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.