We thank Genethon&#39;s &#34;Imaging and Cytometry Core Facility&#34;, as well as the histology service, which are supported in part by equipment funds from the Region Ile-de-France, the Conseil General de l&#39;Essonne, the Genopole Recherche of Evry, the University of Evry Val d&#39;Essonne and the INSERM, France. We are also grateful to Dr. Rashmi Kothary for providing the Smn2B/2B mouse line (University of Ottawa, Canada) and Dr. Eric Krejci for the ColQDex2/+ mouse line (unpublished, University of Paris, France). We thank Guillaume Corre for his support in statistical analysis. The 2H3 (developed by Jessel, T.M. and Dodd, J.) and SV2 (developed by Buckley, K.M.) monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was supported by the Association Fran&#231;aise contre les Myopathies (AFM-Telethon), the INSERM and the University of Evry Val d&#39;Essonne.

<ol>
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The authors declare no conflicts of interest related to this work.

The described video protocol provides a detailed method to quantify the 3D structure of neuromuscular junctions by combining confocal and STED microscopy that can be used to characterize pathological changes at the pre- and postsynaptic levels. The high resolution of STED microscopy allows visualization and morphometric analysis of nanostructures that are not identifiable by conventional confocal imaging. This procedure enabled us to measure structural alterations of NMJs in two appendicular muscles, tibialis anterior and gastrocnemius, of SMA and ColQ-related CMS mice.
To obtain reliable results with this technique, it is critical to dissect and tease muscles properly, paying particular attention to the fascia surrounding the muscle and the applied strength to separate muscle bundles; otherwise, the innervation pattern could be disrupted impeding proper presynaptic NMJ assessment. Although detailed information is provided to analyze NMJs from TA and GA, in principle, this protocol could be adapted to other muscles, including flat muscles, such as the diaphragm or transverse abdominis37, which do not require the teasing step. Tissue fixation is also crucial to ensure good quality staining; therefore, it is recommended to use high-quality PFA at an appropriate volume (15-20 times that of the muscle). In addition, the exposure time to the fixative is an important step because artifacts, such as shrinkage and clumping, may appear due to over-fixation and influence NMJ features. Given the size of the samples and the penetration rate of the paraformaldehyde solution in tissues38, a fixation time of 18-24 h is recommended for this type of muscle. In case the staining step is planned more than a week after tissue harvesting, it is suggested to keep PFA-fixed muscles in PBS supplemented with sodium azide at 4 &#176;C to prevent bacterial proliferation.
This protocol presents an approach using &#945;-BTX-F488 for confocal and &#945;-BTX-F633 for STED imaging. These fluorophores were chosen to fit with the described experimental design but can be modified according to the available equipment and materials. For instance, &#945;-BTX F488 labeling can be selected when using a STED CW 592 nm laser for image acquisitions and quantification. However, it appears that the configuration that was applied in the present study (pulsed excitation gated STED, 775 nm depletion) exhibits higher performance and better resolution than other approaches, such as continuous wave STED39, making it more suitable for the current application. It is also important to select carefully the laser power settings, especially for STED (both excitation and depletion), since the characteristics of an intensity profile cannot be measured in case of saturation, and therefore any saturated signal in a NMJ image could jeopardize the whole analysis.
This detailed workflow, including image acquisitions and analysis using microscope software and ImageJ macros, was developed to facilitate autonomous NMJ morphometric analysis by confocal and STED microscopy from a single muscle. Previously described workflows for NMJ confocal analysis, such as NMJ-morph2 or NMJ-Analyser14, paved the way for the design of semi-automatic methods that facilitate morphological analysis of NMJs and comparative studies. NMJ-morph (and its updated version aNMJ-morph15) is a free ImageJ-based platform that uses the maximal intensity projection to measure 21 morphological features, and NMJ-Analyser uses a script developed in Python that generates 29 relevant parameters from the entire 3D NMJ structure. Manual thresholding is the only step during image processing in these two methods that require user analysis. This integrated protocol details steps for tissue preparation, 3D confocal image acquisitions, and ImageJ-based processing of NMJs from entire skeletal muscles and provides a simplified overview of five important parameters of the postsynaptic (volume, maximal projection area, and tortuosity) and presynaptic (axon terminal occupancy and neurofilament accumulation) endplates. An additional parameter of biological relevance, the AChR organization pattern of the postsynaptic junctional folds, was incorporated for morphometric analysis at the nanoscale level by super-resolution STED microscopy (resolution 20-30 nm)40. Interestingly, tissue preparation for STED imaging is simpler than other methods used for NMJ ultrastructural studies, such as conventional transmission electron microscopy (TEM)9, which is a rather complex and time-consuming procedure that requires a skilled manipulator in order to obtain ultrathin sections of the appropriate muscle region. In addition, quantitative data from multiple junctional folds can be obtained automatically by using the STED-associated software.
This protocol was applied to illustrate previously known NMJs defects in SMN and ColQ deficient muscles20,36,41,42. Common changes were found in the two mouse models by confocal microscopy, such as decreased postsynaptic endplate volume, MIP area, and relative tortuosity, and increased neurofilament accumulation, whereas some more specific findings (decreased NMJ occupancy), were observed only in SMA mice, as an indicator of impaired vesicles trafficking36. Finally, an increase in AChR stripe distance and width were detected in ColQ-KO by STED analysis, which are signs of ultrastructural defects in the postsynaptic junctional folds, as previously observed by TEM20. Importantly, this protocol may help in a more in-depth morphological characterization of neuromuscular junctions during development, maintenance, and under various pathological conditions.

