerve, its destabilization represents a pivotal point in the origin of the disease that is relevant to be analyzed.
The mammal neuromuscular system is functionally organized into discrete motor units, consisting of a motor neuron and the muscle fibers that are exclusively innervated by its nerve terminal. Each motor unit has fibers with similar or identical structural and functional properties25. Motor neuron selective recruitment allows optimizing muscle response to functional demands. Now it is clear that mammalian skeletal muscles are composed of four different fiber types.&#160;Some muscles are named according to the characteristics of their most abundant fiber type. For example, the soleus (a posterior muscle of the hind limb involved in the maintenance of the body posture) bears a majority of slow-twitch units (type 1) and is recognized as a slow muscle. Instead, extensor digitorum longus&#160;(EDL) is essentially composed of units with similar fast-twitch properties (type 2 fibers) and is known as a fast muscle specialized for phasic movements needed for locomotion. In other words, although adult muscles are plastic in nature due to the hormonal and neural influences, its fiber composition determines the capacity to perform different activities, as seen in soleus that experiences continuous low-intensity activity and EDL that exhibits a more rapid single twitch. Other features that are variable among different types of muscle fibers are related to their structure (mitochondrial content, extension of sarcoplasmic reticulum, thickness of the Z line), myosin ATPase content, and myosin heavy chain composition26,27,28,29.
For rodent NMJs, there are significant differences among muscles28,29. Morphometric analyses performed in soleus and EDL from rats revealed a positive correlation between the synaptic area and fiber diameter (i.e., the synaptic area in soleus slow fibers is greater than in EDL fast fibers) but the ratio between NMJ area and fiber size is similar in both muscles30,31. Also, in relation to the nerve terminals, the endplate absolute areas in type 1 fibers were lower than in type 2 fibers, whereas the normalization by fiber diameter made areas of nerve terminals in type 1 fibers the largest32.
However, very few studies focus on morphometric analysis to show the evidence of changes in some of the NMJ components33,34. Thus, due to the relevance of the NMJ in the function of the organism, whose morphology and physiology are altered in various pathologies, it is important to optimize dissection protocols of different types of muscles with quality enough to allow the visualization of the whole NMJ structure. It is also necessary to evaluate the occurrence of pre or postsynaptic changes in different experimental situations or conditions such as aging or exercise35,36,37,38. In addition, it can be helpful to evidence more subtle alterations in NMJ components such as altered neurofilament phosphorylation in the terminal nerve endings as reported in ALS39.

