Name: visualizing the morphological characteristics of neuromuscular junction in rat medial gastrocnemius muscle
Uploaded: 2022-05-17T14:00:00
Description: Watch this Scientific Journal Video about Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle at JoVE.com

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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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 ...

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Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties

Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide ...