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Since the evidence indicates that Brd U might affect the studied cell population, when its use as a birth-date marker was established, much attention was given to its dosage and the frequency of exposure, in order to minimize toxicity and avoid misleading results (e.g., [10, 11, 22–27]).In adult mammals, a dose of 50 mg/kg given daily for up to 12 days and doses of up to 300 mg/kg did not cause adverse physiological effects or toxic effects in dividing cells in the dentate gyrus [11, 28].The marker, which is retained in the postmitotic cell, enables the subsequent detection of a cohort of dividing cells that were labeled at a known time and therefore provides a tool by which to quantify neurogenesis, the effect of various conditions on it, and the fate of the new neurons at a later date (reviewed in [1]).The traditional approach to detection of cell proliferation is by incorporation of [H]-thymidine, using autoradiography [2].This technique has been used for decades to investigate various processes in the CNS (e.g., [3–6]) and has been invaluable for the study of the time of origin of neurons in various species, including birds [1], rodents [7], and nonhuman primates [8, 9].

Brd U can be immunocytochemically detected in vitro and in vivo, allowing the identification of cells that were dividing the period of Brd U exposure.In one of our previous studies, we encountered surprising results in a study with zebra finches (Taeniopygia guttata).To provide evidence for neuronal replacement, we used two cell birth markers in the same birds: Brd U, to label neurons born 1 or 3 months prior to social manipulation [32], and [ neurons to appear also in the recent study, but this was not the case.Furthermore, Brd U has been found to cause abnormal proliferation in vivo [19], although others found no cytotoxic effects on adult neurogenesis [20].In vitro, it has been shown to be toxic to neuronal precursors at the concentrations used in most labeling studies [21].

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