Summary
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.
Abstract
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 (α-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 α-BTX, and in the second group, samples were stained with NF200, α-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.
Introduction
As three essential structural components of the neuromuscular junction (NMJ)1,2,3,4, morphological aspects of the pre-synaptic motor axon terminals, post-synaptic membrane containing nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs) have extensively been investigated. Thin sections and whole-mount specimens of the skeletal muscles have been examined with different histological techniques, such as electron microscopy5,6, confocal microscopy7,8, and light-sheet microscopy9,10. Although the morphological characteristics of NMJ have been demonstrated by these techniques from different aspects, as a comparison, confocal microscopy is still an ideal choice for imaging of the detailed morphology of NMJ.
Recently, many new technologies have been developed for showing the structural components of NMJ. For example, thy1-YFP transgenic fluorescent mice have been directly used to observe motor axons and motor end plates in vivo and in vitro10,11. Additionally, intravenous injection of fluorescent α-BTX has been applied to reveal the spatial distribution of motor end plates in whole-mount skeletal muscles of wild-type and transgenic fluorescent mice, by using tissue optical clearing treatment for examination with light-sheet microscopy9,12. However, besides the pre- and post-synaptic elements that can be viewed by these advanced methods, the PSCs cannot be demonstrated at the same time.
Accumulating evidence indicates that PSCs, as the peripheral glial cells, are closely associated with the pre-synaptic terminals contributing to the development and stability of NMJ, the modulation of synaptic activity of the NMJ under the physiological condition, and the regeneration of the NMJ after nerve injury13,14,15. Considering the cellular architecture of NMJ, this protocol is a proper candidate to simultaneously label the PSCs, pre- and post-synaptic elements, and is potentially used to evaluate the integrity and plasticity of the NMJ under normal and pathological conditions. For example, compare the intensity of NMJ, morphology and volume of post-synaptic motor endplates, innervation and denervation of NMJ, and number of PSCs in muscles of physiological and pathological status.
The gastrocnemius muscle is the largest muscle forming the bulge in the calf, which is easily dissected out by removing the skin and the biceps femoris muscle from the limb. The muscle is often chosen to assess muscle atrophy, neuromuscular degeneration, muscle performance, and motor unit force ex vivo or in vivo16,17,18. However, the technique is also suitable for revealing the morphological characteristics of NMJ from various skeletal muscles. At the same time, the thick muscle sections can reveal more complete morphology and quantity of NMJ compared to thin sections7,8 and teased muscle fibers19.
In line with these studies, the rat medial gastrocnemius muscle was selected as the target-tissue in this study and was sliced at a thickness of 80 µm for multiple fluorescent staining with various kinds of biomarkers according to the structural components of NMJ. Here, neurofilament 200 (NF200)20,21, vesicular acetylcholine transporter (VAChT)22, alpha-bungarotoxin (α-BTX)23,24, and S10025,26 were used to label nerve fibers, pre-synaptic terminals, post-synaptic AchRs, and PSCs respectively. In addition, the background of muscular tissue and cellular nuclei were further counterstained with phalloidin and DAPI.
In this study, we expect to develop a refined protocol for simultaneously staining the cellular architecture of NMJ with their corresponding biomarkers on thicker fixed specimens, which is more convenient for use in confocal microscopy and helps to obtain much more information on the detailed structure of the PSCs, pre- and post-synaptic elements, as well as their spatial correlation to one another. From the perspective of methodology, this protocol may benefit to evaluate the morphological characteristics of NMJ under normal and pathological conditions.
Subscription Required. Please recommend JoVE to your librarian.
Protocol
This study was approved by the Ethics Committee of Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (approval No. 2021-04-15-1). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996). Three adult male rats (Sprague-Dawley, weight 230 ± 15 g) were used. The rats were housed in a 12 h light/dark cycle with controlled temperature and humidity and with free access to food and water. The instruments and materials for this study are shown in Figure 1.
1. Perfusion
- Inject 250 mg/kg tribromoethanol solution intraperitoneally to induce euthanasia of the rats.
- Once breathing stops, open the thoracic cavity to access the heart using scissors and forceps. Insert an intravenous catheter from the left cardiac ventricle toward the aorta, and then cut the right auricle.
- Perfuse the experimental rats in the hood. Start by perfusing with 100 mL of 0.9% normal saline until the blood exiting from the right auricle is clear, then continue to perfuse with 250-300 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for about 10 min until the tail is hard to bend.
