Addition of New Cells to the Olfactory Bulb of Adult Zebrafish

We have been examining patterns of cell proliferation in the brain of adult zebrafish. Understanding this process in fish may lead to important insights due to the tremendous regenerative capabilities of these animals. Fish were exposed to a 1% solution of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) for 1 hr before being returned to small aquaria that received frequent water changes. Animals were overanesthetized and perfused with Bouin's fixative solution after two survival periods (4 hr or 3-4 weeks). Paraffin immunohistochemistry using a monoclonal antibody against BrdU was used to visualize the newly generated cells. Quantitative analyses were performed on serial, 10-micron sections from 4 animals for each survival group. Statistical determinations were based on the nonparametric Mann-Whitney U test. The average number of BrdU-labeled nuclear profiles in the bulbs, from analysis of every 5th section, was significantly different between the two groups (22.0 +/- 6.8 [SEM] for the 4-hr group and 136.7 +/- 11.3 for the 4-wk group; p < 0.05). The volumes of the bulbs, however, were not different between the two groups (p > 0.5). These data indicate that either cells divided repeatedly during the longer survival period or cells migrated into the bulb from other brain regions. To examine this phenomenon further, the location of the new cells was analyzed in three mid-bulb sections (20 microns apart) from each animal. Both the area and number of labeled nuclei in each lamina were measured to obtain an average profile density. Comparison of the 4-hr and the 4-wk groups showed that density was significantly greater in all bulb layers in the long survival group (p < 0.05 for all). In the 4-hr survival group, cells were found mainly in the olfactory nerve layer. When examined after 4 wk, proportionately more labeled cells were found in the internal cell layer. This addition of new cells could be a result of neurogenesis, gliogenesis, and/or angiogenesis. We are currently performing double-labeling experiments to determine the types of cells that are added to the adult bulb. In addition, our future plans include investigating the origin of these cells and the signals that direct their formation.