To determine whether a sex difference exists in the production of hippocampal cells during adulthood, we examined proliferating cells and their progeny in adult rats using the thymidine analog bromodeoxyuridine (BrdU) combined with immunohistochemistry for markers of neurons and glia. Additionally, to determine whether ovarian hormones affect cell proliferation, we examined the numbers of BrdU-labeled cells at different estrous cycle stages and after ovarian steroid manipulation. Stereological analyses of the numbers of BrdU-labeled cells revealed that females produced more cells than males in the dentate gyrus but not in the subventricular zone. The production of new hippocampal cells in females appears to be affected by ovarian hormone levels; ovariectomy diminished the number of BrdU-labeled cells, an effect reversed by estrogen replacement. A natural fluctuation in cell proliferation was also noted; females produced more cells during proestrus (when estrogen levels are highest) compared with estrus and diestrus. Many of these cells acquired neuronal characteristics, including the formation of dendrites and expression of Turned-On-After-Division 64 kDa, a marker of immature granule neurons, and the calcium-binding protein calbindin, a marker of mature granule neurons. However, examination of the numbers of pyknotic cells and the numbers of BrdU-labeled cells at longer survival times revealed that many new cells in the dentate gyrus eventually degenerate. Consistently the number of labeled cells in females is no longer higher than that observed in males by 2 weeks after the last BrdU injection. These findings suggest that estrogen-enhanced cell proliferation during proestrus results in more immature neurons in the hippocampal formation of females compared with males and present the possibility that these new cells exert an important influence on hippocampal function.

The dentate gyrus continues to produce granule neurons throughout adulthood. The present study examined the extension of axons by adult-generated granule neurons into hippocampal area CA3. We injected the fluorescent retrograde tracers Fast blue (FB) and FluoroRuby (FR) into area CA3 of adult male rats at various times after the administration of 5Ј-bromo-2Ј-deoxyuridine (BrdU), a marker of proliferating cells and their progeny. We report that immature granule cells extend axons into CA3 as rapidly as 4-10 days after mitosis. A significant increase in the percentage of BrdU-labeled cells that were labeled with FB or FR was observed by 2 weeks after BrdU administration. This proportion remained roughly constant up to 3 weeks after BrdU-labeling, a time at which markers of a mature neuronal phenotype are expressed. BrdU-labeled cells that contained either FB or FR often were located far from the tracer injection site, indicating that these cells had extended relatively long axons. Collectively these results suggest that adult-generated granule neurons may influence normal hippocampal function, even at a very early stage after their production. J.

To determine whether exposure to fox odor alters granule neuron production, we examined proliferating cells and their progeny in the dentate gyrus of adult male rats exposed to trimethyl thiazoline, a component of fox feces. Additionally, to determine whether this effect is adrenal hormone-mediated, we examined animals exposed to fox odor after bilateral adrenalectomy and replacement with low levels of the endogenous glucocorticoid corticosterone. Stereologic analyses of the number of 5-bromo-2'deoxyuridine (BrdU) -labeled cells revealed that exposure to fox odor but not other, nonthreatening, odors (mint or orange) rapidly decreased the number of proliferating cells in the dentate gyrus. This effect is dependent on a stress-induced rise in adrenal hormones; exposure to fox odor resulted in an increase in circulating corticosterone levels and prevention of this increase (by means of adrenalectomy plus low-dose corticosterone replacement) eliminated the suppression of cell proliferation. Examination at longer survival times revealed that the decrease in the number of new granule cells in fox odor-exposed animals was transient; a difference was still detectable at 1 week after BrdU labeling but not at 3 weeks. In both fox and sham odor-exposed animals, many new cells acquired morphologic and biochemical characteristics of mature granule neurons. The majority of these cells expressed a marker of immature granule neurons (TuJ1) by 1 week after BrdU labeling and markers of mature granule neurons (calbindin, NeuN) by 3 weeks after labeling. These findings suggest that stressful experiences rapidly diminish cell proliferation by increasing adrenal hormone levels, resulting in a transient decrease in the number of adult-generated immature granule neurons.

In previous work, we have demonstrated that cell proliferation in the adult hippocampal formation is regulated by estrogen under both natural and experimental conditions. To determine the extent to which this regulation is affected by the dose or schedule of hormone treatment, or progesterone administration, we examined the impact of different acute and chronic ovarian hormone replacement regimens on cell production using the S-phase marker bromodeoxyuridine. Additionally, we investigated the long-term impact of surgical ovarian hormone depletion on the capacity of estrogen to stimulate cell proliferation and the production of new cells that express either TuJ1 (a marker of neuronal phenotype) or glial fibrillary acidic protein (GFAP; a marker of astroglial phenotype). Acute treatment with a moderate, but not a low or a high, dose of estrogen rapidly increased cell proliferation in ovariectomized (OVX) animals, an effect that was reversed by the administration of progesterone. In contrast, OVX animals that were chronically replaced with either estrogen alone (continuous or cyclic) or estrogen plus progesterone (cyclic) did not exhibit an estrogen-induced increase in cell proliferation 3 weeks following the onset of hormone replacement. In animals that were subjected to a prolonged absence of ovarian hormones, acute treatment with the moderate dose of estrogen failed to stimulate cell proliferation, and a decrease in the number of new cells expressing a neuronal phenotype was evident. Collectively, these results indicate that a prolonged reduction in ovarian hormones results in 1) a diminished responsiveness to estrogen over time in this system and 2) a decrease in neuron production that is unlikely to be reversible by standard regimens of hormone replacement.

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