Elsevier

Neuroscience

Volume 146, Issue 1, 25 April 2007, Pages 108-122
Neuroscience

Cellular neuroscience
Determination of key aspects of precursor cell proliferation, cell cycle length and kinetics in the adult mouse subgranular zone

https://doi.org/10.1016/j.neuroscience.2006.12.064Get rights and content

Abstract

Neurogenesis studies on the adult mouse hippocampal subgranular zone (SGZ) typically report increases or decreases in proliferation. However, key information is lacking about these proliferating SGZ precursors, from the fundamental—what dose of bromodeoxyuridine (BrdU) is appropriate for labeling all S phase cells?—to the detailed—what are the kinetics of BrdU-labeled cells and their progeny? To address these questions, adult C57BL/6J mice were injected with BrdU and BrdU-immunoreactive (IR) cells were quantified. Initial experiments with a range of BrdU doses (25–500 mg/kg) suggested that 150 mg/kg labels all actively dividing precursors in the mouse SGZ. Experiments using a saturating dose of BrdU suggested BrdU bioavailability is less than 15 min, notably shorter than in the developing mouse brain. We next explored precursor division and maturation by tracking the number of BrdU-IR cells and colabeling of BrdU with other cell cycle proteins from 15 min to 30 days after BrdU. We found that BrdU and the Gap2 and mitosis (G2/M) phase protein pHisH3 maximally colocalized 8 h after BrdU, indicating that the mouse SGZ precursor cell cycle length is 14 h. In addition, triple labeling with BrdU and proliferating cell nuclear antigen (PCNA) and Ki-67 showed that BrdU-IR precursors and/or their progeny express these endogenous cell cycle proteins up to 4 days after BrdU injection. However, the proportion of BrdU/Ki-67-IR cells declined at a greater rate than the proportion of BrdU/PCNA-IR cells. This suggests that PCNA protein is detectable long after cell cycle exit, and that reliance on PCNA may overestimate the length of time a cell remains in the cell cycle. These findings will be critical for future studies examining the regulation of SGZ precursor kinetics in adult mice, and hopefully will encourage the field to move beyond counting BrdU-IR cells to a more mechanistic analysis of adult neurogenesis.

Section snippets

Animals

Adult, male C57BL/6J mice (initial weight 23–27 g, 9–11 weeks old; Jackson Laboratories, Bar Harbor, ME, USA) were used. Mice were group housed (maximum five/cage) in a facility approved by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) at the University of Texas Southwestern Medical Center, with a 12-h light/dark cycle and with free access to food and water. Mice were acclimated to vivarium conditions for at least 1 week prior to

Dose response of BrdU in adult C57BL/6J mice

To determine the relationship between BrdU dose and BrdU-IR cell counts in the adult mouse SGZ, we performed two separate dose response experiments. In the first experiment (Fig. 1a, experiment I; Fig. 1b), we compared 50 and 150 mg/kg doses (Hayes and Nowakowski 2002, Kronenberg et al 2003, Mandyam et al 2004) with lower (25 mg/kg) and higher (300, 500 mg/kg) doses. In the second experiment (Fig. 1a, experiment II; Fig. 1c), we directly compared 50, 100 and 150 mg/kg doses. In both dose

Discussion

Key aspects of precursor cell division from the adult mouse SGZ are revealed in this study. We identify a dose of BrdU sufficient to saturate the S phase cohort of SGZ precursors in the adult mouse. Using a saturating dose of BrdU, we highlight the tendency of SGZ cells and other proliferative subregions of the dentate gyrus to divide in clusters. Our time course analysis shows the number of cells, clusters, and cells per cluster increasing and then decreasing from 0.25–720 h after BrdU

Acknowledgment

This work was supported by a postdoctoral Ruth Kirschstein National Research Service F32 Award from NIDA (DA018017; C.D.M.), a Ruth Kirschstein National Research Service T32 Award from NIDA (DA007290, G.C.H.), and grants from the National Institute on Drug Abuse (DA016765), National Institute of Mental Health (MH66172), National Institute of Aging and National Alliance for Research on Schizophrenia and Depression to A.J.E. Other acknowledgments: The authors thank Rebecca Norris and Jessica Yee

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    Present address: Committee on the Neurobiology of Addictive Disorders at the Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

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