Evaluation of 5-ethynyl-2′-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system

Recently, a novel method for detection of DNA synthesis has been developed based on the incorporation of 5-ethynyl-2′-deoxyuridine (EdU), a thymidine analogue, into cellular DNA and the subsequent reaction of EdU with a fluorescent azide in a copper-catalyzed [3 + 2] cycloaddition ("Click" reaction). In the present study, we evaluated this method for studying cell proliferation in the adult central nervous system in comparison with the "gold standard" method of 5-bromo-2′-deoxyuridine (BrdU) staining using two behavioral paradigms, voluntary exercise and restraint stress. Our data demonstrate that the number of EdU-positive cells in the dentate gyrus of the hippocampus (DG) slightly increased in an EdU dose-dependent manner in both the control and voluntary exercise (running) mouse groups. The number of EdU-labeled cells was comparable to the number of BrdU-labeled cells in both the control and running mice. Furthermore, EdU and BrdU co-localized to the same cells within the DG. Voluntary exercise significantly increased the number of EdU- and BrdU-positive cells in the DG. In contrast, restraint stress significantly decreased the number of EdU-positive cells. The EdU-positive cells differentiated into mature neurons. EdU staining is compatible with immunohistochemical staining of other antigens. Moreover, our data demonstrated EdU staining can be combined with BrdU staining, providing a valuable tool of double labeling DNA synthesis, e.g., for tracking the two populations of neurons generated at different time points. In conclusion, our results suggest that EdU staining is a fast, sensitive and reproducible method to study cell proliferation in the central nervous system.