2. Regional anatomy
- After perfusion, remove the skin of the bilateral hind limb using a surgical blade (Figure 2A) and expose the bilateral sciatic nerve and medial gastrocnemius muscle (Figure 2B).
- Dissect out the bilateral medial gastrocnemius muscle carefully from the hind limb using a blade (Figure 2C) and post-fix the entire muscle in 10 mL of 4% paraformaldehyde in 0.1 M PB for 2 h.
- Remove the muscle from the solution and cryoprotect the muscle in 10 mL of 25% sucrose in 0.1 M PB for 1 day at 4 °C until the muscle is completely immersed in the solution.
3. Cryosectioning of the muscle
- Once the muscular tissue is immersed in the solution, fix it on a freezing stage of a sliding microtome system using frozen section medium. Slice the muscle horizontally along the long axis of the muscle at the thickness of 80 µm.
- Place seven cryo-sections per well in an orderly fashion in a six-well culture plate with 10 mL of 0.1 M PB (pH 7.4) using a brush.
NOTE: The sections are placed in order so that the sections in different wells are adjacent, which makes the sections used for different stains more consistent.
4. Multiple fluorescent staining with NF200, VAChT, α-BTX, and Pha
NOTE: Multiple fluorescent staining with NF200, VAChT, α-BTX, and Pha was applied to reveal the nerve fibers, pre-synaptic terminals, post-synaptic nicotinic acetylcholine receptors, and muscular fibers, respectively.
- Incubate four sections per well with 1.5 mL of 0.1 M PB containing 75 μL of 10% Triton X-100 (0.5%) and 45 μL of normal donkey serum (3%) on an orbital shaker at 20 rpm for 30 min.
- Transfer the sections into 1.5 mL of mixed solution of primary antibodies containing rabbit anti-NF200 (1:1,000), goat anti-VAChT (1:500) in 0.1 M PB (pH 7.4) with 1% normal donkey serum, and 0.5% Triton X-100, and incubate overnight at 4 °C. The next day, wash the sections 3x for 5 min each in 0.1 M PB (pH 7.4) on an orbital shaker at 20 rpm.
- Incubate the sections in 1.5 mL of mixed solution of secondary antibodies containing donkey anti-rabbit AF488 (1:500) and donkey anti-goat AF546 (1:500), as well as the biomarkers of α-BTX, AF647 (1:500), and phalloidin 350 (1:500) in 0.1 M PB (pH 7.4) containing 1% normal donkey serum and 0.5% Triton X-100 for 1 h at room temperature. Protect from light.
- After washing 3x for 5 min each in 0.1 M PB (pH 7.4), mount the sections on microscope slides. Apply cover glass to the sections using 50% glycerin in distilled water for observation.
5. Multiple fluorescent staining with NF200, S100, α-BTX, and DAPI
NOTE: The procedures are similar to that of step 4; the main difference is between the primary antibodies and their corresponding secondary antibodies.
- Use the following primary antibodies: chicken anti-NF200 (1:6,000), rabbit anti-S100 (1:2,500) in step 4.2 and their corresponding secondary antibodies: donkey anti-chicken AF488 (1:500) and donkey anti-rabbit AF546 (1:500), as well as the biomarkers of α-BTX AF647 (1:500) and DAPI (1:50000) in step 4.3.
6. Observation and analyses
- Observe the specimen and take images using a confocal imaging system equipped with the following objective lenses: 20x lens with NA of 0.75 and 40x lens with NA of 0.95.
- Use excitation and emission wavelengths of 350 nm (Pha), 488 nm (NF200), 546 nm (VAChT and S100), 647 nm (α-BTX), or DAPI corresponding to the multiple fluorescent staining. Set the resolution of image capture to 640 x 640 pixels.
NOTE: The 640 x 640 pixels resolution is chosen because the images were captured in Z-stacks. A 1024 x 1024 pixel resolution results in slow imaging and photobleaching in Z-stacks. - Set start focal plane and end focal plane. Set step size to either 1 µm or 2 µm. Choose depth pattern, image capture, and Z series.
- For the lower magnification images taken at 20x, capture 40 images (Z-stacks) in 2 µm step size from each 80 µm thick section using a 105 µm pinhole. Choose the projection/topography mode and intensity projection over Z axis to integrate all images in a single in-focus.
- For the higher magnification images taken at 40x, capture 80 images (Z-stacks) in 1 µm step size from each 80 µm thick section with zoom set to 2 and a 105 µm pinhole. Integrate all images in a single in-focus.
- Reconstruct the images in the three-dimensional pattern with the image processing system as previously described in27.
