Fig. 1.

Expression of neuron-specific markers and division rate of neonatal anterior subventricular zone (SVZa) progenitor cells in culture. (A) Diagram of a parasagittal section of the P1 rat forebrain depicting the SVZa (dark gray), the beginning of the rostral migratory stream (RMS; light gray) that was microdissected, dissociated and cultured for immunohistochemical and videomicroscopic analysis. Other subdivisions of the RMS [vertical limb of the RMS (RMSvl), elbow of the RMS (RMSe), horizontal limb of the RMS (RMShl) and subependymal zone (SEZ)] as well as the layers of the olfactory bulb are also indicated. SVZa-derived progenitor cells are fated to become the granule and periglomerular cell interneurons of the olfactory bulb. To reach their destination, SVZa-derived progenitor cells migrate along the RMS to the SEZ. At the SEZ they exit from the RMS and migrate radially into the overlying granule cell and glomerular layers of the olfactory bulb. Anterior is to the right and dorsal is up. AOB, accessory olfactory bulb; CC, corpus callosum; CTX, cerebral cortex; epl, external plexiform layer; gcl, granule cell layer; gl, glomerular layer; LV, lateral ventricle; mcl, mitral cell layer. (B and C) Representative fluorescent photomicrographs of SVZa progenitor cells cultured in serum-free medium for 2 (B) or 4 (C) DIV and then double-labeled with an antibody to neuron-specific tubulin (TuJ1) and an antibody to glial fibrillary acidic protein (GFAP); DAPI was used to label nuclei (not shown). In the fields shown all cells were TuJ1-positive and GFAP-negative. The images shown were captured using fluorescein optics to visualize TuJ1. The results are consistent with our previous analysis of SVZa progenitor cells cultured in serum-containing medium demonstrating that nearly all cultured neonatal SVZa progenitor cells were TuJ1-positive and GFAP-negative (see Luskin et al., 1997). As shown here, at 2 DIV, the majority of the TuJ1-positive cells have extended one or two processes (e.g. arrowheads in B) and, at 4 DIV, TuJ1-positive cells are observed with processes that are longer and more branched (e.g. arrowheads in C). Collectively, these findings demonstrate that the SVZa progenitor cells retain a neuronal phenotype and undergo differentiation in culture. (D) The distribution of cell cycle times for cultured SVZa neuronal progenitor cells (n = 110 cell cycles) as a function of the DIV. The data are presented as box plots. The median value (50th percentile) is marked by an ‘X’. The top and bottom of the box indicate the 75th and 25th percentiles, respectively. The horizontal bars above and below the box represent the maximum and minimum values observed. The DIV (X-axis) to which a cell cycle time is assigned is the day on which the cell under consideration divided. At 1 DIV, the cells were allowed to equilibrate in the incubator. 2 DIV was the first day on which videomicroscopic images were recorded and 3 DIV was the first day for which sufficient numbers of cell cycles ended to allow meaningful display of cell cycle time distribution. The median cell cycle times observed in this culture are similar to the cell cycle times measured in vivo for the neonatal RMS (Smith & Luskin, 1998). Scale bar, 10 µm in B (also applies to C).