Testosterone has been shown to enhance hippocampal neurogenesis through increased cell survival, but which stages of new neuron development are influenced by testosterone remains unclear. Therefore, we tested the effects of sex steroids administered during three different periods after cell division in the dentate gyrus of adult male rats to determine when they influence the survival of new neurons. Adult male rats were bilaterally castrated. After 7 days of recovery, a single injection of bromodeoxyuridine (BrdU) was given on the first day of the experiment (Day 0) to label actively dividing cells. All subjects received five consecutive days of hormone injections during one of three stages of new neuron development (days 1–5, 6–10, or 11–15) after BrdU labeling. Subjects were injected during these time periods with either testosterone propionate (0.250 or 0.500 mg/rat), dihydrotestosterone (0.250 or 0.500 mg/rat), or estradiol benzoate (1.0 or 10 µg/rat). All subjects were euthanized sixteen days later to assess the effects of these hormones on the number of BrdU-labeled cells. The high dose of testosterone caused a significant increase in the number of BrdU-labeled cells in the hippocampus compared to all other groups, with the strongest effect caused by later injections (11-15 days old). In contrast, neither DHT nor estradiol injections had any significant effects on number of BrdU-labeled cells. Fluorescent double-labeling and confocal microscopy reveal that the majority of BrdU-labeled cells were neurons. Our results add to past evidence that testosterone increases neurogenesis, but whether this involves an androgenic or estrogenic pathway remains unclear.
