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 ...

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

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

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. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

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

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

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

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

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 calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

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 impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

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

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

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

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

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 limited information about muscle fiber membrane properties and underlying disease mechanisms. Muscle velocity recovery cycles (MVRCs) illustrate how the velocity of a muscle action potential depends on the time after a preceding action potential. MVRCs are closely related to changes in membrane potential that follow an action potential, thereby providing information about muscle fiber membrane properties. MVRCs may be recorded quickly and easily by direct stimulation and recording from multi-fiber bundles in vivo. MVRCs have been helpful in understanding disease mechanisms in several neuromuscular disorders. Studies in patients with channelopathies have demonstrated the different effects of specific ion channel mutations on muscle excitability. MVRCs have been previously tested in patients with neurogenic muscles. In this prior study, muscle relative refraction period (MRRP) was prolonged, and early supernormality (ESN) and late supernormality (LSN) were reduced in patients compared to healthy controls. Thereby, MVRCs can provide in vivo evidence of membrane depolarization in intact human muscle fibers that underlie their reduced excitability. The protocol presented here describes how to record MVRCs and analyze the recordings. MVRCs can serve as a fast, simple, and useful method for revealing disease mechanisms across a broad range of neuromuscular disorders.

Presented here is a protocol for the recording of muscle velocity recovery cycles (MVRCs), a new method of examining muscle membrane properties. MVRCs enable in vivo assessment of muscle membrane potential and alterations in muscle ion channel function in relation to pathology, and it enables the demonstration of muscle depolarization in neurogenic muscles.

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