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).

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 &#177; 15 g) were used. The rats were housed in a 12 h light/dark cycle with controlled temperature and humidity and with free acc...

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The authors declare no conflicts of interest.

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 bi...

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 &#945;-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 &#181;m 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 (&#945;-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole dihydrochloride</td>
			<td>ThermoFisher</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-chicken AF488</td>
			<td>Jackson</td>
			<td>149973 (703-545-155)</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat AF546</td>
			<td>ThermoFisher</td>
			<td>A11056</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit AF488</td>
			<td>ThermoFisher</td>
			<td>A21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit AF546</td>
			<td>ThermoFisher</td>
			<td>A10040</td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen Section Medium</td>
			<td>ThermoFisher</td>
			<td>Neg-50</td>
			<td>Colorless</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>Citotest</td>
			<td>10212450C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Yamato</td>
			<td>REM-710</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurofilament 200</td>
			<td>Sigma-Aldrich</td>
			<td>N4142</td>
			<td>Rabbit</td>
		</tr>
		<tr>
			<td>Neurofilament 200</td>
			<td>Abcam</td>
			<td>ab4680</td>
			<td>Chicken</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>017-000-12</td>
			<td>10 ml</td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>Shandong Hualu Pharmaceutical Co.Ltd</td>
			<td>H37022750</td>
			<td>250 ml</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Macklin</td>
			<td>P804536</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Phalloidin AF350</td>
			<td>ThermoFisher</td>
			<td>A22281</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision peristaltic pump</td>
			<td>Longer</td>
			<td>BT100-2J</td>
			<td></td>
		</tr>
		<tr>
			<td>S100-&#946;</td>
			<td>Abcam</td>
			<td>ab52642</td>
			<td>Rabbit</td>
		</tr>
		<tr>
			<td>Sodium phosphate dbasic dodecahydrate</td>
			<td>Macklin</td>
			<td>S818118</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic dihydrate</td>
			<td>Macklin</td>
			<td>S817463</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Macklin</td>
			<td>S818046</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Superfrost plus microscope slides</td>
			<td>ThermoFisher</td>
			<td>4951PLUS-001E</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio Life Sciences</td>
			<td>9002-93-1</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Vesicular Acetylcholine Transporter</td>
			<td>Milipore</td>
			<td>ANB100</td>
			<td>Goat</td>
		</tr>
		<tr>
			<td>&#945;-bungarotoxin AF647 conjugate</td>
			<td>ThermoFisher</td>
			<td>B35450</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing the morphological characteristics of neuromuscular junction in rat medial gastrocnemius muscle

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

After multiple fluorescent staining, the corresponding labeling was orderly demonstrated on the 80 &#181;m thick sections of the rat medial gastrocnemius muscle with NF200-positive nerve fibers, VAChT-positive pre-synaptic terminals, &#945;-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 ra...

Watch this Scientific Journal Video about Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle at JoVE.com

