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 ...

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...

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.

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			<td>Nikon</td>
			<td>SMZ-10A</td>
			<td></td>
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			<td>Rocking platform</td>
			<td>Biometra (WT 16)</td>
			<td>042-500</td>
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			<td>Cover glasses (24 x 32 mm)</td>
			<td>Deltalab</td>
			<td>D102432</td>
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			<td>Premium (Plus) microscope slides</td>
			<td>PORLAB</td>
			<td>PC-201-16</td>
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			<td>Tweezers</td>
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			<td>11253-20</td>
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			<td>Uniband LA-4C Scissors 125mm</td>
			<td>E.M.S</td>
			<td>77910-26</td>
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			<td>Disponsable surgical blades #10</td>
			<td>Sakira Medical</td>
			<td>1567</td>
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			<td>Disponsable sterile syringe (1 ml)</td>
			<td>Sakira Medical</td>
			<td>1569</td>
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			<td>Super PAP pen</td>
			<td>E.M.S</td>
			<td>71310</td>
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			<td>100 &#956;l or 200 &#956;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>
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		<tr>
			<td>NTac:SD-TgN(SOD1G93A)L26H rats</td>
			<td>Taconic</td>
			<td>2148-M</td>
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			<td>1X PBS (Dulbecco)</td>
			<td>Gibco</td>
			<td>21600-010</td>
			<td></td>
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			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
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			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
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			<td>Glycine</td>
			<td>Amresco</td>
			<td>167</td>
			<td></td>
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			<td>BSA</td>
			<td>Bio Basic INC.</td>
			<td>9048-46-8</td>
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			<td>Glycerol</td>
			<td>Mallinckrodt</td>
			<td>5092</td>
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			<td>Tris</td>
			<td>Amresco</td>
			<td>497</td>
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			<td>Purified anti-Neurofilament H (NF-H), Phosphorylated Antibody</td>
			<td>BioLegend</td>
			<td>801601</td>
			<td>Previously Covance # SMI 31P</td>
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			<td>Purified anti-Neurofilament H (NF-H), Nonphosphorylated Antibody</td>
			<td>BioLegend</td>
			<td>801701</td>
			<td>Previously Covance # SMI-32P</td>
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			<td>Alexa Fluor 488 goat anti-Mouse IgG (H+L)</td>
			<td>Thermo Scientific</td>
			<td>A11029</td>
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		<tr>
			<td>&#945;-Bungarotoxin, biotin-XX conjugate</td>
			<td>Invitrogen</td>
			<td>B1196</td>
			<td></td>
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		<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>
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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 wer...

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 ...

Dissection of single skeletal muscle fibers for immunofluorescent and morphometric analyses of whole-mount neuromuscular junctions. This video will demonstrate a protocol to effectively and securely dissect EDL and soleus muscles with a repeatable approach. This video will also teach how to obtain whole-mount neuromuscular junctions with the aim of recognizing synaptic components with 3D integrity to allow an accurate morphometric analysis.To dissect the muscles, these surgical instruments are needed. To start the procedure, place the animal with the abdomen facing upwards. Make an initial incision using a surgical blade between the toes towards the hind limbs.Peel off the skin, pulling upward until the right knee is exposed. To find the EDL, follow the foot tendons to the annular ligament. This ligament circles two tendons.Cut the ligament with the Uniband scissors between the two tendons. Identify the EDL tendon by lifting both and seeing which one makes the toes move upwards. Cut the tendon with scissors.While holding the tendon with biological tweezers, begin to slowly separate the EDL muscle from the other hind limb muscles. Separation of the muscle without causing damage can be done by making various cuts on the rest of the lateral muscles to open a path between them, while holding and lifting the muscle from the splitting section of the EDL tendon. To completely isolate the muscle, cut the tendon that is attached to the rat knee using Uniband scissors.Immerse the dissected muscle in the fixative solution, and leave it for 24 hours at four degrees Celsius. In general, it is useful to have an elongated muscle. To do this, use cardboard and staples to attach the muscle for fixation.To isolate the soleus muscle, flip the animal. Through the skin, cut the calcaneal tendon using a surgical blade. With the help of the biological tweezers and Uniband scissors, separate the gastrocnemius muscle from the bones, creating a muscular lid.The soleus will be on the internal side of the muscular lid. It can be identified because it is red and flat. With a pair of biological tweezers, reach and lift the soleus tendon that lies above the above the gastrocnemius.Cut the tendon with Uniband scissors. Lift the whole muscle while cutting some of the weak attachment points. Finally, to completely free the soleus muscle, cut the soleus vesicle that forms the calcaneal tendon.Repeat the fixation step as done with the EDL muscle. To use this method, it is important to secure the staples to the tendons and not to the muscle. Once the 24-hour period of fixation has passed, rinse the muscles in DPBS solution before beginning to isolate the muscle fibers.In order to isolate the fibers, gently hold one of the tendons with one pair of biological tweezers. Then with the other pair of biological tweezers, begin to slowly pinch the tendon to separate the muscle fibers. To isolate the fibers, slowly pull upwards towards the opposite muscle tendon.It will be necessary to repeat this action several times until obtaining multiple, small, isolated bundles. Once separated in small isolated bundles, place them carefully in a pre-treated silanized slide. It is necessary to keep all the fibers orderly so that they do not overlap.At this point, the fibers can be left to air dry for 24 hours. After 24 hours, begin the immunostaining. To create a waterproof barrier, use a PAP pen to encircle the isolated muscle fibers in the pretreated slide.This is an example of a high-resolution confocal image that can be obtained following the protocol described here. On the upper left, the post-synaptic element is shown, and on the upper right, the pre-synaptic element, while the bottom images show the synaptic components schematized in order to illustrate the parameters that will be used for morphometric analysis. The second slide shows the merging of the pre-and post-synaptic signals with the nuclei stain of the API.Differences among the neuromuscular junctions of transgenic animals and non-transgenic animals are clearly visible. The morphometric analysis of neuromuscular junctions shows evidence of the post-synaptic signal in transgenic animals and appears to be more compact, mainly due to reduced neuromuscular junction total area. As shown here, the neuromuscular junction pre-synaptic area of the transgenic animal is reduced.The smallest detected was the one found with anti-phosphorylated neurofilament signal, as shown in image B, for example. The last image shows the results pertaining to the status of whole-mount neuromuscular junctions. Using the coverage index, it was established that the transgenic neuromuscular junctions were found be partially denervated.Also, it could determine that the coverage and nexus of the phosphorylated neurofilament is lower than obtained when a non-phosphorylated neurofilament is detected. The proposed protocol allows obtaining high-quality neuromuscular junction preparations from slow and fast muscle fibers. Pre-and post-synaptic neuromuscular junction components were identified by specific probes or antibodies, and then imaged in confocal or super-resolution confocal microscopy, enabling morphometric analysis at a micrometric scale.

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JoVE Journal

Neuroscience

7.9K visualizações.  Universidad de la República. Dissecção de fibras musculares esqueléticas únicas para análises imunofluorescentes e morfométricas de junções neuromusculares de montagem inteira. Este vídeo demonstrará um protocolo para dissecar os músculos EDL e soleus de forma eficaz e segura com uma abordagem repetível. Este vídeo também ensinará como obter junções neuromusculares de montagem total com o objetivo de reconhecer componentes sinápticos com integridade 3D para permitir uma análise morfométrica precisa.