Subscription Required. Please recommend JoVE to your librarian.
Representative Results
After multiple fluorescent staining, the corresponding labeling was orderly demonstrated on the 80 µm thick sections of the rat medial gastrocnemius muscle with NF200-positive nerve fibers, VAChT-positive pre-synaptic terminals, α-BTX-positive post-synaptic AchRs, S100-positive PSCs, phalloidin-positive muscular fibers, and DAPI-labeled cellular nuclei (Figure 3 and Figure 4).
It was shown that NF200-positive nerve fibers ran in bundle and gave out branches to the VAChT-positive pre-synaptic terminals that formed a mirror relationship with the α-BTX-positive post- synaptic AchRs (Figure 3). In addition, S100-positive PSCs were detected around the NF200-positive nerve fibers close to the pre-synaptic terminals (Figure 4).
By taking advantage of the image-reconstruction, the spatial correlation of the nerve fibers, pre-synaptic terminals, PSCs, and post-synaptic AchRs was further exhibited in a three-dimensional pattern showing the detailed morphological characteristics of NMJ from different perspectives (Figure 3C,C1 and Figure 4C,C1).
Figure 1. Images of the various protocol steps. (A) Perfuse the rat in the hood. (B) Dissect out the rat medial gastrocnemius muscle. (C) Cut the muscular tissue on a freezing stage of sliding microtome system. (D) Collect tissue sections orderly in a six-well dish. (E) Fluorescent staining on the shaker. (F) Mount the sections on microscope slides. (G) Apply coverslips to the sections with 50% glycerin. (H) Observe the samples with a confocal imaging system. Please click here to view a larger version of this figure.
Figure 2. Procedure of regional anatomy for dissecting out the rat medial gastrocnemius muscle. (A) Outside view of muscles in the rat hind limb. (B) Inside view of the medial gastrocnemius muscle and its innervation. (C) Dissecting out the rat medial gastrocnemius muscle. Please click here to view a larger version of this figure.
Figure 3. Spatial correlation of the nerve fibers, pre-synaptic terminals, post-synaptic nicotinic acetylcholine receptors, and muscular fibers on the rat medial gastrocnemius muscle. (A) Representative image from the rat medial gastrocnemius muscle showing the multiple fluorescent staining with neurofilament 200 (NF200), vesicular acetylcholine transporter (VAChT), alpha-bungarotoxin (α-BTX), and phalloidin (Pha). (B) The magnified image from panel A (arrowhead) showing the various labeling in detail. B1-B4: panel B shown separately with NF200 (B1), VAChT (B2), α-BTX (B3), and Pha (B4). (C) The adjusted images from panel B with the frame showing the front view of the 3D pattern in (C) and the back view in (C1). Same scale bar for B1-B4 as in B. Please click here to view a larger version of this figure.
Figure 4. Spatial correlation of the nerve fibers, peri-synaptic Schwann cells, post-synaptic nicotinic acetylcholine receptors, and cellular nuclei on the rat medial gastrocnemius muscle. (A) Representative image from the rat medial gastrocnemius muscle showing the multiple fluorescent staining with neurofilament 200 (NF200), Schwann cell marker (S100), alpha-bungarotoxin (α-BTX), and cellular nucleus marker 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). (B) The magnified image from panel A (arrowhead) showing the various labeling in detail. B1-B4: show panel B separately with NF200 (B1), S100 (B2), α-BTX (B3), and DAPI (B4). (C) The adjusted images from panel B with the frame in a 3D pattern in (C) and the view without S100 labeling in (C1). Same scale bar for B1-B4 as in B. Please click here to view a larger version of this figure.
Subscription Required. Please recommend JoVE to your librarian.
Discussion
We have described the technical details required for performing successful multiple staining of muscle slices and use of fluorescent immunohistochemistry for revealing the morphological characteristics of NMJ on the thick sections of the rat medial gastrocnemius muscle. By using this approach, the fine details and spatial correlation of the PSCs and pre- and post-synaptic elements can be analyzed and appreciated under confocal microscopy, and further reconstructed in a three-dimensional pattern. Here, various kinds of biomarkers were used to reveal the morphological characteristics of NMJ, in which, NF200 is highly expressed on the myelinated axons20,21, VAChT accumulated at the pre-synaptic terminals22, α-BTX is exhibited on the post-synaptic AchRs23,24, and S100 is shown on the PSCs25,26. Taking together our results and those from previous studies indicates that these biomarkers can be combined freely to label the structural elements of NMJ according to the experimental design for assessing their spatial relationship to one another1,28,29.
