Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons
Luppi P.H., Fort P., Jouvet M.
Brain Res. 534 (1-2) pages : 209-224 (1990)


Materials and Methods

Materials and Methods


(A) Injection sites

(B) Retrograde labeling

(C) Artefactual labeling due to uptake by fibers of passage

(D) Anterograde tracing

(E) Double immunostaining technique



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(D) Anterograde tracing

As described above, we observed intense retrograde and anterograde immunostaining of lesioned fibers particularly after CTb pressure injections in fiber bundles. However, many features indicated that a specific anterograde transport of CTb arose from the neurons localized in the sites of injection to their terminals. Indeed, after iontophoretic injections without any destruction of fibers of passage, immunostained fibers formed bundles which could sometimes be followed over several millimeters terminating with many arborizations in the well-known efferents of the injected region. The morphology of the labeled axons and axon terminals was clearly demonstrated including boutons en passant, fine collateral branches and various terminal specializations.

For example, as illustrated in Fig. 2, after an iontophoretic injection in the nucleus reticularis parvicellularis of the medulla oblongata (Pc) and 72 h of survival including 48 h of colchicine treatment, we observed a bundle of fibers arising from the site taking a ventral route in the rostral medulla (dorsal to the facial nucleus, Fig. 2E) and caudal pons, ascending ventrally to the trigeminal motor nucleus and terminating with many arborizations in the nuclei parabrachialis lateralis and Kolliker-Fuse at the level of the pons (not illustrated). We also found numerous varicose labeled fibers in the hypoglossal (Fig. 2E), facial (Fig. 2D) and trigeminal motor nuclei. These fibers are likely to be anterogradely labeled because previous anterograde20 as well as retrograde 14 15 tracing studies demonstrated a massive efferent projection from the Pc to these cranial motor nuclei as well as to the pontine nuclei parabrachialis lateralis and Kolliker-Fuse.

On the other hand, we never detected bundles of retrogradely labeled fibers in the well characterized pathways from the bed nucleus of the stria terminalis and the central nucleus of the amygdala to the Pc despite the considerable number of retrogradely labeled cell bodies found in these nuclei. These results demonstrate that, although necessarily present in the fibers arising from the retrogradely labeled cells, CTb is below the detectable level in these fibers.

Taken together, these findings prove that CTb can be used as an anterograde tracer. We further determined that the extent of this anterograde labeling depended on the time of survival. Indeed, in cats with short survivals (24-72 h), we detected anterograde labeling only 6-8 mm away from the site. For example, after pressure or iontophoretic injections of CTb in the nucleus raphe dorsalis (NRD) and 72 h of survival including 48 h of colchicine treatment, anterograde labeling was observed only for 6-8 mm in the midbrain and pons. In contrast, as shown in Fig. 3E and F, in a cat with an iontophoretic application of CTb in the NRD, 18 days of survival and no colchicine treatment, we observed a very extensive anterograde labeling of fibers in the median forebrain bundle (Fig. 3E) and many terminal arborizations in forebrain and frontal cortical areas (Fig. 3F). These results corroborate the previous autoradiographic anterograde study of Bobillier et al. 7 and demonstrate that CTb is anterogradely transported via a slow axonal mechanism.

It must also be noted that the quality of this anterograde labeling highly depended on the immunohistochemical technique used. Indeed, we determined that it was much more extensive when using streptavidin-HRP rather than ABC-HRP. With ABC-HRP, the staining of axons and axon terminals was limited to their varicosities and therefore looked granular, whereas it completely filled them when using streptavidin-HRP (see Fig. 3E and F). This result confirms the greater sensitivity of streptavidin-HRP over ABC-HRP.

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