- First, immunostain Drosophila larvae with markers that highlight different aspects of NMJs or neuromuscular junctions. This is the region where the ending of a motor neuron axon contacts a muscle, and its morphology is used as a readout of synaptic function.
The axon terminates in several branches that form bulges called boutons. Neurotransmitters are released from boutons and cause muscle contraction. Areas of neurotransmitter release are called active zones. Here one marker is specific for a synaptic protein that outlines the NMJ, and the other marker is for a protein present in active zones.
Next, acquire NMJ images using microscopy and quantitatively assess their morphology with semi-automated image analysis software. Apply a preset series of software commands to the images. The output files contain analyzed NMJ images and morphology results. The features that can be measured include number and surface area of boutons, NMJ length and branch number, number of unconnected NMJ compartments, and number of active zones.
In the following example, we will analyze the morphology of Drosophila NMJs stained with DLG1, a postsynaptic marker, and BRP, an active zone marker.
- For this protocol, generate image stacks of NMJs and save them as individual TIFF files where channel 1 shows DLG1 staining or similar marker and channel 2 shows BRP staining. To begin, create Z projections and hyperstacks of the NMJ image files. Open the plugin options and select Drosophila NMJ morphometrics.
Now, identify the unique file string that the microscope has assigned to the image series when storing them as TIFF. This will be at the end of the image name. Copy and paste the string given to the lowest plane and channel number into the unique file string setting window. Then select the sub-macro 'Convert to stack' and choose the directory or folder where the images are located.
For each image file, two new files are made with the default names stack and flat stack, followed by the original image name. The original files can then be deleted to save storage space. Next, from the Drosophila NMJ morphometrics interface, select the defined ROI sub-macro and choose the image file directory.
As the first projection opens, select the freehand selections tool, then use the mouse to define a region containing a complete NMJ terminal of interest. Once selected, click OK in the defined terminal window. Continue doing this until the NMJ terminals are defined in all of the projections. The macro advances the process automatically. For each image file, one new file is made with the default names ROI followed by the original image name.
To quantify the NMJ features, first go to the Drosophila NMJ morphometrics interface and set the scale. For example, if one pixel of the image corresponds to 0.72 microns, set scale pixels to 1 and scale distance to 0.072. Then select the sub-macro Analyze, and if there are two channel images, also toggle Wait. Press OK and when prompted, select the image file directory. The processing time can be several minutes per synapse.
After the analysis, new image files for each analyzed synapse are stored in the parental folder, and the quantitative measurements are in the results.txt file. Inspect all the images and exclude pictures with segmentation errors.
For example, parts of the synaptic terminal might not be included in the yellow outline. Parts of the background might be included in the synaptic terminal. A blue skeleton line may extend beyond the synaptic terminal. There may be too many active zones, or some active zones may remain undetected.