The neuromuscular junction (NMJ) is a complex structure composed of a motor axon terminal, a perisynaptic Schwann cell, and a skeletal myofiber portion involved in the transmission of chemical information and coupling of lower motor neuron activity to muscle contraction. In mammals, the morphology of the neuromuscular junction changes during development, adopting a typical pretzel-like shape after maturation, with differences in shape and complexity between species, and shows some degree of plasticity in response to physiological processes such as exercise or aging1,2,3,4. The postsynaptic motor endplate forms membrane invaginations named junctional folds, where the upper part containing acetylcholine receptors (AChR) is in close contact with the presynaptic terminal axon branch5.
Morphological and functional changes in neuromuscular junctions contribute to the pathophysiology of several neurodegenerative disorders such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), myopathies like Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), myasthenia gravis (MG) and centronuclear myopathies (CNM), and aging-associated sarcopenia3,6,7,8,9,10,11,12. In these diseases, NMJ structural alterations such as endplate fragmentation, reduced postsynaptic junctional fold size and/or denervation are observed. The pathology of NMJs can be a primary or early event during disease progression or appear more lately as a secondary event contributing to the clinical manifestations. In any case, monitoring the morphology of NMJs in animal models of these diseases represents a valuable parameter to study pathological changes and assess the efficacy of potential treatments.
The morphology of neuromuscular junctions is usually analyzed by techniques using confocal microscopy2,13,14,15 or electron microscopy5,16, with their inherent limitations such as resolution or technical difficulties, respectively. More recently, super-resolution microscopy was also used to visualize particular regions of the NMJ, such as presynaptic active zones or AChR distribution on the postsynaptic membrane16,17,18, as an alternative or complementary approach to ultrastructural analysis by electron microscopy.
This protocol aims to provide a detailed and reproducible method to assess NMJ morphological parameters by combining fluorescence confocal and stimulated emission depletion (STED) microscopy. Important features of the presynaptic and postsynaptic endplates, such as volume, area, relative tortuosity, AChR stripe width, and axon terminal distribution in innervated teased muscle fibers of mouse&#160;gastrocnemius and tibialis anterior were quantified in the context of normal and diseased conditions. In particular, NMJ defects were exemplified in the Smn2B/- mouse model of spinal muscular atrophy, a neuromuscular disease with motor neuron degeneration caused by mutations in the SMN1 gene11,19, and in a collagen-like tail subunit of asymmetric acetylcholinesterase knockout (ColQDex2/Dex2 or ColQ-KO) mice, as a model of the congenital myasthenic syndrome20,21,22.

