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 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.
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 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.
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 °C to prevent bacterial proliferation.
This protocol presents an approach using α-BTX-F488 for confocal and α-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, α-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 authors have nothing to disclose.
We thank Genethon's "Imaging and Cytometry Core Facility", 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'Essonne, the Genopole Recherche of Evry, the University of Evry Val d'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 Française contre les Myopathies (AFM-Telethon), the INSERM and the University of Evry Val d'Essonne.
Buffers and Reagents | |||
Alexa Fluor 488 goat anti-mouse IgG (F488) | Life Technologies, Thermofisher | A-11001 | |
Alexa Fluor 488 α-bungarotoxin (F488-a-BTX) | Life Technologies, Thermofisher | B13422 | |
Alexa Fluor 594 goat anti-mouse IgG (F594) | Life Technologies, Thermofisher | A-11032 | |
ATTO-633 α-bungarotoxin (F633-a-BTX) | Alomone Labs | B-100-FR | |
Bovine serum albumin (BSA) | Sigma | A2153 | |
DAPI Fluoromount-G | Southern Biotech | 00-4959-52 | |
DPBS | Gibco, Invitrogen | 14190-169 | |
Ethanol Absolute | VWR | 20821.296 | |
Immersion Oil, n = 1.518 | THORLABS | MOIL-10LF | Low autofluorescence |
Neurofilament (NF-M) antibody | DSHB | AB_531793 | |
Paraformaldehyde (PFA) | MERCK | 1.04005 | |
Synaptic vesicle glycoprotein 2 (SV2) antibody | DSHB | AB_2315387 | |
Triton X-100 | Sigma | T8787 | |
Materials | |||
Alnico Button cylindrical magnets | Farnell France | E822 | diameter of 19.1 mm with maximal pull of 1.9 Kg |
63x 1.4 NA magnitude oil immersion HCX Plan Apo CS objective | Leica Microsystems | ||
100x 1.4 NA HC PL APPO CS2 Objective | Zeiss | ||
Curved thin forceps-Moria iris forceps | Fine Science Tools | 11370-31 | |
Extra thin scissors – Vannas-Tübingen Spring Scissors | Fine Science Tools | 15-003-08 | |
Fine serrated forceps | Euronexia | P-95-AA | |
Gel loading tip round 1-200 µL | COSTAR | 4853 | |
Leica laser-scanning confocal microscope TCS SP8 | Leica Microsystems | ||
Leica Laser-scanning confocal microscope TCS SP8 Gated STED 775 nm | Leica Microsystems | ||
Lens Cleaning Tissue | Whatman (GE Healthcare) | 2105-841 | |
Medium serrated forceps | Euronexia | P-95-AB | |
Microscope cover glasses 24×50 nm No 1.5H 170±5 µm | Marienfield | 107222 | High precision |
Nunclon delta surface (12-well plates) | Thermo Scientific | 150628 | |
Nunclon delta surface (24-well plates) | Thermo Scientific | 142475 | |
Safeshield scalpel | Feather | 02.001.40.023 | |
Sharp-blunt scissors – fine Scissors – Martensitic Stainless Steel | Fine Science Tools | 14094-11 | |
Superfrost plus slides | Thermo Scientific | J1800AMNZ | |
Software | |||
GraphPad | Prism, San Diego (US) | Release N°6.07 | Statistical software |
ImageJ software | National Institutes of Health | Release N° 1.53f | |
Leica Application Suite X software | Leica Microsystems | Release N°3.7.2.2283 | Free microscope software available at https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/downloads/ |