<table><tbody><tr><td>Stereomicroscope with cool light illumination</td><td>Nikon</td><td>SMZ-10A</td><td></td></tr><tr><td>Rocking platform</td><td>Biometra (WT 16)</td><td>042-500</td><td></td></tr><tr><td>Cover glasses (24 x 32 mm)</td><td>Deltalab</td><td>D102432</td><td></td></tr><tr><td>Premium (Plus) microscope slides</td><td>PORLAB</td><td>PC-201-16</td><td></td></tr><tr><td>Tweezers</td><td>F.S.T</td><td>11253-20</td><td></td></tr><tr><td>Uniband LA-4C Scissors 125mm</td><td>E.M.S</td><td>77910-26</td><td></td></tr><tr><td>Disponsable surgical blades #10</td><td>Sakira Medical</td><td>1567</td><td></td></tr><tr><td>Disponsable sterile syringe (1 ml)</td><td>Sakira Medical</td><td>1569</td><td></td></tr><tr><td>Super PAP pen</td><td>E.M.S</td><td>71310</td><td></td></tr><tr><td>100 &amp;mu;l or 200 &amp;mu;l pipette</td><td>Finnpipette</td><td>9400130</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM 800 - AiryScan</td><td></td></tr><tr><td>NTac:SD-TgN(SOD1G93A)L26H rats</td><td>Taconic</td><td>2148-M</td><td></td></tr><tr><td>1X PBS (Dulbecco)</td><td>Gibco</td><td>21600-010</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Glycine</td><td>Amresco</td><td>167</td><td></td></tr><tr><td>BSA</td><td>Bio Basic INC.</td><td>9048-46-8</td><td></td></tr><tr><td>Glycerol</td><td>Mallinckrodt</td><td>5092</td><td></td></tr><tr><td>Tris</td><td>Amresco</td><td>497</td><td></td></tr><tr><td>Purified anti-Neurofilament H (NF-H), Phosphorylated Antibody</td><td>BioLegend</td><td>801601</td><td>Previously Covance # SMI 31P</td></tr><tr><td>Purified anti-Neurofilament H (NF-H), Nonphosphorylated Antibody</td><td>BioLegend</td><td>801701</td><td>Previously Covance # SMI-32P</td></tr><tr><td>Alexa Fluor 488 goat anti-Mouse IgG (H+L)</td><td>Thermo Scientific</td><td>A11029</td><td></td></tr><tr><td>&amp;alpha;-Bungarotoxin, biotin-XX conjugate</td><td>Invitrogen</td><td>B1196</td><td></td></tr><tr><td>Streptavidin, Alexa Fluor 555 conjugate</td><td>Invitrogen</td><td>S32355</td><td></td></tr><tr><td>Diaminophenylindole (DAPI)</td><td>Sigma</td><td>D8417</td><td></td></tr></tbody></table>

dissection of single skeletal muscle fibers for immunofluorescent and morphometric analyses of whole-mount neuromuscular junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