<table><tbody><tr><td>&lt;strong&gt;Buffers and Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG (F488)</td><td>Life Technologies, Thermofisher</td><td>A-11001</td><td></td></tr><tr><td>Alexa Fluor 488 &amp;alpha;-bungarotoxin (F488-a-BTX)</td><td>Life Technologies, Thermofisher</td><td>B13422</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-mouse IgG (F594)</td><td>Life Technologies, Thermofisher</td><td>A-11032</td><td></td></tr><tr><td>ATTO-633 &amp;alpha;-bungarotoxin (F633-a-BTX)</td><td>Alomone Labs</td><td>B-100-FR</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>DAPI Fluoromount-G</td><td>Southern Biotech</td><td>00-4959-52</td><td></td></tr><tr><td>DPBS</td><td>Gibco, Invitrogen</td><td>14190-169</td><td></td></tr><tr><td>Ethanol Absolute</td><td>VWR</td><td>20821.296</td><td></td></tr><tr><td>Immersion Oil, n = 1.518</td><td>THORLABS</td><td>MOIL-10LF&amp;nbsp;</td><td>Low autofluorescence</td></tr><tr><td>Neurofilament (NF-M) antibody&amp;nbsp;</td><td>DSHB</td><td>AB_531793</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>MERCK</td><td>1.04005</td><td></td></tr><tr><td>Synaptic vesicle glycoprotein 2 (SV2) antibody</td><td>DSHB</td><td>AB_2315387</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alnico Button cylindrical magnets</td><td>Farnell France</td><td>E822</td><td>diameter of 19.1 mm with maximal pull of 1.9 Kg</td></tr><tr><td>63x 1.4 NA magnitude oil immersion&amp;nbsp; HCX Plan Apo CS objective</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>100x 1.4 NA HC PL APPO CS2 Objective</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Curved thin forceps-Moria iris forceps</td><td>Fine Science Tools</td><td>11370-31</td><td></td></tr><tr><td>Extra thin scissors - Vannas-T&amp;uuml;bingen Spring Scissors</td><td>Fine Science Tools</td><td>15-003-08</td><td></td></tr><tr><td>Fine serrated forceps</td><td>Euronexia</td><td>P-95-AA</td><td></td></tr><tr><td>Gel loading tip round 1-200 &amp;micro;L</td><td>COSTAR</td><td>4853</td><td></td></tr><tr><td>Leica laser-scanning confocal microscope TCS SP8</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leica Laser-scanning confocal microscope TCS SP8 Gated STED 775 nm</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Lens Cleaning Tissue&amp;nbsp;</td><td>Whatman (GE Healthcare)</td><td>2105-841</td><td></td></tr><tr><td>Medium serrated forceps</td><td>Euronexia</td><td>P-95-AB</td><td></td></tr><tr><td>Microscope cover glasses 24x50 nm No 1.5H 170&amp;plusmn;5 &amp;micro;m</td><td>Marienfield</td><td>107222</td><td>High precision</td></tr><tr><td>Nunclon delta surface (12-well plates)</td><td>Thermo Scientific&amp;nbsp;</td><td>150628</td><td></td></tr><tr><td>Nunclon delta surface (24-well plates)</td><td>Thermo Scientific&amp;nbsp;</td><td>142475</td><td></td></tr><tr><td>Safeshield scalpel</td><td>Feather</td><td>02.001.40.023</td><td></td></tr><tr><td>Sharp-blunt scissors - fine Scissors - Martensitic Stainless Steel</td><td>Fine Science Tools</td><td>14094-11</td><td></td></tr><tr><td>Superfrost plus slides</td><td>Thermo Scientific&amp;nbsp;</td><td>J1800AMNZ</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GraphPad&amp;nbsp;</td><td>Prism, San Diego (US)</td><td>Release N&amp;deg;6.07</td><td>Statistical software</td></tr><tr><td>ImageJ software</td><td>National Institutes of Health</td><td>Release N&amp;deg; 1.53f</td><td></td></tr><tr><td>Leica Application Suite X software</td><td>Leica Microsystems</td><td>Release N&amp;deg;3.7.2.2283</td><td>Free microscope software available at https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/downloads/&amp;nbsp;</td></tr></tbody></table>

characterization of neuromuscular junctions in mice by combined confocal and super-resolution microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

