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

This protocol helps quantitatively analyze the 3D structure of neuromuscular junctions at a resolution close to electron microscopy using a simple procedure. The main advantage of this protocol is that muscle samples are processed similarly and immunolabeled for confocal and STED microscopy, reducing the time for accurate neuromuscular junction structure evaluation in animal models. The morphological quantification that we developed can be easily adapted to analyze other subcellular structures.

It is my pleasure to introduce Dr.Martina Marinello, a scientist from my team who will be showing how muscle teasing and immunostainings are performed and give some recommendations for the successful application of this protocol. I am pleased to introduce Dr.Jeremie Cosette from Genethon's imaging platform who will be showing image acquisition and analysis using confocal and STED microscopes. Post-euthanization, begin teasing the tibialis anterior muscles in small fiber bundles of about one-millimeter wide using two fine serrated forceps.

Then, transfer these muscle bundles into a 24-well plate containing 1%Triton X-100 prepared in PBS and allow it to gently agitate for one hour at room temperature or five hours at four degrees Celsius. After washing the samples three times for five minutes with PBS at room temperature, incubate the samples with a blocking solution composed of 4%BSA prepared in PBS with 1%Triton X-100 for four hours at four degrees Celsius under gentle agitation. Then, incubate the samples overnight with the same blocking solution containing primary monoclonal antibodies against either neurofilament M or synaptic vesicle glycoprotein 2 to label presynaptic axon terminals or active zones, respectively.

The following day, wash the labeled muscle bundles three times for five minutes with PBS under gentle agitation. Then, incubate them with appropriate secondary antibodies by placing them on a shaker for two hours at room temperature. After washing the muscle bundles again, place them on a slide with mounting medium.

Then, place a 1.5-grade glass cover slip on the top, followed by cylindrical magnets on both sides of the slide to apply pressure and flatten the muscles. To acquire images using the confocal microscope, launch the microscope software and choose machine as configuration mode and collect image stacks of the neuromuscular junctions in each experimental group as per the settings mentioned in the text manuscript. At the end of the session, click on Open in 3D viewer and choose a neuromuscular junction representative of an experimental group to visualize the 3D labeling.

To calculate post-synaptic neuromuscular junction and plate volume, maximum intensity projection area, and relative tortuosity, drag and drop the macro for neuromuscular junction volume quantification to the Image J window, and when the macro opens in a second window, click on Macros and Run macro. Select the native folder containing the junction subfolders to be analyzed and click on Select. Then, in the saving folder popup menu, select the storage folder and click on Select.

In the new pop-up menu called Image Type, select the format of the Z stack acquisitions. Select the RGB channel corresponding to the staining of interest and indicate X, Y pixel size, and Z step. If proprietary file formats are selected, the macro directly reads pixel size and Z step.

To quantify presynaptic neurofilament accumulation, drag and drop the macro for neuromuscular junction accumulation quantification to the Image J window, and click on Macros and Run macro. Select the junction subfolders, storage folder, and the format of the Z stack acquisitions as demonstrated previously. Then, in the Staining Infos popup, indicate the pre-synaptic and post-synaptic label and color and click on OK.In the Pixel Size popup, indicate the X, Y pixel size as 0.072 micrometers, the Z step as 0.5 micrometers, and click OK.If proprietary file formats are selected, the macro directly reads pixel size and Z step.

To calculate the distance between the acetylcholine receptor stripes, select the Quantify menu on top of the central window. Click on the Tools tab, select intensity, and click on the line profile icon. Set Oversampling to one and tick Sort Channels.

Click on the Open Projects tab and select the filtered image to be analyzed. Then, click the draw line icon and trace a line crossing perpendicularly several stripes or junctional folds. Click on the top of the first peak and move the mouse pointer while pressing the left mouse button until the next maximum peak is reached.

Note the DX value, which corresponds to the distance between the two peaks. Right-click on the mouse while in the image of the right window and select Save ROIs. After clicking on the arrow icon, click on the ROI and delete it by clicking on the bin icon.

For calculating the acetylcholine receptor stripe's width, select intensity in the top left panel under the tools tab, click on the determine FWHM icon, and tick the sort channels. Then click on the Open Projects tab and select the filtered image to be analyzed. Next, click on the draw rectangle icon in the top menu of the right window.

Select a stripe that is either horizontal or vertical and draw a rectangle perpendicularly to the stripe. A profile appears in the central window. Click on either vertical or horizontal of the Average Projection menu located in the left panel, depending on whether the stripe orientation is vertical or horizontal.

Click on the Statistics in the central window to read the FWHM value and then save and delete the ROIs as demonstrated previously. The post-synaptic motor end plate, labeled with fluorescent alpha-Bungarotoxin, appeared smaller and fragmented in the mutants of two mouse lines. Quantification of the neuromuscular junction Z stacks using the customized Image J macros revealed a marked decrease in the end plate volume, maximum intensity projection, and relative tortuosity in both disease mouse models compared to controls, indicating neuromuscular junction maturation defects.

Quantifying the presynaptic axon terminal branch distribution using the Image J custom macro revealed an altered pattern in neurofilament M distribution in the two animal models with increased immunolabeling. Through synaptic vesicle glycoprotein 2 staining, a 43%reduction in the occupancy ratio of the acetylcholine receptor-containing regions with adjacent nerve terminal active zones was observed in Smn 2B/mice. The aspect of the neuromuscular junction postsynaptic end plates was clearly visualized by fluorescent alpha-Bungarotoxin labeling and intensity profile analysis.

Evaluating the neuromuscular junction parameters revealed an increase in the junctional fold distance and width of acetylcholine receptor stripes in the gastrocnemius muscle of the mutants. To obtain reliable results, it's crucial to tease muscles properly, paying a particular attention to the applied strength to separate muscle bundles. This protocol can help obtain a more in-depth morphological characterization of neuromuscular junction, which was previously accounted for only by complex and time-consuming procedures, such as transmission electron microscopy.

The STED imaging procedure paved the way for the investigation of new insights concerning pre-and post-synaptic markers, which contribute to the pathophysiology of diseases affecting neuromuscular junction.