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

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

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

This protocol can be used to evaluate integrity and plasticity of a neuromuscular junction under normal and pathological conditions. This technique is a to simultaneously label the Schwann cells and pre and post lab animals on single muscle sections. After euthanizing the adult male rat, place the rat in the hood and open the thoracic cavity to access the heart using scissors and forceps.Insert and intravenous catheter from the left cardiac ventricle toward the aorta and cut the right oracle. Next, start the perfusion with 100 milliliters of 0.9%normal saline until the blood exiting from the right oracle is clear. Then continue to perfuse with 250 to 300 milliliters of 4%paraformaldehyde in 0.1 molar phosphate buffer or PB pH 7.4 for 10 minutes.After perfusion, remove the skin of the bilateral hind limb using a surgical blade and expose the bilateral sciatic nerve and medial gastrocnemius muscle. Dissect out the bilateral medial gastrocnemius muscle carefully from the hind limb using a blade and postfix the entire muscle in 10 milliliters of 4%paraformaldehyde. After two hours, remove the muscle from the solution and cryoprotect it in 10 milliliters of 25%sucrose in 0.1 molar PB for one day at four degrees celsius.Once the muscular tissue is immersed in the sucrose solution, fix it on a freezing stage of a sliding microtome system using a frozen section medium. Slice the muscle horizontally along the long axis of the muscle at the thickness of 80 micron. Using a brush, place seven cryo sections per well in an orderly fashion in a six well culture plate with 10 milliliters of 0.1 molar PB.For fluorescent staining, incubate four sections per well with 1.5 milliliters of 0.1 molar PB containing 75 microliters of 10%Triton X 100 and 45 microliters of normal donkey serum on an orbital shaker at 20 rpm.After 30 minutes, transfer the sections into 1.5 milliliters of a mixed solution of primary antibodies containing rabbit anti-neuro filament 200, goat anti-vesicular acetylcholine transporter in 0.1 molar PB, in 1%normal donkey serum, and 0.5%Triton X 100 and incubate overnight at four degrees Celsius. The next day, wash the sections three times for five minutes each in 0.1 molar PB on in orbital shaker at 20 rpm. After the last wash, incubate the section in a mixed solution of secondary antibodies containing donkey anti-rabbit AF 488 and donkey anti-goat AF 546, as well as the biomarkers of alpha bungaro toxin AF 647 and phalloidin 350 containing in 0.1 M PB containing 1%normal donkey serum and 0.5 Triton x 100 for one hour, Protecting them from light.After incubation, wash the sections in PB and mount them on a microscope slide. Apply cover glass to the section using 50%glycerin in distilled water. Observe and image the specimen using a con focal imaging system equipped with a 20 x subjective lens with a numerical aperture of 0.75 and a 40 x lens with a numerical aperture of 0.95.For multiple fluorescent imaging, set the excitation and emission wavelengths of each biomarker as described in the text manuscript. Set the resolution of image capture to 640 by 640 pixels. Next, set the start and end focal plane.Set step size to either one or two microns. Then choose depth pattern, image capture, and Z series. For the lower magnification images at 20 X, capture 40 Z stack images in two micron step size using a 105 micron pinhole.Choose the projection or topography mode and intensity projection over Z axis to integrate all images in a single in focus. For the higher magnification images at 40 X, capture 80 Z stack images in one micron step size with zoom set to two and a 105 micron pinhole. Integrate all images in a single in focus.Reconstruct the images in the three dimensional pattern with the image processing system. The representative spacial correlation of the medial gastrocnemius muscle with neuro filament 200 positive nerve fibers, vesicular acetylcholine transporter positive pre-synaptic terminal, alpha bungaro toxin positive post-synaptic acetylcholine receptors, S100 positive perisynaptic Schwann cells, phalloidin positive muscular fibers, and DAPI labeled cellular nuclei are demonstrated here. The neurofilament 200 positive nerve fibers ran in bundle.These nerve fibers gave branches to the vesicular acetylcholine transporter positive presynaptic terminals, which formed a mirror relationship with the alpha bungaro toxin positive post-synaptic acetylcholine receptors. In addition, the S100 positive perisynaptic Schwann cells were detected around the neurofilament 200 positive nerve fibers close to the pre-synaptic terminals. The spatial correlation was further exhibited in a three dimensional pattern showing the detailed morphological characteristics of the neuromuscular junction from different perspectives.In order to observe more imaging in muscular sections, it is critical to slice the muscle horizontally along the long axis at a thickness of 80 micron.

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Research

JoVE Journal

Neuroscience

3.0K Views.  China Academy of Chinese Medical Sciences. This protocol can be used to evaluate integrity and plasticity of a neuromuscular junction under normal and pathological conditions. This technique is a to simultaneously label the Schwann cells and pre and post lab animals on single muscle sections. After euthanizing the adult male rat, place the rat in the hood and open the thoracic cavity to access the heart using scissors and forceps.Insert and intravenous catheter from the left cardiac ventricle toward the aorta and cut the right ...