In order to facilitate the morphological analysis of neuromuscular junctions at the pre- and postsynaptic level in a reproducible manner, a workflow was developed from muscle harvesting to imaging and quantification using the microscope software and ImageJ custom macros (Figure 1). To exemplify the utility of this protocol, the morphology of NMJs in two mouse models of genetic disorders, Smn2B/- and ColQDex2/Dex2 mice affected by spinal muscular atrophy (SMA) and a congenital myasthenic syndrome (CMS) form, respectively, were evaluated and data were compared to age-matched control littermates.
The NMJ structure was assessed from tibialis anterior and gastrocnemius muscles of 3- and 6-week-old Smn2B/- (C57Bl/6 background) and ColQDex2/Dex2 (B6D2F1/J background) mice, respectively, when signs of the disease are already present in these animals. At 3 weeks of age, Smn2B/- mice show signs of delayed skeletal muscle development and denervation, such as NMJ atrophy and loss35,36. CMS mice have a primary pathology in NMJs and manifest a reduction in body weight from the first week of life and marked muscle weakness20 (data not shown). As shown in Figure 2A, the postsynaptic motor endplate labeled with fluorescent &#945;-bungarotoxin appeared smaller and/or fragmented in mutants of the two mouse lines by confocal microscopy. Quantification of NMJ Z-stacks using this customized ImageJ macros revealed marked decreases in endplate volume, maximum intensity projection (MIP), and relative tortuosity in both SMA and CMS mice compared to controls, as signs of NMJ maturation defects32 (Figure 2B-D). Postsynaptic endplate volume and MIP were decreased in diseased animals (fold-change of 2.7 and 2.0 for volume, and 2.5 and 2.0 for MIP, in Smn2B/- and ColQDex2/Dex2 mice, respectively). The relative tortuosity was also smaller in SMN and ColQ deficient muscles&#160;than WT (16.97% &#177; 1.33% in SMA&#160;versus 48.84% &#177; 5.90% WT mice, and 13.29% &#177; 2.79% in CMS versus 30.20% &#177; 4.44% control mice). In addition, the quantification of the distribution of presynaptic axon terminal branches using the ImageJ custom macro revealed an altered pattern in neurofilament M distribution in the two animal models, with increased immunolabelling (84.65% &#177; 0.32% versus 16.57% &#177; 2.03% and 23.64% &#177; 2.78% versus 18.77% &#177; 1.73% in Smn2B/- and ColQDex2/Dex2 mice compared to controls, respectively) (Figure 3A-D). By SV2 staining, a 43% reduction in the occupancy ratio, i.e., percent of AChR-containing regions with adjacent nerve terminal active zones, was also observed in Smn2B/- mice (49.36% &#177; 3.76% in SMA versus 85.69% &#177; 2.34% WT mice) (Figure 3E,F). This NMJ parameter was also calculated in GA of ColQDex2/Dex2 mutants, but no statistically significant difference was found compared to control littermates (data not shown).
We further analyzed postsynaptic membrane characteristics by quantifying the distance between junctional folds and the width of AChR stripes, which are located at the crest of these folds, in ColQ-deficient muscle using super-resolution stimulated emission depletion (STED) microscopy. As shown in Figure 4, the aspect of these structures can be clearly visualized by fluorescent &#945;-bungarotoxin labeling and intensity profile analysis. We&#160;evaluated these NMJ parameters and found an increase in the junctional fold distance (d) and width (w) of AChR stripes in the gastrocnemius muscle of mutants (358.3 nm &#177; 11.97 nm and 320.8 nm &#177; 10.90 nm for the distance, and 216.9 nm &#177; 10.51 nm and 186.3 nm &#177; 7.015 nm for the width, in ColQDex2/Dex2 as compared to wild-type mice, respectively, p &#60; 0.05) (Figure 4C,D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63032/63032fig01.jpg" /> 
Figure 1: Flowchart of the video protocol for 3D multiscale NMJ characterization by confocal and STED microscopy. Tibialis anterior (TA) and gastrocnemius (GA) muscles were collected from mice, and muscle fibers were teased before labeling with &#945;-bungarotoxin-F488 or &#945;-bungarotoxin-F633, DAPI, primary antibodies directed against neurofilament M (NF-M) and synaptic vesicle glycoprotein 2 (SV2), and fluorophore (F488 or F594)-conjugated secondary antibodies. Image stacks were acquired by confocal microscopy and processed to measure postsynaptic NMJ volume, presynaptic NF-M accumulation, NMJ axon terminal occupancy, postsynaptic maximum intensity projection (MIP) endplate area, and tortuosity (dObj(AB) is the distance between A and B along the perimeter of the object (red line), whereas dEuc(AB) is the Euclidian distance between A and B (green line)). For STED microscopy analysis, the width of acetylcholine receptor (AChR) stripes and the distance between junctional folds were quantified from intensity profiles of &#945;-BTX-F633 staining. <a href="https://www.jove.com/files/ftp_upload/63032/63032fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63032/63032fig02.jpg" /> 