This protocol offers a straightforward method to isolate and immunostain muscle fibers from two different types of muscles (fast- and slow-twitch muscles, see Figure 1). Using the correct markers and / or probes, NMJ components can be detected and evaluated since a quantitative point of view to assess some of the morphological changes that can occur as consequence of illness progression or a specific drug treatment. In the present study, presynaptic and postsynaptic components of the NMJ were fluorescently labeled using anti-Nf or anti-Phos-Nf antibodies and Btx respectively (Figure 2, upper panels). Each high-resolution confocal image of a NMJ obtained after the immunostaining was used to determine the morphometric measurements and indexes specified in the Protocol section (Table 1) applying the parameters described in the bottom panels of the Figure 2.
To demonstrate that this whole-mount NMJ preparation perfectly retained the synapse architecture, allowing the visualization of the effect that have a progressive nervous system disease on it, teased fibers of transgenic rats expressing human gene SOD1G93A (an ALS model) were immunostained using the protocol (Figure 3). The merging images of pre and postsynaptic signals with the nuclei stained with DAPI showed that differences among the NMJs between transgenic and non-transgenic age-matched animals are clearly visible and compatible with the results expected by literature.
In addition, the quality of images made it possible to quantify the changes observed by performing the morphometric analysis. For example, Figure 4 shows evidence that the postsynaptic signal in transgenic animals appears to be more compact (approximately 20%), mainly due to the reduction of the NMJ total area.
On the other hand, Figure 5 shows that the presynaptic area of the NMJs in transgenic animals is also reduced. The quality of this whole-mount NMJ preparation was evidenced by analyzing the extent of the nerve terminal retraction and by detecting changes concerning neurofilament phosphorylation states. This fact will be important to get a deep insight for the NMJ disassembly process related to ALS disease in the model employed. The smallest presynaptic area detected was the one labeled with anti-phosphorylated neurofilament (Figure 5B, E, C, F).
The use of the coverage index (Figure 6) made it possible to establish that the transgenic NMJs were partially denervated. Also, it could determine that the coverage indexes of the phosphorylated neurofilament are lower than that obtained with non-phosphorylated neurofilament. These results show the pertaining to the status of whole-mount NMJ and that it could be useful to introduce the study of neurofilament phosphorylation state (an important determinant of filament plasticity and stability) using this whole-mount NMJ preparation.
Finally, methods presented in this work offer the greatest NMJ quality to identify, assess and evaluate even subtle changes in NMJ as a whole or in one of its component.
<img alt="Rat muscle dissection; diagrams showing tibialis anterior, gastrocnemius; anatomical illustration." class="xfigimg" src="/files/ftp_upload/62620/62620fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: EDL and soleus anatomical landmarks. Image highlight (A) EDL and (B) soleus location among the other muscles of the rat lower hind limb. Insets show schemes of the hind limb muscles close to EDL (red) and soleus (green) muscles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62620/62620fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Postsynaptic and presynaptic elements; fluorescent microscopy diagram; neural structure analysis." class="xfigimg" src="/files/ftp_upload/62620/62620fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Whole-mount neuromuscular junction of EDL muscle fibers. (A) Postsynaptic component of NMJ detected using alpha-bungarotoxin (Btx, magenta), a probe that binds specifically to the acetylcholine receptors (AChR). (B) Motor terminal detected using anti-neurofilaments (anti-Nf, green). (C,D) Synaptic components are schematized in order to illustrate the parameters (external and internal borders) that defined the measurements (Total Area, Presynaptic and Postsynaptic Area) used for the morphometric analysis detailed in the protocol. Scale bar = 50 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62620/62620fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy of Phos-Nf and Nf distribution in non-Tg and hSOD1G93A mouse tissue." class="xfigimg" src="/files/ftp_upload/62620/62620fig3v4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Pre and postsynaptic alterations observed in NMJs of an ALS model. Representative confocal images obtained from whole-mount NMJs obtained in preparations from EDL muscle fibers stained to detect neurofilaments (green), AChR (magenta), and nucleus (blue) from either non-transgenic (-/-) or transgenic (hSOD1G93A/-) rats. (A,C) Non-transgenic rats have normal NMJ morphology (pretzel shape) both when the motor end is visualized using anti-Phos-Nf (A) or anti-Nf (C). (B,D) Transgenic rats present more compact postsynaptic area and smaller presynaptic terminals with a reduction in the neurofilament phosphorylation signal. Scale bar = 50 µm.
