Cytokine regulation of adult neurogenesis: The immune system provides neuronal precursors for adult neurogenesis

.......................................................................................................5 Introduction................................................................................................6-17 Adult neurogenesis: a controversial history.............................................................7-8 BrdU labeling to track adult neurogenesis..............................................................8-9 Crayfish as a non-vertebrate model system for adult neurogenesis.................................9-11 Figure 1: Confocal microscopical image of the ventral side of the crayfish brain.........9 Figure 2: Schematic showing the neuronal precursor lineage................................10 The immune system as the likely source of neuronal precursors.................................11-12 The crayfish immune system...........................................................................12-14 Figure 3: Schematic of the crayfish immune system...........................................13 Regulation of the crayfish immune system...........................................................14-15 Current Experiments....................................................................................15-17 Materials and Methods................................................................................18-22 Animals.........................................................................................................18 Injection protocols.......................................................................................18-19 Experimental series 1: BrdU only experiment....................................................18 Experimental series 2: Simultaneous BrdU and r-AST1 injection paradigm................18 Experimental series 3: 24-hour delay between BrdU and r-AST1 injection paradigm....19 Dissection protocols..........................................................................................19 Immunohistochemical labeling.........................................................................19-20 Data Collection...............................................................................................20 Figure 4: Schematic of dissected tissue regions and labeling sites..........................20 Data analysis..................................................................................................21 Determining the number of BrdU-labeled cells in a niche....................................21 Figure 5: Representative image of a niche with a BrdU-labeled cell................21 Determining BrdU-labeling in the APC and HPT..........................................21-22 Figure 6: Representative 2D, stacked 4 μm think APC/HPT image............. ...22 Determining EdU-labeling in Hemocytes........................................................22 Statistical Analysis..................................................................................22 Results.....................................................................................................23-31 Figure 7: Injection of r-AST1, an immune system modulator, alters the timeline by which BrdU-labeled cells appear in the niche...................................................26 Figure 8: The percent of BrdU-labeled cells in the immune tissues (APC and HPT) decreases prior to an increase of EdU-labeled cells in the circulating 3 hemolymph and the subsequent reappearance of BrdU-labeled cells in the niche.........27 Figure 9: Figure 9: The percent of BrdU-labeled cells in the immune tissues (APC and HPT) remains constant on days 1 and 2 and then decreases sharply prior to the second, third and fourth labeling peaks in the niche...................28 Figure 10: Figure 10: Following a BrdU injection on day 0 and a r-AST1 injection on day 1 the percent of BrdU-labeled cells in the immune tissues (APC and HPT) fluctuates on days 1-7 before dropping on day 8, prior to the peak in BrdU labeling in the niche on day 14...................................................................29 Figure 11: BrdU labeling patterns in the APC across all three experimental protocols...........30 Figure 12: Figure 12: BrdU labeling patterns in the HPT across all three experimental protocols.......................................................................................................31 Discussion................................................................................................32-43 Conclusions.............................................................................................. 42-43 Future experiments...........................................................................................43