Figure 2: Multi-parameter postsynaptic NMJ characterization in mouse models of spinal muscular atrophy (SMA) and ColQ-related congenital myasthenic syndrome (CMS). (A) Representative images of postsynaptic motor endplates from TA and GA muscles labeled with &#945;-bungarotoxin-F488 (&#945;-BTX). (B) Quantification of NMJ postsynaptic endplate volume, (C) maximal intensity projection (MIP) area and (D) relative tortuosity in TA of 3 week-old wild-type (WT) and Smn2B/- mice (left graphs, N = 3 animals per genotype, n = 37 and n = 56 NMJs, respectively) and 6 week-old WT and ColQDex2/Dex2 mice (right graphs, N = 5 mice per genotype, n = 89 and n = 97 NMJs, respectively). Data are expressed as the mean per mouse (dot) &#177; SEM. Differences between groups were analyzed by Mann-Whitney test (* p &#60; 0.05). Scale bar is 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63032/63032fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63032/63032fig03.jpg" /> 
Figure 3: Morphometric analysis of presynaptic axon terminal distribution in muscles of WT and mutant mice. NMJ innervation pattern in tibialis anterior (TA) and gastrocnemius (GA) muscles of wild-type, SMA and ColQ-related CMS mice. (A, B) Representative neuromuscular junctions from TA of WT and Smn2B/- mice at 21 days of age labeled with antibodies against neurofilament M (NF-M, red) and &#945;-bungarotoxin-F488 (&#945;-BTX, green) (A), and results from quantitative analysis of neurofilament accumulation (B); (C, D) Representative neuromuscular junctions from GA of 6 week-old WT and ColQDex2/Dex2 mice labeled with antibodies against neurofilament M (NF-M, red) and &#945;-bungarotoxin-F488 (&#945;-BTX, green), showing fragmented and immature postsynaptic endplates (C), and results of neurofilament accumulation in the two groups of animals (D). N= 4 (n = 34 NMJs) (B) and N = 3 (n = 54 NMJs) (D) WT animals, and N=3 (n = 36 NMJs) Smn2B/- and N = 3 (n = 55 NMJs) ColQDex2/Dex2 mice were analyzed in the experiments (B, D). (E, F) Representative images of axon terminal occupancy in NMJs from TA of 3 week-old WT and Smn2B/- mice labeled with antibodies against synaptic vesicle glycoprotein 2 (SV2, red) and &#945;-bungarotoxin-F488 (&#945;-BTX, green) (E), and results of NMJ occupancy (SV2/AChR volume ratio) (F). Muscles from N = 3 (n = 50 NMJs) wild-type and N = 4 (n = 62 NMJs) Smn2B/- mice were analyzed. Data are expressed as the mean value per mouse (dot) &#177; SEM. Differences between groups were analyzed by Mann-Whitney test (* p &#60; 0.05). Scale bars are 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63032/63032fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63032/63032fig04.jpg" /> 
Figure 4: STED imaging of NMJ postsynaptic endplates. (A) Representative STED image of a NMJ labeled with &#945;-bungarotoxin-F633 (&#945;-BTX) from gastrocnemius of a 6 week-old wild-type mouse showing postjunctional AChR stripes (scale bar is 5 &#181;m). (B) Higher magnification of a region with AChR stripes (bottom panel) that was used to generate the intensity profile. The width (w) of AChR stripes and the distance between two adjacent stripes (d) of this region were quantified and presented in the bar graph. Schematic representation of the postsynaptic endplate to illustrate AChR stripe width (w) and distance (d). These parameters, (C) AChR stripe distance and (D) width, were measured in ColQDex2/Dex2 mice and control littermates at 6 weeks of age. NMJs from 5 WT (total n = 29 NMJs) and 6 ColQDex2/Dex2 (total n = 43 NMJs) animals were analyzed blindly. Data are expressed as the mean per mouse (dot) &#177; SEM. Statistical differences between groups were analyzed by using the Mann-Whitney test (* p &#60; 0.05). <a href="https://www.jove.com/files/ftp_upload/63032/63032fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: Launch of LAS X software and parameters for confocal acquisitions. The various steps to acquire confocal images are described in sections 3.1.2 to 3.1.7 of the protocol. For each NMJ stack acquisition, a project is opened (step 3.1.4) and the parameters of image