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First published online March 31, 2007
Journal of Experimental Biology 210, 1311-1324 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02758

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Social domination increases neuronal survival in the brain of juvenile crayfish Procambarus clarkii

Cha-Kyong Song^*, Laurel M. Johnstone, Manfred Schmidt, Charles D. Derby and Donald H. Edwards

Department of Biology, Program in Brains and Behavior, and Center for Behavioral Neuroscience, Georgia State University, Atlanta, GA 30302-4010, USA

View larger version (55K): [in this window] [in a new window]   Fig. 1. Experimental design. (A–E) Animal setup. (A) A juvenile crayfish, 2.0 cm from rostrum to telson. (B) Individual cages in an aquarium. Each cage is 5.5 cm in diameter. (C) Diagrams illustrating setup for animals in the Isolate and Pair groups. Rectangles and circles represent plastic aquaria and cages, respectively. Small ovals in each circle in the Isolate groups represent animals in isolation, while small triangles and small squares in each circle in the Pair group represent dominant and subordinate animals, respectively. Each gray object inside a circle represents a shelter. For the Pair group, 20 animals were transferred to new cages, two animals per cage, and only one shelter was provided in each cage. For the Isolate group, ten animals were individually transferred to new cages and a shelter was provided in each new cage. (D) Two animals in the Pair group (arrows) in a cage with one shelter. (E) One animal in the Isolate group (arrow) in a cage with one shelter. (F,G) Protocols for Cell Proliferation Test (F) and Cell Survival Test (G). Following the protocol in F and G, the animals in the Isolate and Pair groups were treated for cell proliferation and cell survival tests. A dotted line indicates an isolated condition; a broken line indicates a pairing condition; gray bars indicate immersion of animals in BrdU solution for 24 h. (F) Cell Proliferation Test. Animals were isolated for 6 weeks, paired or isolated for 1, 7 or 14 days, immersed in BrdU for 24 h on the last day of the pairing period, and sacrificed. (G) Cell Survival Test. All animals were isolated either for 5 or 9 weeks, paired or isolated for 7 or 14 days, immersed in BrdU for 24 h at the beginning of the pairing period, and sacrificed. A separate group of animals (age-control groups 1 and 2) was treated similarly (isolated for 5 or 9 weeks, immersed in BrdU for 24 h, and sacrificed immediately) but without any pairing. The age-control groups show the level of cell proliferation at the end of the isolation period, while the animals in the Isolate group show the level of cell survival after 7 days or 14 days of isolation. (H) Diagram of the cell division cycle. BrdU, a thymidine analog, labels cells in the S phase during which DNA replication occurs. H3P, a mitosis marker, labels cells in the M phase during which histone 3, a protein mediating the folding of DNA, is phosphorylated.

View larger version (63K): [in this window] [in a new window]   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.

