A method for measuring colocalization of presynaptic markers with anatomically labeled axons using double label immunofluorescence and confocal microscopy.

Information concerning the location and distribution of presynaptic neurotransmitter release sites within anatomically labeled axons would be of value for a large number of studies in functional anatomy, development, and plasticity. Here we report a method for localizing presynaptic sites within identified arbors of interest using anterograde anatomical tracer injections to label axonal projections and synaptic vesicle protein (SVP) antibodies to label presumptive presynaptic terminals. The axons and presynaptic sites are independently visualized with double label immunofluorescence and confocal microscopy. Stacks of images representing adjacent focal planes are collected, and image processing techniques are applied to identify the location of each axonal branch segment and each cluster of SVP label in three-dimensional space. Segmentation of the SVP label into distinct pixel clusters in three-dimensional space, followed by colocalization of these clusters with the labeled axons (object-based analysis), yields much more reliable and sensitive measures of colocalization than a simple determination of the number (or summed intensities) of colocalized pixels in a single optical section (pixel-based analysis). The method has been extended to measure the colocalization of antigens that are not located at the presynaptic terminal with a labeled population of axons.