(C) Artefactual labeling due to uptake by fibers of passage
After pressure applications in fiber bundles such as the lateral vestibulo-spinal
and olivo-cerebellar tracts, the medial lemniscus and medial longitudinal
fasciculus, we observed CTb immunostaining of fibers belonging to these
tracts not only in the anterograde direction up to the terminals but also
in the retrograde direction all the way to their retrogradely labeled
parent cell bodies. For example, following pressure injections in the
olivocerebellar tract, axons were immunostained on their way back to their
retrogradely labeled parent cell bodies in the contralateral inferior
olivary complex but also as far as their terminals in the cerebellum.
These findings clearly demonstrate that CTb is taken up, and anterogradely
but also retrogradely transported by fibers of passage. However, many
points suggest that this uptake occurred only in damaged fibers of passage.
Indeed, after CTb pressure injections centered on the lateral vestibulospinal
tract with its destruction by the needle of the Hamilton Syringe, we detected
a number of retrogradely labeled cells in the Deiters nucleus as well
as immunostained fibers in the tract rostral and caudal to the injection
site. In contrast, when the site involved the tract but without mechanical
injury to it, no labeled cells were observed in the Deiters nucleus nor
any labeled fibers in the tract. Furthermore, in contrast with pressure
injections, 2- uA iontophoretic applications with 20-um micropipettes
centered on the olivocerebellar tract gave rise only to occasional retrogradely
labeled neurons in the contralateral olivary complex and no or only a
few labeled fibers in the tract. Taken together, these results indicate
that CTb is taken up and transported by damaged but not by intact fibers
of passage, a drawback nearly eliminated by iontophoretic applications.
Importantly, in contrast to the specific anterograde labeling (see below),
the artefactual labeling of lesioned fibers was observed for long pathways
even after the shortest survival (24 h), suggesting that CTb is transported
in lesioned axons by a fast axonal transport mechanism. For example, after
CTb injections in the fasciculus longitudinalis medialis at the level
of the raphe dorsalis and 24-72 h of survival, we detected anterogradely
labeled lesioned fibers 13 mm caudally in the inferior olivary complex.