The PHA-L Anterograde Axonal Tracing Method

The plant lectin Phaseolus vulgaris leukoagglutinin (PHA-L) has unique properties that have made it particularly useful for tracing the axonal projections of neurons (Gerfen and Sawchenko, 1984, 1985). When PHA-L is injected by iontophoresis into the brain and subsequently localized by standard im-munohistochemical techniques, neurons at the injection site are labeled in their entirety, revealing the morphology of dendritic arborizations and both local and distant axonal projections. PHA-L appears to label only those neurons that incorporate tracer at the injection site; i.e., there is negligible uptake of the tracer by fibers of passage and negligible retrograde axonal transport of the tracer. In addition, neurons and their axonal projections are labeled with the PHA-L method in a manner that rivals the best Golgi impregnation techniques. With this method the amplification of the signal is generated by the immunohistochemical procedure and both the fluorescent and peroxidase-coupled diaminobenzidine labels provide an accurate visualization of cellular and axonal morphology. Furthermore, because PHA-L is localized with standard immunohistochemical techniques, it may be combined with other neuroanatomical and neurohistochemical methods. Such combined methods generate information not only concerning the interrelations between separate populations of neurons but also about the neurochemical characteristics of such neurons.