<img alt="Comparative synapse microscopy, postsynaptic area analysis; bar graph shows statistical results." class="xfigimg" src="/files/ftp_upload/62620/62620fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Morphometric evaluation of the NMJ postsynaptic element in EDL muscles from 180 days old male hSOD1G93A rats. Representative confocal images of postsynaptic components in (A,D) non-transgenic and (B,E) transgenic animals. Evaluation of (C) postsynaptic area and (F) postsynaptic density index in NMJs of non-transgenic (solid columns) and transgenic (striped columns) animals. While the postsynaptic area was slightly reduced in transgenic animals, the postsynaptic index increased in greater proportion highlighting the degree of NMJ compaction as indicated by reduced total area in transgenic animals as shown in Table 1. Scale bar = 50 µm. Data represented is mean ± SEM. (*) and (***) indicated p&lt;0.05 and p&lt;0.001, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62620/62620fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Neurofilament microscopy images and bar graphs compare phos-Nf in Non-Tg and hSOD1G93A samples." class="xfigimg" src="/files/ftp_upload/62620/62620fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Morphometric evaluation of the NMJ presynaptic element in EDL muscles from 180 days old male hSOD1G93A rats. Given the importance of NMJ disassembly in ALS disease, the quality of the preparation was evaluated by analyzing, not only the extent of the nerve terminal retraction, but also the possibility of detecting changes in the phosphorylation of the NMJ presynaptic element. Representative confocal images of the presynaptic component in (A,D) non-transgenic and (B,E) transgenic animals detected using (A,B) anti-Phos-Nf or (D,E) anti-Nf antibodies. Evaluation of Presynaptic Area delimited by (C) phosphorylated and (F) non-phosphorylated neurofilaments in NMJs of non-transgenic (solid columns) and transgenic (striped columns) animals. Note that these areas are very similar in non-transgenic animals whereas they present a significant reduction in the NMJs of transgenic animals. Scale bar = 50 µm. Data represented is mean ± SEM. (****) indicated p&lt;0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62620/62620fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Phosphorylation vs non-phosphorylation in neurons, immunofluorescence images and histogram analysis." class="xfigimg" src="/files/ftp_upload/62620/62620fig06.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Evaluation of innervation status at whole-mount NMJs of EDL hSOD1G93A fibers. The coverage index is widely used to evaluate the state of innervation. It is considered that "fully innervated" NMJs present a coverage index <img alt="Approximate equal to symbol used in equations and formulas for mathematical expressions." src="/files/ftp_upload/62620/graphic2.jpg" style="margin:0px;" data-altupdatedat="1747911167000" data-alt="Equation 1"> 50%, while those "partially innervated" have coverages between ≥20% and ≤50% and those vacant are considered "denervated". Representative confocal images of (A,D) non-transgenic and (B,E) transgenic NMJs showing presynaptic and postsynaptic elements detected using (A,B) anti-Phos-Nf or (D,E) anti-Nf antibodies to observe the motor terminal. Evaluation of coverage using (C) phosphorylated and (F) non-phosphorylated neurofilaments in NMJs of non-transgenic (solid columns) and transgenic (striped columns) animals. The images and graphics show evidence that NMJs from transgenic animals present a lower coverage index than non-transgenic ones. In addition, in transgenic animals, the coverage indexes of phosphorylated neurofilaments is lower (<img alt="Approximate equal to symbol used in equations and formulas for mathematical expressions." src="/files/ftp_upload/62620/graphic2.jpg" style="opacity: 0.9; font-size: 14px; margin: 0px;" data-altupdatedat="1747911167000" data-alt="Equation 1"> 18%) than that obtained when the non-phosphorylated form (<img alt="Approximate equal to symbol used in equations and formulas for mathematical expressions." src="/files/ftp_upload/62620/graphic2.jpg" style="opacity: 0.9; font-size: 14px; margin: 0px;" data-altupdatedat="1747911167000" data-alt="Equation 1"> 25%) is detected. This fact is corroborated with the relationship between both indexes as shown in Table 1. Note that the quality of the preparations allows obtaining <img alt="Approximate equal to symbol used in equations and formulas for mathematical expressions." src="/files/ftp_upload/62620/graphic2.jpg" style="opacity: 0.9; font-size: 14px; margin: 0px;" data-altupdatedat="1747911167000" data-alt="Equation 1"> 50% of the coverage index stipulated for the NMJs expected as "fully innervated". Scale bar = 50 µm. Data represented is mean ± SEM. (***) and (****) indicated p&lt;0.001 and p&lt;0.0001, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62620/62620fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1. Morphometric parameters and ratios obtained from whole-mount NMJs of non-transgenic and transgenic EDL fibers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62620/Table%201_JoVE62620.doc" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions at JoVE.com

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.4K Views.  Universidad de la República. Dissectie van enkelvoudige skeletspiervezels voor immunofluorescente en morfometrische analyses van neuromusculaire verbindingen met hele montering. Deze video toont een protocol om EDL- en soleusspieren effectief en veilig te ontleden met een herhaalbare aanpak. Deze video leert ook hoe je neuromusculaire verbindingen met een hele montering kunt verkrijgen met als doel synaptische componenten met 3D-integriteit te herkennen om een nauwkeurige morfometrische analyse mogelijk te maken.O...

Video: Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

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 ...

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 ...

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. ...

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

Medicine

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 ...

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (&#945;-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and &#945;-BTX, and in the second group, samples were stained with NF200, &#945;-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of ...

Viewing Microtubule Networks in Drosophila Neuromuscular Junctions and Muscle Cells

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

Author Spotlight: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Types

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

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