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

The overall goal of this procedure is to objectively quantify synaptic parameters, including synapse size, fragmentation, and the relative density of proteins in the post synaptic membrane. This is accomplished by first dissecting the muscle of interest from the experimental animals. Next, the dissected muscle is cryo sectioned, and the sections are immuno stain for fluorescence microscopy.

Then on phos, images of neuromuscular junctions from the different treatment groups are objectively sampled, and the relative intensity of staining for synaptic specializations is quantified. Ultimately, reproducible quantitative measures of the effects of a treatment or genetic modification on the structural organization of the neuromuscular junction are obtained by confocal microscopy. Well, this method provides standardized objective protocols using confocal microscopy to assess changes in the structure of the neuromuscular junction in mouse models of disorders such as myasthenia gravis and motor neuron disease.

Generally, people who attempt this method may find it difficult because sampling NPLS on the confocal microscope in a unbiased and objective manner requires planning After sampling analysis of NPLS needs to be conducted in a consistent and reproducible fashion where animal to animal variability between nplate parameters can make this difficult. After isolating cryosectioning and immuno fluorescently staining, mouse neuromuscular junctions or njs, according to the text protocol, blind the slides by labeling each one with a random number that remains known only to a second researcher until after quantification of NMJ parameters is complete. Place a slide on the microscope stage and use a trit C filter set to view it under wide field illumination.

Move progressively through the fields left to right and back until an endplate appears in the field with the confocal pinhole set to 1.0 airy unit, and using low laser power, optimize the gain and offset levels for trit C red BGT at the end plate to be imaged. Next, using the 488 nanometer laser, optimize fit C fluorescence, which represents synaptophysin, then collect a Z stack of the end plate with a 0.7 micron interval between each optical slice. Save the images with a file name that includes the date of the imaging session, the code name of the slide, and the number of the end plate.

Repeat the sampling and imaging procedure just demonstrated until 20 end plates are collected from the slide or sample. And then repeat the process for the remaining coded slides under the same confocal settings. Take a few images from the control slide.

Use image J freeware to open a Zack and in one fluorescent channel, prepare a maximum projection image. Save the projections as TIF files with the file name, including the image session date, the sample code, the endplate number, and the fluorescent channel. Now use image J to open the Z projection.

Select the acetylcholine receptor or A CHR image channel and select image type eight bit to convert the 24 bit RGB colored image into one eight bit gray scale image on the screen with the polygon tool. In the red BGT stain channel, draw a rough outline around the endplate of interest, excluding any staining that does not originate from the endplate of interest. Select image adjust threshold to apply a minimum intensity threshold to the image.

Adjust the threshold level to isolate the A CHR stained portions while excluding the surrounding background signal as sub-threshold. Then open a second window with the original full tone image for comparison. To confirm the threshold settings with the polygon outline still present, select analyze, analyze particles in the popup menu.

Specify the range of sizes in number of pixels as 50 to infinity, which eliminates selection of tiny artifacts arising from electrical noise. In the photo multiplier, click on analyze particles to generate a window with a list of discreet supra threshold areas and their fluorescence intensity values numbered as they appear in the binary image. Copy this data into a labeled spreadsheet to measure the total end plate area.

Within the polygon, select Analyze measure, copy and paste the data for A CHR areas and intensities into a spreadsheet with labeled columns. Switch to the synaptophysin channel. Prepare a maximum projection image.

Select an area of interest, and set the minimum intensity threshold as just demonstrated. To measure the area of overlap, apply the following steps. Open the original file containing the two channel images and select image stacks.

Stack to images to split it into two separate images. Using the image J colocalization plugin, select plugin colocalization and input the threshold values previously recorded for the A CHR and nerve channels into the respective channel query box. The result is an overlap image in white pixels.

Convert the newly created overlap image into a gray scale format and apply a threshold to the maximum value, which will only select the white pixels corresponding to the overlap area of the two previous channels. Record in the spreadsheet the resulting area value of colocalization, which represents the area of overlap in pixels, prepare a spreadsheet of data. Sample means calculate and plot standard deviations and standard errors as histograms or scatter plots.

Plot and plate A CHR areas as scatter plots or frequency histograms to determine whether the data is normally distributed before statistical testing. After preparing immunofluorescent staining and imaging of transverse muscle fiber sections as outlined in the text protocol, open an original image file and while viewing the A CHR channel, select image stacks stack to images to split the channels select image type eight bit to convert eight bit gray scale format to the screen. Do this for both fluorescence channels.

Select image stacks images to stack and open a new stack from two previously separated eight bit images, which enables switching between the two fluorescence channels within the single window with the polygon tool. Draw a line tightly around the boundary of the A CHR staining, select analyze measure to measure the average intensity for A CHR within the enclosed area. Copy this value into a labeled spreadsheet while retaining the polygon outline.

Switch to the second fluorescence channel and select analyze measure. This will yield the average staining for the protein of interest within the synaptic area defined by A CHR staining. Choose an area away from visible endplate staining.

Then select analyze measure to measure the average background fluorescence intensity. Repeat the measurement for the other fluorescence channels and copy the background values into the spreadsheet of fluorescence values. Subtract the average background values from endplate values to obtain the corrected intensities for A CHR and the protein of interest at each endplate.

Finally, divide the corrected nplate intensity values for the protein of interest by the corrected BGT fluorescence intensity to yield the fluorescence intensity ratios as shown here. Fluorescence intensity for both A CHR and synaptophysin rises sharply at the boundary between the PERS synaptic and synaptic portion of the motor end plate. For such images, a minimum threshold will readily isolate the A CHR rich or synaptophysin rich area of the endplate.

As demonstrated in this figure in aged mice and in some disease states, endplate staining for synaptophysin may be less intense. A CHR cluster edges may appear blurred, and there may be higher levels of extra synaptic Autofluorescence scatterplots reveal a considerable range in the A CHR rich area among end plates of the tibials anterior muscle of any individual mouse. Nevertheless, the average A CHR area based on 15 to 20 on face endplate images was similar among seven sample mice In transverse sections, the endplate achr typically appear as a crescent shape, but this shape is often irregular as illustrated here.

Low intensity background fluorescence as shown here on the Synaptophysin channel, usually reveals whether a patch of A CHR staining represents a single end plate or two separate end plates located on adjacent muscle fibers. In addition, many synaptic proteins such as rapson, musk, and beta distro glycan are co localized with a CHR at the nplate. While attempting this procedure, it is important that experimenters remain blind to treatment and genotype groups during the sampling and analysis of nplate images.

After watching this video, you should have a good understanding of how to gather, sample, and analyze confocal fluorescent images of neuromuscular junctions. Ultimately, the researchers should be able to consistently and objectively compare different structural parameters of the nplate amongst different treatment groups and genotypes.