Time course of morphine's effects on adult hippocampal subgranular zone reveals preferential inhibition of cells in S phase of the cell cycle and a subpopulation of immature neurons.

Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (experiment 2). In experiment 1, mice received sham or morphine pellets (s.c., 0 and 48 h) and bromodeoxyuridine (BrdU) 2 h prior to sacrifice (24, 72 or 96 h). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 h of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 h. In experiment 2, nestin-green fluorescent protein (GFP) mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 h, sacrifice 96 h) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells was less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage.

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

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.

Knockout of the mu opioid receptor enhances the survival of adult-generated hippocampal granule cell neurons.

Recent evidence suggests that mu opioid receptors (MOR) are key regulators of hippocampal structure and function. For example, exogenous MOR agonists morphine and heroin negatively impact hippocampal function and decrease adult hippocampal neurogenesis. Here we explored the role of MOR in the birth and survival of hippocampal progenitor cells by examining adult neurogenesis in mice that lack MOR. Adult male mice lacking exon 1 of MOR were injected with the S phase marker bromodeoxyuridine (BrdU) and killed either 2 hours or 4 weeks later to evaluate proliferating and surviving BrdU-immunoreactive (IR) cells, respectively, in the adult hippocampal granule cell layer. Wild-type (WT), heterozygote, and homozygote mice did not differ in the number of BrdU-IR cells at a proliferation time point. However, 4 weeks after BrdU injection, heterozygote and homozygote mice had 57% and 54% more surviving BrdU-IR cells in the hippocampal granule cell layer as compared with WT mice. A decrease in apoptosis in the heterozygote and homozygote mice did not account for the difference in number of surviving BrdU-IR cells since there were no alterations in number of pyknotic, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive, or activated caspase 3-IR cells compared with WT. In concordance with the increased numbers of granule cells maturing into neurons, heterozygote and homozygote mice had larger hippocampal granule cell layers and increased numbers of granule cells. These findings indicate that MOR may play a role in regulating progenitor cell survival and more generally encourage further exploration of how MOR activation can influence hippocampal structure and function.