Quantitative analysis of axonal branching using the retrograde transport of fluorescent latex microspheres

A method is described for the quantitative analysis of double retrograde labelled neuronal cell bodies following labelling of branched axonal projections. This exploits the known ability of retrograde translocator proteins to transport latex microspheres following their uptake at nerve terminals. Conditions necessary for uptake and transport include small bead diameter (0.05-micron) and carboxylation of the latex particle. Using coumarin- and rhodamine-labelled microspheres a reliable, sensitive, rapid method has been developed, which results in double retrograde cell labelling in branched axonal pathways from the frontal cortex, basal forebrain, and brainstem. The technique has several advantages over currently available double retrograde labelling methods and yields repeatable quantitative estimates of populations of neurones bearing branched axons.