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

Carmen Bolatto; Silvia Olivera-Bravo; Sof&#237;a Cerri

doi:10.3791/62620

JoVE Journal > Neuroscience

Neurociência

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

Published: August 14, 2021

doi:

10.3791/62620

Carmen Bolatto*¹, Silvia Olivera-Bravo*², Sofía Cerri^1,2

¹Developmental Biology Laboratory, Histology and Embryology Department, Faculty of Medicine,Universidad de la República, ²Cell and Molecular Neurobiology Laboratory, Clemente Estable Biology Research Institute (IIBCE),Ministerio de Educación y Cultura

Summary

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.

Abstract

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.

Introduction

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 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. 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 (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.

Protocol

All animal procedures were performed according to the guidelines of the National Law N° 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´s phosphate saline (DPBS). Optionally, make 20 mL of 4% PFA. Prepare 5 mL aliquots and freeze at -20 °C. On the day of dissection, defrost a 4% aliquot and add 35 mL of DPBS to obtain 40 mL of 0.5% PFA.

Isolation of EDL (fast-twitch muscle)
1. 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.
2. Peel off the skin, pulling it upwards until the rat knee is exposed.
3. 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.
4. 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.
5. To completely isolate the muscle, cut the tendon attached to the rat knee with uniband scissors.
6. Immerse the dissected muscle in 5 mL of 0.5% PFA and leave for 24 h at 4 °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.
Isolation of soleus (slow-twitch muscle)
1. Flip the animal (the abdomen is now facing downwards). Through the skin, cut the calcaneal tendon using a surgical blade.
2. 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 in color and has a flat morphology.
3. With a pair of biological tweezers, reach and lift the soleus tendon that lies above the gastrocnemius (see Figure 1B).
4. 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.
5. Repeat step 1.1.6 to fix dissected soleus muscle.

2. Teased fiber preparation (Day 2)

After 24 h of fixation, rinse the muscles with 6 mL of DPBS solution 3x for 10 min each before isolating the muscle fibers.
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 upwards towards the opposite muscle tendon. It will be necessary to repeat this action several times until obtaining multiple small, isolated bundles.
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.

3. Immunofluorescence

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.
1. To start the immunostaining process, encircle the dried isolated muscle fibers with a pap pen to create a waterproof barrier.
2. To hydrate the muscle fibers, add 200 µL of distilled water for 5 min, and then change the water to 200 µL of DPBS for the next 5 min incubation. At this point, start the immunofluorescence labeling.
3. Incubate fibers with 300 µL of a blocking buffer (BB) containing 50 mM glycine, 1% BSA, and 1% Triton X-100 for 30 min.
4. Replace BB with the primary antibody at a concentration suggested by manufacturers. Use the BB to dilute the antibody. This experiment used 400 µL of SMI 32 or SMI 31 that recognized non-phosphorylated (Nf) or phosphorylated (Phos-Nf) neurofilament H 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.
5. Incubate the fibers with the primary antibody dilution at 4 °C overnight (optionally, incubation can be performed at 37 °C for 1 h). In both cases, it will be important to perform the incubation using a humid chamber.
Immunofluorescence: Secondary antibody incubation (Day 4)
1. Remove the primary antibody and rinse the fibers 3x for 10 min each with 500 µL of DPBS and the last time with BB.
2. Remove this last wash and add the fluorescently labeled secondary antibody diluted in BB. For this experiment, 500 µL 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 α-Bungarotoxin (Btx) biotin-XX conjugate was added to the secondary antibody dilution.
  NOTE: The addition of Btx-biotin XX 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.
3. Incubate the secondary antibody and the Btx for 2 h at room temperature or 1 h at 37 °C protected from light.
4. Remove the secondary antibody and the Btx. Wash 3x for 10 min each with 500 µL of DPBS and the last rinse with BB.
5. Incubate with 500 µL 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 µg/mL.
6. Remove the incubation solution and wash fibers 3x for 10 min each with 500 µL of DPBS. Then, mount the fibers.
Mounting
1. 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.
2. Remove the last DPBS wash and add enough mounting media (~ 200 µL) 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).
3. Use a 200 µL 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.
4. Place the coverslip onto the samples trying to prevent the generation of air bubbles. This movement can be done with the aid of forceps.

4. Microscopy and morphometric analysis

Image the teased fiber preparations using laser confocal microscopy.
Take a series of optical sections (30 µm 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.
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.
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 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.
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.
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.

Representative Results

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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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.

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<0.05 and p<0.001, respectively. Please click here to view a larger version of this figure.

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<0.0001. Please click here to view a larger version of this figure.

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 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 ( 18%) than that obtained when the non-phosphorylated form ( 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 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<0.001 and p<0.0001, respectively. Please click here to view a larger version of this figure.

Table 1. Morphometric parameters and ratios obtained from whole-mount NMJs of non-transgenic and transgenic EDL fibers. Please click here to download this Table.

Discussion

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 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 °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 °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 "tuft" of muscle fibers) and carry out incubations in microcentrifuge tubes by the "free-floating" 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 "tease" 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.

Declarações

The authors have nothing to disclose.

Acknowledgements

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 Nicolás Bolatto for lending his voice for it.

Materials

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

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Bolatto, C., Olivera-Bravo, S., Cerri, S. Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions. J. Vis. Exp. (174), e62620, doi:10.3791/62620 (2021).