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We have studied the expression of the NG2 chondroitin sulfate proteoglycan on bipotential glial precursor cells in cultures of postnatal rat optic nerve. Purified populations of these precursor cells were prepared by panning dissociated optic nerve cells on dishes coated with monoclonal A2B5 antibody. Using immunofluorescence double staining, we found that NG2 was present on almost 95% of the purified A2B5+ precursor cells. The NG2 core protein from optic nerve cells was identified by immune precipitation and PAGE and was found to be identical to the 300,000 Da NG2 core protein from a clonal rat cell line B49. Over a culture period of 5 d in medium containing 10% fetal calf serum, more than 80% of the NG2+ precursor cells acquired the glial fibrillary acidic protein (GFAP), an astrocyte-specific marker. Under these conditions, fewer than 10% of the NG2+ cells expressed galactocerebroside (GC), an oligodendrocyte-specific marker. These GFAP+GC- type II astrocytes continued to express the NG2 antigen for up to 10 d in culture. During a 5 d culture period in hormonally supplemented, serum-free medium, fewer than 15% of the NG2+ cells expressed GFAP, while up to 40% expressed GC. The NG2 antigen continued to be expressed for only a short period of time by these GFAP-GC+ oligodendrocytes, so that mature oligodendrocytes in the cultures became NG2-. These results support our previous suggestion that the NG2 antigen is found on a class of neural cells that can differentiate along more than one pathway.

The nerve growth factor-inducible large external glycoprotein (NILE) has been found only on the surface of neuronal cells and Schwann cells. Since NILE seems to be concentrated on neurites, we have investigated its possible role in the development of neurites in primary cultures of rat brain. Cultures of embryonic day 14 (E14) whole brain and cultures of postnatal day 5 (P5) cerebellum were grown in the presence of Fab' fragments of antibody against NILE in an attempt to perturb the normal pattern of neurite development. For comparison, cultures were treated with two other reagents that recognize neuronal cell surface molecules: tetanus toxin, which binds to the GD1b and GT1 gangliosides, and Fab' fragments of antibody against neural cell adhesion molecule (N-CAM). Under the conditions used, none of the exogenous reagents affected neurite outgrowth, but specific effects on neurite fasciculation were observed. Anti-NILE inhibited fasciculation in cultures of E14 whole brain but had no effect on fasciculatiyn in cultures of P5 cerebellum. Conversely, anti-N-CAM inhiBited fasciculation in cultures of P5 cerebellum, which contain the adult form of N-CAM, but had little effect on fasciculation in cultures of E14 whole brain, which contain the embryonic form of N-CAM. Tetaiius toxin had no effect on fasciculation in either culture system. Our results imply that NILE-mediated neurite-neurite interactions are stronger than N-CAM (embryonic)-mediated interactions in the E14 brain cultures, whereas N-CAM (adult)-mediated interactions are stronger than NILE-mediated interactions in the P5 cerebellar cultures.The nerve growth factor-inducible large external glycoprotein (NILE) was originally identified as a cell surface component of PC12 cells (1) which incorporated increased amounts of fucose and glucosamine upon exposure of the cells to nerve growth factor (2). Subsequently, it was shown that this 230-kDa glycoprotein was one of the components recognized by polyclonal antisera raised against PC12 cells (3). Closely related glycoproteins have now been found on the surfaces of all neuronal cell lines examined and all neurons in primary culture, including neurons of central, sensory, and sympathetic origin (4-7). Schwann cells also express a NILE-related component (5, 6). Although they are immunologically cross-reactive, these neuronal glycoproteins are not identical. Depending on the neuronal cell type from which they are derived, glycoproteins of the NILE family range in size from 215 to 230 kDa aM judged by their mobility in NaDodSO4/ PAGE (5-7).Anti-NILE antibodies are useful not only for biochemical studies of this family of neuronal glycoproteins but also for immunohistochemical identification of neuronal cells in primary culture (5, 6) and in tissue sections (8). In central nervous system tissue, astrocytes, oligodendrocytes, and fibroblasts do not express a NILE-related component, so that the anti-NILE antibody can be used with confidence to identify neurons. In our hands, NILE-related glycoproteins appear to...

A PCR-based strategy for amplifying putative receptors involved in murine olfaction was employed to isolate a member (OR3) of the seven-transmembrane-domain receptor superfamily. During development, the first cells that express OR3 appear adjacent to the wall of the telencephalic vesicle at embryonic day 10. The OR3 receptor is uniquely expressed in a subset ofolfactory cells that have a characteristic bilateral symmetry in the adult olfactory epithelium. This receptor and its specific pattern of expression may serve a functional role in odor coding or, alternatively, may play a role in the development of the olfactory system. The olfactory system combines broad selectivity with extreme sensitivity for the detection and discrimination of a large number of odorants. A combination of two cellular mechanisms might form the basis for odor discrimination: (i) selectivity by a limited set of olfactory receptors in each sensory neuron and (ii) spatio-temporal patterns of action potentials in cells projecting from the olfactory epithelium to the olfactory bulb. In vertebrates, odorants exert their actions by modulating the excitability of sensory neurons located in an epithelium in the nasal airways (1-4). Peripheral odor-induced activity is transmitted directly to the mitral cells in the olfactory bulb where it is integrated and relayed to other regions in the central nervous system (CNS) (5). Olfactory cilia on the apical ends of neuronal dendrites are embedded in a mucus layer that separates the olfactory epithelium from the external air. Signal transduction is initiated when odorants interact with specialized receptors in the ciliary membranes (6-8). In vitro, odorants can rapidly stimulate guanine nucleotide binding (G) protein-coupled second messenger pathways for both cAMP and inositol trisphosphate (9), suggesting that at least two classes of G-protein-coupled receptors are expressed by olfactory neurons. A group of 10-18 candidate olfactory receptors that appear to define a class in the G-protein-coupled receptor superfamily was identified in the olfactory epithelium (10). The tissue specificity and the large number of genes for these receptors suggest that they are responsible for the detection of numerous odorants, a hypothesis that awaits confirmation. Recently, members of a putative olfactory receptor gene family have been identified in mammalian germ cells, suggesting a broader range of function for these receptors (11).To understand the molecular and cellular basis of odor discrimination, we adopted a strategy to isolate olfactoryspecific receptor genes and to study their expression in the olfactory epithelium. In this report, we have characterized a G-protein-coupled receptor (OR3) ¶ and its gene expression.The OR3 pattern of expression is symmetric within and specific to the olfactory epithelium. We present evidence that the first OR3-positive cells appear to originate from the CNS, suggesting that this receptor might serve a functional role not only in odor coding but also in the elaboration ...

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