Life-long neurogenesis is a characteristic feature of many vertebrate and invertebrate species. are influenced by environmental factors (Sandeman and Sandeman 2000 Ayub et al. 2011 seasonality (Hansen and Schmidt 2004 and time of day (Goergen et al. 2002 Endogenous signals such as serotonin and nitric oxide play important roles in regulating the numbers of labeled cells (Beltz et al. 2001 Benton et al. 2007 2008 indicating that BX-795 these BX-795 factors also influence the cell cycle of the neuronal precursors and/or the survival of the newborn cells. Figure 1 Neurogenesis in the adult crayfish (daughter cells migrate away from the niche towards the proliferation zones (Benton et al. 2011 2013 BX-795 Nevertheless the niche is not depleted as the animals grow and age. Based on our latest studies we have therefore concluded that the niche is not a closed system and that there must be an extrinsic source of first-generation neuronal precursors. Experimental evidence to date indicates that the innate immune (hematopoietic) system may be the source of these neuronal precursor cells (Benton et al. 2011 2012 2013 Beltz et al. 2011 Chaves da Silva 2013 In vertebrate and invertebrate species adult-born neurons differentiate and are incorporated into brain circuitry. The survival and incorporation of newborn cells into brain circuits can be explored with long-survival time experiments. Animals are exposed to BrdU for a specific time period and then left for FACD several months before sacrifice and examination of the brains for the presence of BrdU-labeled cells. The persistence of labeled cells in the brain many months after exposure to BrdU is an BX-795 indication that the cells may have differentiated and become incorporated into the brain. Differentiation of BrdU-labeled cells can then be assessed by examining the anatomical development of axons and dendrites acquisition of physiological properties and expression of transmitters normally found in mature neurons of the same type. Studies in a number of crustacean species have provided strong evidence that the surviving adult-born neurons are incorporated as new functional units. In adult shore crabs (of newborn neurons and thus their availability for incorporation into the brain circuitry. It is worth noting that was measured in these experiments which is not comparable to the wheel running used in prior studies in rodents that examined the effects of locomotion on adult neurogenesis. Our results in crayfish therefore suggest that fewer BX-795 neurons survive in “couch potatoes” than in physically active individuals even if this activity does not include aerobic exercise. The results presented here provide the first quantitative data in support of a period of cell death following cell proliferation in the adult crustacean brain. Further a timeline for differentiation of the new neurons beginning at 4 weeks after BrdU labeling is established. Finally these studies test the hypothesis that physical activity in crayfish as in mammalian species influences the number of cells that survive and differentiate into neurons. MATERIALS AND METHODS Proliferation survival and differentiation studies Animal Care and Treatment Freshwater crayfish (Malacostraca Decapoda Parastacidae; supplied by Yabby Growers and Traders Bulahdelah Australia) were bred in the Wellesley Animal Care Facility. Egg-bearing female crayfish were housed at 21± 1°C on a 12:12 light:dark cycle in aquaria supplied with artificial pond water adjusted for mineral balance and pH (ddH2O with NaHCO3 Seachem Equilibrium trace elements from Seachem Laboratories Inc Coving GA pH = 7.6). Eggs from the same mother hatched and developed into adult stage II (ADII) crayfish which are not yet sexually differentiated (Sandeman and Sandeman 1991 ADII crayfish in these studies had a carapace length (CL) of 3-4 mm; using siblings from the same brood ensured the least possible genetic variability among the crayfish. BrdU labeling Crayfish were incubated in 2 mg/mL BrdU in artificial pond water for 5 days after which the crayfish were transferred into an aquarium of artificial pond water containing gravel artificial plants and pieces of 1″ diameter plastic tubing for shelter. Previous research has.