Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons.

In this report, we demonstrate that cholera-toxin B subunit (CTb) is a very sensitive retrograde tracer in the central nervous system when recognized by streptavidin-peroxidase immunohistochemistry. We further show that: (1) injection of a small volume of CTb gives rise to small sharply defined injection sites limited to the cell group of interest associated with the labeling of all the known afferent projections, (2) CTb is taken up, and anterogradely as well as retrogradely transported in damaged but not intact fibers of passage, (3) CTb can be applied iontophoretically, allowing us to study the afferents to small cell groups without any evidence of tissue necrosis in the sites and therefore without artefactual labeling due to uptake by damaged fibers of passage, (4) the use of 4% paraformaldehyde fixative ideally suited for the preservation of most neural antigens, the addition of a 48 h colchicine treatment and the development of a double immunohistochemical method allow the biochemical characterization of the cell of origin of particular pathways in the CNS, (5) CTb is also anterogradely transported with an extensive filling of axons and axon terminals and thereby opens up the possibility of identifying simultaneously the afferents as well as the efferents of the group of cells studied and finally (6) the very long conservation of the preparation, the possibility of counterstaining it and of making camera lucida drawings allow easy and precise localization of the retrogradely labeled cells.