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The localization of vesicular glutamate transporter 2 (VGLUT2) was examined by immunohistochemistry and in situ hybridization histochemistry in the developing rat olfactory region with special relation to the spatiotemporal location of NCAM, a neural cell adhesion molecule expressed in differentiated neurons, and the calcium-binding protein calbindin D-28k, a marker of neurons migrating from the vomeronasal organ anlage (Y. Toba et al. (2001) J. Neuroendocrinol., 13, 683-694). Both VGLUT2 and NCAM immunoreactivities were first detected at embryonic day 11.5 (E11.5) in the neuronal cell mass beneath the telencephalic vesicle. After E12.5, VGLUT2-immunoreactive cells were detected in the migratory pathways from both medial and lateral olfactory pits, anlagen of the vomeronasal organ and olfactory epithelium. Between E15.5 and E19.5, moderate to intense VGLUT2 immunoreactivity was observed in cell clusters situated along NCAM-bearing vomeronasal nerves, and frequently colocalized with calbindin D-28k immunoreactivity. Using in situ hybridization histochemistry, VGLUT2 mRNA signals were detected in the clustered cells as well as in cells of the vomeronasal and olfactory epithelium. After E20.5, migrating cells gradually decreased in number and VGLUT2 immunoreactivity attenuated in the clustered cells, although calbindin D-28k immunoreactivity in these residual cells was still intense. The presence of intense VGLUT2 immunoreactivity in neurons actively migrating from the olfactory placode suggests that this transporter is involved in the migratory process of these neurons.

In rat fetuses, neurons generated in the lower rhombic lip on embryonic day 14 (E14) specifically express the transcription factor Pax-6 and migrate circumferentially in the subpial region along axons which are immunoreactive for TAG-1, a neural cell adhesion molecule. These neurons ultimately settle from E17 in the contralateral medulla oblongata to form the precerebellar nuclei, the lateral reticular and external cuneate nuclei. We have examined this migratory process in rat small eye (rSey2) which has a mutation in the Pax-6 gene. Both the migration and settlement of neurons labelled with 5-bromo-2'-deoxyuridine (BrdU) at E14 delayed in the mutant. Furthermore, in the ventrolateral part of the E16 mutant medulla oblongata, cells both expressing Pax-6 mRNA and prelabelled with BrdU at E14 were ectopically localized in the deep zone of the medullary parenchyma in addition to the normal subpial location. These ectopic neurons remained in close contact with ectopic TAG-1-immunoreactive axons. These results indicate that Pax-6 plays a role in the migration of medullary precerebellar neurons, although neurons generated in the lower rhombic lip can nevertheless migrate and settle to form the external cuneate nucleus in the absence of Pax-6.