Table 1.

Suggested parameters for in vivo neurogenesis studies using thymidine analogs to detect newly generated cells

	Thymidine analog injection frequency	Postinjection examination interval	Cells identified	Key considerations
Proliferation	50 mg/kg BrdU or equimolar equivalent of IdU or CldU	2 h after last injection	Cohort of proliferating cells	Validate against false-positive artifacts as described in text
	1–3 injections at 2-h intervals			Conditions of low proliferation (such as aging) may require more injections to establish a detectable cohort
Cell-cycle reentry	1–3 injections of IdU at 2-h intervals, then:	Variable interval between analogs based on study question	Subpopulation of rapidly dividing cells	Administration of analogs must be equimolar to allow comparison between each cohort of proliferating cells
	1–3 injections of CldU at 2-h intervals	2 h after last injection		Order of analog administration is unimportant and could be reversed to CldU first, then IdU
				Interval between delivery provides an index of cycle length
Fate specification	50 mg/kg BrdU or equimolar equivalent of IdU or CldU	Approximately 3–10 days after last injection	Recently generated cells coexpressing early lineage markers	Validate that detection is in appropriate cell compartments for confirming true coexpression as described in text
	1–3 injections at 2-h intervals			Lineage commitment may produce a transient overlap of phenotypic markers
Neuronal maturation	50 mg/kg BrdU or equimolar equivalent of IdU or CldU	2–4 wk after last injection	Newly generated cells differentiating into phenotypically mature neurons	Cells observed may express various differentiation markers, requiring multiple labeling approaches
	1–3 injections at 2-h intervals			Cells observed reflect the net result of continued proliferation following labeling and cell loss
Survival	50 mg/kg BrdU or equimolar equivalent of IdU or CldU	Varies depending on study question	Intervals of <10 days address short- and long-term survival	Study could combine IdU/CldU delivery to provide baseline data for comparison to proliferation within the same animal under the same conditions
	1–3 injections at 2-h intervals		Intervals >30 days assess integration	Assessment of integration should be combined with a functional readout
Rare/valuable subjects	1–3 injections of IdU at 2-h intervals, then:	2–4 wk after IdU injection (first cohort)	Differentiation from cells in the first cohort	Permits efficient use of rare or valuable subjects for measuring both proliferation and differentiation
	1–3 injections of ldU at 2-h intervals	2 h after CldU injection (second cohort)	Proliferation from cells in the second cohort	Permits use of same animal for increased statistical power

BrdU, Bromodeoxyuridine; IdU, iododeoxyuridine; CldU, chlorodeoxyuridine.