An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L)

Abstract

A new neuroanatomical method for tracing connections in the central nervous system based on the anterograde axonal transport of the kidney bean lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L) is described. The method, for which a detailed protocol is presented, offers several advantages over present techniques. First, when the lectin is delivered iontophoretically, PHA-L injection sites as small as 50-200 micron in diameter can be produced, and are clearly demarcated since the neurons within the labeled zone are completely filled. Second, many morphological features of such filled neurons are clearly demonstrated including their cell bodies, axons, dendritic arbors and even dendritic spines. Third, there is some evidence to suggest that only the neurons at the injection site that are filled transport demonstrable amounts of the tracer, raising the possibility that the effective injection site can be defined quite precisely. Fourth, even with the most restricted injections, the morphology of the labeled axons and axon terminals is clearly demonstrated; this includes boutons en passant, fine collateral branches, and various terminal specializations, all of which can be visualized as well as in the best rapid Golgi preparations. Fifth, when introduced iontophoretically, PHA-L appears to be transported preferentially in the anterograde direction; only rarely is it transported retrogradely. Sixth, PHA-L does not appear to be taken up and transported effectively by fibers of passage. Seventh, there is no discernible degradation of the transported PHA-L with survival times of up to 17 days. Finally, since the transported marker can be demonstrated with either peroxidase or fluorescent antibody techniques, it may be used in conjunction with other neuroanatomical methods. For example, double anterograde labeling experiments can be done using the autoradiographic method along with immunoperoxidase localization of PHA-L, and the retrogradely transported fluorescent dyes can be visualized in the same tissue sections as PHA-L localized with immunofluorescence techniques.

Figure 1

Examples of types of labeling obtained with PHA-L as localized by an indirect immunofluorescence technique. PHA-L-lableed neurons at an in jection site in the lateral hypothalamus (A) show the typical morphology of labeled neurons, including their dendritic arbors. Projections of these labeled neurons in the lateral septal area (B, low magnification, and D, high magnification) and in the supramammilary nucleus (C, low magnification). Labeled hypothalamic afferents in the lateral septal area target the cell bodies and proximal dendrites of neurons (B) that at higher magnificaction display large boutons (D). On the other hand, afferents in the supramammilary nucleus ramify extensively and are distributed to more distal dendrites (C). (E) A high-power photomicrograph of one neuron labeled at an injection site in the striatum shows the detailed morphology of dendrites and their spines obtained with PHA-L labeling. (F) PHA-L labeled projections of striatal neurons in the globus pallidus demonstrate the fine details axonal and terminal morphology.