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 neurotr ansmitter release sites within anatomically labeled axons would be of value for a large number of studies in functional anatomy, development, and plas ticity. Here we report a method for localizing presynaptic sites within ide ntified arbors of interest using anterograde anatomical tracer injections t o label axonal projections and synaptic vesicle protein (SVP) antibodies to label presumptive presynaptic terminals. The axons and presynaptic sites a re independently visualized with double label immunofluorescence and confoc al microscopy. Stacks of images representing adjacent focal planes are coll ected, and image processing techniques are applied to identify the location of each axonal branch segment and each cluster of SVP label in three-dimen sional space. Segmentation of the SVP label into distinct pixel clusters in three-dimensional space, followed by colocalization of these clusters with the labeled axone (object-based analysis), yields much more reliable and s ensitive measures of colocalization than a simple determination of the numb er (or summed intensities) of colocalized pixels in a single optical sectio n (pixel-based analysis). The method has been extended to measure the coloc alization of antigens that are not located at the presynaptic terminal with a labeled population of axons. (C) 2000 Elsevier Science B.V. All rights r eserved.