To obtain ideal imaging with this technique, the post-fix time is an important issue to be paid attention to because over-fixation may cause high background. Therefore, it is recommended that the post-fix time should not be over 2 h in the fix solution. In addition, in order to observe more NMJ in muscular sections, it is critical to slice the muscle horizontally along the long axis at a thickness of 80 µm, which can comprehensively demonstrate the spatial correlation of the pre-synaptic terminals, post-synaptic AchRs, and PSCs in the NMJ. Here, it should be emphasized that a sliding microtome is a convenient tool for slicing the muscular tissue. Similarly, a cryostat and vibratome can also be used to get thick sections.
In summary, the morphological characteristics of NMJ have been widely studied from two-dimensional histological sections to three-dimensional whole-mount tissue by using different techniques. Considering the laborious and time-consuming tissue treatments for examination with electron microscopy and light-sheet microscopy, multiple fluorescent staining on the thicker tissue section is still a convenient approach to effectively explore the stereological architecture of NMJ by using confocal microscopy.
Subscription Required. Please recommend JoVE to your librarian.
Disclosures
The authors declare no conflicts of interest.
Acknowledgments
This study was funded by the CACMS Innovation Fund (No. CI2021A03407), National Natural Science Foundation of China (No. 82004299), and the Fundamental Research Funds for the Central Public Welfare Research Institutes (No. ZZ13-YQ-068; ZZ14-YQ-032; ZZ14-YQ-034; ZZ201914001; ZZ202017006; ZZ202017015).
Materials
| Name | Company | Catalog Number | Comments |
| 4',6-diamidino-2-phenylindole dihydrochloride | ThermoFisher | D3571 | |
| Confocal laser scanning microscope | Olympus | FV1200 | |
| Donkey anti-chicken AF488 | Jackson | 149973 (703-545-155) | |
| Donkey anti-goat AF546 | ThermoFisher | A11056 | |
| Donkey anti-rabbit AF488 | ThermoFisher | A21206 | |
| Donkey anti-rabbit AF546 | ThermoFisher | A10040 | |
| Frozen Section Medium | ThermoFisher | Neg-50 | Colorless |
| Microscope cover glass | Citotest | 10212450C | |
| Microtome | Yamato | REM-710 | |
| Neurofilament 200 | Sigma-Aldrich | N4142 | Rabbit |
| Neurofilament 200 | Abcam | ab4680 | Chicken |
| Normal donkey serum | Jackson ImmunoResearch Laboratories | 017-000-12 | 10 ml |
| Normal saline | Shandong Hualu Pharmaceutical Co.Ltd | H37022750 | 250 ml |
| Paraformaldehyde | Macklin | P804536 | 500g |
| Phalloidin AF350 | ThermoFisher | A22281 | |
| Precision peristaltic pump | Longer | BT100-2J | |
| S100-β | Abcam | ab52642 | Rabbit |
| Sodium phosphate dbasic dodecahydrate | Macklin | S818118 | 500g |
| Sodium phosphate monobasic dihydrate | Macklin | S817463 | 500g |
| Sucrose | Macklin | S818046 | 500g |
| Superfrost plus microscope slides | ThermoFisher | 4951PLUS-001E | |
| Triton X-100 | Solarbio Life Sciences | 9002-93-1 | 100 ml |
| Vesicular Acetylcholine Transporter | Milipore | ANB100 | Goat |
| α-bungarotoxin AF647 conjugate | ThermoFisher | B35450 |
References
- Kawabuchi, M., et al. The spatiotemporal relationship among Schwann cells, axons and postsynaptic acetylcholine receptor regions during muscle reinnervation in aged rats. TheAnatomical Record. 264 (2), 183-202 (2001).
- Nishimune, H., Shigemoto, K. Practical anatomy of the neuromuscular junction in health and disease. Neurologic Clinics. 36 (2), 231-240 (2018).
- Guarino, S. R., Canciani, A., Forneris, F. Dissecting the extracellular complexity of neuromuscular junction organizers. Frontiers in Molecular Biosciences. 6, 156 (2020).
- Cruz, P. M. R., Cossins, J., Beeson, D., Vincent, A. The neuromuscular junction in health and disease: molecular mechanisms governing synaptic formation and homeostasis. Frontiers in Molecular Neuroscience. 13, 610964 (2020).
- Matthews-Bellinger, J., Salpeter, M. Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions. The Journal of Neuroscienc : the Official Journal of the Society for Neuroscience. 3 (3), 644-657 (1983).
