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

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 cells^1,2,3. This synapse exhibits high morphological and functional plasticity^4,5,6,7,8, 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 activity^{4,10,11,12,13,14,15}. 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 branching^{14,16,17,18,19,20}.

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 somas^23,24. 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 composition^26,27,28,29.

For rodent NMJs, there are significant differences among muscles^28,29. 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 muscles^30,31. 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 components^33,34. 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 exercise^35,36,37,38. 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.

1. 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.
2. 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)

1. After 24 h of fixation, rinse the muscles with 6 mL of DPBS solution 3x for 10 min each before isolating the muscle fibers.
2. 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.
3. 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

1. 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.
2. 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.
3. 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

1. Image the teased fiber preparations using laser confocal microscopy.
2. 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.
3. 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 morphology^33,34.
4. 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.
5. 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.
6. 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.

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 wer...

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 models^41,42 to evaluate NMJ during physiological or pathological processes as exemplified here in a model of...

Materials

Name	Company	Catalog Number	Comments
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