Proximal trajectory of the brachium conjunctivum in rat fetuses and its early association with the parabrachial nucleus. A study combining in vitro HRP anterograde axonal tracing and immunocytochemistry

The proximal course of the developing brachium conjunctivum (BC) in the rat is described from embryonic day 16 (E16) to one day postnatal (P1). Axons of the cerebellar deep nuclear neurons entering this bundle were identified by anterograde axonal tracing after in vitro horseradish peroxidase (HRP) injections in the cerebellar plate. At all ages, the main ascending limb of the BC can be followed from its emergence, dorsal to the cerebellar plate where it assumes an almost vertical course, up to its decussation. Close to the ventricle at E16, the decussating fibers are progressively displaced ventrally probably because of the fusion, on the midline, of bilaterally produced raphe neurons. In E16 and E17 embryos, labeled BC fibers extend beyond the decussation in the caudal part of the red nucleus. Decussating BC axons, in some E16 early embryos, end with large and complicated growth cones, as described previously in ‘decision regions’ for chick embryo motoneurons. Growth cones were never observed in this region in older embryos. In addition to the main ascending limb of the BC, we also traced its ipsilateral descending limb and the cerebello-olivary projections. In parallel, the development of a nucleus immunoreactive for the vitamin D-dependent calcium-binding protein (CaBP) is reported. By E16, its neurons migrate rostrally and settle in the region where the BC is demonstrated by tracing experiments. At E17 and thereafter this isthmic nucleus is composed of a shell of CaBP-immunoreactive neurons ensheathing an immunonegative cylinder. Between E17 and birth, in spite of the profound modifications of the isthmic region, this CaBP-immunoreactive nucleus remains in close proximity to the BC. This nucleus is identified as the marginal nucleus of the BC or parabrachial nucleus, by double-labeling experiments combining the visualization of the retrogradely labeled axons and neurons of the deep cerebellar nuclei inside the CaBP immunofluorescently labeled parabrachial nucleus. Subsequently the deep cerebellar neurons translocate caudoventrally moving away from the parabrachial nucleus inside which their axons become visible. This pattern of migration could indicate that a few neurons of the deep nuclei remain ectopic, wedged between the restiform body and the BC while receiving an appropriate Purkinje cell (PC) projection.