- Desaki, J., Uehara, Y. Formation and maturation of subneural apparatuses at neuromuscular junctions in postnatal rats: a scanning and transmission electron microscopical study. Developmental Biology. 119 (2), 390-401 (1987).
- Marques, M., Santo Neto, H. Imaging neuromuscular junctions by confocal fluorescence microscopy: individual endplates seen in whole muscles with vital intracellular staining of the nerve terminals. Journal of Anatomy. 192, 425-430 (1998).
- Magill, C. K., et al. Reinnervation of the tibialis anterior following sciatic nerve crush injury: a confocal microscopic study in transgenic mice. Experimental Neurology. 207 (1), 64-74 (2007).
- Yin, X., et al. Spatial distribution of motor endplates and its adaptive change in skeletal muscle. Theranostics. 9 (3), 734-746 (2019).
- Cai, R., et al. Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections. Nature Neuroscience. 22 (2), 317-327 (2019).
- Feng, G., et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron. 28 (1), 41-51 (2000).
- Chen, W. T., et al. In vivo injection of -bungarotoxin to improve the efficiency of motor endplate labeling. Brain and Behavior. 6 (6), 00468 (2016).
- Sugiura, Y., Lin, W. Neuron-glia interactions: the roles of Schwann cells in neuromuscular synapse formation and function. Bioscience Reports. 31 (5), 295-302 (2011).
- Alvarez-Suarez, P., Gawor, M., Proszynski, T. J. Perisynaptic schwann cells - The multitasking cells at the developing neuromuscular junctions. Seminars in Cell & Developmental Biology. 104, 31-38 (2020).
- Walker, C. L. Progress in perisynaptic Schwann cell and neuromuscular junction research. Neural Regeneration Research. 17 (6), 1273-1274 (2022).
- Michaud, M., et al. Neuromuscular defects and breathing disorders in a new mouse model of spinal muscular atrophy. Neurobiology of Disease. 38 (1), 125-135 (2010).
- Sharma, S., et al. Heat-induced endoplasmic reticulum stress in soleus and gastrocnemius muscles and differential response to UPR pathway in rats. Cell Stress Chaperones. 26 (2), 323-339 (2021).
- Raikova, R., Celichowski, J., Angelova, S., Krutki, P. A model of the rat medial gastrocnemius muscle based on inputs to motoneurons and on an algorithm for prediction of the motor unit force. Journal of Neurophysiology. 120 (4), 1973-1987 (2018).
- Marinello, M., et al. Characterization of neuromuscular junctions in mice by combined confocal and super-resolution microscopy. Journal of Visualized Experiments: JoVE. (178), e63032 (2021).
- Perrot, R., Berges, R., Bocquet, A., Eyer, J. Review of the multiple aspects of neurofilament functions, and their possible contribution to neurodegeneration. Molecular Neurobiology. 38 (1), 27-65 (2008).
- Yuan, A., Rao, M. V., Nixon, R. A. Neurofilaments and neurofilament proteins in health and disease. Cold Spring Harbor Perspectives in Biology. 9 (4), 018309 (2017).
- Petrov, K. A., Proskurina, S. E., Krejci, E.
- Karlin, A. Emerging structure of the nicotinic acetylcholine receptors. Nature Reviews. Neuroscience. 3 (2), 102-114 (2002).
- Rudolf, R., Straka, T. Nicotinic acetylcholine receptor at vertebrate motor endplates: Endocytosis, recycling, and degradation. Neuroscience Letters. 711, 134434 (2019).
- Barik, A., Li, L., Sathyamurthy, A., Xiong, W. C., Mei, L. Schwann cells in neuromuscular junction formation and maintenance. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 36 (38), 9770-9781 (2016).
- Kang, H., Tian, L., Thompson, W. J. Schwann cell guidance of nerve growth between synaptic sites explains changes in the pattern of muscle innervation and remodeling of synaptic sites following peripheral nerve injuries. Journal of Comparative Neurology. 527 (8), 1388-1400 (2019).
- Wang, J., et al. A new approach for examining the neurovascular structure with phalloidin and calcitonin gene-related peptide in the rat cranial dura mater. Journal of Molecular Histology. 51 (5), 541-548 (2020).
- Hughes, B. W., Kusner, L. L., Kaminski, H. J.
- Boehm, I., et al. Comparative anatomy of the mammalian neuromuscular junction. Journal of Anatomy. 237 (5), 827-836 (2020).