size, acquisition speed, X, Y and Z axes are selected (step 3.1.7), with each sequential scan indicated (Seq.1, laser 405 for DAPI; Seq.2, laser 488 for &#945;-BTX-F488; and Seq.3, laser 552 for F594 conjugated secondary antibodies). <a href="https://www.jove.com/files/ftp_upload/63032/Supplemental Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure 2: Launch of LAS X software and parameters for STED acquisitions. The steps to acquire STED images are described in sections 3.2.2 to 3.2.8 of the protocol. The microscope is launched in configuration mode STED ON (step 3.2.2), and a project is opened (step 3.2.3). The parameters for image acquisition (step 3.2.7) (image size, acquisition speed, Zoom factor, X axis), with each sequential scan are indicated (Seq.1 for &#945;-BTX-F633; Seq.2 for F488 conjugated secondary antibodies). <a href="https://www.jove.com/files/ftp_upload/63032/Supplemental Figure 2.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure 3: Images of &#945;-BTX-stained junctional folds obtained by STED microscopy. Image examples of a postsynaptic endplate labeled with&#160;&#945;-BTX-F633 from a 6 week-old wild-type mouse that were acquired with either a correct (left) or incorrect focus (right). <a href="https://www.jove.com/files/ftp_upload/63032/Supplemental Figure 3.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure 4: Windows pop-ups to describe the input and output data obtained by the custom ImageJ macros. Input data examples (.tif and .lif files) of NMJ images are shown on the left column. The output data from the macros (right column) are saved in folders (Save_Volume, Save_Accu) that contain images of the junction (.tif) and datasheets containing the results (.csv files). <a href="https://www.jove.com/files/ftp_upload/63032/Supplemental Figure 4.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure 5: AChR stripe distance and width analysis from a STED acquisition using the LAS X software. The steps to analyze NMJ STED images are described in section 5 of the protocol. A) Image of a labeled postsynaptic endplate region containing AChR stripes. The region of interest for stripe analysis is selected by drawing a perpendicular line (green line, for stripe distance), or a perpendicular rectangle (purple rectangle, for stripe width). (B, C) Intensity profiles of the selected regions and measurements to calculate the distance between AChR stripes (B) and the AChR stripe width (C) are shown. <a href="https://www.jove.com/files/ftp_upload/63032/Supplemental Figure 5.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 1: Macro_NMJ_VOL_Marinelloetal. ImageJ custom macro to extract NMJ parameter measurements (NMJ volume, MIP endplate area, and NMJ tortuosity). <a href="https://www.jove.com/files/ftp_upload/63032/Macro_NMJ_VOL_Marinelloetal_63032.ijm" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 2: Macro_NMJ_ACCU_Marinelloetal. ImageJ custom macro to extract NF-M accumulation and SV2 staining. <a href="https://www.jove.com/files/ftp_upload/63032/Macro_NMJ_ACCU_Marinelloetal_63032.ijm" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy at JoVE.com

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

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JoVE Journal

Neuroscience

3.6K Views.  Genethon, 91000, Evry, France. Dit protocol helpt kwantitatief de 3D-structuur van neuromusculaire juncties te analyseren met een resolutie die dicht bij elektronenmicroscopie ligt met behulp van een eenvoudige procedure. Het belangrijkste voordeel van dit protocol is dat spiermonsters op dezelfde manier worden verwerkt en immunoactief worden gelabeld voor confocale en SOA-microscopie, waardoor de tijd voor nauwkeurige neuromusculaire junctiestructuurevaluatie in diermodellen wordt verkort. De morfologische kwantificering ...

Video: Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

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 ...

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. ...

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

Developmental Biology

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

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

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...

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.

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 ...

Viewing Microtubule Networks in Drosophila Neuromuscular Junctions and Muscle Cells

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

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