Fig. 2. `Comma-shaped' neurogenic areas in the juvenile crayfish brain. (A) Diagram of the juvenile crayfish brain identifying the neuropils and cell clusters [modified from Sandeman et al. (Sandeman et al., 1992)] as well as the neurogenic areas in the deutocerebrum (this study). Primary sensory inputs from olfactory receptor neurons (ORN) in olfactory organs enter the olfactory lobe (OL). The cell bodies of the olfactory local and projection interneurons comprise cell clusters 9 and 10, respectively. Groups of BrdU+ cells (green) appear in cell clusters 9 and 10 and lines of BrdU+ cells, surrounded with a strand-like structure (violet), meet at a patch of cells (blue) near the posterior accessory lobe (AL). 9, cell cluster 9; 10, cell cluster 10; AMPN, anterior median protocerebral neuropil; AN, antennal neuropil; DCN, deutocerebral commissure neuropil; LAN, lateral antennular neuropil; PMPN, posterior median protocerebral neuropil. (B) Juvenile crayfish brain showing robust cell proliferation in cell clusters 9 and 10. The image is taken with the camera attached to the epifluorescence microscope. Each dotted area (orange for cluster 9 head and white for cluster 10 head) indicates regions where a ball of BrdU+ cells is present within the cluster boundary. Each broken area indicates a region around the LAN (orange for cluster 9 tail) or around the posterior AL (white for cluster 10 tail) where `tails' of BrdU+ cells are located. Scale bar, 50 µm. (C–F) Collapsed stacks of confocal images taken at 1.0 µm intervals in (C) and 0.5 µm intervals in (D–F). The entire stack for each image was approximately 60 µm. (C) A strand labeled with anti-tubulin (violet, arrows), surrounds lines of BrdU+ cells (green) from clusters 9 and 10, and a patch of cells (arrowhead). Scale bar, 50 µm. (D) High-resolution image of the patch of cells (green, anti-BrdU; blue, Hoechst, nuclear staining; violet, anti-tubulin) indicated by an arrowhead in (C). The patch of cells and a few BrdU+ cells are surrounded by a tubulin+ strand. Scale bar, 50 µm. (E) Lines of BrdU+ cells (green) in the tail of cluster 9 are surrounded by a strand faintly labeled with anti-H3P (red, arrows). BrdU+ cells outside the strand (arrowheads) were not included in our counts of labeled cells. Asterisks indicate H3P+ (mitotic) cells. Scale bar, 50 µm. (F) Lines of BrdU+ cells (green) in the tail of cluster 10 are surrounded by a strand faintly labeled with anti-H3P (red, arrows). A BrdU+ cell on the border of AL (arrowhead) was not included in our counts of labeled cell. Asterisk indicates H3P+ (mitotic) cells. Scale bar, 50 µm.