Cellular and Molecular Studies on Olfactory Bulb Ensheathing Cells

Olfactory bulb ensheathing cells (OBECs) are a specialised type of glial cell that reside in the outer layer of the olfactory bulb, where they are thought to play a role in the exceptional ability of this central nervous system tissue to support re-enervertion throughout life. This thesis reports on the characterisation of the antigenic expression and growth and functional properties of OBECs in vitro. OBECs were purified from perinatal rat olfactory bulbs by fluorescence activated cell sorting using the O4 antibody (Barnett et al., 1993a). Immunofluorescence labelling of the cultures and of tissue sections of the olfactory bulb with a large panel of neural marker antibodies showed that OBECs represent a heterogeneous population of cells. The antigenic profile of the sorted cell population initially correlated with their antigenic profile in vivo, although expression of some of the markers was either lost or gained during time in culture. These changes were influenced by the culture conditions, with a greater loss of "typical" OBEC markers in serum-containing medium. In serum-free medium, which maintained the cells in a phenotype that closely resembled their in vivo counterparts, two antigenically and morphologically distinct cell types were observed, reminescent of non-myelinating Schwann cells and type-1 astrocytes, respectively. Two similar phenotypes were detected in a clonal OBEC cell line generated by retroviral infection with the temperature sensitive mutant gene of the large T antigen, supporting the view that the astrocyte-like and Schwann cell-like cells belong to the same lineage. A detailed immunocytochemical analysis of the developing olfactory system suggested that the diversification of OBEC antigenic phenotype occurs prior to embryonic day 14, along both, the peripheral and central portion of the olfactory nerves, is maintained throughout development, and is controled by microenvironmental cues. Astrocyte conditioned medium (ACM) was shown to exibit potent mitogenic activity for cultured OBECs but most of the known growth factors present in ACM were not mitogenic when applyed individually using recombinant or highly purified preparations. The mitogenic activity of ACM could be reproduced with a glial growth factor (GGF)-enriched preparation of bovine pituitary extract. However, preliminary biochemical characterisation of the ACM-derived mitogenic activity did not correlate with that of any of the previously purified neuregulins and suggested that it may be confered by a novel member of this family of polypeptide growth factors. The ability of OBECs to promote neuronal attachment/survival, neurite initiation and neurite elongation was compared to that of astrocytes and Schwann cells in an in vitro system consisting of plating cerebellar granule neurons onto monolayers enriched for each glial cell type. OBEC monolayers grown in the presence or absence of serum exerted similar neurite-outgrowth promoting activity. They were similar than Schwann cells and astrocytes at supporting neuronal attachment/survival and at initiating neurite outgrowth. However, the extent of neurite elongation on OBECs was greater than on Schwann cells and smaller than on astrocytes. When neurons were plated onto sparse cultures of OBECs, neurite initiation and elongation was greater among neurons in contact with glial cells, suggesting a role for surface interactions