View larger version (123K): [in this window] [in a new window]   Fig. 3. Cell proliferation and cell survival in `comma-shaped' neurogenic areas in cell clusters 9 (A–C) and 10 (D–F) after 1, 7 and 14 days of social interaction. BrdU+ cells are shown in green and H3P+ cells are shown in yellow or in orange, depending on the amount of BrdU/H3P colocalization (asterisks). Arrows indicate strand surrounding BrdU+ cells. For clarity, counted BrdU+ cells are outlined in white in B,C and E,F. Collapsed stacks of confocal images were taken at 0.5 µm intervals. The entire stack for each image was approximately 60–80 µm. Scale bar, 50 µm in all images. (A,B) Lines of BrdU+ cells are present in the ventral posterior part of cell cluster 9 and around the lateral antennular neuropil (LAN) after 1 day (A) and 7 days (B) of social interaction. BrdU+ cells were present in the same location in all cell proliferation tests irrespective of the length of social interaction. (C) After 14 days of survival time, a ball of BrdU+ cells was present in the posterior part of cell cluster 9 with a faint line of BrdU+ cells around the anterior edge of the LAN. (D–F) A ball of BrdU+ cells was present in cell cluster 10 near the olfactory lobe (OL) and accessory lobe (AL) after 1 day (D), 7 days (E) and 14 days (F) of social interaction. Regardless of the length of social interaction, all cell proliferation tests produced a ball of BrdU+ cells in the same location of cell cluster 10. As the survival time increased, the center of the ball of BrdU+ cells was fainter than the outer ring of BrdU+ cells, indicating the dilution of BrdU due to cell divisions. AL, accessory lobe; DCN, deutocerebral commissure neuropil; LAN, lateral antennular neuropil; OL, olfactory lobe.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Cell proliferation test for cell clusters 9 and 10. Number of BrdU+ cells in cluster 9 (A–C) and in cluster 10 (D–F) after 1 (A,D), 7 (B,E) and 14 (C,F) days of social interaction. Values are the sum of labeled cells from the two bilateral cell clusters 9 or 10 of a brain, and expressed as median ± interquartile range. (A,D) After 1 day, the number of BrdU+ cells did not differ for dominant (D), subordinate (S) and isolate (I) animals (Kruskal–Wallis test for overall difference: Pcluster9=0.87; Pcluster10=0.32). (B,E) After 7 days, isolated animals had significantly more BrdU+ cells in cluster 9 than subordinate animals but not more than dominant animals (Kruskal–Wallis test for overall difference: *P<0.05 (P=0.02); multiple comparison tests: PIvsS<0.05; PIvsD and PDvsS >0.05). There was a non-significant trend for the isolates to have more BrdU+ cells in cluster 10 than subordinate and dominant animals (Kruskal–Wallis test for overall difference: P=0.03; multiple comparisons test: PIvsD is >0.05 and PIvsS <0.10; PDvsS>0.10. (C,F) After 14 days, the number of BrdU+ cells did not differ among the groups (Kruskal–Wallis test for overall difference: Pcluster9=0.64; Pcluster10=0.91). (Numbers in parentheses indicate the number of animals examined.)

View larger version (12K): [in this window] [in a new window]   Fig. 5. Cell survival test for cell clusters 9 and 10. Number of BrdU+ cells in cluster 9 (A,B) and in cluster 10 (C,D) after 7 days and 14 days of social interaction. Values are the sum of labeled cells from the two bilateral cell clusters 9 or 10 of a brain, and expressed as median ± interquartile range. (A) 7 days of social interaction did not affect the number of surviving BrdU+ cells in cluster 9 (Kruskal–Wallis test for overall difference: P=0.28). The number of BrdU+ cells in the three experimental groups doubled relative to that of the age-control animals. (B) After 14 days of social interaction, dominant animals (D) had significantly more surviving BrdU+ cells in cluster 9 than subordinate animals (S) but were not different from isolate animals (I) (Kruskal–Wallis test for overall difference: *P<0.05 (P=0.029); multiple comparison tests: PDvsS<0.05; PIvsD and PIvsS>0.10). Over 14 days of survival time, the number of BrdU+ cells in the three experimental groups approximately doubled twice relative to that of the age-control animals. (C,D) The number of cluster 10 BrdU+ cells did not differ among social groups after surviving 7 days (C) or 14 days (D) of social interaction (Kruskal–Wallis test for overall difference: P7days=0.98, P14days=0.93). The number of BrdU+ cells in the 7-day survival groups was similar to that of the age-control animals (C), while the number of BrdU+ cells in the 14-day survival groups approximately doubled (D). (Numbers in parentheses indicate the number of animals examined.)

View larger version (10K): [in this window] [in a new window]   Fig. 6. Effect of social status on body growth rate. Values are median ± interquartile range of the average body growth rate from the beginning to the end of the experimental period, expressed as the increase in wet body mass per week in the experiment (g week–1). (A,B) Body growth rate in the Cell Proliferation Test groups. Body growth rate of animals was not influenced by 7 days (A) or 14 days (B) of social interaction (Kruskal–Wallis test for overall difference: P7days=0.052, P14days=0.36). (C,D) Body growth rate in the Cell Survival Test groups. (C) 7 days of social interaction did not alter the body growth rate of animals (Kruskal–Wallis test for overall difference: P=0.72). (D) After 14 days of social interaction, body growth rate was significantly higher for dominant than subordinate animals (Kruskal–Wallis test for overall difference: *P<0.05 (P=0.03); multiple comparisons test: PIvsD=0.39, PIvsS=0.065; PDvsS=0.02).