Multiple subcellular mRNA distribution patterns in neurons: a nonisotopic in situ hybridization analysis.

Previous studies have established that most of the mRNAs that neurons express are localized in the cell body and very proximal dendrites, whereas a small subset of mRNAs is present at relatively high levels in dendrites. It is not clear, however, whether particular mRNAs have the same subcellular distribution in different types of neurons or whether different types of neurons sort mRNAs in different ways. The present study was undertaken to address these questions. Nonisotopic in situ hybridization techniques were used to define the subcellular localization of representative mRNAs including beta-tubulin, low-molecular-weight neurofilament protein (NF-68), high-molecular-weight microtubule-associated protein (MAP2), growth-associated protein 43 (F1/GAP43), the alpha subunit of calcium/calmodulin-dependent protein kinase II (alpha CaMII kinase), and poly (A+) mRNA. The mRNAs for beta-tubulin, neurofilament 68, and F1/GAP43 were restricted to the region of the cell body and very proximal dendrites in most neurons. In some neuron types, however, labeling for NF-68 extended for considerable distances into dendrites. In some neurons that express MAP2, the mRNA was present at the highest levels in the proximal third to half of the dendritic arbor, whereas in other neurons the highest levels of labeling were in the cell body. In most neurons that express alpha CaMII kinase, the highest levels of the mRNA were in the cell body, but labeling was also present throughout dendrites. However, in a few types of neurons, alpha CaMII kinase mRNA was largely restricted to the cell body. The fact that there are no general rules for mRNA localization that apply to all neuron types implies the existence of neuron type-specific mechanisms that regulate mRNA distribution.

Correspondence between L7-lacZ-expressing Purkinje cells and labeled olivocerebellar fibers during late embryogenesis in the mouse.

It has been suggested that Purkinje cells (PC) play a role in organizing topographic relationships of several cerebellar afferent systems, including olivocerebellar fibers. This hypothesis is based on the observation that PC in the rat express biochemical heterogeneities during the presumptive period of olivocerebellar fiber ingrowth to the cerebellum. Previous studies designed to investigate the organization of murine olivocerebellar fibers during embryogenesis have suggested that interactions with PC may play a role in segregating olivocerebellar fibers after they enter the cerebellum. To determine whether PC heterogeneities are related to olivocerebellar fiber organization, transgenic mice carrying a beta-galactosidase (beta-gal) reporter gene linked to the promoter from the PC-specific gene L7/pcp-2 were used in neuroanatomical tracing experiments. Expression of the transgene mirrors endogenous L7/pcp-2 expression, which is upregulated earliest in parasagittal strips of the vermal cortex. Studies were conducted in vitro by using brainstem-cerebellar explants from embryonic day 17/18 (E17/18) and 18/19 mice. Applications of neuroanatomical tracer (horseradish peroxidase or neurobiotin) were made in either the caudal medial accessory olive (cMAO) or the rostral olive. These studies indicate that groups of olivocerebellar fibers and clusters of L7/lacZ+ and L7/lacZ-Purkinje cells respect common distribution boundaries during late embryogenesis. The strong correspondence between the distribution patterns generated by these two markers suggests that expression of L7/pcp-2 and the topographic organization of olivocerebellar (OC) fibers are not interdependent, but may be regulated by a common event or interaction, of a presently unknown nature, which occurs earlier during cerebellar development.

Evidence of early topographic organization in the embryonic olivocerebellar projection: a model system for the study of pattern formation processes in the central nervous system.

Many projection systems within the peripheral and central nervous system are topographically organized, and it has become increasingly clear that interactions which occur during development determine the projection patterns these systems exhibit in the adult. The olivocerebellar system was chosen as a model system for this study of afferent pattern formation because it has several characteristics which lend themselves to a study of this type. Applications of horseradish peroxidase were made to both the cerebellar primordium and to the inferior olive of embryonic and neonatal mice using an in vitro perfusion system to support the tissue during the transport period. Fibers labeled after restricted olivary applications are limited to particular mediolateral regions of the cerebellum. Similarly, olivary cells retrogradely labeled after discrete cerebellar applications are restricted to particular olivary subdivisions. The results indicate that the olivocerebellar projection displays elements of topographic organization as early as E15 and that the pattern displayed is roughly comparable to that of the adult mammal. The observed trajectories of olivocerebellar fibers and their concomitant association with both Purkinje and cerebellar nuclear cells during embryonic development suggests a role for either or both cell types in the pattern formation process.